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Contents
Unit I: General Microbiology 1-84

1. Historical Introduction 3
2. Microscopy and Morphology of Bacteria 9
3. Growth, Nutrition and Metabolism of Bacteria 22
4. Sterilisation and Disinfection 28
5. Culture Media 40
6. Culture Methods 46
7. Identification of Bacteria 51
8. Bacterial Taxonomy 59 ·-:,<
9. Bacterial Genetics 61
10. Microbial Pathogenicity 77
Unit II: Immunology 85-170

,...H. Immunity 87
12. Antigen 93
13. Antibodies-Immunoglo bulins 97
14. Antigen-Antibody Reactions 105
15. Complement System 121
16. Structure and Functions of Immune System 127
17. Immune Response 136
18. Immunodeficiency Diseases 147
19. Hypersensitivity 152
20. Autoimmunity 160
21. Transplantation and Tumour Immunity 163
22. Immunohaematology 168
Unit III: Systemic Bacteriology 171-408

23. Staphylococcus 173
24. Streptococcus and Enterococcus 184
25. Pneumococcus 196
26. Neisseria and Moraxella 201
27. Corynebacterium 210
28. Bacillus 219

29. Clostridium 226
30. Nonsporing Anaerobes 237
31. Enterobacte riaceae 243
32. Shigella 257
33. Salmonella 262
_34. Vibrio 276
35. Campylobac ter, Helicobacter , Mobiluncus 287
. 36. Pseudomona s, Stenotropho monas, Burkholderi a 292
37. Yersinia, Pasteurella, Francisella 300
38. Legionella 308
39. Haemophilu s and Gardnerella 311
40. Bordetella 319
41. Brucella 324
42. Mycobacter ium Tuberculosis 330
43. Non-Tuberc ulous Mycobacteri a 346
44. Mycobacter ium Leprae 352
I
45. Spirochaetes 360
46. Mycor,lasma and Ureaplasma 375
47. Actino~yce tes 381
48. Miscellaneo us Bacteria 386
49. Ricketfsia, Orientia, Coxiella, Ehrlichia, Bartonella 393
50. Chlamydia and Chlamydop hila 402
Unit IV: Virology 409-540

51. General Properties of Viruses 411
52. Virus-Host Interactions 4-25
53. Bacteriopha ge 434
54. Poxviruses 439
55. Herpesvirus es 443
56. Adenovirus es 452
57. Picornaviruses 455
58. Orthomyxov iruses 464 /
59. Paramyxovi ruses 472
60. Arboviruses 479
61. Rhabdoviru ses 489
62. Hepa ti tis Viruses 497
63. Retroviruses : HIV 509
64. Miscellaneo us Viruses 523
65. Oncogenic Viruses 536

Unit V: Mycol ogy _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __ 541-566
66. Medica l Mycolo gy 543
Unit VI: Clinical Microbiology _ _ _ _ _ _ _ _ _ _ _ _ _ _ __ 567-646

67. Norma l Microb ial Flora of the Human Body 569
68. Sore Throat and Pneum onia 573
rv-'69. Urinary Tract Infectio ns 577
\~ O. Diarrho eal Disease s 581
71. Mening itis 589
72. Bactera emia, Septica emia and Infectiv e Endoca rditis 593
73. Fever of Unkno wn Origin (FUO) 597
74. Sexuall y Transm itted Disease s 600
75. Health Associ ated Infectio n 605
76. Prophy lactic Immun isation 610
77. Antimi crobial Therap y 615
78. Antimi crobial Sensiti vity Testing 621
79. Molecu lar Detecti on of Microo rganism s 628
80. Bacteri ology of Water, Milk and Air 632
81. Hand Hygien e 638
82. Biomed ical Waste Manag ement 640
83. Vehicles and Vectors 644
Appendix
Index lll

GENERAL MICROBIOLOGY
1. Historical Introduction ................................................................................... 3
2. Microscopy and Morphology of Bacteria ...................................................... 9
3. Growth, Nutrition and Metabolism of Bacteria .......................................... 22
4. Sterilisation and Disinfection ..................................................................... 28
5. Culture Media .. .......................................................................................... 40
6. Culture Methods ........................................................................................ 46
7. Identification of Bacteria ............................................................................. 51
8. Bacterial Taxonomy .................................................................................... 59
9. Bacterial Genetics ...................................................................................... 61
10. Microbial Pathogenicity ............................................................................... 77

Chapter 1
HISTORICAL INTRODUCTION
I. Introduction
II. Scientific Development of Microbiology
A. Louis Pasteur B. Joseph Lister
C. Robert Koch D. Paul Ehrlich
Ill. Virology
IV. Immunology
V. Chemotherapeutic Agents
I. INTRODUCTION of bacteria and communicated his findings to the Royal

Microbiology is the science of living organisms that are Society of London. The significance of these observations
only visible under the microscope. Medical microbiology was not then realised. In 1678, Robert Hook developed
deals with the causative agents of infectious diseases compound microscope and confirmed Leeuwenhoek's
of man, his reaction to such infections, the ways in observations.
which they produce disease and the methods for their To Leeuwenhoek the world of those · "little
diagnosis. animalcules" represented only a curiosity of nature and
The disease is transmfrted by contact, from man to their role in disease was not realised. Their observations
man, was known since biblical times. Vara and Columel/a lay dormant almost for a century. It was only some two
(first century B.C.) postulated that diseases were caused by centuries later that the importance of microorganisms in
invisible organisms which they called 'Animalia minuta'. medicine was recognised.
Fracastorius of Verona (1546) proposed a contagium The earliest discovery of pathogenic • role of
vivum (living germs) as a possible cause of infectious microorganisms probably goes to Augustina Bassi
disease. Von Plenciz (1762) suggested that each disease (1835), who showed that the muscardine disease of silk
was caused by a separate agent. Kircher (1659) reported worms was caused by a fungus. Oliver Wendell Holmes
finding minute worms in the blood of plague patients, (1840) and Ignaz Semmelweis (1846) had independently
but it is more likely that he observed only blood cells concluded that puerperial sepsis was transmitted by the
with the equipment available to him. contaminated hands of obstetricians, nurses and medical
As microbes are invisible to the unaided eye, definitive students and this could be prevented by washing hands
knowledge about them had to await till microscopes were in antiseptic solution.
developed. The credit for observation and description of Needham (1745), an Irish priest, believed in
bacteria goes to Antony van L eeuwenhoek (1632-1723), a spontaneous generation (abiogenesis) of microorganisms
draper from Delft, Holland, whose hobby was grinding from the decomposing organic matter. He had shown
lenses. He described three major morphological forms in his experiments· that microbes developed in closed
3
4 --------------------------------,:c-P--1
· '&nit I: General Microbiology
flasks of previously heated putrescible fluids. This view .-e-:-Live vaccin~. He introduced attenuated live vaccine
was opposed by Spallanzani (1769) who found no such for prophyl ctic use. An accidental observation that
generation of microorganism after long heating of the chofera bacillus cultures left for several~
putrescible fluids. He proved that organic contents of l ~ h ~ t y but retained their ability to
flask supported the microbial growth when exposed to protectTiiebirds against subsequent infection by them
air. Later on, Pasteur proved conclusively that all forms led to the concept of attenuation and development
of life, even microbes, arose only from their like and of live vaccines. He attenuated the anthrax bacillus
not de novo. by incubation at high temperature (42°-43°C) and
proved that inoculation of such bacilli in animals
induced specific protection against anthrax. He
II. SCIENTIFIC DEVELOPMENT convincingly demonstrated the protective role of
OF MICROBIOLOGY anthrax vaccine in a public experiment (1881) where
Scientific development of microbiology was ushered by vaccinated sheep, goats and cows were challenged
Louis Pasteur, perfection on microbiological studies by with a virulent anthrax bacillus culture. All the
Robert Koch, the introduction of antiseptic surgery by vaccinated animals survived while an equal number
Lord Lister and the contributions of Paul Ehrlich m of unvaccinated control animals died. Pasteur coined
chemotherapy. the term vaccine for such prophylactic preparations to
commemorate the first of such preparations, namely,
A. Louis Pasteur (1822-1895) cowpox, employed by Jenner for protection against
He was a trained chemist of France. smallpox.
His studies on fermentation led him Pasteur's de:elopme_nt of a vaccine f o ~
to take interest in microbiology. marked a milestone m development of 1mmum 1 ,
He established that fermentation in ~ e . He obtained the fixed virus of rabies
was caused by microbial agents. h5yserial intracerebral passage in rabbits. The rabies
He further noted that different vaccine was prepared by drying pieces of spinal cord
types of fermentations_ were from rabbits infected with fixed virus. This work
associated with gjfferent kinds of was acclaimed throughout the world. The Pasteur
microorganisms He is also known Fig. 1.1 Louis Pasteur Institute, Paris, was built by public contribution for
asfath~ ogy. the preparation of vaccines and for the investigation
of infectious diseases.
Important Contributions of Louis Pasteur B. Joseph Lister (1827-1912)

in Microbiology He was a Professor of Surgery in Glasgow Royal Infirmary.
y-Development of methods and techniques of He applied Pasteur's work and introduced antiseptic
bacteriology. techniques in surgery (1867) effecting a pronounced drop
2. Proved conclusively that all forms of life, even in mortality and morbidity due to surgical sepsis. It was
microbes, arose only from their like and not de a milestone in the evolution of surgical practice from the
novo. era of 'laudable pus' to modern aseptic techniques. His
3. Pasteur also had to face challenge from Pouche!, who antiseptic surgery involved the use of carbolic acid. He _/4i
was a proponent of spontaneous generation theory of
microbe. Pasteur disproved the view by demonstrating
.!'.'1M as father of antiseptic surgery.
is • .'!-';::; \
the ubiquity of microorganisms in the air by his C. R~ Koch (1843-1910) \'-f
experiments performed in the swan-necked flasks. Robert Koch was a German
Introduction of sterilisation techniques and
7 development of steam steriliser, autoclave and hot-
general practitioner. He is
also known as the father of
air oven. bacteriology. His contributions
5. Studies on anthrax, chicken cholera and hydrophobia. are as follows:
During studies on rabies, though Pasteur could not 1. Perfected bacteriological
isolate any microorganism from man and dog but techniques and introduced
suggested that the causative agent of rabies was too methods for isolation of
Fig. 1.2 Robert Koch
small to be seen by microscope. pure strains of bacteria.

---------------------- ----- --
,;:~
Ch 1: Historical Introduction ------------'th!~------------------ 5
2. Introduced methods of obtaining bacteria in pu"(e Important Discoveries by other Scientists

cultures using solid media. / Lepra bacillus Hansen (1874)
3. Introduced staining techniques. Gonococcus Neisser (1881)
4. Discovered the anthrax bacillus (1876), tubercle Diphtheria bacillus Klebs (1883), Loeffler (1884)
... '""==== -
~ s (1882) and the ch;?_lera v1brios ,0883). Pneumococcus Frankel ( 1886)
5. Koch) postulates: According to Koch s postulates, Meningococcus Weichselbum (1887)
a microor_ganism can be accepted as the causative Diphtheria toxin Roux and Yersin (1888)
a~ent cl an infectious disease only if the following Tetanus bacillus Kitasato (1889)
~a. Plague bacillus Yersin (1890)
,)!,) The organism should be constantly associated
with the lesions of the disease.
(ii) It shouldbe possible to ISolate the organism in III. VIROLOGY
/ pure culture from the lesions of the disease. The existence of viruses became evident during the
(iii) The isolated organisnr(mpure cultureywhen closing years of nineteenth century, when many infectious
/ inoculated in suitable lap.Qfatory animals should diseases had been proved to be caused by bacteria. But
produce a similar disease. - there remained a large number of infectious diseases for
(iv) It sliould be possible o re-isolate the organism which no bacterial cause could be established. During
/ in pure cultli're from he lesions produced in the studies on rabies, Pasteur suspected that the causative
x erimental animal agent could be too small to be seen under microscope. The
An a ditional criterion introduced subsequently existence of such ultramicroscopic microbes was proved
requires that specific antibodies to the organisms should be when Ivanovosky (1892) reproduced mosaic disease in
demonstrable in the serum of patients. These postulates tobacco plant by applying the juice of diseased plants
have proved to be useful in confirming doubtful claims to the healthy leaves. Loeffler and Frosch (1898) observed
made regarding the causative agents of infectious diseases. that the causative agent of foot and mouth disease in
However, it may not always be possible to satisfy all the cattle could also pass through a bacterial filter. The first
postulates in every case. An important example of not human disease proved to be of viral origin was yellow fever.
fulfilling the Koch's postulates is lepra bacillus. It was discovered by Walter Reed (1902) in Cuba and he
6. Ifg_clfs phenoll1£llon: Robert Koch observed that established that the disease is transmitted through the bite
guineapigs already infected with tubercle bacillus of infected mosquitoes. Landsteiner and Popper (1909)
respoiUied with an exaggerateci inffammatory demonstrated that poliomyelitis was due to filterable
response when injected with tbe tubercle bacillu@ virus and they also transmitted the disease experimentally
its protein. This hypersensitivity reaction is called to monkeys. Goodpasture (1930) developed techniques
Koch's phenomenon. of viral cultivation in chick embryos. The application
-------=------ of tissue culture expanded the scope of virological
techniques considerably. Though the larger viruses could
D. Paul Ehrlich (1854-1915) be seen under light microscope after appropriate staining,
Paul Ehrlich was a German scientist and is also known detailed morphological study of viruses became possible
as father of chemotherapy. His contributions are as only with the introduction of electron microscope by
follows: Ruska (1934).
I. Applied stains to cells and tissues for study of their The possibility of virus causing cancer was first put
functions. forth by Ellerman and Bang (1908) in fowl leukaemia.
2. Reported the acid-fast nature of tubercle bacillus. Rous (1911) isolated a virus causing fowl sarcoma.
3. Proposed 'side chain theory' of antibody production. Several viruses have since been isolated which cause
4. Discovered salvarsan, an arsenical compound, tumours in animals and birds. Viruses can also cause
sometimes called the 'magic bullet'. It was capable malignant transformation of infected cells in tissue
of destroying the spirochaete of syphilis. Later on cultures. The discovery of viral and cellular oncogenes
he discovered 'neosalvarsan' and thus created a new was a breakthrough for possible mechanisms of viral
branch of medicine known as chemotherapy. oncogenesis. The positive proof of viral causation of
5. Introduced methods of standardising toxin and human malignancy was established when the human
antitoxin. T-cell leukaemia virus was isolated in 1980.

8 _ _ _ __ _ _ _ _ _ _ _ _ _ _ _ _ __ _ __ _ _ _ _ _ __ Unit I: General Microbiology
KEY POINTS
1. Microbiology is the science of living organisms that are only visible under microscope.
2. Scientific development of microbiology is contributed by several scientists.
3. A microorganism can be accepted as the causative agent of infectious disease only if it fulfills the Koch's
postulates.
4. Nobel prize has been awarded to various scientists for their discoveries.
YOU MUST KNOW

1. Contributions of Robert Koch , Louis Pasteur, Joseph Lister and Paul Ehrlich.
2. Koch's postulates and Koch's phenomenon.
STUDY QUESTIONS
1. Write short notes on:
(a) Robert Koch (b) Louis Pasteur
(c) Koch's postulates (d) Koch's phenomenon
(e) Paul Ehrlich
MU TIPLE CHOICE QUESTIONS (MCQs)

1. Who is known as the Father of Microbiology?
(a) Paul Ehrlich (b) ,oseph Lister (c) Louis Pasteur (d) Kitasato
2. Who introduced the techniques of sterilisation?
(a) Robert Koch (b) Louis Pasteur (c) John Needham (d) Joseph Lister
3. The term vaccine was coined by:
(a) Louis Pasteur (b) Robert Koch (c) Joseph Lister (d) Edward Jenner
4. Who introduced methods of obtaining bacteria in pure cultures using solid media?
(a) Robert Koch (b) Louis Pasteur (c) Joseph Lister (d) Paul Ehrlich
5. Who discovered Mycobacterium tuberculosis?
(a) Louis Pasteur (b) Robert Koch (c) Loeffler (d) Yersin
6. Who is known as the 'Father of Chemotherapy'?
(a) Robert Koch (b) Louis Pasteur (c) Paul Ehrlich (d) Joseph Lister
7. Who proposed 'side chain theory' of antibody production?
(a) Paul Ehrlich (b) Edward Jenner (c) Robert Koch (d) Joseph Lister
8. Who introduced vaccination method for prevention of small pox?
(a) Edward Jenner (b) Joseph Lister (c) Robert Koch (d) Louis Pasteur
9. Which is the first human disease proved to be of viral origin?
(a) Japanese encephalitis (b) Yellow fever (c) Dengue (d) Small pox
Answers (MCQs):
1. (c) 2. (b) 3. (a) 4. (a) 5. (b)
6. (c) 7. (a) 8. (a) 9. (b)
---++• - - -
Chapter 2
MICROSCOPY AND MORPHOLOGY OF BACTERIA
I. Introduction
II. Microscopy
A. Optical or Light Microscope B. Phase Contrast Microscope
C. Dark Field (Dark Ground) Microscope D. Interference Microscope
E. Fluorescent Microscope F. Electron Microscope
Ill. Study of Bacteria
A. Unstained (Wet) Preparations B. Stained Preparations
C. Morphology of Bacteria D. Bacterial Anatomy
I. INTRODUCTION Table 2.1 Differences between Prokaryotic and

Microorganisms are living structures of microscopical Eukaryotic Cells
size. These were originally classified under plant and Structure Prokaryotes Eukaryotes
animal kingdoms. This classification proved unsatisfactory,
therefore, a third kingdom Protista was formed. Nucleus
Nuclear membrane Absent Present

Protista
Nucleolus Absent Present
It is divided into two groups- prokaryotes and eukaryotes
(Table 2.1) . Chromosome One More than
one
l. Eukaryotes Deoxyribonucleoprotein Absent Present
F ungi, algae (except blue green algae), protozoa and slime
moulds are included in this group. Division By binary By mitosis
fission
2. Prokaryotes Cy toplasm
Bacteria and blue green algae belong to this group.
Bacteria are unicellular without any true branching Mitochondria, Golgi All are absent All are
apparatus, Lysosomes, present
except in the higher bacteria (Actinomycetales). They Pinocytosis, Endoplasmic
don't contain chlorophyll but in contrast blue green algae reticulum
possess chlorophyll.
Chemical Composition
Bacteria Sterols Absent Present
The size of bacteria is measured in unit of a micron
Muramic acid Present Absent
(micrometre).

10 Unit I: General Microbiology
1 micron (µ) or micrometre (µm) = 1/1000 B. Phase Contrast Microscope

millimetre Phase contrast microscopy improves the contrast and
1 millimicron (mµ) or nanometre (nm) = 1/1000 µm makes evident the internal structures of cells which differ
(micron) or one millionth of a millimetre in thickness or refractive index. The different parts of
1 Angstrom unit (A = 1/10 nm (nanometre)
0
) a cell and its surrounding medium have got differept
Bacteria of medical importance measure 2-5 µm (L) refractive indices. When rays of light are passed through
x 0.2-1.5 µm r.:w). The resolution power of an unaided an object, they emerge in different phases depending on
eye is about 200 microns. Bacteria, being much smaller the difference of the refractive indices between the object
than the resolution limit, can be visualised only under and its surrounding medium. A special optical system
magnification. Hence, the study of bacteria requires the (special condenser and objective lens) converts difference
use of microscopes. in phase into difference in intensity of light, producing
light and dark contrast in the image. A light microscope
II. MICROSCOPY can be converted into phase contrast microscope by using
Microscopy is done for following purposes: a special condenser and objective lens.
1. magnification of an object
2. maximisation of resolution C. Dark Field (Dark Ground) Microscope
3. optimisation of the contrast between structures, Reflected light is used instead of the transmitted light
organisms and background. used in the light microscope. A dark field condenser with
The following types of microscope are being employed a circular stop is fitted with a light microscope. This
for the study of bacteria. condenser lens system is arranged in such a way that no
light reaches the eye, unless reflected from the object. The
A. Optical or Light Microscope object or bacterium appears self-luminous against a dark
The light microscope contains a light source and a background. The contrast gives an illusion of increased
compound lens system. The lens system consists of a resolution, so that extremely slender organisms such as
number of objective lenses in conjunction with a fixed spirochaetes, not visible under ordinary microscope, can
(usually XlO), ocular (eye piece) lens. Light microscopes be seen under the dark field microscope.
are equipped with objective lenses of low power (XlO),
high power (X40) and oil immersion (Xl00), which will D. Interference Microscope
result in final magnifications, in conjunction with the It is useful for revealing cell organelles and for quantitative
ocular lens, of Xl00, X400, and Xl,000, respectively. measurements of the chemical constituents of cell such
Besides magnification, resolving power is another essential as, lipids, proteins and nucleic acids.
component of microscopy. Resolving power is the ability .;--- .sM ~3
of the lens system to distinguish two objects as separate, E. Flu~ scent Microscope
rather than one. Resolving power is dependent on the Fluorescence is a phenomenon that occurs when an object
wavelength of the light used to illuminate the object and is impacted by a given wavelength of light and emits
the numerical aperture of the microscope. Numerical light at a wavelength longer than the one to which it was
aperture is defined as a light gathering power of microscope. exposed. In fluorescent microscope, specimens are exposed
Resolution power can be optimised through proper use to a light of shorter wavelength (ultraviolet light), which
of the condenser, which focuses light into the plane of results in emission of longer wavelength visible light. Due
the object. The commonly used condensers can produce to shorter wavelength of UV light, the resolving power
a numerical aperture of 1.25. Resolution power can be can be proportionately extended. Bacteria stained with
further increased by adjusting the medium through which fluorescent dye (e.g. auramine, rhodamine) become visible
light passes between the object and the objective lens. as brightly glowing objects in a dark background.
Special oils, termed immersion oils, have a refractive index · o y has also been ~ d
similar to glass, thus, use of these oils permit more light fo detect-c--io_n_o-.i--~ · en direct fluorescent antibody
to be incorporated in the image resulting in improving technique) an antibo ie (indirect fluorescent antibody
the resolution power. Visualisation of bacteria generally methods). The.,c--,u.=t-tIUuS are described in Chapter 14.
requires the use of immersion oil with Xl00 objective. This
combination results in resolution of approximately 0.2 F. Electron Microscope
microns. However, X40 objective is used for examination of A beam of electrons is employed instead of the beam of
wet preparations e.g. hanging drop, stool for ova and cyst. light used in optical microscope. The electron beam is

Ch 2: Microscopy and Morphology of Bacteria _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __ 11
focussed by circular electromagnets (magnetic condenser), Common Staining Techniques

which are analogous to the lenses of light microscope. I. Simple stains
The wavelength of electrons is approximately 0.005 nm, Basic dyes such as methylene blue or basic fuchsin
as compared to 500 nm with visible light. The resolving are used as simple stains. They provide the colour
power of any microscope is directly related to the contrast, but impart the same colour to all the
wavelength, thus, the resolving power of the electron bacteria in a smear.
microscopes should be theoretically 100,000 times that 2. Negatfre staining
of light microscopes but in practice, it is about 0.1 nm. Bacteria are mixed with dyes such as India ink or
Shadow casting is an important technique in electron nigrosin. The background gets stained and unstained
microscopy. This is achieved by depositing a thin layer bacteria stand out in contrast. This is very useful in
of metal (e.g. platinum) on the object. Such metal- the demonstration of bacterial capsules which do
coated object held in the path of the beam, scatters the not take simple stains.
electrons and produces an image which is focussed on a 3. Impregnation 111etliods
fluorescent viewing screen. Another technique in studying Bacterial cells and structures that are too thin to be
the fine structures of the object is negative staining with seen under the light microscope, are thickened by
phosphotungstic acid. impregnation of silver on the surface to make them
The scanning electron microscope is a recent visible e.g. demonstration of bacterial flagella and
development which provides a higher resolution and spirochaetes.
three dimensional image of the object. 4. Differential stains
Although the substantial increase in resolution power They impart different colours to different bacteria or
of electron microscope has led to significant discoveries, bacterial structures. The most commonly employed
a major disadvantage is the inability to examine living differential stains are the Gram stain, the acid-fast
cells. A method introduced to overcome this disadvantage t? ,,,stain and the Albert stain.
is freeze-etching. This method involves rapid cooling ¾J;;_c' L 111
of specimens by deep-freezing in liquid gas and the GRAM STAIN
subsequent formation of carbon-platinum replicas of It is the most widely used stain in bacteriology. The
the material. Since such frozen cells may remain viable, stain was originally devised by the histologist Christian
it enables the study of cellular ultra structure as it appears Gram (1884) as a technique of staining bacteria in
in the living state. tissues. Nowadays, modification of original Gram stain
Electron microscopy is especially used for rapid is used.
detection of viruses in clinical samples. It is particularly
useful for detecting non-cultivable viruses. Ultrastructural Method
study of various microorganisms may also be done by 1. Heat fixed smear of specimen or bacterial culture
this method. is stained w1th..c.crysta.J3iolet (prima;y stain) for one
_minu~. Other pararosaniline dyes such as gentian
III. STUDY OF BACTERIA violet or methyl violet may also be used as primary
A. Unstained (Wet) Preparations stain.
Unstained preparations are examined mainly for 2. Pour Gram's iodine (dilute solution of iodine) over
bacterial motility (e.g. hanging drop preparation) and the slide for one min_!!):e.
for demonstration of spirochaetes (e.g. dark ground 3. smear with water.
microscopy). 4. Decolourise with a"-.etone for 10-30 seconds. Alcohol
can be substituted for acetone.
B. Stained Preparations 5. Wash~with water.
Structural detail of bacteria cannot be seen under light 6. Counterstain with a dye safrnnin for 30 seconds.
microscope due to lack of contrast. Hence it is necessary Dilute carbol fuchsin ~r neutral red may also be
to use staining methods to produce colour contrast. used as counterstain.
Smear made from bacterial culture or specimen is first
dried and then fixed with heat by flaming the slide from Differentiation on Gram Staining
underneath. Heat kills and fixes the bacteria on slide due Two broad groups
to coagulation of bacterial proteins. The fixed smear is - Gram positive
stained by appropriate staining technique. - Gram negative

r 12 Unit I: General Microbiology
Gram positive 3. The stained smear is decolourised with 20% sulphuric

- resist decolourisation and retain the colour of primary acid and washed with water. This step should be
stain i.e. vj olet repeated till the pink/red colour stops coming out.
Gram negative In case of lepra bacilli 5% sulphuric acid is used as
- are decolourised by acetone/alcohol and, therefore, M. /eprae is less acid-fast. Another alternative for
take counterstain and appear red. decolourisation is acid-alcohol (3 ml HCl and 97 ml
~G,.- ,...~,.,.,.,..
ethanol).
Mechanism of Gram Staining .- 4. The smear is counterstained with 2% methylene
The exact mechanism is not understood. It may, however, blue for 1-2 minute. Malachite green can also be
be attributed to following. used as counterstain instead of methylene blue.
1. Permeability of bacterial cell wall and 5. Wash with water and air dry.
cytoplasmic membrane
The Gram positive cells have a more acidic pratapla.srn, Microscopic Examination of the Smear
which accounts for retaining the basic dye more strongly Acid-fast bacilli appear red (colour of carbol fuchsin) in
than the Gram negative bacteria. Iodme makes the blue (colour of methylene blue) background of pus cells
protoplasm more a ~ r v e s as mordant, i.e. iodine and epithelial cells.
combines with dye to form a dye-iodine 'Complex and
fixes the dye in bacterial cell. The Gram positive cell wall Principle
or cytuptasnuc membrant: being less permeable, the dye- Acid-fastness is due to the high content of lipids, fatty
iodine complex gets trapped within the cell. The Gram acids and higher alcohols found in the cell wall of
negative cell wall has increased permeability to acetone mycobacterium. Mycolic acid (a wax), acid-fast in the
or alcohol, permitting the outflow of complex during free state, is found in all acid-fast bacteria. Besides lipid
decolourisaton. This is not all-or-none phenomenon. The contents, acid-fastness depends also on the integrity of
Gram positive cells may be decolourised by prolonged the cell wall.
treatment with acetone/alcohol. In contrast, inadequate
decolourisation may cause cells to appear Gram ALBERT'S STAIN
positive. Staining of Corynebacteria ( Corynebacterium diphtheriae
and other corynebacteria is done by this technique.
2. Integrity of cell wall
The integrity of the cell wall is essential for a positive Method
stain. The Gram positive bacteria become Gram negative 1. The smear is heated gently by flaming the slide from
when cell wall is damaged. underneath. It will fix the smear. Do not overheat.
2. Cover the smear with Albert I (Albert's stain) for 5
minutes.
ACID-FAST STAIN (ZIEHL-NEELSEN STAIN) 3. Drain off the whole stain without washing.
The acid-fast stain was discovered by Ehrlich and 4. Pour Albert II (iodine solution) over the smear so
subsequently modified by Ziehl and Neelsen. Staining as to cover it completely, leave it for 2 minutes.
of Mycobacteria (usually tubercle and lepra bacilli) is 5. Drain off the Albert II solution without washing.
done by this technique. 6. Blot dry the smear with the help of filter paper.
Method Reagents
1. The carbol fuchsin stain is poured on a slide containing A. Albert I or Albert stain
fixed smear. Gentle heat is applied to the underside 1. Toludine blue .. 0.15 gm
of the slide, by means of a spirit flame, until the 2. Malachite green .. 0.20 gm
stain just commences to steam. The carbol fuchsin 3. Glacial acetic acid 1 ml
is left on the slide for 5-10 minutes with intermittent 4. Alcohol (95 per cent ethanol) .. 2ml
heating during that period. Care must be taken to 5. Distilled water to make 100 ml
ensure that the stain does not dry out, to counteract
drying more solution of stain is added to the slide B. Albert II or Albert's iodine solution
and the slide reheated. Heating of the stain is required 1. Iodine 2 gm
for penetration of the dye into the cell wall. 2. Potassium iodide 3 gm
2. Wash in tap water. 3. Distilled water to make 300-ml
Ch 2: Microscopy an d Morphology of Bacteria _ _ _ __ __ __ _ _ _ _ _ _ _ __ _ __ _ 13
Microscopic Examination of the Smear filaments. They are very small in size (50-300 nm in
Corynebacterium diphtheriae appear as green coloured diameter). They can reproduce in cell-free medium.
bacilli with bluish black metachromatic granules. These 6. Rickettsiae and Chlamydiae: These are very small,
bacilli are arranged in Chinese letter or cuneiform obligate parasites. Due to their inability to grow
arrangement. outside living cells, they were previously considered
+.J.!dJ!:1-.. as viruses. Now they are classified as bacteria because
C. Morphology of Bacteria of typical bacterial cell wall, possession of various
Depending on their shape, bacteria are classified into bacterial enzymes and structural similarities with
several types f- g 2 ): bacteria.
1. Cocci (from kokkos, meaning berry): These are oval
or spherical cells. These cocci may be arranged in D. Bacterial Anatomy
pairs (diplococci), chains (streptococci), clusters The outer layer or cell envelope of a bacterial cell consists
(staphylococci) and groups of four (tetrads) or eight of two components-
(sarcina). 1. a rigid cell wall.
2. Bacilli (bacillus, meaning rod): These are rod shaped 2. underlying cytoplasmic or plasma membrane.
cells. Some of these bacilli may be having peculiar The cell envelope encloses the protoplasm which
arrangement or shape as follows: comprises cytoplasm, cytoplasmic inclusions (mesosomes,
(i) Cocco bacilli- length of bacteria is approximately ribosomes, inclusion granules, vacuoles) and a single
same as its width e.g. Brucella. circular chromosome of deoxyribonucleic acid (DNA)
(ii) Streptobacilli- These bacilli are arranged in (Fig. 2.2) . Besides these essential components, some
chains e.g. Streptobacillus. bacteria may possess additional structures, such as,
(iii) Chinese letter or cuneiform pattern- arranged capsule, flagella and fimbriae.
at angles to each other e.g. Corynebacterium.
(iv) Comma-shaped- curved appearance e.g. Vibrio.
(v) Spirilla- rigid spiral forms e.g. Spirillum. 1. THE CELL WALL (TABLE 2.2)
3. Spirochaetes (from spiera meaning coil; chaite The cell wall is a tough and rigid structure, surrounding
meaning hair): These are slender, flexous spiral forms the bacterium like a shell. It weighs about 20-25% of the
e.g. Treponema. dry weight of the cell. The thickness of Gram negative cell
4. Actinomycetes (from actis, meaning ray, mykes wall is 10-25 nm. The cell wall has following functions:
meaning fungus): These are branching filamentous 1. Accounts for the shape of the cell.
bacteria resembling fungi. They have a rigid cell 2. Provides protection to the cell against osmotic
wall. damage.
5. Mycoplasmas: These bacteria are cell wall deficient 3. Confers rigidity upon bacteria.
and hence do not possess a stable shape. They may 4. It takes part in cell division .
occur as round or oval bodies and as interlacing 5. It possesses target site for antibiotics, lysozymes and
,,.,
I Cocci I
,.,,. f(
'!?~
,~
-~- --s~
~\JJ~
•• II
•
- ..
Cocci in chains Cocci in clusters Tetrad Sarcina
Diplococci
-J.t\
~c,lu"
,_
I Bacilli I
_,,,. •••
~\(:)-'
-\\-.#' ~ ) ~;\,JP
-u
½/1I\ "
[p,'-f
!:7Y tR
Bacilli Coccobacilli Bacilli in chains Comma shaped Chinese letter pattern
Fig. 2.1 Shapes and different arra ngements of bacteria

14 - - -- - -- - -- -- -- -- -- - - -- - - - - - - - Unit I: General Microbiology
,- ---+-4- Ribosomes
Cell wall
-- 1
Flagellum
--
///-
-Pili
Fig. 2.2 Bacterial cell
bacteriophages. It carries bacterial antigens that are

important in virulence and immunity. N-acetyl muramic
-l-l-4..r<.. -H-l-t,--1...-H 4-1,.L acid
N-acetyl
Table 2.2 Differences between Cell Wall of Gram positive glucosamine
and Gram negative Bacteria
Character Gram positive Gram negative
Thickness Thicker Thinner
i:,:ripl~ c s~ Absent Present
Lipids Absent or small Present
Teichoic acid Present Absent
Peptidoglycan 16-80 nm 2nm
Fig. 2.3 Chemical structure of bacterial cell wall
The rigid part of the cell wall is a peptidoglycan
which is a mucopeptide (murein) composed of N-acetyl named as outer membrane proteins (OMP). These are
murarnic acid and N-acetyl glucosamine molecules target sites for phages, antibiotics and bacteriocins.
alternating in chains, cross linked by peptide subunits (c) Lipopolysaccharide (LPS): This layer consists of
(Fig. 2.3). The cell walls of Gram positive bacteria have lipid A, to which is attached a polysaccharide. LPS
simpler chemical nature than those of Gram negative constitutes t h ~ o f gram-negative bacteria.
bacteria. The polysaccharide determines a major surface
antigen, the O antigen. The toxicity (pyrogenicity,
(i) Gram-negative Cell Wall lethal effect, tissue necrosis) of bacteria is associated
The Gram negative cell wall is a complex structure with with lipid A.
th ollowing components (Fig. 2.4). (d) The periplasmic space: It is the space in between
Lipoprotein layer: It connects the peptidoglycan to the inner and outer membranes. It contains various
outer membrane. binding proteins for specific substrates.
(b) Outer membrane: rtain proteins Peptidoglycan.
Teichoic acid
Lfi Lipopolyic~ de - -
Gram positive Gram negative
Fig. 2.4 Structure of Gram positive and Gram negative bacterial cell wall

l
Ch 2: Microscopy and Morphology of Bacteria---..~-- - - - - - - - - -- -- - - - -- - 15
(ii) Gram-positive Cell Wall presence of penicillin or other agents that interfere
(a) Peptidoglycan: This layer in Gram positive bacteria with synthesis of cell wall. These are difficult to
is thicker (16-80 nm) than that in Gram negative cultivate and require agar containing solid medium
bacteria (2 nm) (Fig. 2.4). having right osmotic strength. L-forms are sometimes
(b) Teichoic acid: Gram positive cell wall contains spontaneously formed in patients treated with
significant amount of teichoic acid which is absent in penicillin. L-forms are more stable than protoplasts
Gram negative bacteria. The teichoic acids constitute and spheroplasts.
major surface antigens of Gram positive bacteria. (iii) Protoplasts: These are derived from Gram positive
They are water soluble polymers, cont~ining ribitol bacteria. They contain cytoplasmic membrane and
or glycerol polymers. Teichoic acids are of two types, cell wall is totally lacking. These are produced
cell w ~ i d and m e ~ a c i d . artificially by lysozyme in a hypertonic medium.
The cell wall teichoic acid is covalently linked to These are unstable. Hypertonic condition is necessary
pept'ictoglycan; and tne membra°ile te1choic acid to for their maintenance.
cytoplasmic membrane. (iv) Spheroplasts: These are derived from Gram negative
(c) Other components: Certain Gram positive bacteria. They are produced in presence of penicillin.
cells also contain antigens such as protein and They are osmotically fragile and must be maintained
polysaccharides. in hypertonic culture medium. They differ from
the protoplast in that some cell wall material is
Demonstration of Cell Wall retained.
The cell wall cannot be seen by light microscope and
does not stain with simple dyes. Demonstration of cell Pleomorphism and Involution Forms
wall can be done by following methods: Certain species of bacteria which exhibit great variation in
(a) Plasmolysis- When bacteria is placed in a hypertonic shape and size of individual cells are called pleomorphism.
saline, shrinkage of the cytoplasm occurs, while cell Some bacteria show swollen and aberrant forms in ageing
wall retains original shape and size. laboratory cultures and are known as involution forms.
( b) Microdissection Defective cell wall synthesis is often responsible for
(c) Differential staining development of these two abnormal forms.
(d) Reaction with specific antibody
(~) Electron microscopy.
2. CYTOPLASMIC MEMBRANE
Bacteria with Defective Cell Wall It is 5-10 nm thick elastic semipermeable layer which lies
The synthesis of cell wall may be inhibited or interfered beneath the cell wall separating it from the cell cytoplasm
by many factors such as, antibiotics, bacteriophages and (Fig. 2.2). Electron microscopy shows the presence of
lysozyme. Lysozyme, an enzyme normally present in many three layers constituting a unit membrane. Chemically,
tissue fluids, lyses susceptible bacteria by splitting the it consists of lipids and protein molecules. Sterols are
linkage of peptidoglycan in the cell wall. When lysozyme absent, except in mycoplasma.
acts on a Gram positive bacterium in a hypertonic solution, Cytoplasmic membrane act as an osmotic barrier.
a protoplast is formed. In case of Gram negative bacteria, It is the site of numerous enzymes (permease, oxidase,
the result is a spheroplast. Bacteria with defective cell wall polymerase) involved in the active transport of selective
may probably have a role in the persistence of certain nutrients. It acts as a semipermeable membrane controlling
chronic infections such as pyelonephritis. Bacteria without the inflow and outflow of metabolites to and from the
cell walls or with deficient cell walls are of four types: protoplasm. It also contains cytochrome oxidase, enzymes
(i) Mycoplasma: This is a naturally occurring bacteria of tricarboxylic acid cycle and enzymes necessary for the
without cell walls. Mycoplasma is classified as an cell wall synthesis.
independent bacterial genus. They don't require
hypertonic environment for maintenance and are 3. CYTOPLASM
stable in culture medium. The bacterial cytoplasm is a colloidal system containing
(ii) L -forms: Kleineberger-Nobel, while studying a variety of organic and inorganic solutes in a viscous
Streptobacillus moniliformis in the Lister Institute, watery solution. It lacks mitochondria and endoplasmic
London, observed abnormal forms of the bacteria and reticulum of eukaryotic cell. It contains ribosomes,
named them L-forms after the Lister Institute. mesosomes, vacuoles and inclusions. The cytoplasm stains
L-forms develop either spontaneously or in the uniformly with basic dyes in young cultures.

(i) Ribosomes
These are the centres of protein synthesis. These are Tnt~ l+llJW:.;i,W,).U,l,-VI· s cid bacterial secretion which
composed of ribosomal RNA (rRNA) and ribosomal surrounds some bacteria as their outermost layer (Fig.
proteins. Ribosomes are integrated in linear strands of 2.2) . When it diffuses into the surrounding medium
mRNA to form polysomes and it is at this site that code and remains as a loose undemarcated secretion as in
of mRNA is translated into peptide sequences. They L euconostoc, it is known as slime layer. When this
arel0-20 nm in size with a sedimentation constant of secretion is organised into a sharply defined structure,
70 S (S for Svedberg units). Each 70 S unit consists of as in Stre tococcus pneumoniae (pneumococcus), it is
a 30 S and a 50 S subunit. known as the capsule. Capsules which are very thin and
cannot be demonstrated under the light microscope are
(ii) Intracytoplasmic inclusions ca11ed microcapsules e.g. Ne zsserza meningitidis. The slime
These are sources of stored energy and present in some layer or capsule is generally-polysacchanae in nature but
species of bacteria. Their function and significance is it is polypeptide in anthrax bacillus. Some bacJeria may
uncertain. They may be present as polymetaphosphate have both a capsule and a slime layer (e.g. Streptococcus
(volutin), lipid, polysaccharide (starch or glycogen) and salivarius). Bacteria like Klebsiella secreting large amount
granules of sulphur. They are most frequent in bacteria of slime produces mucoid growth on agar, with a stringy
grown under conditions of nutritional deficiency and consistency when touched with the loop.
disappear when the deficient nutrients are supplied.
Functions
(iii) Mesosomes (chondroids) (i) Capsule enhances bacterial virulence by inhibiting
Mesosomes are vesicular, multilaminated or convoluted phagocytosis. Loss of capsule may render the
structures formed as invaginations of the plasma bacterium avirulent. Bacteria tend to lose capsules
membrane into the cytoplasm (Fig. 2.2) . They are the on re ea ures.
principal centres of respiratory enzyme and are analogous (ii) It acts as protective coyerj.g,g against antibacterial
to mitochondria of eukaryotes. These are more prominent substances such as, bacteriophages, phagocytes,
in Gram positive bacteria. There are two types of enzymes.
mesosomes- septal and lateral. The septa! mesosome (iii) Capsular antige is specific for bacteria and can be
is attached to bacterial chromosome and is involved in used for identification and typing of bacteria.
DNA segregation and in the formation of cross-walls
during binary fission. Capsulated Organisms
Streptococcus pneumoniae, Klebsiella sp. , Bacillus anthracis,
Haemophilus infiuenzae, Cryptococcus neoformans ( a
4. NUCLEUS fungus ) .
Bacterial nucleus has no nuclear membrane or nucleolus. It
cannot be demonstrated by ordinary microscope but needs Demonstration of Capsule
an electron microscope. The nuclear deoxyribonucleic acid Capsule has little affinity for ~asic dyes, therefore, it can't
(DNA) doesn't appear to contain any basic protein. The be stained by Gram staining. The following methods have
genomic DNA is double stranded in the form of a circle. been used for demonstration of capsule.
It measures about 1 mm (1000 µm) when straightened.
The bacterial DNA is haploid, replicates by simple fission · ndia ink staining (negative staining)
and maintains bacterial genetic characteristics. Capsule appears as a clear halo around bacterium as the
Some bacteria may possess extranuclear genetic i~ can't penetr~te the capsule (Fig. 2.5) .
material in the cytoplasm consisting of DNA named
as plasmids or episomes. The plasmid replicates
autonomously. They are not essential for the life of the Capsule
cell, but may confer on the bacteria certain properties,
such as drug resistance and toxigenicity which constitute
a survival advantage to the bacteria. These plasmids can
be transmitted from one bacterium to another either by
conjugation or by the agency of bacteriophage. Besides
these methods, plasmids may be transferred to daughter
cells during cell division by binary fission . Fig. 2.5 Pneumococci stained with India ink to show capsule

Ch 2: Microscopy and Morphology of Bacteria _ _ __ _ _ _ _ __ _ _ _ _ _ __ _ __ _ __ 17
(ii) Serological methods different. Specific flagellar antibodies are produced in

Capsular material is antigenic and can be demonstrated high titres in response to antigenic stimulation of flagella.
by mixing it with a specific an,ticapsu}p.r serum. When These antibodies are useful in serodiagnosis but are not
a suspension of capsulated bacterium is mixed with its protective.
specific anticapsular serum and examined under the
microscope, the capsule appears 'swollen' due to increase Arrangement/Types (Fig. 2.7)
in its refractivity. This phenom?n'on &called as capsule ~onotrichous- Single polar flagellum (at one end)
swelli~ion or Quellung phenomenon. This is e.g. Vibrio cholerae.
also named as Neufeld reaction after the person who _0l. Amphitrichous-Single flagellum at both the ends
described it. This was widely employed for the typing e.g. Alcaligenes faecalis .
of pneumococci. .Jjiir Lophotrichous-Tuft of flagella at one or both ends
e.g. Spirilla.
(iii) Special capsule staining -fWY Peritrichous-Flagella arranged all round the cell
These techniques employ copper salts as mordants for e.g. Salmonella typhi .
staining of capsule. ~ - - -____, ./¥..,3/vJ
.. :e.s.3 15eiiiiii'istration 0 t,)
I
6. FLAGELLA o-1 F lagella are about 0.02 µmin thickness and hence beyond
Flagella are c)'.!cn;ilasmic appendages protruding through the resolution limit of the light microscope. The following
cell wall. These are thread like structures composed of methods are used for its demonstration:
a protein (flagellin), 5-20 µm in length and 0.01-0.02 (i) DarJs.,grouJ}d illumination.
µm in diameter. They are organ of locomotion. All (ii) Special staining techniques in which tlii9kness of
motile bacteria, except spirochaetes, pos~ess one~ r more flagella is increased by mordanting.
flagella.
Parts and Composition

-
(iii) Elect ro ti ,mic " copy.
(iv) Indirect methods by which motility of bacteria can
be seen or demonstrated.
Each flagellum consists of three parts (Fig. 2.6) . (a) Spreading type of growth on a medium e.g.
(i) filament _. swarming growth of Proteus sp.
(ii) hook - (b) Motility under the microscope e.g. hanging drop
(iii) basal body preparation.
,.-
Tlie filamentJies external to the J;cl.l and is connected
to the h~bk at the cell surface. The hook-basal body
portion is embedded in the cell envelope. The basal body
contains outer and inner rings by which the basal body
is attached to the cytoplasmic membrane. Outer rings Monotrichous
are absent in Gram positive bacteria.
The flagella is made up of protein...(fggcllin) which
is s~ilar to myosin. Although chemical composition of
diffhent genera of bacteria is similar, they are antigenically Amph itrichous
"Y\ ,,
Lophotrichous
Peritrichous
Fig. 2.6 Structure of bacterial flagellum Fig. 2.7 Arrangement of flagella

r sporrQ__,,c.- \ f>~
(c) Spreading of bacteria in semisolid agar e.g. spores are formed within the parent cell, these are called
--
Craigie's tube metl!Q.d.
7. FIMBRIAE
'l'hese are hair-like. appendages projecting from the cell
endospores. Sporulation is not a method of reproduction.
In the process of sporulation, each vegetative cell forms
only one spore and during subsequent germination, each
spore gives rise to only one vegetative bacterium.
surface as shaight filaments. They are also called pil"
They are 0.1 to 1.0 µm in length and less than 10 nm Sporulation (Fig. 2.8)
thick (shorter and thinner than flagella). Fimbriae are Bacterial cell undergoes spore formation in nutritionall
found in some Gram negative bacteria. Each bacterium de rived conditions and this process is called sporulation.
possesses 100-500 fimbriae peritrichously. They are more Spore develops from a portion of protoplasm (forespore)
numerous than flagella. They are antigenic. They are best near one end of the cell. The remaining part of cell is
developed in freshly isolated strains an m 1quid cultures. called sporangium. Bacterial DNA replicates and divides
They tend to disappear when subcultures are made on into two DNA molecules. One of these is incorporated
solid media. Fimbria is composed of protein called pilin. into forespore and other into sporangium. A transverse
They are unrelated to motility and are found on motile septum grows across the cell from the cell membrane.
as well as non-motile bacteria. It divides forespore and sporangium. The forespore is
completely encircled by this septum as a double layered
Types membrane. The inner layer becomes the spore membrane
There are three main types of fimbriae. and the outer layer becomes thickened spore coat. Between
(!),Common pili- These are of six types depending the two layers is spore cortex.
/ on their morphology, number per cell, adhesive
properties and antigenic nature. Morphology of Spore
(u) Sex or F (fertility) pili The clear area in the protoplasm of cell becomes
iii) Col I (colicin) pili gradually more opaque with condensation of nuclear
chromatin forming the forespore. The cell membrane
Functions grows inward and forms spore wall around the core
(i) Adhesion- Fimbriae are or a esion. This (forespore). The inner-most layer of the spore wall forms
property e9hances th virulence f bacteria. the spore membrane from which the cell wall of future
(iz) Transfer of genetic mate · - ex pili are present in vegetative bacterium develops. Outside this membrane
male bacteria. 'these p1h a?e"longer (18-20 µm) and is thick layer, the cortex and a multilayered tough spore
1-4 in number. They help the male cells to attach coat. Some spores _have an additional apparently rather
with non-male (female) cells in forming "conjugation loose, outercovering called exosporium (Fig. 2.9).
tubes" through which genetic material is believed to
be transferred from the donor (male) to the recipient Shape and Position of Spores
(female) cell. The precise position, shape and relative size of spore
are constant within a particular species. Spores may be
Detection of Fimbriae / central, subterminal or terminal (Fig. 2.10). They may be
(i) Electron microscopy. oval or spherical in shape. The diameter of spore may be
(ii) ,Haemagglutination- Many fimbriated bacteria (e.g. same or less than the width of bacteria (Bacillus) , or may
7 ~ erichia coli, Klebsiella) strongly agglutinate red be wider than the bacillary body producing a distension
blood~ells of guin~~' fowl , horses and pigs; or bulge in the cell ( Clostridiufn ).
human and sheep ce ls weakly and ox cells scarcely.
This property of haemagglutination is a simple
method for detecting the presence of fimbriae. The Resistance
haemagglutination can be specifically inhibited by Bacterial spores are extremely resistant to ordinary
D-mannose. .5.3 5 (V) boiling, disinfectants and heating. The high resistance
. of s · due to hi content o calcium nd dipicolinic
~ TERIALSPORE acid; low water content; he thick impervious cortex and
Spores are highly resistant resting stage formed in spore coats; their low metabolic and enzymatic activity.
urµavourableenvironmental conditions presumed to However, spores of all medically im ort
related to the depletion of exogenous nutrients. As bacterial are destroyed ·ng at 121 °C for

Ch 2: Microscopy and Morphology of Bacteria _ _ _ _ _ _~..,,........<>---- ------------ 19
6-~
( J1s ) DNA
/
Germination of
endospore
Vegetat;,e cell
I GD DNA replication
Free endospore
released
Sporangium
~Forespore
Formation of sporangium
and forespore
:::::==:f===::=:;:::---- Spore coat
/
~ - ~ - Spore cortex
,.___.,,._,,___ Spore membrane
Mature endospore
inside the cell
Formation of a double
layer membrane
Sporangium with completely enclosed forespore
Fig. 2.8 Sporulation
Germination
The process of conversion of a spore into vegetative cell
under suitable conditions is known as germination. The
spore loses its refractivity, and swells when transferred
to conditions conducive to vegetative growth. The spore
wall is shed and the germ cell appears by rupturing the
spore coat. The formation of vegetative bacterium occurs
by elongation of the germ cell.
Demonstration
_09f:am staining Spores appear as an unstained
Fig. 2.9 Structure of bacterial spore refractile body within the cell.
(ij}Modified ·Ziehl-Neelsen (ZN) staining- Spores
Methods of disinfection and sterilization should ensure appear as acid-fast (red colour). Ziehl-Neelsen
destruction of spores also. staining with 0.25-0.5% sulphuric acid (instead of

20% sulphuric acid as used in conventional method)

as decolouring agent is used spore staining.
Central
/
r<1on-bulging
spores Uses of Spores
Subterminal
Spa certain of ba teria are employed
as ·nd·cator for e.g. Bacillus
Terminal stearothermop i us w 1c 1s estro a temperature
of 121 °C for 10-20 minutes (same temperature and time
as used in autoclaving). These spores may be kept in
autoclave prior to its use. Absence of the spores after
Central
autoclaving indicates proper sterilisation.
Bulging
/ spores
Subterminal
Spore Forming Bacteria
Obligate aerobes- genus Bacillus e.g. B. anthracis and
B. subtilis
Obligate anaerobes- genus Clostridia e. g. Cl. tetani,
Free Spore Cl. welchii, Cl. botulinum
Both Bacillus and Clostridia
Fig. 2.10 Types of bacterial spores
bacilli.
-- ~ -fan~
KEY POINTS
CJ-b~tv-V-!j
1. Microorganisms are living structures of microscopical size. They belong to prokaryotes.
2. Microscopy can be done to observe these microorganisms.
3. Unstained (wet) and stained preparations are examined for the study of bacteria.
4. Bacteria are either Gram positive or Gram negative on Gram staining.
5. Broadly bacteria are classified into cocci or bacilli.
6. Bacterial cell possesses several components such as the cell wall, the cytoplasmic membrane, the cytoplasm , the nucleus,
the bacterial capsule, the flagella, the fimbriae and the bacterial spore.
YOU MUST KNOW

1. Differences between prokaryotes and eukaryotes.
2. Principles of different microscopes.
3. Principles of various staining techniques.
4. Differences between cell wall of Gram positive and Gram negative bacteria.
5. Bacterial capsule and its functions.
6. Types of flagella.
7. Functions of fimbriae.
8. Different types of bacterial spores.
STUDY QUESTIONS
(a) Phase contrast microscope (b) Dark field microscope
(c) Fluorescent microscope (d) Electron microscope.

Ch 2: Microscopy and Morphology of Bacteria _ _ _ _ _ __ _ _ __ _ _ _ _ __ _ __ __ _ 21
2. Draw a labelled diagram of bacterial cell.

(a) Bacterial cell wall (b) Cytoplasmic membrane
(c) Bacterial capsule (d) Flagella
(e) Fimbriae (f) Bacterial spore.
MULTIPLE CHOICE QUESTIONS (MCQs)

l . Peptidoglycan layer of cell wall is thicker in:
(a) Gram positive bacteria (b) Gram negative bacteria
(c) Fungi (d) Parasites
2. Lipopolysaccharide is a major component of cell wall in:
(a) Gram positive bacteria (b) Gram negative bacteria
(c) Fungi (d) Parasites
3. A tuft of flagella present at one or both ends of bacterial cell, is known as:
(a) Monotrichous (b) Arnphitrichous
(c) Lophotrichous (d) Peritrichous
4. Which one of the following bacteria is cell wall deficient?
(a) Escherichia coli (b) Salmonella typhi
(c) Mycoplasma (d) Treponema pallidum
5. All of the following are spore forming bacteria except:
(a) Clostridium botulinum (b) Bacillus subtilis
(c) Bacillus anthracis (d) Pseudomonas aeruginosa
6. Indian ink staining is used to demonstrate:
(a) Cell wall (b) Bacterial capsule
(c) Bacterial flagella (d) Bacterial spore
Answers (MCQs):
1. (a) 2. (b) 3. (c) 4. (c) 5. (d) 6. (b)
____ ... ____

Chapter 3
GROWTH, NUTRITION AND METABOLISM OF BACTERIA
I. Bacterial Growth
A. Bacterial Cell Division B. Generation Time
C. Bacterial Count D. Bacterial Growth Curve
E. Batch Culture and Continuous Culture F. Bacterial Growth in Vivo
II. Bacterial Nutrition
Ill. Bacterial Metabolism
A. Oxidation B. Fermentation
C. Oxidation-Reduction (O-R) potential
The minimum nutritional requirement for growth and

multiplication of bacteria includes sources of carbon,
nitrogen, hydrogen, oxygen and some inorganic salts.
Bacteria can be classified on the basis of nutrition as
follows:
1. Autotrophs : Bacteria which can synthesise their
essential metabolites (organic compounds) from the
atmospheric carbon dioxide and nitrogen are known
- - -- - - Septu m
as autotrophs or lithotrophs. Autotrophs obtain their
energy either photosynthetically (photolithotrophs
or photoautotrophs) or by oxidation of chemical
compounds (chemolithotrophs or chemoautotrophs).
2. H eterotrophs : These bacteria are unable to synthesise
their own metabolites. They depend on preformed - - Daughter cell
organic compounds. Majority of the pathogenic

bacteria are heterotrophs and parasitic. They have Fig. 3.1 Binary fission
evolved with the animal body where readymade
nutrients are freely available.
or chromosome is a circular double stranded DNA
I. BACTERIAL GROWTH molecule. The nuclear division precedes bacterial cell
A. Bacterial Cell Division
division. During replication, the two strands of DNA
Bacteria divide by binary fission (Fig. 3.1). When a are separated and new complementary strands are formed.
bacterial cell reaches a critical mass in its cellular Thus two identical double stranded DNA molecules are
constituents, the cell division starts. Bacterial nucleus formed.
22
Ch 3: Growth, Nutrition and Metabolism of Bacteria _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ 23
The replicated DNA molecules are distributed to the In the plating method, appropriate dilutions of
two daughter cells. A transverse septum grows across the specimen are inoculated on solid media, either on
the cell from the cell membrane. This is followed by the the surface of plates or as pour plates. The number of
deposition of cell wall material and the two daughter cells colonies that grow after incubation gives an estimate of the
separate. In some bacterial species, the daughter cells may viable count. Each colony represents a clone of bacteria
remain partially attached even after cell division. derived from a single parent cell. Therefore one colony
is equivalent to one bacterium. Number of colonies are
Clinical Significance equal to number of bacteria in the specimen.
Some bacteria are present as 'diplococci' ( e1ssena,
Pneumococcus) and others in chains (Streptococcus). Clinical Significance
Morphology of 'diplococci' and 'in chains' are due to Viable count is useful in clinical microbiology for testing
non-separation of daughter cells after cell division. These of water by presumptive coliform count (Ref. Chapter
morphological features are very useful in identification 80) and also in diagnosis of urinary tract infection (Ref.
of these bacteria in the laboratory. Chapter 69).
B. Generation Time D. Bacterial Growth Curve

The time required for a bacterium to give rise to two When a bacterium is inoculated into a suitable culture
daughter cells under optimum conditions, is known as liquid medium and incubated, its growth follows a
the generation time. The generation time in most of definite course. When bacterial count of such culture is
the medically important bacteria is about 20 minutes. determined at different intervals and plotted in relation
In Mycobacterium tuberculosis it is about 20 hours and to time, a growth curve is obtained (Fig. 3.2). The growth
in lepra bacilli it is 20 days. curve has four phases. ct
As bacteria grow so rapidly and by geometric
progression, a single bacterium can theoretically give rise
to 1021 progeny in 24 hours. In actual practice, bacterial ,··...
1 •••••- Viable cell count
multiplication is arrested after a few cell divisions due to
exhaustion of nutrients and accumulation of toxic products.
! · ·· ... .-:-
Ol
·;:: 1
I I
I I
Clinical Significance Ol 1 Stationary 1
When generation time is about 20 minutes (most of the
.0
15 l phase - - Phase of decline
Q) Lag
medically, important bacteria), bacteria take 18-24 hrs to .0 phas~
E I
grow on culture media. In contrast M. tuberculosis take z
::i
I
I
longer duration (in weeks) to grow as its generation time I
I
is longer i.e. 20 hrs. M. leprae takes months to grow in
animal models as its generation time is 20 days. c,dl
C. Bacterial Count Time •
1. Total count: This indicates total number of bacteria

in the specimen, irrespective of whether they are
living or dead. Total count may be made by counting
the bacteria under microscope using counting 1. Lag Phase
chamber. After inoculation of the culture medium, multiplication
2. Viable count: It measures the number of living usually does not begin immediately. The period between
(viable) cells. Viable counts are obtained by dilution inoculation and beginning of multi ~icati
or plating methods. In the dilution method, several as ag phase. unng this period the organisms adapt to
tubes with liquid culture media are inoculated with tfre"'new environment, during which necessary enzymes
varying dilutions of sample and the viable count and intermediate metabolites are built up in adequate
calculated statistically from the number of tubes quantities for multiplication to proceed. There is increase
showing growth. Though this method does not give in the size of the cells but there is no appreciable increase
accurate values, but is still used widely for finding in numbers. The duration of lag phase varies with the
out presumptive coliform count in drinking water. species, nature of culture medium and temperature etc.

24 - - - - ----,--- - -- - - - - - - - - - - - - - - -- -- - Unit I: General Microbiology
3M
2. Log (Lo a r i t ~ Ex onential Phase in human body and artificial culture medium. When
The cell division starts and their num ers mcrease bacteria multiply in host tissues, the situation may be
exponentially or by geometric progression with time. If intermediate between a batch culture and a continuous
the logarithm of the viable count is plotted against time, culture. The source of nutrients may be plenty, but the
a straight line is obtained. defence mechanisms of the body influence the bacterial
growth in-vivo.
3. Stationary Phase
After log phase, the bacterial growth ceases almost II. BACTERIAL NUTRITION
completely due to e of nutrients and The principal constituent of bacterial cell is water, which
accumulation of toxic products. The nu..,..,..,,.,.-"'-'- progeny is about 80% of the total weight. For growth of bacteria,
cells formed is just enough to replace the number of cells the minimum nutritional requirement is water, a source
that die. The number of viable cells remain stationary of carbon, a source of nitrogen and some inorganic salts.
as there is almost a balance between the dying cells and These nutritional elements may be supplied in suitable
the newly-formed cells. artificial culture media. In addition to these, bacteria
require sulphur, phosphorus and other elements such as
4. Phase of Decline sodium, potassium, magnesium, iron and manganese in
After a period of stationary phase, the bacterial population small amounts.
decreases due to the death of cells. The decline phase starts Some bacteria are fastidious and will not grow unless
due to exhaustion of nutrients, accumulation of toxic certain organic compounds are added to the medium.
products and autolytic enzymes. There is decline in viable These are known as growth factors or bacterial vitamins.
count and not in total count. With autolytic bacteria, In many cases, bacterial vitamins are same as the vitamins
even the total count shows a phase of decline. necessary for mammalian nutrition, particularly those
belonging to the vitamin B group-thiamine, nicotinic
Morphological and physiological alterations of cells acid, riboflavine, pyridoxine, folic acid and vitamin
during growth curve B 12 . These growth factors may be 'essential' for some
The end of the Bacteria have maximum bacteria when growth does not occur in their absence,
lag phase cell size. or 'accessory' when they enhance growth, without being
In the log phase Cells are smaller and stai absolutely necessary for it.
uniformly.
In the stationary • Cells are Gram- Significance
phase variable and show irregular Knowledge of compounds required by different bacteria
staining (due to intracellular is helpful in preparation of culture media for growing
sJ9,age granules). that particular bacterium. Some bacteria may require
~__prulation occurs. some special nutrients to be incorporated in the culture
~ · ---=-~
any bact:.;e:.=-r_ media.
-
produc exotoxi~
In the phase of Involution forms are ENVIRONMENTAL FACTORS AFFECTING
decline c ommon. GROWTH
1. Moisture and Desiccation
E. Batch Culture and Continuous Culture Moisture is an essential requirement for the growth
When bacteria are grown in a vessel of liquid medium of bacteria because 80% of the bacterial cell consists
(batch culture), after sometime the cell division ceases and of water. However, the drying has varying effects in
multiplication is arrested due to depletion of nutrients different organisms. Some organisms like Treponema
or accumulation of toxic products. By the use of special pallidum and N gonorrhoeae die quickly after drying
devices for replenishing nutrients and removing bacterial while M. tuberculosis and Staph. aureus may survive
cells, continuous culture of bacteria is maintained. The drying for several weeks. Drying in cold and vacuum
techniques namely chemostat and turbidostat are in (lyophilisation or freeze drying) is used for preservation
use. Maintenance of continuous culture of bacteria is of microorganisms.
sometimes required for industrial and research purposes.
Clinical Significance
F. Bacterial Growth in Vivo Transport media are used for transport of those clinical
There is a _significant difference of bacterial growth specimens which may contain delicate organisms such

Ch 3: Growth, Nutrition and Metabolism of Bacteria _ _ _ _ __ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ 25
as Neisseria gonorrhoeae. In contrast, specimens of is useful for sterilisation (destruction of microorganisms)

pus suspected to have Staph. aureus do not require any by heat e.g. autoclave (121 °C) and hot-air oven (160°C).
transport medium. Lyophilisation is an important method In contrast, some bacteria die rapidly at low temperature
of preservation of microorganisms and many biological (e.g. Haemophilus infiuenzae) but most survive well. Low
materials such as vaccines, antibiotics etc. temperature is used for preservation of bacterial cultures
in refrigerator (3°C to 5°C) or the deep freezer (- 30°C
2. Oxygen to - 70°C).
On the basis of requirement of oxygen, bacteria are
divided into aerobes and anaerobes. 5.pH
(a) Aerobes: They require oxygen for their growth. They Most of the medically important bacteria can grow at pH
may be obligate aerobes, which grow only in the presence 7.2-7.6. However, some bacteria such as lactobacilli and
of oxygen, or facultative anaerobes that are ordinarily cholera vibrio grow at acidic and alkaline pH respectively.
aerobes but can also grow without oxygen. Most of Strong solutions of acid or alkali (5% hydrochloric acid
the pathogenic bacteria are facultative anaerobes. or sodium hydroxide) kill most bacteria but mycobacteria
Example of obligate aerobe is Pseudomonas aeruginosa. are resistant to these agents.
Microaerophilic bacteria (microaerophiles) can grow in
the presence of traces of oxygen. Campylobacter and Clinical Significance
Helicobacter pylori are examples of microaerophilic Culture media having alkaline pH are used for growing
bacteria. Vibrio cholerae (Ref. Chapter 34). Strong solutions of acid
(b) Anaerobes: The obligate or strict anaerobes can grow or alkali are used in Petroff's concentration method for
only in the absence of oxygen. Clostridium tetani is sputum specimen for M . tuberculosis). (Ref Chapter 42)
one such example. Obligate anaerobes cannot grow
even in the presence of traces of oxygen. 6. Light
Bacteria (except phototrophic species) grow well in
3. Carbon Dioxide darkness. They are sensitive to ultraviolet rays and other
Small amount of carbon dioxide is required by all radiations. Photochromogenic mycobacteria produce
bacteria. This requirement is usually made available by pigment only on exposure to light.
the carbon dioxide present in the atmosphere, or produced
endogenously as a product of cellular metabolism. Some 7. Osmotic Effect
organisms such as Bruce/la abortus require much higher Bacteria are able to withstand a wide range of external
level of carbon dioxide (5-10 per cent) for their growth, osmotic variation because of the mechanical strength of
especially for primary isolation. These bacteria are called the cell wall. Sudden exposure of bacteria to hypertonic
capnophilic bacteria. solutions may cause p/asmolysis- osmotic withdrawal of
water leading to shrinkage of protoplasm. This occurs
4. Temperature more readily in Gram-negative than in Gram-positive
The temperature at which growth occurs best, is known bacteria. Sudden transfer of bacteria from concentrated
as the optimum temperature. The optimum temperature solution to distilled water may cause plasmoptysis-
range varies with different bacterial species. Most of the excessive osmotic imbibition of water leading to swelling
pathogenic bacteria grow optimally at 37°C (our body and rupture of the cell.
temperature). On the basis of optimum temperature range
for growth, bacteria are grouped as follows: 8. Mechanical and Sonic Stresses
(a) Mesophiles - between 25°C and 40°C e.g. majority Though bacteria have tough cell walls, they may be
of the pathogenic bacteria. ruptured and disintegrated by vigorous shaking with glass
(b) Psychrophiles - below 20°C e.g. soil and water sapro- beads and by exposure to ultrasonic vibrations.
phytes.
(c) Thermophiles - between SS°C and 80°C e.g.
Geobacillus stearothermophilus (formerly Bacillus III. BACTERIAL METABOLISM
s tearothermophilus). The series of changes of substance (carbohydrate,
protein or fat) within the bacterial cell from absorption
Clinical Significance to elimination is known as metabolism of the substance.
Most of the pathogenic bacteria die between 55°C to 65°C Aerobic bacteria obtain their energy only through
and most spores between 100°C to 120°C. This knowledge oxidation involving oxygen as the ultimate hydrogen

acceptors, while the anaerobes use hydrogen acceptors by the process of fermentation and aerobic respiration.
other than oxygen. They derive their energy by fermentation in the absence
of oxygen but switch over to aerobic respiration in the
A. Oxidation presence of ·oxygen.
In case of aero bes, where the ultimate hydrogen acceptor is
oxygen, the carbon and energy source may be completely Clinical Significance
oxidised to carbon dioxide and water. The conversion of Biochemical reactions are used for identification of Gram
adenosine diphosphate (ADP) to adenosine triphosphate negative bacilli. Carbohydrates are fermented to produce
(ATP) provides energy in this process. This process is acid (lactic acid, pyruvic acid etc.) and gas (hydrogen,
called oxidative phosphorylation. Small amount of carbon dioxide).
hydrogen peroxide produced in the process is disposed off
by enzymes catalase or peroxidase present in aerobes. C. Oxidation-Reduction (O-R)
Potential (Redox potential)
B. Fermentation Oxidising agent is a substance which can accept electrons
In anaerobic respiration, hydrogen acceptor compounds while reducing agent is able to part with electrons (Fig.
are nitrates or sulphates, instead of oxygen. In fermen- 3.3). The oxidising or reducing condition of a system is
tation, a series of oxidoreductions occur in which the indicated by the net readiness of all the components in
carbon and energy source acts as both the electron that system to accept or to part with the electrons. This
donor and electron acceptor. The organic end products is called oxidation-reduction potential or redox potential.
of fermentation include acids (lactic acid, formic acid, The redox potential of a medium is determined by
pyruvic acid), gas (hydrogen, carbon dioxide) and recording the electrical potential difference set up between
alcohols. the medium and an 'unattackable' electrode immersed
During fermentation, energy rich phosphate bonds in it. The electrode potential (Eh) can be measured in
are produced by the introduction of organic phosphate millivolts. This depends upon the state of oxidation or
into intermediate metabolite. This process is known reduction of the solution.The more oxidised the system,
as substrate-level phosphorylation. These energy-rich the higher is the potential and in reduced system potential
phosphate groups so formed help in conversion of ADP is lower. The redox-potential of the medium can also
to ATP. Fermentation is carried out by both obligate and be assessed by the use of certain indicator dyes such as
facultative anaerobes. methylene blue or resazurin, which become coloured in
The oxygen and hydrogen peroxide are toxic to oxidised state. It is a simple but less accurate method.
anaerobes. The reason for this toxicity is not well Anaerobic organisms require low redox potential for their
understood. It is suggested that in the presence of oxygen, growth.
hydrogen peroxide accumulates in the media and inhibits
the growth of anaerobes The enzyme catalase, which splits Clinical Significance
hydrogen peroxide, is present in aerobic bacteria but is Anaerobic jar is used for growing anaerobic bacteria in
absent in the anaerobes. Another reason might be that it. Methylene blue solution is kept in the anaerobic jar
anaerobes possess essential enzymes that are active only to acertain anaerobic conditions in the jar. Methylene
in the reduced state. blue solution is colourless in anaerobic conditions and
The facultative anaerobes may act in both ways i.e. blue in aerobic conditions. (Ref. Chapter 6).
Electron
Electron
Reducing Oxidising Oxidised Reduced

agent agent product product
Fig. 3.3 Exchange of electrons in an oxidation - reduction process

Ch 3: Growth, Nutrition and Metabolism of Bacteria _ _ _ _ __ _ _ _ __ __ _ _ __ __ _ _ 27
KEY POINTS
1. The minimum nutritional requirement for growth and multiplication of bacteria includes sources of carbon, nitrogen,
hydrogen, oxygen and some inorganic salts.
2. Bacterial cell division occurs by binary fission .
3. The time required for the bacterium to give rise to two daughter cells under optimum conditions is known as the
generation time.
4. Viable count measures the number of living (viable) bacteria while total count indicates total number of bacteria in
the specimen, irrespective of whether they are living or dead.
5. Bacterial growth curve has four phases namely lag phase, log phase, stationary phase and phase of decline .
6. On the basis of requirement of oxygen, bacteria are divided into aerobes and anaerobes.
7. Some organisms require higher level of carbon dioxide (5 -10%), for their growth, they are n amed as capnophilic
bacteria.
8. Majority of bacteria grow between temperature range of 25°C to 40°C i.e. mesophiles.
9. T he oxidising or reducing condition of a system is indicated by the net readiness of all the components in that system
to accept or to part with the electrons. This is called oxidation reduction potential or redox potential.
10. Anaerobic organisms require low redox potential for their growth.
YOU MUST KNOW

1. Generation time.
2. Various phases of bacterial growth curve.
3. Microaerophilic bacteria, capnophilic bacteria and their examples.
4. Redox potential.
STUDY QUESTIONS
(a) Bacterial growth curve (b) Redox potential.

1. Generation time of Mycobacterium tuberculosis is about:
(a) 20 seconds (b) 20 minutes (c) 20 hours (d) 20 days
2. Bacteria which can grow at temperature between 25°C and 40°C are known as:
(a) Mesophiles (b) Psychrophiles (c) Thermophiles (d) None of the above
3. Which one of the following bacteria can grow in acidic pH?
(a) Klebsiella sp. (b) Lactobacilli (c) Pseudomonas aeruginosa (d) Vibrio cholerae
4. The bacteria which require higher level of carbon dioxide for their growth are known as:
(a) Microaerophilic bacteria(b)Capnophilic bacteria (c) Aerobic bacteria (d) None of the above
5. Low redox potential of culture medium is required for growth of:
(a) Anaerobic bacteria (b) Escherichia coli (c) Pseudomonas aeruginosa (d) None of the above
Answers (MCQs):
1. (c) 2. (a) 3. (b)
____ ... ____

4. (b)

5. (a)

Chapter 4
STERILISATION AND DISINFECTION
I. Introduction: Some Definitions

II. Methods of Sterilisation
A. Physical Methods B. Chemical Methods
Ill. Testing of Disinfectants
I. INTRODUCTION: SOME DEFINffiONS 2. For media and reagents used in the microbiology
Microorganisms are responsible for contamination and laboratory.
infection. They are present all around. The aim of 3. In food and drug manufacturing to ensure safety
sterilisation is to remove or destroy them from materials from contaminating organisms.
or from surfaces.
) 'O~ II. METHODS OF STERILISATION
~ rilisation A. Physical methods
It is a process by which an article, surface or medium is 1. Sunlight ~ ~v-"~-~ ~
-~ ).~
made free of all microorganisms either in the vegetative 2. Heat /
or spore form. --- (a) Dry heat • . /4
(b) Moist heat
Disinfection 3. Ozone
It means the destruction of all pathogens or organisms 4. Filtration
capable of producing infections but not necessarily spores. 5. R adiation
All organisms may not be killed but1file:m:mrl:reris7roaceu-
to a level that is no longer harmful to health. B. Chemical met hods
1. Alcohols
Antiseptics 2. Aldehydes
These are chemical disinfectants which can safely be 3. Phenols
applied to living tissues and are used to prevent infection 4. Halogens
by inhibiting the growth of microorganisms. 5. Oxidising agents
6. Salts
Asepsis 7. Surface active agents
The technique by which, the occurrence of infection 8. Dyes
into an uninfected tissue is prevented. 9. Vapour phase disinfectants
Uses of Sterilisation/Disinfection A. Physical Methods

1. Sterilisation of materials, instruments used in surgical 1. Sunlight
and diagnostic procedures. Sunlight has an active germicidal effect due to its content
28
Ch 4: Sterilisation and Disinfection _ _ _ _ _ _ _ _ _ _ _ __ _ _ _ __ _ _ _ __ __ _ _ 29
of wtravia)et xa,zs. It is a natural method of sterilisation passed through bunsen flame without allowing them to
in cases of water in tanks, rivers and lakes. become red hot.
2. Heat (iii) Incineration

Heat is the most reliable and commonly employed method By this method, infective material is reduced to ashes by
of sterilisation. It should be the method of choice unless burning. Instru ·. cinerator may be used for
contraindicated. Two types of heat are used, dry heat this purpo . oiled dressin s animal carcasses, bedding
and moist heat. and atholog1ca materials are dealt with this method.
<If, K g /1,; ~ 5'/v}
Principle --..,;. ---------
(i) Dry heat kills the organisms by _genatu.r,ation of It is y used method of sterilisation by dry
bacterial protein, oxidative damage and by the toxic heat. The oven is electrically heated and is fitted with a
effect of elevated levels of electrolytes. However, the fan to ensure adequate and even distribution of hot air
possibility of DNA damage is also incriminated in the chamber (Figs. 4.1 and 4.2) . It i ~fitted with
as one of the mechanisms of inactivations of a t12sfpostat that.maintains the chamb~t a chosen
microbes. temperature. •
(ii) Moist heat kills the microorganisms by denaturation
and s,oagulation of proteil!s.
Factors influencing
(i) Nature of heat: D ry heat or moist heat
t oe,!_
-f i
(ii) Temperature and duration: The time required for
sterilisation is inversely proportional to temperature
to which organisms are exposed.
(iii) Characteristic of microorganisms and spores present:
Bacterial spores are killed by moist heat at j 21 °C
for 15 minutes. Most vegetative bacteria, fungi and
viruses are killed in 30 minutes at 65°C by moist Fig. 4.1 Hot air oven- Diagramatic
heat.
(iv) Type of material: A high content of organic
substances tend to protect the vegetative form and
spores against the lethal action of heat. Materials
containing organic substances require more time
for sterilisation. Proteins, sugars, fats and starch
are some of the organic substances.
DRY HEAT STERILISATION I

The following procedures are used for sterilisation by
dry heat.
(i) Red heat
(ii) Flaming
(iii) Incineration
f cP.tf --
IU:.&.:I
(iv) Hot air oven
Fig. 4.2 Hot air oven
(i) Red heat
and needles
urner till they become Temperature and time
160° for w urs (holding time) is required for
sterilisation (previous y it was 160°C for one hour).
However, alternative temperatures and holding time
include 170°C for one hour and 180°C for 30 minutes.

30 - - - - - - - - - - - - - - - - - - - - - - - - - - - Unit I: General Microbiology
Uses sterile by heating at 80-85°C temperature for half an

It is used for sterilisation of hour daily on three consecutive days. This process
(i) Glasswares like glass syrin_,ges, petridishes, flasks, of sterilisation is called inspissation. The instrument
,.gipettes and test tubes. used is called inspissator.
(ii) Surgical ius1:;!Jments like scalpels, (c) Vaccine bath- Bacterial vaccines are sterilised in
etc. special vaccine baths at 60°C for one ho r. Serum
(iii) Chemicals such as liquid paraffin, fats, sulphonamides or body fluids can be sterilised by heating for one
powders etc. hour at 56°C in a water bath on several successive
days.
Precautions (d) Low temperature steam formaldehyde ( LTSF)
(i) It should.,got be over oaded . sterilisation- Items which cannot withstand the
(ii) The material should he arranged in a manner which temperature of 100°C may be sterilised by a method
allows free circulation of air. known as low temperature steam-formaldehyde (LTSF)
(iii) Material to be sterilised should be erfec y dr . sterilisation. In this method steam at subatmospheric
(iv) Test tubes, flasks etc. shou d be fitted witli cotton pressure at the temperature of 75°C with formaldehyde
lu vapour is used. Bacillus stearothermophilus has been
(v) P~tr!dishes and pipettes should be ed in used as biological control to test the efficacy of LTSF
paper. sterilisers.
( vi) '@bee uyiterials ex6e't silj.cnne--rubber) or any
inflammable material should not be kept inside the
-<oven.
( vii) The oven must be allowed t<_?. cool fQ[ two hours
before opening the doors, since the glasswares may
(ii) At a Temperature of 100°C
(a) Boiling Boiling for 10 to 30 minutes may kill most
of the vegetative forms but many spores withstand
crack by sudden cooling . boiling for a considerable time. When better methods
.- !'2-.<;'1-- are not available, boiling may be used for glass
Sterilisation control syringes and rubber stopper . It is not recommenaed
(i) The spores of BacillzisfzF rlls ubsp. niger (NCTC for the sterilisation of instruments for surgical
10075 or ATCC 9372) are kept inside the oven. procedures.
These spores should be destroyed if the sterilisati.9.~~ (b) Tyndallisation- Steam
is proper. ,W ,, ,A'o \P- ' • on three successive days · is known
(ii) Thermocouples may also be used. ,;J £.-lP · as tyndallisation or intermittent sterlisation. The
(iii rowne' tu6e with green spot is available. After principle is that the first exposure kills all the vegetative
proper sterilisation green coJo ·s produced after forms, and in the intervals between the heatings the
two hours at l 60°q. remaining spores germinate into vegetative forms
which are killed on subsequent heating. It is used
MOIST HEA STERILISATION for sterilisation of egg, serum or sugar containing
---.UllS-lll-e-1~rer of sterilisation may be used at different media which are da at higher temperature of
temperatures as follows. ;.,-----=-~;,::.o-=n~ly:......:used is Koch's
(i) At a temperature below 100°C or Arnold's steam nliser.
(ii) At a temperature of 100°C (c) team sten iser t 100°C for 90 min es-Koch's or
(iii)'-At a temperature above 100°C Arnold's steam st · iser (Fig. 4. ually used for
media which are decomposed at high temperature of
(i) At a Temperature below 100°C autoclave. The articles are kept on a perforated tray
(fl' fasteurisation o mil Two types of method,._ holder through which steam can pass. They are exposed to
method (63°C for 30 minutes) and~ metl!gd (72°C steam at atmospheric pressure for 90 minutes. Most
for 20 seconds followed by coolingqwckly to l3°C of the vegetative forms are killed by this method
or lower) are used. All nonsporing pathogens such except thermophiles.
as mycobacteria, brucellae and salmonellae are killed
except Coxiella burnetii which being relatively heat (iii) At a Temperature above 100°C n er ressure)
resistant may survive in holder method. Water boils when its vapour pressure equals that of the
(b) ~ 'g]J"SS£.1..U£!lJ--,Some serum or egg media, such as surrounding atmosphere. When the atmospheric pressure
Lowenstein-Jensen's and Loeffier's serum, are rendered is raised then the boiling temperature is also raised. At

Ch 4: Sterilisation and Disinfection _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ 31
- - - - - - -- - - Perforated tray
- - - f - - -- Water Fig. 4.4 Vertical autoclave
Fig. 4.3 Steam steriliser
normal pressure water boils at 100°C but when pressure

inside a closed vessel increases, the temperature at which Chamber
water boils also increases. Thi principle has een applie Door
Perfor~
in autoclave and ressure cooker. (E_?Y,
~W' sM Water
AUTOCLAVE c:;;.;.--
100°C or satu am has a better killing Fig. 4.5 Autoclave (inner view)
power than dry heat. Bacteria are more susceptible to
moist heat as bacterial protein coagulates ra.e,idly.
Saturated
- - -
steam can penetrate porous material easily.>
Procedure
When steam comes into contact with a cooler surface it The cylinder is filled with sufficient water and the material
condenses to water and liberates its latent heat to that to be sterilised is placed on tpe tray. The lid is closed
surface, for example, 1600 ml of steam at 100°C and at with the discharge tap open. The heater is put o~ Safety_
atmospheric pressure condenses into one ml of water valve is adjusted to the required pre·ssure: After boiling
at 100°C and releases 518 calories of heat. The large of w a t e ~ m and air mixture is allowed' to escape
reduction in volume sucks in more steam to the same till all the air has been displaced. This can be tested by
site and the process continues till the temperature of leading the escaping steam-air mixture into a pail of water
the article is raised to that of steam._ The condensed through rubber tubing. When the air bubbles stop coming
water produces moist conditions for killing the m1cro6es in the pail it indicates that all the air has been displaced.
----
present.
Components of autoclave
Autoclave is a modified pressure cooker or boiler. It
The discharge tap is now closed. The steam pressure
rises inside and when it reaches the desired set level
(15 pounds (lbs) per square inch), the safety valve opens
and the excess steam escapes. From this point, the holding
a vertical or horizontal cylinder of gunmetal period (15 minutes) is counted. After the holding period,
or stainless steel in 'a supporting iron case. The lid is the heating is stopped and autoclave allowed to cool till the
fastened by screw clamps and rendered air tight by an pressure gauge indicates that the pressure inside is equal
asbestos washer. The lid bears a discharge tap for air and to the atmospheric pressure. The discharge tap is opened
steam, a pressure gauge and a safety valve.. Heating is slowly and air is allowed to enter the autoclave. The lid
~ al)y done by electric_i!y. The steam circulates wiiliin is now opened and the sterilised material removed.
the jacket and is supplied under high pressure to the Note: The domestic pressure cooker works on the
inner chamber where materials are kept for sterilisation same principle and may ~sed for sterilisation of small
(Figs. 4.4 and 4.5).
·-
articles in clinics.

J.-.1 .<1----v y ~
\1,,ol- 1vv-, I
32 - - - - - - - - - - - - + - - - - ~ f - - - - - - - - - - - - - - - Unit I: General Microbiology
Sterilisation conditions (0) combines with other oxygen molecules (0 2) to form

Temperature - 121 °C ozone (0 3). The ozone provides a sterility assurance of
Chamber pressure - 15 lb per square inch I0----6 in approximately 4 hours.
Holding time - 15 minutes
These conditions are generally used, however, 4. Filtration
sterilisation can also be done at higher temperatures, at This method of sterilisation is useful for substances which
126°C (20 lbs/square inch) for 10 minutes or at 133°C get damaged by heat process e.g. sera, sugars, anti ioti
(30 lbs/square inch) for 3 minutes. solutions tc.
Uses Uses o filtration

(i) To sterilise culture media i:_ubber material, gowns, i sterilise sera, sugars and antibiotic solutions.
dressing, gloves etc. (ii) Separation of toxins and bacteriophages from
(ii) It is particularly useful for materials which cannot bacteria.
withstand the igher temperature o Ii oven. (iii) To obtain bacte · - ree tilt ates of clinical samples
(iii) For all glass syringes, hot air oven is a better for virus isolation.
sterilising method. (iv) Sterilisation of hydatid fluid.
(v) Filter discs retain the organism which can then be
Precautions cultured e.g. esfin of water samples for holera
(i) The air must be allowed to escape from the chamber vibrios or tx: hoid bacilli.
as temperature of air-steam mixture is lower than , vi) rificafom o
that of pure steam.
(ii) Materials should b~ =rr~a~n~g~edT"".:m , such a manner Limitation
as to ensure free circulation of steam inside the The pore size of filter is around .75 µm in diameter
chamber. Jl,J:, r,:\'0 ., which etains bacteria but al ows viruses an mycop as a
t ()'.P i1 \vJ Un ,\1.& to pass through filter, therefore, filtered preparations are
Sterilisation control not safe for clinical us .
(i) Thermocouple-It is to record the emperature .::ii-~ \2._s; 3 M
directly by a potentiometer. Types of filters
(ii) acterial s[!_ore Spores of aci us stearo- (i) Candle filters e.g. Berkefeld, Chamberland filter
t ermo hilus are used as the test organism. This ~sbestos disc flit s e.g. Seitz filter
organism has an optimum growth temperature of 55- (iii) Sintered glass filte
600C and its spores are killed in 12 minutes at 121 °C. (iv) Membrane filters
An envelope containing a filter paper strip impregnated ( v) Air filters
6
with 10 spores is placed inside the autoclave during (vi) Syringe filters
sterilisation. After sterilisation is over the strip is It is normal practice to use positive or more
inoculated into a suitable recovery medium and usually negative pressure by suction pump to enhance
incubated at 55°C for five days. Spores are destroyed filtration. ~--=-
if sterilising conditions of autoclave are proper. (i) anale filter These have been used widely for
(iii) Chemical indicators rowne's tube contains red urification of water for industrial and drinking
solution w ich turn re n , when exposed to purposes. These filters are usually made in the
temperature of l21 °C for 15 minutes in autoclave. form of hollow candles. The liquid to be filtered
(iv) Autoclave tapes " -1JJ\ is passed through the candle. They are available
"( l r ~ --- " in different grades of porosity. Candle filters are
3. Ozone of two types:
Low Temperature sterilisation by ozone (a) Diatomaceous earth filters e.g. , Berkefeld
Ozone steriliser uses oxygen, water and electricity (b) Unglaze porce ain filter e.g.,
to produce ozone within the steriliser and provide Chamberland.
sterilisation without producing toxic chemicals. It runs (ii) Asbestos disc filters ( Seitz lter)- These are made
at lower temperature i. . 25°C-35° In this device, oxygen up of asbestos magnesium silicate . The filter
molecules (0 2) are separated into atomic oxygen (0) in disc is supported on a metal mount. The filter
the presence of intense electrical field. This atomic oxygen is attached to a vacuum flask through a silicone

.T0 d---,,--..._
, -vvy~
- -- J" ( ,Y-:.
Ch 4: Sterilisation and Disinfection --------'------- 1-------0-=---- --'V'---___-+-_ _ _ _ _ 33
rubber bung. After use, the filter disc is discarded . High energy electron radiation is not used widely
Each time a fresh disc is used and the outfit is in medicine.
sterilised by autoclaving. Th se filter discs are (ii) Non-ionising ra iations- The-se inclu~e infrared and
available in different grades. ultraviolet (UV) radiations. Infrared is used for
(iii) Sinterea g ass flit s- These are prepared by fusing ~ e r i l i s a t i o n of syringes and catheters.
finely powdered glass particles. These are available U ~ radiation with wavelength of 240 to
in different pore sizes. 280 nm has marked. gactericidal activity. It acts
(iv) embr e lter - Membrane filters are made by <ieoaturation of bacterial protein and interference
up o ~ These are routinely used with DNA replication. UV radiation is used for
in water analysis, sterility testing, and for the disinfecting enclosed areas such as bacteriological
solutions for parenteral use. laboratory, inoculation hoods, laminar flow and
it ocellulose illipore) membrane filters are operation theatres. Most vegetative bacteria are
>f"' widely used. Membrane filters are available in susceptible to UV radiation but spores are highly
pore sizes of 0.015 to 12 µm. The 0.22 µm filter -.r- resistant. Susceptibility of viruses is variable.
is the most commonly used because the pore size is Sf11
smaller than that of bacteria. For bacterial counts B Chemical Methods -=:::::::::::i_
of water, a known amount of water is filtered A vane ents are used as antise tics and
through the membrane filter disc. The upper side disinfectants. A§lideal antiseptig o disinfectant ould
of filter disc is placed on a culture medium and (i) have wide spectrum of activity and e e ective
incubated. The colonies develop on the medium. against all microorganisms including bacteria (both
Viable counts can be calculated by counting the vegetative and spore forms), viruses, protozoa and
colonies. fungi,
(v) ir 7ilters- T hese filters are used to deliver (iz) act in the presence of organic matters,
clean bacteria-free air to a cubicle or a room. (iii) have high P.enetration power and quick action,
High efficiency particulate air HEPA filters are (iv) be stable and effective in ~Ldi&- as well as in alkaline
used in air filtration in laminar air flow system mnditioos,
in microbiology laboratories. HEPA filters can (v) not co e metals,
remove articles of 0.3 µm or larger. (vi) be ati with other disinfectants,
( vi) rin efilter - Syringes fitted with membrane of (vii) not caJISe local irritation,
different diameters are available. For sterilisation, ( viii) not be toxic if absorbed into circulation,
the fluid is forced through the disc (membrane) (ix) be safe and easy to use, and
. , p by pressing the piston of the syringe. (x) be easil~ available =;;d cheap.
C fv'\ y-. ~J The disinfectant which possesses all the above criteria
5. Radiations X' - ,, ' is yet to be found.
Two types of radiations are used for sterilisatio~bii1fui g "'fhe following factors influence the potency of a
and non- ·onising. · -:,,- disinfectant :
(t) o ising radiations - Ionising radiations include (i) concentration
gamma rays, X-rays and cosmic rays. They have (ii) time of action
very high penetrating power. They are highly lethal (iii) pH
to all cells including bacteria. They damage DNA by (iv) temperature
yarious mecbaoism.s...Gamma radiaiions from a Cobalt (v) nature of organism and
60 source are commercially used 'lisation ( vi) presence of organic matter.
of disposable items such las ·c s r· swabs, tants can be divided into three
culture plates, cannulas cat rs tc. This method ....-i. !:!,!gh level disinfectants.
is also known a cold sterilisat · n ecause there is no ~!,o.ulrmediate level disinfectants.
appreciable incr em temperature. The advantages J ~Low level disinfectants.
of this method include speed, high penetrating power
(it can sterilise materials, through outer packages and 1. High level disinfectants
wrappings), and the absence of heat. Bacillus pumilis ~tiveness of high level disinfectants may be
has been used for testing the efficacy of ionising · to that of sterilisation. These disinfectants
radiations. are used for certain types of endoscopes, cystoscopes
(
and surgical instruments with plastic com o GLUTARALDEHYDE

which cannot wit stand the sterilisation procedures It is effective against bacteria (including M. tuberculosis) ,
such as autoclaving. The high level disinfectants include fungi and viruses (including human immunodeficiency
glutaraldehyde, peroX:1~eracetic acid and viruses, hepatitis B virus and enteroviruses). It also kills
chlorine compou~ (9
d-l-J) f spores. It is less toxic and irritant to the eyes and skin
than formaldehyde. It is rapid, broad spectrum, and is
2. Intermediate level disinfectants ~ J..J: · one of the few chemicals accepted as a sterilant and high
Intermediate level disinfectants may effective level disinfectant. It can act in the presence of organic
agai bacteria spore , hence these disinfectants are used matter and does not damage plastics. It is used as 2%
for inst ents (e.g. laryngoscopes, fiberoptic endoscopes) buffered solution. It is available commercially as 'cidex'.
where contamination with spores and other highly resistant It can be used for delicate instruments having lenses.
organisms is unlikely. The intermediate level disinfectants
include alcohols, iodophores and phenolic compounds. JJ,~
C) 0 rf (Y (i) For sterilisation of cystoscopes, endoscopes and
3. Low level disinfectants ® bronchoscope.
Many organisms can survive on exposure to low level (ii) To sterilise plastic endotracheal tubes, face masks,
disinfectants. These disinfectants are used for items corrugated rubber anaesthetic tubes and metal
which come in contact with the patients but they do instruments.
not penetrate into tissue. S~OPfS, electrocardiogram
electrodes etc. are examples ot strc'h items. ORTHO-PHTHALALDEHYDE
Ortho-phthalaladehyde (OPA) is a high level disinfectant.
l. Alcohols It is more stable during storage and more rapidly
Ethyl alcohol and isopropyl alcohol are the most frequently mycobactericidal than glutaraldehyde. 0.5% OPA is slowly
used. They act by denaturing bacterial proteins. They sporicidal. OPA vapours may irritate the respiratory tract
rapidly kill bacteria including tubercle bacilli but they and eyes, therefore, it must be handled with appropriate
have no sporicidal or virucidal activity. However, human safety precautions. It can be used for materials where
immunodeficiency v1ru IV) is susceptible to 70% ethyl glutaraldehyde is used such as endoscopes. This new
alcohol and 35% isopropyl alcohol in the absence of disinfectant may be useful for glutaraldehyde resistant
organic matter. They are used mainly as skin antiseptics. mycobacteria.
To be effective, they should be used at a concentration of
60-70 per cent in water. Isopropyl alcohol is preferred to 3. Phenols
ethyl alcohol as it is a better fat solvent, more bactericidal Lister, the father of antiseptic surgery, first introduced use
and less volatile. of phenol (carbolic acid) in surgery (1867). Phenols are
Methyl alcohol is effective against fungal spores and produced by distillation of coal tar between temperatures
is used for treating cabinets affected by them. Methyl of l 70°C and 270°C. The lethal effect of phenols is due
alcohol vapour is toxic and inflammable. to cell membrane damage, thus releasing cell contents
and causing lysis. Phenol (1 %) has bactericidal action.
2. Aldehydes It is readily absorbed by skin and causes toxicity.
FORMALDEHYDE I Certain phenol derivatives like cresol, chlorhexidine,
It is markedly actericidal, sporicidal and virucidal. It is chloroxylenol and hexachlorophane are commonly used
used both as aqueous solution an m gii:!t~~~rm. A as antiseptics.
10% aqueous solution of formalin is routinely used. It is (i) Cresols- Lysol is a solution of cresols in soap. It
active against amino group in the protein molecule. is most commonly used for sterilisation of infected
Uses glasswares, cleaning floors, disinfection of excreta.
Preservation of tissue for histological examination.
(i) They are not readily inactivated by the presence of
To sterilise bacterial vaccines.
(ii) organic matter. They are active against a wide range
To prepare toxoid from toxin.
(iii) of organisms.
For killing of bacteripl cultures and suspensions.
(iv) (ii) Chlorhexidine-§J:X,lon (chlorhexidine and cetrimide)
For destroying a nthrax spores in hair and wool.
(v) is widely used in wounds, pre-operative disinfection
Formalin gas has been described under "Vapour of skin, as bladder irrigant etc. It is bactericidal at
phase disinfectants". a high dilution. They are more active against Gram

---co 01J
~-
~s ,~ D~ ~kc ~
Ch 4: Sterilisation and Disinfection
h
a<; P~ .-------- m sc..."" to') _
J
ll-iLoib' ¥'•"&,obL
t)
@ Sea;,., I
'1 , ~ < >t ~
-
i,..Yf>~
s~ I) olb,~
positive than Gram negative bacteria with no action radical on decompos ition of H 20 2 . These free radicals
against tubercle bacilli or spores and have very little are the active ingredient in the disinfection process. H 0
2 2
activity against viruses. They have a good fungicidal is used to disinfect contact lenses, surgical prostheses and
activity. plastic implants. It is used for high level disinfectio n and
(iii) Chloroxy lenol-It is an active ingredient of dettol. It sterilisation.
is less toxic and less irritant. It is readily inactivated
by presence of organic matter. It is inactive against ~ ~id
Pseudomo nas. Peracetic acid is an oxidising agent. It is one of the
(iv) Hexachlorophane- It is more active against Gram high level disinfecta nts. It is effective in resence of
positive than Gram negative bacteria. It is applied organic matter. It is a more poten errnicidal age t than
on skin as prophylax is against staphyloco ccal hydrogen peroxide. The end pro uc s ace 1c acid and
infections. It is bacteriost atic at very high dilutions. It oxygen) of this agent are non-toxic. It is also used in
is potentially toxic and should be used with care. plasma sterilisatio n procedure.
4. Halogens U 4- !-- (iii) Plasma sterilisati on

Chlorine and iodine are two commonly used disinfectants. Plasma refers to any gas which consists of electrons, ions
These are bactericid..gl and are effective against sporing or neutral particles. The formation of a low temperatu re
bacteria and viruses. plasma requires several devices. Chemical disinfecta nt
Chlorine is used in water supplies, swimming pools, such as H 2O 2 alone or a mixture of H O and peracetic
2 2
food and dairy industries. Chlorine compound s in the acid is used in these devices to induce the plasma. The
form of bleaching powder, sodium hypochlor ite and resulting UV radiation causes destructio n of vegetative
chloramin e are also used. The disinfectio n action of all organisms and spores. Two commerci al plasma sterilisers
the chlorine compound s is due to release of free chlorine. available are Sterrad JOOS steriliser and Plazlyte steriliser.
When hypochlorites are added to water, the chlorine reacts It is used for sterilisatio n of surgical instrumen ts.
with water to form hypochlor ous acid which is a strong
oxidising agent and effective disinfecta nt. The activity 6. Salts
of chlorine is markedly influenced by the presence of Salts of heavy metals have toxic effect on bacteria. The
organic matter. Bleaching powder or hypochlorite solution salts of copper, silver and mercury are used as disinfectant.
are the most widely used for human immunode ficiency They are protein coagulant s and act by combining with
virus (HIV) infected material. The ~chlori t;s have a sulphydryl (SH) groups of bacterial proteins and other
b~tericidaL...f.ungicidal, virucidal and rapidly sporicidal essential intracellul ar compound s. Merthiola te (sodium
action. Hypochlo rite solution decays rapidly and should ethyl mercurithi osalicylate ) is used in a dilution of 1:
be prepared daily. It should not be used in the presence 10,000 for preservati on of sera.
of formaldeh yde as one of the reaction products is found Mercuric chloride, once used as a disinfecta nt is
to be carcinogenic. Chloramin es are used as antiseptics highly toxic. Thiomersa l and mercuroch rome are less toxic
for dressing wounds. and are used as mild antiseptics. They have
)f l ~ n alcoholic and aqueous solutions is used as a bacteriost atic but limited fungicidal action. opper salt
skin disinfectant. It is actively bactericid al, with moderate
~es.
action against spores. It is active against M. tuberculosis
and viruses. Like chlorine, it is also inactivated by organic 7. Surface Active Agents
matter. Compoun ds of iodine with surface active agents Substance s which alter energy relationsh ips at interfaces,
known as iodophore s, are claimed to be ~re active than producing a reduction of surface tension, are known as
the a_queous or ~lcoholic _solutions of iodine. Betadine is
-
surface active agents or surfactants. They are classified into
one example of commonly used iodophore s. anionic, cationic, nonionic and amphoter ic compound s.
Of these, the cationic surfactant s have been the most
5. Oxidising Agents important antibacterial agents. These act on the phosphate
(i) Hydrogen peroxide groups of the cell membrane and also enter the cell.
Hydrogen peroxide (H 20 2) is effective against most This results in loss of membr,.ane semiperm eability and
organisms at concentra tion of 3-6%, while it kills all the cell proteins are denatured . Quaternar y ammoniu m
organisms including spores at higher concentra tion (10- compound s are the most important cationic surfactant s.
25%). Mode of action is by liberation of free hydroxyl Although these compound s are bactericid al for a wide

36 - - - - - - - - -- - - -- A'- ~ - - ~ ~eral Microbiology
------'--~----
range of organisms, Gram positive species are more 9. Vapour Phase Disinfectants
susceptible. They have no action on spores and tubercle FO~
bacilli. They are active against viruses with lipid envelopes This is employed for fumigation of operation theatres,
(e.g. rabies virus) and much less against non-enveloped wards laboratories etc. Forma ehyde gas is generated
viruses (e.g. enteroviruses). The common cationic by adding 150 gm of KMn0 4 to 280 ml formalin for
compounds are acetyl trimethyl ammonium bromide 1000 cu. feet of room volume. This reaction produces
(cetavalon or cetrimide) and benzalkonium chloride. The considerable heat, and so heat resistant containers should
activity of these compounds is greatest at an alkaline pH. be used. The doors should be sealed and left unopened
Acid inactivates them. Antibacterial activity is reduced for 48 hours. Sterilisation is achieved by condensation
in the presence of organic matter. Anionic surface active of gas on exposed surfaces. The gas is irritant and toxic
agents, like ordinary soaps, render them inactive. when inhaled. After completion of disinfection, the effect
The anionic compounds, e.g. common soaps, have of irritant vapours should be nullified by exposure to
strong detergent but weak antimicrobial properties. These ammonia vapour.
agents are most active at acidic pH. They are effective
against Gram positive organisms but are relatively ETHYLENE OXIDE (ETO)
ineffective against Gram negative species. It is a colourless liquid with a boiling point of 10.7°C. It
The amphoteric compounds, known as 'Tego' is effective against all types of microorganisms including
compounds possess detergent properties of anionic and viruses and spores. It is a highly penetrating gas and it
antimicrobial activity of cationic compounds. They are readily penetrates some plastics. It acts by alkylating the
active against a wide range of Gram positive and Gram amino, carboxyl, hydoxyl and sulphydryl groups in protein
negative organisms and some viruses. They are active molecules. In addition, it reacts with DNA and RNA.
over a wide range of pH. The presence of organic matter It has a potential toxicity to human beings, including
reduces their activity. mutagenicity and carcinogenicity. It is highly inflammable.
It forms explosive mixture when its concentration in air
8. Dyes is greater than 3 per cent. By mixing it with inert gases
Two groups of dyes, aniline dyes and acridine dyes have such as carbon dioxide, its explosive tendency can be
been used extensively as skin and wound antiseptics. eliminated.
Both are bacteriostatic in high dilution but have It is specially used for sterilising plastic and rubber
low bactericidal action. Aniline dyes include crystal articles, respirators, heart-lung machines, sutures, dental
violet, brilliant green and malachite green. They are equipments and clothing. It is commercially used to
more active against Gram positive bacteria than sterilise disposable plastic syringes, petridishes etc. It has a
Gram negative bacteria. They have no activity against high penetrating power and thus can sterilise prepackaged
M . tuberculosis, and hence the use of malachite green materials. Ethylene oxide steriliser called a chemic/ave is
in the Lowenstein-Jensen medium makes it selective for used for sterilisation. It is unsuitable for fumigation of
isolation of tubercle bacilli. They are non-toxic and non- rooms because of its explosive nature. It is irritant, and
irritant to the tissues. Their activity is inhibited by organic personnel working with it have to take precautions.
material such as pus. They interfere with the synthesis Bacillus globigi (a red pigmented variant of Bq.cillus
of peptidoglycan component of the cell wall. These dyes subtilis) has been used as biological control for testi~g
are used in the laboratory as selective agents in culture of ethylene oxide sterilisers.
media.
Acridine dyes include acriflavine, euflavine, proflavine
and aminacrine. They are affected very little by the BETAPROPIOLACTONE (BPL)
presence of organic material. They are also more active This is a condensation product of ketane and formaldehyde.
against Gram positive bacteria than Gram negative It has a boiling point of 163°C. Though it is a gas, it has
bacteria but are not as selective as the aniline dyes. These a low penetrating power. It has a rapid action and is used
dyes are used in clinical medicine. They interfere with in 0.2 per cent. It is effective against all microorganisms
the synthesis of nucleic acids and proteins in bacterial including viruses. It is more efficient for fumigation than
cells. formaldehyde. Unfortunately it has some carcinogenic
Gentian violet and acriflavine are two widely used activity. BPL is used for inactivation of vaccines.
dyes for skin disinfection especially in Gram positive Methods of sterilisation and disinfection of some
bacterial infections. important materials are shown in Table 4.1.

Ch 4: Sterilisation and Disinfection _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _
_ _ _ _ __ _ 37
Table 4.1 Methods of Sterilisation and Disinfection of Some Important Materials

Materials Methods
1. Inoculating wires or loops Red heat
2. Glasswares- syringes, petridishes, test tubes, flasks, universal container, oily fluids Hot-air oven
(Paraffin)
3. Disposable syringes and other disposable items Gamma radiations
4. Culture media Autoclaving
5. Culture media containing serum, egg Tyndallisation
6. Toxin, serum, sugar and antibiotic solutions Filtration
7. Milk Pasteurisation
8. Cystoscope and endoscope GI u taraldehyde
9. Sterilisation of operation theatre 3 /\1 ~S Formaldehyde gas
10. Infective material like soiled dressings, beddings, animal carcasses Incineration
11. Skin Tincture iodine, 70% ethanol, savlon
12. Aprons, gloves, catheters, surgical instruments Autoclaving
except sharp instruments
13. Sharp instruments 5% Cresol
14. Rubber, plastic and polythene tubes Glutaraldehyde
Recommended concentrations of some commonly sterilises the suspension in a given time is divided by the
used disinfectants are given in Table 4.2. corresponding dilution of phenol. This gives the phenol
coefficient. The phenol coefficient of 1.0 means that the
Table 4.2 Recommended Concentrations of Some test disinfectant has been as effective as phenol. Higher the
Disinfectants phenol coefficient, more effective is the disinfectant. The
Disinfectant Concentration phenol coefficient does not, however, give any indication
Ethyl alcohol 70% of how the test disinfectant will function practically in
G lutaraldehyde 2%
the presence of organic matters.
Lysol 2.5%
2. Chick Martin Test
Savlon (chlorhexidine and 2%, 5% Chick Martin test is a modification of Rideal Walker test.
cetrimide)
In this test , disinfectant acts in the presence of organic
Dettol (chloroxylenol) 4% matter to simulate natural conditions. Organic matter, in
Bleaching powder (calcium 14 gm in one litre of water the form of dried yeast or faeces, is included .
hypochlorite)
Sodium hypochlorite 1%, 0.1 % 3. Kelsey-S ykes Test
Betadine (Iodophore) 2% This test gives a measure of the capacity of a disinfectant to
retain its activity when repeatedly used microbiologically.
III. TESTIN G OF DISINFE CTANTS It is also named as capacity test. The standard organism
(Staphylococcus aureus, Esch. coli, Ps. aeruginosa) is
1. Rideal Walker test added to the disinfectant in three successive lots at 0,
2. Chick Martin test 10 and 20 minutes. These three lots are in contact with
3. Kelsey-Sykes test disinfectant for eight minutes and samples are transferred
4. In- Use tests at 8, 18 and 28 minutes respectively to a recovery medium.
The disinfectant is judged by its ability to kill bacteria
1. Rideal Walker Test (growth or no growth on recovery medium) and not by
In Rideal Walker test, suspensions contammg similar comparison with phenol. The test is carried out under
quantities of organisms are submitted to the action of both 'clean' and 'dirty' conditions. Thus it also measures
varying concentrations of phenol and of the disinfectant the effectiveness of a disinfectant in the presence of an
to be tested. The dilution of the test disinfectant which organic matter.

Unit I: General Microbiology
38 - - - - - - - - - - - -- - - - - - - - - - - - - - - - - -
4. In-Use Tests incubation period (Refer 'Slow Viral Diseases' in Chapter

The liquid phase of disinfectant solutions in actual use 64).
in hospital practice is examined quantitatively for viable l. Heat: They are extremely resistant to dry heat. A
organisms. A use-dilution is then determined which temperature of 360°C for one hour has not been
only very rarely yields a positive culture. The efficiency found to be effective completely. They are more
of a new disinfectant is determined by its ability to resistant to moist heat than bacteria, spores, fungi
inactivate a known number of a standard strain of a and viruses. Steam at a temperature of 134-138°C
pathogenic staphylococcus on a given surface within a for 18 minutes is found to be effective.
certain time. The results of such tests are generally more
useful than those of the phenol coefficient test and its 2. Chemicals: Prions are inactivated by sodium
modifications. hypochlorite (25% available chlorine) if treated for
one hour. They are also sensitive to household bleach,
STERILISATION OF PRIONS phenol (90%) and iodine disinfectants. Chemicals
Prions are infectious proteins without any detectable such as aldehydes, hydrogen peroxide, ethylene
nucleic acid. They are highly resistant to physical and oxide, ethanol and ionising radiations are found to
chemical agents. They produce slow infections with long be ineffective.
KEY POINTS
1. Sterilisation is a process by which an article, surface or medium is made free of all microorganisms either in the
vegetative or spore form.
2. Disinfection means the destruction of all pathogens or organisms capable of producing infections but not necessarily
spores.
3. Asepsis is the technique by which, the occurrence of infection into an uninfected tissue is prevented.
4. Methods of sterilisation include physical methods and chemical methods.
5. Hot-air oven and autoclave are two commonly used methods of sterilisation. Hot-air oven is a dry heat sterilisation
method while autoclave is a moist heat sterilisation.
6. Temperature and time period for hot-air oven is 160°Cfor two hours (holding time) whereas it is 121°Cfor 15 minutes
at pressure of 15 pounds per square inch for autoclave.
7. Other important methods of sterilisation include filtration, radiations, glutaraldehyde and ethylene oxide.
8. Filtration is used to sterilise sera, sugars and antibiotic solutions.
9. Gamma radiations and ethylene oxide (ETO) are the methods commercially used for sterilisation of disposable syringes
and other disposable items.
l 0. Glutaraldehyde is used for sterilisation of cystoscopes, endoscopes and bronchoscopes. It is effective against bacteria
(including M. tuberculosis), fungi and viruses (including human immunodeficiency viruses and hepatitis B virus).
YOU MUST KNOW

1. Difference between sterilisation and disinfection.
2. List of physical methods and chemical methods of sterilisation.
3. Principle, temperature and time period; uses of hot-air oven, autoclave, pasteurisation, inspissation and tyndallisation.
4. Uses of filtration and types of filters.
5. Types of radiations and their uses.
6. Uses of formaldehyde and glutaraldehyde as disinfectants.
7. Uses of gases as chemical method of sterilisation and role of ethylene oxide in sterilisation of disposable items.
8. Methods used for testing of disinfectants.

of free jee neet and Engineering PDFs •
Ch 4: Sterilisation and Disinfection _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ 39
STUDY QUESTIONS
1. Define sterilisation. How does it differ from disinfection? Classify the various agents used in sterilisation. Add a note
on the principle and functioning of autoclave.
(a) Hot air oven (b) Autoclave
(c) Filtration (d) Sterilisation by radiation
(e) Vapour phase disinfectants or Gaseous sterilisation.
3. Write briefly about:
(a) Sterilisation by moist heat (b) Tyndallisation
(c) Inspissation (d) Pasteurisation.
(e) Sterilisation of prions

1. The process by which an article, surface or medium is made free of all microorganisms either in the vegetative or
spore form is known as:
(a) Sterilisation (b) Disinfection (c) Asepsis (d) None of the above
2. Chemical disinfectants which can be safely applied to living tissues to prevent infection are known as:
(a) Antiseptics (b) Formaldehyde (c) Ethylene oxide (d) None of the above
3. Temperature and time period used in hot air oven is:
(a) 160°C for two hours (b) 140°C for two hours (c) 160°C for one hour (d) None of the above
4. Temperature and time period used in holder method of Pasteurisation is:
(a) 63°C for 30 minutes (b) 63°C for 50 minutes (c) 72°C for 20 seconds (c) 72°C for 40 seconds
5. Which of the following bacteria can survive in holder method of Pasteurisation?
(a) Bordetella pertussis (b) Coxiella burnetii (c) Salmonella typhi (d) Mycobacterium bovis
6. Steam sterilisation at 100°C for 20 minutes on three successive days is known as:
(a) Tyndallisation (b) Inspissation (c) Pasteurisation (d) Vaccine bath
7. Which of the following sterilisation conditions are used in autoclave?
(a) 121°C, 15 pounds pressure/sq inch, 15 minutes (b) 160°C, 20 pounds pressure/sq inch, 20 minutes
(c) 140°C, 10 pounds pressure/sq inch, 15 minutes (d) None of the above
8. Which bacterial spores are used as sterilisation control in autoclave?
(a) Clostridium perfringens (b) Bacillus cereus (c) Bacillus stearothermophilus (d) Pseudomonas aeruginosa
9. Gamma radiations can be used for sterilisation of:
(a) Plastic syringes (b) Catheters (c) Swabs (d) All of the above
10. Glutaraldehyde is used for sterilisation of:
(a) Cystoscopes (b) Endoscopes (c) Bronchoscopes (d) All of the above
11 . The most widely used disinfectant for human immunodeficiency virus (HIV) infected material is:
(a) Hypochlorite solution (b) Lysol (c) Formaldehyde (d) Mercuric chloride
12. Which of the following is used commercially for sterilisation of disposable plastic items?
(a) Ethylene oxide (b) Autoclave (c) Glutaraldehyde (d) Ethyl alcohol
Answers (MCQs):
I. (a) 2. (a) 3. (a) 4. (a) 5. (b) 6. (a) 7. (a) 8. (c) 9. (d) 10. (d)
11. (a) 12. (a)
____ ... ____

Chapter 5
CULTURE MEDIA
I. Introduction
II. Types of Media
~ v, I. INTRODUCTION II. TYPES OF MEDIA

(}Culture media are required to grow the or anisms from Media are classified in many ways:
infected mate al,to identify t e causative agent. he basic 1. Based on physical state
C constituents of u ture me ia are:

1. Water: Source of hydrogen and oxygen.
2. Electrolyte: Sodium chloride or other electrolytes.
/3~ e : It is a complex mixture of partially digested
( ~ n s . It is obtained from lean meat or other protein
(i) Liquid media
(ii) Semisolid media
(iii) Solid media
2. On the basis of presence of molecular oxygen and
reducing substances in the media
material such as heart muscle, casein or fibrin , usually (i) Aerobic media
by digestion with proteolytic enzymes. It contains (ii) Anaerobic media
proteoses, amino-acids, polypeptides, phosphates, 3. Based on nutritional factors
minerals (K, Mg), and accessary growth factors (i) Simple media
like nicotinic acid and riboflavin . Special brands of (ii) Complex media
peptone such as neopeptone, proteose peptone are (iii) Synthetic media
av le for special uses. (iv) Special media f\1~
Meat extra t: It is available commercially as 'Lab- (a) Enriched media R~ ,-..,</'
r-,:ew~'-,---n contains protein degradation products, (b) Enrichment
inorganic salts, carbohydrates and growth factors.
(c) Selective media
5. Blood or serum: These are used for enriching culture
(d) Differential m e ~ (b0fl
media. Usually 5-10% defibrinated sheep blood is
. In certain media, serum is used. (e) Indicator media
: It is prepared from sea weed (Algae- geladium (j) Transport media
es). It contains mainly long-chain polysaccharide, (g) Sugar media
a small amount of protein-like material and a variety
of inorganic salts. It is available either in long shreds
or powder form. It is used in concentration of 2-3%. Simple Media
It melts at 98°C and usually solidifies at 42°C. Agar Nutrient broth is an example of simple medium. It
does not provide any nutrition to the bacteria but contains peptone water and meat extract 1%. When
acts as a solidifying agent only. New Zealand agar glucose (0.5%) is added to nutrient broth, it becomes
has twice the jellifying capacity than that of Japanese glucose broth. Peptone water and glucose broth are shown
agar. in Figs. 5.1 and 5.2.
40
Ch 5: Culture Media _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ 41
Synthetic Media
These are prepared from pure chemicals and the exact
composition of the medium is known. These are used for
special studies such as metabolic requirements. Dubo's
medium with tween 80 is one example of a synthetic
medium.
Special Media
(a) Enriched Media
When basal medium is added with some nutrients such
as blood, serum or egg, it is called enriched medium.
Some examples of enriched media are:
Blood agar (Fig. 5.4) - Blood is added to nutrient
agar. It may be used for gro ·n a number of bacteria
but one specific example is Streptococcus mres
blood for its growth.
Fig. 5.1 Peptone water Fig. 5.2 Glucose broth
Fig. 5.4 Blood agar
Chocolate agar (Fig. 5.5) - It is a heated blood

...
agar used for isolation of Neisseria and Haemophilus_
Fig. 5.3 Nutrient agar

----
infiuenzae.
When 2-3% agar is added to nutrient broth, it

becomes nutrient agar (Fig. 5.3). This is the simplest and
routinely employed medium in laboratory for diagnostic
purposes. If concentration of agar is reduced (0.2 to
0.4%), semisolid medium is obtained which enables motile
bacteria to spread.
Complex Media
All media other than simple media are complex. Complex
media have added ingredients for bringing out certain
properties or providing special nutrients required for the Fig. 5.5 Chocolate agar
growth of the bacterium in question.

Loeffier's serum slope (Fig. 5.6) - Serum is added for (d) Differential Media
enriching the medium. This medium is used for grouping When a medium contains substances which help to
Corynebacterium diphtheriae. distinguish differing characteristics of bacteria, it is called
These media are employed to grow organisms which "differential medium" e.g. MacConkey's medium, which
are more exacting in their nutritional needs. contains peptone, lactose, agar, sodium taurocholate
and neutral red. The lactose fermenters (LF) form pink
coloured colonies whereas non-lactose fermenters (NLF)
produce colourless or pale colonies.
(e) Indicator Media

These media contain an indicator which changes colour
when a bacterium grows in them. Salmonella typhi grow
as black colonies on Wilson and Blair medium containing
sulphite.
MacConkey's agar (Fig. 5. 7) is also an indicator
Fig. 5.6 Loeffler's serum slope medium. Due to fermentation of lactose, there is acidic
pH which forms the pink colonies in the presence of
neutral red indicator. The blood agar is an enriched
""'~
(b) Enrichment Media )J-~_f medium but bacteria lysing red blood cells give clearing
Some substances are incorporated in the liquid medium (haemolysis) around their colonies, thus, it is also an
which have a stimulating effect on the bacteria to be grown indicator medium. Therefore, some of these terms
or inhibits its competitors. es Its in an absolute like indicator, differential, selective or enriched are
increase in the number of nted acteria · related to interchangeable and more than one term may be applied
other acteria. Such media are calle enrichment media. for a single medium.
These are very useful for culture of faeces where the
nonpathogenic or commensal bacteria tend to overgrow
the pathogenic ones, e.g. , Salmonella being overgrown
i by Esch. coli. Some examples of enrichment media are
as fo 11 ows:"""
Tetrathionate broth - Tetrathionate is added which
inhibits coliforms while allows typhoid-paratyphoid
bacilli to grow.
Selenite 'F' broth - Selenite has similar action as that /
of tetrathionate in tetrathionate broth. vf.~>
'Alkaline peptone water - It is used to grow Vibrio l
erae.
@..gs ~/ ~vl>\
(cl. Selective Media / 0-tf'
Selective media contain substances that inhibit all 6;t
Fig. 5.7 MacConkey's agar
a few types of bacteria and facilitate the isolation of ~ - - - - - - - . + - - - "}I.
a particular species. These media are used to isolate a
articular bacteria from--;s~ecc1irmnie~rwh'e:re-11'H-Hlfetl-n:i'!denm-- (f) T~ por~::;:: 7
_flora 1s c e . elective me ia are so 1 in contrast to These are used in the case of delicate organisms (e.g.
media are: -
.. enrichment meoia which are liquid. Examples of selective gonococci) which may not survive the time taken for
transit or may be overgrown by nonpathogenic bacteria
Deoxycholate citrate agar ( DCA ) - Addition of (e.g. cholera organisms). For transport of specimens to
deoxycholate acts as a selective agent for enteric bacilli the laboratory, special media are devised and these are
(Salmonella, Shigella). termed transport media. Examples of transport media
/ Bile salt agar ( BSA ) - Bile salt is a selective agent. are:
It favours the growth of only Vibrio cholerae whereas (a) uart's transport medium is a non-nutrient soft agar
inhibits the growth of other intestinal organisms. gel conta· · ucing agent to prevent oxidation,

Ch 5: Culture Media _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __ _ __ _ __ 43
and charcoal to neutralise bacterial inhibitors. It may gas production is indicated by gas bubbles accumulated
be used for organisms such as gonococci. in Durham's tube. Certain bacteria are exacting in their
(b) Buffered glycerol saline transport medium for enteric growth requirements and need serum for their growth
bacilli. e.g. Hiss's serum sugars for pneumococcus.
(g) Sugar Media ANAEROBIC MEDIA

Sugar media (Fig. 5.8) help in identification of bacteria. These are used for cultivation of anaerobic bacteria
The term sugar in microbiology denotes any fermentable e.g.
substance. Glucose, lactose, sucrose and mannitol are (a) Cooked meat broth (CMB)
routinely employed for fermentation tests. Sugar media (b) Thioglycollate broth.
contain l % sugar in peptone water alongwith an indicator For more details about these media, refer to
(Andrade's indicator--0.005% acid fuchsin in IN NaOH). Chapter 6.
A small tube (Durham's tube) is kept inverted in the
larger sugar tube to detect gas production. The colourless
medium turns pink with production of acid by bacteria and MEDIA FOR TESTING SPECIAL PROPERTIES
Urea medium is used to test the property of urease
production. Phenylpyruvic acid (PPA) is employed for
identification of Proteus sp. These miscellaneous tests
are discussed in Chapter 7.
r " DEHYDRATED MEDIA

I
I
I
Dehydrated media are commercially available. These are
. f", . simply reconstituted in distilled water and sterilised before
use. With dehydrated media, the process of media making
has become simpler and its quality more uniform.
Table 5.1 and Table 5.2 show some of the liquid
Fig. 5.8 Sugar media
media and solid media respectively.
Table 5.1 Composition and Uses of Liquid Media

Medium Composition Uses
1. Peptone water Peptone - 1% Routine culture, Sugar
NaCl - 0.5% fermentation tests
Water - 100 ml (pH 7.4)
2. Nutrient broth Peptone water Routine culture
+
Meat extract- !%
3. Glucose broth Nutrient broth Blood cul ture, Culture of certain

+ organisms e.g. Streptococci
Glucose - 0.5%
4. Enrichment media
(i) Alkaline peptone water Peptone water (pH 9.0) Culture of Vibrio
(ii) Selenite F broth Peptone water, Sodium selenite Culture of faeces for salmonellae
and shigellae
(iii) Tetrathionate broth Nutrient broth, Sodium thiosulphate, Calcium Culture of faeces especially for
carbonate, Iodine solution salmonellae
(iv) Cooked meat broth (CMB)/ Nutrient broth, Predigested cooked meat of ox Culture of anaerobic bacteria
Robertson's cooked meat broth heart
(RCM)

44 - - - - - - - - - - - - - - - - -- -- - - - - - - - - Un it I: General Microbiology
Table 5.2 Composition and Uses of Solid Media
Medium Composition Uses
I. Simple medium
(t) Nutrient agar Nutrient broth, Agar (2-3%) Routine culture

2. Enriched media
(t) Blood agar Nutrient agar, Sheep blood (5-10%) Routine culture
(it) Chocolate agar Heated blood agar (55°C x 2hr) Culture of Neisseria, H. injl.uen::.ae
(iit) Loeffier's serum slope (LSS) Nutrient broth, Glucose, Horse serum Culture of C. diphtheriae
3. Indicator medium
(i) MacConkey's medium Peptone Culture of Gram negative bacilli
Lactose
Sodium taurocholate
Agar
Neutral red
4. Selective media
(t) Deoxycholate citrate agar Nutrient agar, Sodium deoxycholate, Culture of Salmonellae and Shigellae
(DCA) Sodium citrate, Lactose, Neutral red
(it) Bile salt agar (BSA) Nutrient agar, Sodium taurocholate Culture of Vibrio cholerae
(0.5%) pH 8.2
KEY POINTS
I. Culture media are required to grow the organisms from infected material to identify the causative agent.
2. The basic constituents of culture media are water, electrolyte, peptone, meat extract, blood or serum and agar.
3. When basal medium is added with some nutrients such as blood, serum or egg, it is called enriched medium.
4. Some substances are incorporated in the liquid medium which have a stimulating effect on the bacteria to be grown
or inhibits the competitors. Such media are called enrichment media, e.g., selenite broth.
5. Selective media contain substances that inhibit all but a few types of bacteria and facilitate the isolation of a
particular species. Example is bile salt agar (BSA) .
6. Differential medium contains substances which help to distinguish differing characteristics of bacteria. Example is
MacConkey's medium.
7. Indicator media contain an indicator which changes colour when bacterium grows in them. MacConkey's medium is
differential as well as an indicator medium.
8. Anaerobic media are used for cultivation of anaerobic bacteria, e.g., cooked meat broth (CME).
SK OW
1. List of types of media.
2. Basic differences between enriched media, enrichment media, selective media, differential media, indicator media and
examples of these media.
3. Composition and uses of different liquid media and solid media.

Ch 5: Culture Media _ _ _ _ _ _ _ _ _....!.__ _ _ _ _ _ _ _ _ _ _ __ _ _ __ ____ 45
STUDY QUESTIONS
(a) Enriched media (b) Enrichment media
(c) Selective media (d) Differential media
(e) Indicator media (f) Transport media.

1. All of the foll owing are examples of enriched media except:
(a) Blood agar (b) Chocolate agar
(c) Loeffier's serum slope (d) Bile salt agar
2. When a solid medium contains a substance that inhibits all unwanted bacteria but facilitates the isolation of particular
bacteria, it is known as:
(a) Selective medium (b) Enriched medium
(c) Indicator medium (d) None of the above
3. When a liquid medium contains a substance that inhibits all unwanted bacteria but stimulates the growth of particular
bacteria, it is known as:
(a) Selective medium (b) Enriched medium
(c) Enrichment medium (d) None of the above
4. Which enrichment medium is preferred to grow Vibrio cholerae?
(a) Tetrathionate broth (b) Selenite F broth
(c) Alkaline peptone water (d) All of the above
Answers (MCQs):
1. (d) 2. (a) 3. (c) 4. (c)
- - - - •++----

Chapter 6
CULTURE METHODS
I. Methods of Culture
A. Streak Culture B. Lawn Culture
C. Stroke Culture D. Stab Culture
E. Pour Plate Culture F. Liquid Culture
II. Anaerobic Culture Methods
Ill. Methods of Isolating Pure Cultures
In the clinical laboratory, bacterial cultures are indicated

to Nichrome-
loop
1. is bacteria in pure culture from the clinical
specimens an t eir identification by various tests,
2. determine antibiotic susceptibility, - - Nichrome straight
wire
3. prepare antigens for serodiagm;sis of infective
diseases,
4. maintain stock cultures.
I. METHODS OF CULTURE
A. Streak culture
B. Lawn culture
C. Stroke culture
D. Stab culture
E. Pour plate culture
F. Liquid culture
A. Streak Culture (Surface Plating)

It is the routine method employed for bacterial isolation
in pure culture. A platinum or nichrome wire loop of
2-4 mm internal diameter is used (Fig. 6.1) . Due to high
cost of platinum, loops for routine laboratory work are Fig. 6.1 Inoculating loop and straight wire
made of nichrome wire (24 S.WG. size). This loop is first
sterilised in the bunsen flame by making it red hot and of a dried plate near the peripheral area. This is named
cooled by touching an uninoculated part of the medium. as primary inoculum. From the primary inoculum, it
Then a loopful of specimen is smeared onto the surface is spread thinly over the plate by streaking with the
46
Ch 6: Culture Methods _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __ _ _ _ _ _ _ _ _ _ _ 47
loop in parallel lines (Fig. 6.2) . The loop is flamed and of molten agar, mixed well and the contents of the tube
cooled in between the different set of streaks. It is done poured into a sterile petridish and allowed to solidify.
to obtain isolated colonies over the final series of streaks. These plates are incubated at 37°C for overnight. Colonies
The culture plate is incubated at 37°C for overnight. will be seen throughout the depth of the medium and
Confluent growth occurs at the primary inoculum and can be counted using colony counter.
well separated colonies are obtained on the final streaks.
Single isolated colony is the best to study the various Uses
properties of bacteria. (a) To estimate viable bacterial count in a suspension.
(b) To quantitate bacteria in urine cultures.
F. Liquid Culture
Primary Liquid cultures in test tubes, screw-capped bottles or
inoculum
flasks may be inoculated by touching with a charged
loop or by adding the inoculum with pipettes or syringes.
This type of culture method is adopted for blood culture
and for sterility tests, where the number of bacteria in
the inocula are expected to be small. Liquid cultures are
Fig. 6.2 Streak culture (surface plating) on solid medium preferable for specimens containing antibiotics and other
antibacterial substances, as these become ineffective by
dilution in the medium. Liquid cultures are also preferred
B. Lawn Culture when large yields of bacteria are required.
This type of culture method is employed in antibiotic
sensitivity testing (disc diffusion method) and in Disadvantages
bacteriophage typing. It may also be employed for (a) It does not provide a pure culture from mixed
preparation of bacterial antigens and vaccines where inocula.
a large amount of bacterial growth is required. Lawn (b) Identification of bacteria is not possible.
cultures are obtained by flooding the surface of the plate
with a liquid culture or suspension of the bacterium. Incubation of Culture Media
Culture plate is kept for a minute and then excess material Most of the pathogenic organisms grow best at 37°C
is poured off. Alternatively, the culture plate may be i.e. body temperature of human beings. The inoculated
inoculated by a sterile swab soaked in liquid bacterial culture media are incubated at 37°C in an incubator (Fig.
culture or suspension. Plate is then incubated at 37°C 6.3).
overnight to obtain bacterial colonies. ' S:Jv? /25:"
{}f--'IJf- itr-~
C. Stroke Culture II. ANAEROBIC CULTURE METHODS
Stroke culture is done in tubes containing agar slope and is Anaerobic bacteria grow only in the absence of oxygen
~c/M
employed for providing a pure growth of the bacterium for (anaerobic conditions). These anaerobic conditions or
slide agglutination and other diagnostic tests. Commonly anaerobiosis can be established by various methods.
used agar slope is Nutrient agar slope.
D. Stab Culture
Methods of Anaerobiosis
1. Production of a vacuumJv
®- 0¥J~~
kld.u:
Stab culture is performed by a straight wire, charged with 2. Displacement of oxygen.
culture material (bacteria), by puncturing deep inside the 3. By displacement and combustion o oxygen.
agar. This technique is employed to demonstrate gelatin 4. Absorption of oxygen by chemical or biological
liquefaction, oxygen requirement of the bacterium and methods. r) c.,~(__ r.ll\ I
to maintain stock cultures for preservation of bacteria. 5. By reducirlg a~~hts. LY ro~ lo1
6. Anaerobic chamber.
lJ_..,,.
@~ 0 7
E. Pour Plate Culture
Tubes containing 15 ml of agar medium in each are 1. Production of a Vacuum CJ) 1CA/2,. ftA.l?-
melted and kept to cool in a water bath at 45-50°C. Cultivation in v uum was attempted by incubating
The inoculum to be tested is diluted in serial dilutions. cultures in a acuum desiccato·r but it proved to be
One ml of each diluted inoculum is added to each tube unsatisfactory. This metho 1s not in use now.

·•
~7
'~'l
6~Qj Fig. 6.3 Incubator
2. Displacement of Oxygen the terminals to heat the catalyst for its activity. Nowadays
Displacement of oxygen by inert gases like hydrogen, catalyst (without heating) at room temperature is used.
nitrogen, carbon dioxide or helium is sometimes employed. Culture plates inoculated with specimens are placed
Oxygen can never be removed completely by this method.
---
A popular, but ineffective, method is the use of cc,lJldle. A
inside the anaerobic jar with an indicator. The lid is
clamped tight. The outlet tube is connected to a vacuum
lighted candle is kept in a lar~ir-tight container loaded pump while inlet tube is closed. The air inside is evacuated.
with inoculated plates. It is expected that burning candle The outlet tube is then closed and hydrogen gas is passed
will use up all the oxygen inside before it is extinguished through inlet tube till reduced atmospheric pressure is
but some amount of oxygen is always left behind. brought to normal atmospheric pressure (i.e. 760 mm
Hg) which is monitored on the vacuum gauze as zero.
3. By Displacement and Combustion of Oxygen Electric terminals are switched on to heat the catalyst
Anaerobiosis obtained by "McIntosh and Fi and if ' room temperature catalyst' is used, heating is not
anaerobic jar (Fig. 6.4) is the most reliable and widely required. The c~rst helps ta combine hydro_gen and
~- residual ~yg~ to form water.
Reduced methylene blue is generally used a ~ r
Procedure of anaerobiosis in the jar. It remains col~r~s in
McIntosh and Filde's anaerobic jar consists of a stout anaerobic conditions, but turns blue on exposure to
glass or metal jar with a metal lid which can be clamped oxygen .
air-tight with a screw. The lid is fitted with two tubes with
taps, one acting as inlet for introduction of gas and the 4. Absorption of Oxygen by Chemical or
other as the outlet. The lid also contains two terminals Biological Methods
rical supply. cafa:lys . Clj)i,M
C ICAL METHODS
alla m ) is suspended~ Pyrogallol
y stout wires which are connected with Alkaline pyrogallol absorbs oxygen. This method was first
Fig. 6.4 McIntosh and F ilde's anaerobic jar

Ch 6: Culture Methods _ _ _ _ _ _ _ _ _ _ _ __ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ 49
introduced by Buchner (1888). A large tube containing Principle

solution of sodium hydroxide and pyrogallic acid placed 1. Un.satm ated .fatty acids present in meat utilise
inside air-tight jar produces anaerobic conditions. oxy~n for au!ooxidati.Qn, this reaction is catalysed
by haematin in the meat.
~ omium and sul · "d 2. Glutathione and cysteine (both are reducing
A mi o chromium and sulphuric acid is used for agents) present in meat also utilize oxygen.
producing anaerobiosis. The two chemicals react in the 3. Sulphydryl compounds (present in cysteine) also
~ c e of oxygen and produce chromous sulphate(_~ contribute for a reduced oxidation-reduction (OR)
C~V'----t ¼ OL-\ ~ potential.
(iii) Gas-pak
It is-avaiiable commercially as a disposable packet Procedure
·~ -~5.i-iellets of ~ u m ~ cobalt chloride, Before inoculation, the medium is boiled in water bath
citric acid d sodium bicarbonate. It is now widely used at 80°C for 30 minutes to make it oxygen free. For strict
paring anaerobic jars. These chemicals generate anaerobiosis the surface of CMB medium may be covered
hydrogen and carbon dioxide when water is added. with a layer of sterile liquid paraffin.
Hydrogen combines with oxygen in the presence of a
catalyst. ~
CA'lv-> w~w Interpretation
After the inoculated pl re placed insid~ air! Saccharolytic anaerobes ( Cl. perfringens) turn the colour
tight jar, the packet of " as-pa ' with water added, is of meat pieces into red while it becomes black in case
kept inside and the lid is ti htly closed. The "Gas-pak" of proteolytic anaerobes ( Cl. tetani).
technique is simple as compared to filling of gases by 6. Anaerobic Chamber
cylinders. It is an anaerobic incubation system. It provides oxygen-
~ , _ _ -f ~ e e environment for inoculating culture media and for
BIOLOGICAL METHODS
This ha~ been attempted by ~ncubatinfc
j!long with a~a7::ob1c bacten_a. lwo boo
a:~=uisros e~are
their incubation. It is fitted with airtight rubber gloves to
insert hands for working with specimens. These anaerobic
chambers contain a catalyst, dessicant, hydrogen gas,
taken--one 1s moculated with Pseudomonas aeruginosa carbon dioxide gas, nitrogen gas and an indicator.
(aerobic bacteria) and the other with specimen of
anaerobic bacteria. Two plates are placed one over the
other and sealed along the rims and are incubated. This III. MEmoos OF ISOLATING
~ . \.w.,e--::1 c.- PURE CULTURES
method is slow and ineffective.
5. By Reducing Agents
J rt}_~ y,'L»-'>/ 1. Surface plating
f
\~.N C.- It is routinely employed in the laboratory.
Oxygen in culture media can be redu~d lYy various 2. Use of enriched and selective media
agents such as glucose, thioglycollate, cooked meat pieces, It is employed for isolating pathogens from specimens
cysteine and ascorbic acid. Based on this principle, the with varied flora e.g. faeces.
two most widely employed anaerobic liquid culture media 3. Pretreatment of specimens
are: Suitable bactericidal substances are used for pre-
treatment of specimens to isolate a particular bacteria
THIOGLYCOLLATE BROTH / e.g. concentration and decontamination of sputum
It contains nutrient broth and 1% thioglycollate. before culture for Mycobacterium tuberculosis.
4. By heating liquid medium
COOKED MEAT BROTH (CMB) _ / Specimens are heated at 80°C to destroy vegetative
It is also known as Robertson's cooked meat (RCM)
forms of bacteria but spore bearing bacteria survive
medium.
It contains nutrient broth and pieces of fat-free e.g. isolation of tetanus bacilli from dust and similar
minced cooked meat of ox heart. sources.

50 - - - -- -- - - - -- - - -- - - -- - - -- -- -- -- Unit I: General Microbiology
KEY POINTS
1. Various culture methods namely streak culture, lawn culture, stroke culture, stab culture, pour plate culture and liquid
culture are used in microbiology laboratory.
2. Streak culture is the routine method employed for bacterial isolation in pure culture.
3. Anaerobic bacteria grow only in the absence of oxygen (anaerobic conditions) and hence require anaerobic culture
methods for their growth.
4. McIntosh and Filde's anaerobic jar is the most reliable and widely used method for obtaining anaerobiasis. Gas-pak
is now widely used for preparing anaerobic jars.
5. Commonly used anaerobic culture medium is cooked meat broth (CMB).
YOU MUST KNOW

1. Methods of anaerobic culture.
2. Principle and use of McIntosh and Filde's anaerobic jar.
3. Principle and uses of cooked meat broth (CMB).
STUDY QUESTIONS
1. Write briefly about anaerobic culture methods.
(a) Streak culture (b) Lawn culture
(c) Pour plate culture (d) Cooked meat broth (CMB) or RCM medium
(e) Methods of isolating pure cultures.

I. Anaerobic jar is commonly used to grow:
(a) Clostridium tetani (b) Klebsiella sp.
(c) Pseudomona s aeruginosa (d) None of the above
2. Which of the following bacteria produce saccharolytic reaction in cooked meat broth (CMB)?
(a) Clostridium perfringens (b) Clostridium tetani
(c) Pseudomonas aeruginosa (d) None of the above
Answers (MCQs):
1. (a) 2. (a)
---• ••-- -

Chapter 7
IDENTIFICATION OF BACTERIA
Identification and Characterisation of a Bacterial Strain

I. Conventional Methods
A. Morphology of Bacterial Colony B. Growth in Liquid Media
C. Staining D. Hanging Drop Preparation
E. Biochemical Tests F. Antigenic Structure
G. Typing Methods H. Pathogenicity Tests
I. Antibiotic Sensitivity Tests
II. Rapid Methods
After isolation of bacteria in pure culture from a specimen,

it has to be identified. The following studies are necessary Flat
to characterise a bacterial strain.
Convex
A. Morphology of bacterial colony
B. Growth in liquid media
C. Staining Raised
D. Hanging drop preparation
E. Biochemical tests
F. Antigenic structure Umbonate
G. Typing methods Low convex
H. Pathogenicity tests Fig. 7.1 Different elevation of colonies
I. Antibiotic sensitivity tests
I. CONVENTIONAL METHODS
A. Morphology of Bacterial Colony
0- Uodolate
o-Entire
The appearance of bacterial colony on culture medium
is usually characteristic. The following features of the
colony are studied:
(i) Size-in millimetres e.g. pin head size is characteristic
of staphylococci whereas pin point size is a feature
of streptococci colonies.
(ii) Shape - Circular, irregular
(iii) Surface - Smooth, rough, granular
0 - Crenated
o-cmate
(iv) Elevation - Colony may be flat, raised, low convex,
convex or umbonate (Fig. 7.1).
0-Lobate
(v) Edge-Entire, crenated, lobate, undulate, ciliate Fig. 7.2 Various types of edges of colony
(Fig. 7.2)
51
52 - - - - - -- - - - - - - - - - - - - - - - - - - - - - - - Unit I: General Microbiology
(vi) Opacity- Translucent, transparent or opaque l. Sugar Fermentation

(vii) Colour- Pigment may be produced by certain Principle
bacteria. To determine the ability of an organism to ferment a
(viii) Haemolysis- Haemolysin produced by some specific carbohydrate (sugar) incorporated in a medium
bacteria leads to haemolysis around the colony. producing acid or acid with gas.
(ix) Consistency- Mucoid, friable, firm, butyrous
Procedure
B. Growth in Liquid Media Test organism is inoculated in a sugar medium and
Commonly used liquid media are peptone water and incubated at 37°C for 18-24 hours. Glucose, lactose,
nutrient broth. Bacterial growth appears in following sucrose and mannitol are widely used sugars. Sugar media
forms: contain 1% sugar. Indicator used is Andrade's indicator
(i) Uniform turbidity- Most of the Gram negative (a solution of acid fuchsin to which is added sodium
bacteria grow in this form. hydroxide).
(ii) Deposit at bottom- This occurs in growth of
streptococci, chains of this bacteria being heavier Interpretation
may settle down as deposit. Positive- Pinkish-red (acidic)
(iii) Surface pellicle formation- All aerobes have tendency Negative- Yellow to colourless (alkaline)
to grow on surface of media due to more content Gas production can be seen as bubbles in Durham's tube
of oxygen present on the surface e.g. Pseudomonas (Figs. 7.3 and 7.4).
sp.
Cotton plug
C. Staining
Staining methods are employed to examine smears
prepared from the bacterial colony or liquid culture.
Commonly used stains are as follows:
(i) Gram's staining-It divides bacteria into Gram
positive and Gram negative. It may be used for
almost all the bacteria. Sugar medium
(ii) Albert's staining- It is employed to identify

rv--o- - Bubble due to gas production
Cory nebacterium diphtheriae , a causative agent of Inverted Durham's tube
diphtheria.
(iii) Ziehl-Neelsen (ZN) staining- It differentiates acid-
Fig. 7.3 Inverted Durham's tube showing gas production
fast bacilli (AFB) from non-acid-fast bacilli. This
stain is commonly employed for identification of
M ycobacterium tuberculosis and M. leprae which
are causative agents of tuberculosis and leprosy
respectively. Both these bacteria are acid-fast in
nature. 1 " I
I
D. Hanging Drop Preparation
It helps to distinguish motile bacteria from non-motile I r'' L
ones.
E. Biochemical Tests
Fig. 7.4 Sugar fermentation tests
Bacterial species differ in their capacity to break down
different sugars. Some biochemical tests are based on
presence of specific enzymes such as catalase, coagulase, Examples of fermentative bacteria
oxidase, urease, gelatinase etc. Some of the widely used Glucose fermenters- All members of the
biochemical tests are described below. Enterobacteriaceae

Ch 7: Identification of Bacteria _ _ _ _ _ _ _ _ _ _ _ __ _ _ _ _ _ _ _ _ __ ______ 53
Glucose and lactose fermenters- Escherichia coli, Positive and negative bacteria
Klebsiella sp. Indole positive- Esch. coli, Proteus sp. other than P
Glucose and mannitol fermenter- Salmonella sp . mirabilis, Edwardsiella
, .-:-\ Indole negative- Klebsiella sp., Proteus mirabilis
2. ~n~ole Production ~ · r.t..- h0\ ~,A'
· 3. Urease Production
Principle
To determine the ability of an organism to decompose Principle
aminoacid tryptophan into . Tr tophan is To determine the ability of an organism to produce an
decomposed e,,J:cyptophanase p oduced by enzyme urease which splits urea to ammonia. Ammonia
certain bacteria. + ") __ 1
_; , makes the medium alkaline and thus phenol red indicator
l\tpl.JJ11Af,>'V\_ - j J:nc:W~anges to pink/red in colour.
Procedure
Indole production is detect~ by inoculating the test Procedure
bacterium into~ptone wa~(tryptophan rich) and The test is done in Christensen's urease medium. The test
incubating it at 37°C for 48-96 hours. 0.5 ml of Kovac's organism is inoculated on the entire slope of medium
reagent is added to the bacterial growth and gently and incubated at 37°C. It is examined after 4 hours and
shaken. after overnight incubation.
Kovac's reagent contains Christensen's urease medium contains

Paradimethyl amino- Peptone water
benzaldehyde 10 g Urea (20%)
Amyl or isoamyl alcohol 150 ml Agar
Cone. HCI. 50 ml Phenol red
Interpretation Interpretation
Indole positive (Fig. 7.5)- A red coloured ring near Positive (Fig. 7.6)- Pink colour.
the surface of the medium Negative (Fig. 7.6)- Pale yellow colour.
Indole negative (Fig. 7.5) - Yellow coloured ring
near the surface of the medium
Positive

Positive and negative bacteria Koser's citrate medium

Urease positive-Kleb siella sp., Proteus sp., Yersinia Positive-Turb idity due to growth of bacteria
enterocolitica, Helicobacter pylori Negative-No turbidity
Urease negative-Esch . coli, Providencia sp., Yersinia
pestis. Positive and negative bacteria
Citrate positive- Klebsiella sp., Salmonella sp. except
4. Citrate Utilisation Test S. typhi, Citrobacter sp., Enterobacter sp.
Principle Citrate negative-Esch . coli, Edwardsiella, Salmonella
It is the ability of an organism to utilize citrate as the typhi
sole source of carbon for its growth, with resulting
alkalinity.
. ciple
Procedure Certain bacteria have an enzyme catalase which acts on
Solid (Simmon's) or liquid (Koser's) media can be used. hydrogen peroxide to release oxygen.
A bacterial colony is picked up by a straight wire and
inoculated into either of these media. These inoculated catalase ) 0
media are incubated at 37°C for overnight. Simmon's Nascent oxygen
citrate medium contains agar, citrate and bromothymol Procedure
blue as an indicator. Original colour of the medium is Pick up a few colonies of test bacteria with platinum
green. loop from nutrient agar slope/plate and mix it in a drop
of H 2O2 (10 volumes) on a clean glass slide. A positive
Interpretation catalase reaction produces gas bubbles immediately. Since
Simmon's citrate medium blood contains catalase, culture on blood containing
Positive (Fig.7.7)- Growth with an intense blue colour media may result in false positive reaction. Use of iron
on the slant. Blue colour is due to the alkaline pH which wireloop for picking up bacterial colonies may be another
results from utilization of citrate. Bromothymol blue cause of false positive test.
(indicator) is blue in alkaline conditions.
Negative (Fig. 7.7)- No growth with no change in Interpretation
colour (green). Positive test-Immedia te bubbling, easily observed
(Oz formed)
Negative test- No bubbling (no Oz formed)
Positive and negative bacteria

Catalase positive- All members of enterobacteriaceae
except Shigella dysenteriae type I; Staphylococcus,
Micrococcus, Bacillus
Catalase negative- Shigella dysenteriae type I,
Streptococcus, Clostridium
Catalase test is primarily used to differentiate between
genera Staphylococcus from Streptococcus
6. Oxidase Test
Principle
To determine the presence of an enzyme cytochrome
oxidase which catalyses the oxidation of reduced
cytochrome by molecular oxygen.
Procedure
Freshly prepared solution of 1% tetra-methyl paraphenylene
Negative Positive diamine dihydrochloride (oxidase reagent) is used . There
Fig. 7.7 Citrate utilisation test are different methods to perform this test.
(i) A filter paper strip, soaked in the oxidase reagent,

Ch 7: Identification of Bacteria _ _ _ _ _ _ __ __ _ _ _ __ _ _ _ _ _ _ _ _ _ _ _ ___ 55
is smeared with test organism. In a positive oxidase

reaction the smeared area turns deep purple within 10
seconds. Oxidase positive control (Pseudomonas sp.)
should always be included to find out the working
of oxidase strip.
(ii) Another method is to pour oxidase reagent on the
surface of colonies, the colonies become purple
within 10-30 minutes. This technique is useful to
pick up Neisseria colonies from mixed growth on
culture media. Purple coloured colonies can be
subcultured to get pure cultures.
Interpretation
Positive- Deep purple within 10 seconds
Negative- No colour change

Oxidase positive- Pseudomonas sp., Vibrio sp., Neisseria
sp., Aeromonas sp., Alcaligenes sp., Plesiomonas sp.,
Moraxella sp.
Fig. 7.8 Phenylpyruvic acid (PPA) test
Oxidase negative-All members of enterobacteriaceae,
Stenotrophomonas maltophila
Procedure
The test organism is inoculated in glucose phosphate
7. Phenylalanine Deaminase Test broth and incubated at 37°C for 2-5 days. Then add five
To determine the ability of an organism to deaminate drops of 0.04% solution of methyl red, mix well and read
phenylalanine to phenyl pyruvic acid (PPA). This test is the results immediately.
also commonly called as P PA test.
Interpretation
Procedure Positive-Red colour
A medium containing phenylalanine is inoculated with Negative-Yellow colour
a growth of bacterial culture and incubated at 37°C for
overnight. A few drops of 10% ferric chloride solution is Positive and negative bacteria
added. If PPA is formed , green colour is produced due MR Positive-Esch. coli, Yersinia sp., Listeria
to reaction of ferric chloride with PPA. monocy togenes
MR negative- Klebsiella sp., Enterobacter sp.
Interpretation
Positive- Green colour 9. Voges-Proskauer (VP) Test or Acetoin
Negative- No colour change Production Test
Principle
Positive and negative bacteria This test depends on the production of acetyl methyl
PPA positive (Fig. 7.8~ Proteus sp., Morganella sp., carbinol (acetoin) from pyruvic acid in the media. In
Providencia sp. the presence of alkali and atmospheric oxygen, acetoin
PPA negative- All members of enterobacteriaceae. is oxidised to diacetyl which reacts with a-naphthol to
give red colour.
8. Methyl Red (MR) Test Procedure

Principle Test organism is inoculated in glucose phosphate broth
This test detects the production of sufficient acid during and incubated at 37°C for 48 hours. Then add 1 ml of
fermentation of glucose by bacteria and sustained 40% KOH and 3 ml of a 5% solution of a-naphthol in
maintenance of a pH below 4.5. absolute alcohol.

56 - - - -- -- - - - - - - - - - - - - -- - - - - - - - Unit I: General Microbiology
Interpretation determination of possible hydrogen sulphide (H 2S)

Positive- Pink colour within 2-5 minutes, deepening production.
to crimson colour in 30 minutes.
Negative- Colourless for 30 minutes. Procedure
TSI is a composite medium which contains three
Positive and negative bacteria carbohydrates namely glucose, lactose, sucrose and also
VP positive- Klebsiella sp., Enterobacter sp. , E/Tor ferric salts for testing H2 S production. The concentration
vibrios, Staphy lococcus of lactose and sucrose is 10 times that of glucose in the
VP negative- Esch. coli, Micro coccus medium. Phenol red is incorporated as an indicator. This
Indole, MR, VP and citrate tests are very useful medium is widely used and is in the form of a butt and
in the identification of enteric Gram negative bacteria. slant in the test tube. Medium is inoculated with bacterial
These tests are commonly referred to by the sigla 'IMViC' culture by a straight wire pierced deep in the butt (stab
tests. culture). It is incubated at 37°C for overnight.
Bacterium attacks simple sugar (glucose) first
10. Nitrate Reduction Test and then the lactose and the sucrose. The test is read
Principle after 18 to 24 hours and not before 18 hours. If the
This test depends upon presence of an enzyme nitrate test is read before 18 hours, the glucose fermentation
reductase, which reduces nitrate to nitrite or free nitrogen produces sufficient acid to make both slant and butt
gas. yellow (acidic) thus giving a wrong interpretation of
A/A (refer Interpretation below). After 18 to 24 hours
Procedure the glucose concentration is depleted in the slant and
Test organism is grown for five days at 37°C in a broth the butt. The organism begins oxidative degradation of
containing 1% KN0 3. Then 0.1 ml of the reagent the peptones present in the slant, resulting in alkaline
(sulphanilic acid and a-naphthylamine) is added. byproducts which change the indicator to a red colour.
Fermentation (anaerobic) of glucose in the butt produces
a larger amount of acid, overcoming the alkaline effect
Interpretation of peptone degradation, therefore the butt remains
Positive- Red colour develops within few minutes yellow (acidic).
Negative- No colour development Certain bacteria produce H2S which is a colourless
In case of no colour development, it indicates either gas. H 2S combines with ferric ions (from ferric salts)
nitrate has not been reduced or nitrate has been reduced to form ferous sulphide as black precipitate and thus
beyond nitrite to nitrogen gas, which reagents will not blackening of the medium.
be able to detect. To interpret this, zinc dust is added to
the test. Zinc reduces nitrate to nitrite, so development Interpretation
of red colour after addition of zinc dust means that Yellow colour (acidic)-Fermentation of
the organism was unable to reduce the nitrate to nitrite carbohydrate
(negative test). However, no colour development after Red colour (alkaline)- No fermentation
addition of zinc dust indicates positive test as nitrate has Bubbles in the butt- Gas is also produced during
been reduced to nitrite and further nitrogen gas. fermentation of carbohydrate.
Blackening of the medium (Fig. 7.9)- H 2S
Positive and negative bacteria production
N O 3 reduction positive- All members of Various possible combinations of the different TSI
enterobacteriaceae, Branhamella catarrhalis reactions are listed below; remembering that the slant is
NO 3 reduction negative- Haemophilus ducrey i first, followed by the butt reaction.
KIA (red/yellow)-Glucose only fermented .
AJA (yellow/yellow)- Glucose fermented, lactose
11. Triple-Sugar Iron (TSI) Agar and/or sucrose fermented.
Principle K/K (red/red)- Neither glucose, lactose, nor sucrose
To determine the ability of an organism to attack fermented
specific carbohydrates incorporated in a growth medium, K- Alkaline
with or without the production of gas, along with the A- Acidic

Ch 7: Identification of Bacteria _ _ _ _ _ _ _ _ _ _ _ _ _ __ _ _ __ __________ 57
Interpretation
Positive Turbidity due to growth
egative - Clear (no growth)

Positive KCN test- Klebsiella sp., Citrobacter freundii,
Pseudomonas aeruginosa
Negative KCN test- Salmonella sp., Esch. coli,
Alkaligenes faecalis
F. Antigenic Structure
Biochemically identified organisms are further confirmed
by agglutination or precipitation reaction. Unknown
bacterial antigen is identified by known specific antisera
by slide agglutination test.
Salmonella typhi or other salmonella can be confirmed
Fig. 7.9 Triple sugar iron (TSI) agar by agglutination with the specific antisera.
12. Hydrogen Sulphide Production

Principle G. Typing Methods
To determine whether hydrogen sulphide (H 2S) has been This is useful for epidemiological studies. All the above
liberated, by enzymatic action, from sulphur containing mentioned tests can identify up to species level though
amino acids producing a visible, black colour reaction. intraspecies differentiation is not possible. Hence, typing
methods like bacteriophage typing, bacteriocin typing
Procedure and others are very useful for intraspecies differentiation.
Organisms are cultured in media containing lead acetate, Typing methods help to find out the source of infection
ferric ammonium citrate or ferrous acetate, they turn them in epidemics or sometimes in outbreak of food
black or brown. Another method is to grow the organisms po1somng.
in culture tubes, inserting a filter paper strip impregnated
with lead acetate between the cotton plug and the tube.
Blackening of the paper indicates H 2S production. H. Pathogenicity Tests
Guinea pigs, rabbits and mice are mostly used for
Interpretation pathogenicity tests. These animals may be injected by
Positive Black colour subcutaneous, intramuscular, intraperitoneal, intravenous
Negative No change in colour or intracerebral routes depending upon the organism to
be tested. The identification of the organism is carried
Positive and negative bacteria out on the basis of postmortem findings and cultural
H2S positive- Proteus mirabilis, Proteus vulgaris, characteristics.
Salmonella sp. with some exceptions
H 2S negative- Morganella sp., Salmonella paratyphi I. Antibiotic Sensitivity Tests
A, S. cholerae-suis The isolated bacterium is subjected to antibiotic
sensitivity tests in-vitro for selecting appropriate antibiotic
13. Growth in KCN for therapeutic use. Different techniques for antibiotic
Principle sensitivity testing are described in Chapter 78 .
To determine the ability of an organism to grow in a
medium containing potassium cyanide.
II. RAPID METHODS
Procedure Conventional methods take days to identify and
Inoculate peptone water containing 1 in 13,000 characterise the isolates while rapid automated methods
concentration of potassium cyanide, with test organism. take only hours. Detection of specific enzymes, toxins,
Incubate at 37°C for 24-48 hours. antigens or metabolic end products of the isolates make

the identification simpler and rapid. For example, gas obligate anaerobes. Polymerase chain reaction (PCR) and
liquid chromatography (GLC) is based on identification of nucleic acid probes carrying specific DNA or RNA base
specific fatty acids (metabolic end products) produced by sequences are some molecular methods being widely used
the organisms. This technique is very useful in identifying for identifying microbes.
KEY POINTS
1. After isolation of bacteria in pure culture from a specimen, it can be identified by morphology of bacterial colony,
staining, hanging drop preparation and various biochemical tests.
2. Biochemically identified organisms are further confirmed by slide agglutination with the specific antisera.
3. Typing methods are useful for epidemidogical studies.
4. Antibiotic sensitivity tests are done to select appropriate antibiotic for therapeutic use.
5. Polymerase chain reaction (PCR) and nucleic acid probes are rapid methods for identifying microbes.
YOU MUST KNOW

1. Principle and interpretation of biochemical tests such as sugar fermentation , indole production, urease production,
citrate utilisation test, catalase test, oxidase test phenylalanine deaminase test, triple-sugar iron (TSI) agar.
2. Examples of bacteria which give positive tests for above mentioned biochemical tests.
STUDY QUESTIONS
(a) Indole production (b) Citrate utilisation test (c) Urease test (d) Catalase test (e) TSI
(f) PPA test (g) Nitrate reduction test (h) VP test (i) Oxidase test (j) MR test.
lMC s
1. All of the following bacteria are urease test positive except:
(a) Klebsiel/a sp. (b) Yersinia enterocolitica (c) Yersinia pestis (d) Helicobacter pylori
2. Catalase test is negative in:
(a) Staphy lococcus aureus (b) Salmonella typhi (c) Streptococcus pyogenes (d) Klebsiel/a sp.
3. All of the following bacteria are oxidase test positive except:
(a) Neisseria meningitidis (b) Vibrio cholerae (c) Escherichia coli (d) Pleisiomonas sp.
4. Phenylalanine deaminase test is negative in :
(a) Proteus vulgaris (b) Providencia sp. (c) Morgane//a sp. (d) Klebsie//a sp.
5. Which of the following tests detects the production of acetyl methyl carbinol from pyruvic acid in the media?
(a) Methyl red test (b) Voges-Proskauer test (c) Urease test (d) Citrate utilisation test
6. Triple sugar iron (TSI) agar medium contains all the following carbohydrates except:
(a) Glucose (b) Lactose (c) Sucrose (d) Mannitol
Answers (MCQs):
1. (c) 2. (c) 3. (c) 4. (d) 5. (b) 6. (d)
____ ...
____

Chapter 8
BACTERIAL TAXONOMY
I. Phylogenetic Classification
II. Adansonian Classification
Ill. Genetic Classification
IV. lntraspecies Classification
Taxonomy is the classification of organisms into Genus : Salmonella

ordered groups. Organisms are divided into three main Species : Salmonella typhi
Kingdoms: A phylogenetic classification of bacteria has been
1. Animals published in USA as Bergey's Manual of Systematic
2. Plants Bacteriology. The manual is very useful in compilation of
3. Protista-contains unicellular organisms including names and descriptions and also as an aid to identification
eukaryotes and prokaryotes (bacteria). of newly isolated bacterial types.
There is no universally accepted bacterial classification. For identification and classification of bacteria, a
There are mainly three approaches, Phylogenetic, minimum number of important characters are selected
Adansonian and Genetic. However, bacterial species can so that various members can be distinguished. These
be further sub-classified by intraspecies classification. important characters include:
1. Morphology
2. Staining
I. PHYLOGENETIC CLASSIFICATION 3. Cultural characteristics
Phylogenetic classification groups together types that 4. Biochemical reactions
are related on evolutionary basis where several ranks 5. Antigenic structure
are used: Divisions, Classes, Orders, Families, Tribes, 6. Guanine: Cytosine ratio of DNA.
Genera and Species. Major groups are differentiated by
some characters of special importance such as Gram Nomenclature
reaction, spore formation etc. The genera and species It is the labelling of the groups and of individual
are distinguished by less important properties, such members. Scientific name of bacteria usually consists of
as fermentation reactions, nutritional requirement etc. two words-first is the name of the genus and second
The full taxonomical position of Salmonella typhi is as is that of the species (e.g. Proteus vulgaris). The generic
follows: name is usually a Latin noun and second name indicates
Division Protophyta some property of the species (e.g. a/bus, meaning white),
Class Schizomycetes the disease it causes (e.g. , diphtheriae, from diphtheria),
Order Eubacteriales the person who discovered it (e.g. , we/chii, after Welch),
Family Enterobacteriaceae or the animal in which it is found (e.g., suis, meaning
Tribe Salmonellae pig). The generic name always begins with a capital letter
59
60 - - - - - - - - - - - - - - - - - - - -- - - - - - - - - Unit I: General Microbiology
and the second name with a small letter, even if it refers by their genes. DNA is extracted from pure bacterial
to a person or place (e.g. Salmonella london) . culture and their G + C (guanine + cytosine) content
is estimated. The nucleotide base composition and base
II. ADANSO NIAN CLASSIFICATION
ratio (adenine- thymine: guanine-cytosine) varies widely
The Adansonian classification was originally proposed among different groups of microorganisms, but for any
by Michael Adanson in the 18 th century. It makes no one species, it is constant.
phylogenetic assumption , but takes into account all the
characters with equal importance. It groups organisms on IV. INTRASPECIES CLASSIFICATION
the basis of similarities in large number of characters. The For diagnostic or epidemiological purposes, it is often
computer has extended the scope of this classificatio n by necessary to subclassify bacterial species on the basis
permitting comparison of very large number of properties of biochemical properties (biotypes), antigenic structure
of several organisms at the same time. The computer (serotypes), bacteriophage susceptibility (phage types) or
analyses the observations and prints out a list of strains production of bacteriocins (colicin types). A species may
on the basis of degree of similarity in their properties. be divided first into groups and then into types.
Thus several broad subgroups of bacterial strains are The application of newer molecular techniques has
identified, which are further divided into species. This led to greater discrimination in intraspecies typing. These
is also known as Numerical classification. molecular techniques are of two types, phenotypic and
genotypic. Phenotypic methods include electrophoretic
III. GENETIC CLASSIFICATiON
typing of bacterial protein and immunoblotting.
It is based on the degree of genetic relatedness of different Genotypic methods include Southern blotting, PCR, and
organisms. This classification is the most natural or nucleotide sequence analysis. Some of these methods are
fundamental since all properties of bacteria are regulated described in Chapter 9.
KEY POINTS
1. Taxonomy is the classification of organisms into ordered groups.
2. There are mainly three approaches of bacterial classification namely Phy logenetic, Adansonian and Genetic. Bacterial
species can be further subclassified by intraspecies classification.
YOU MUST KNOW

1. Approaches of bacterial classification.
STUDY UESTIO S
1. Write briefly about bacterial taxonomy.
1 J TJPLE CHOICE UESTIONS (MCQs)

1. Which of the following methods may be used in bacteriology for epidemiological purposes?
(a) Biotyping (b) Phage typing
(c) Serotyping (d) All of the above
2. Molecular techniques employed for intraspecies typing of bacteria include:
(a) Southern blotting (b) Polymerase chain reaction
(c) Nucleotide sequence analysis (d) All of the above
Answers (MCQs):
1. (d) 2. (d)
- - - - ++• ----

b
Chapter 9 ~U'¼- ~::ie

BACTERIAL GENETICS
I. Basic Principles
II. Synthesis of Polypeptide
A. Transcription B. Translation
Ill. Extrachromosomal Genetic Elements
IV. Bacterial Variation
A. Phenotypic Variation B. Genotypic Variation
V. Gene Transfer
A. Transformation B. Transduction
C. Lysogenic Conversion D. Conjugation
VI. Genetic Mechanisms of Drug Resistance
VII. Genetic Engineering
VIII. DNA Probes
IX. Polymerase Chain Reaction (PCR)
A. Principle B. Procedure
C. Appl ication of PCR D. Types of PCR
X. Genetically Modified Organisms
XI. Gene Therapy
Genetics is the study of heredity and vanat1on. All a double stranded D A molecule of approximately 1000
hereditary characteristics are encoded in DNA. The µm (1 mm) long when straightened.
chromosomal DNA plays a major role in the maintenance Each strand has a backbone of de-oxyribose (sugar)
of characters constant from generation to generation. and phosphate groups. There are four nitrogenous bases,
This is accomplished by most accurate replication of its two purines : adenine (A) and guanine (G) and two
DNA, yet, heritable variations in small proportion of pyrimidines: thymine (T) and cytosine (C). One of these
daughter cells occur. four nitrogenous bases is attached to each deoxyribose.
The two strands are held together by hydrogen bonds
I. BASIC PRINCIPLES between the nitrogenous bases on the opposite strands.
The DNA molecule is composed of two strands of This bonding is in such a specific manner that hydrogen
complementary nucleotides wound together in the form of bonds can only be formed between adenine and thymine
a double helix (Fig. 9 1) as deduced by Watson & Crick. (A-T) and between guanine and cytosine (G-C)
The bacterial nucleus contains a circular chromosome of 9 7) . Adenine and thymine thus form one complementary
61
62 - - - - -- -- - - - - - -- -- - -- - - - - - -- - Unit I: General Micf biology
Hence, a molecule of DNA contains as many numbers

of adenine as thymine, and of guanine as cytosine. The
ratio of A + T to G + C is constant for each species
but varies widely from one bacterial species to another.
During replication of the DNA molecule, the two strands
separate at one end, and each strand then acts as a
template for the synthesis of a complementary strand,
with which it then forms a double helix.
RNA is structurally similar to DNA, except for two
differences, sugar ribose is present instead of deoxyribose
and the nitrogenous base uracil replaces thymine. There
are three different types of RNA in a cell, messenger
RNA (mRNA), ribosomal RNA (rRNA) and transfer
RNA (tRNA).
The Important Definitions in Genetics

Gene-A segment of DNA that specifies for a
particular polypeptide is called a gene.
Codon- Genetic information is stored in the DNA as
a code. Codon consists of a sequence of three nucleotide
bases i.e. the code is triplet. Each codon specifies for
production of a single aminoacid but more than one
Fig. 9.1 DNA double helix (Watson-Crick model) codon may exist for the same aminoacid e.g. AGA codes
for arginine but CGU, CGC, CGG, CGA and AGG
base pair, and guanine and cytosine form another. Thus, also code for the same aminoacid, arginine. This is also
when the arrangement of bases along one strand is applicable to other aminoacids as well.
T-C-G-A-T-C, the arrangement on the other strand will Non-sense codons- Three codons (UAA, UGA and
be A-G-C-T-A-G. Double strand of this DNA will be UAG) do not code for any aminoacid and act as 'stop
as follows: codons' for terminating the message for the synthesis of
a polypeptide. These are called nonsense codons.
T - C - G - A-T-C
II. SYNTHESIS OF POLYPEPTIDE
Genetic information in DNA, is transcribed on to RNA
A - G - C - T-A-G and then translated as the particular polypeptide (Fig. 9.3).
Other strand
Deoxyribose Adenine Thymine

/
Deoxyribose
Phosphate( > Phosphate

Deoxyribose
Phosphate "
/
Deoxyribose
I /
Cytosine
Guanine
Guanine
Cytosine
>
Deoxyribose
Phosphate
Deoxyribose
One strand
Double stranded DNA
Fig. 9.2 Chemical structure of a segment of double stranded DNA

" etc.

Ch 9: Bacterial Genetics'- -- - --------+-- - - - - - - - - - - - - - - - -- - - - - 63
/ ' Growing polypeptide chain
t-RNA-- --,
Anti-codon---~
Codon-----
m-RNA-- - - u...J....L...,__._..
Ribosome (r-RNA +protein)

Fig. 9.3 Synthesis of polypeptide
A. Transcription of bacteria). Plasmids are circular DNA molecules and

RNA polymerase attaches itself to the beginning of a can replicate autonomously (independent replicons). It is
gene on DNA and synthesises m-RNA, using one of the often not possible to differentiate between plasmids and
strands in DNA as a template. This process is known episomes, therefore, the two terms are frequently used
as transcription. DNA acts as a template for synthesis synonymously. Plasmids and episomes are not essential
of m-RNA, therefore, the bases in m-RNA will be for function of the host bacterium but they may carry
complementary to one strand of DNA. properties of drug resistance, toxigenicity, conjugation
and others.
B. Translation Some plasmids have an ability to transfer themselves
The m-RNA passes into cytoplasm and then m-RNA to other bacteria of the same and also of different species.
and t-RNA come together on the surface of ribosome These are called self transmissible plasmids. This transfer
containing r-RNA. The triplet base sequence on m-RNA occurs usually by method of conjugation. There are some
is known as codon . The base sequences on m-RNA other non-conjugative (non-transmissible) plasmids which
are recognised by the anti-codon (corresponding cannot transfer themselves but can be transduced.
complementary) sequences of t-RNA. The t-RNA By their ability to transfer DNA from one cell to
molecule contains a triplet at one end and aminoacid another, plasmids have become important vectors in
at the other end. The ribosome moves along them-RNA recombinant DNA technology or genetic engineering.
until the entire m-RNA molecule has been translated into
corresponding sequences of aminoacids. This process is
called translation. IV. BACTERIAL VARIATION
There are two types of variation
A. Phenotypic variation
III. EXTRACHROMOSOMAL B. Genotypic variation
GENETIC ELEMENTS The phenotype ('Phaeno'; display) is the expression
Besides the chromosomal DNA, some bacteria may also of various characters by bacterial cells in a g1ven
possess extrachromosomal DNA which may be plasmids environment.
(when these are situated in the cytoplasm as free state) The genotype is the sum total of the genes. It is the
or episomes (when integrated with chromosomal DNA genetic constitution of an organism. All the genes of the
64 - - - -- -- - - - - - -- -- - - - - - - - - - -- - Unit I: General Microbiology
bacterial cell may or may not be expressed in a given The regulator gene is lac I, which codes for a 'repressor',
environmental situation. which is an allosteric protein molecule. This repressor
can combine either with the operator region or with the
A. Phenotypic Variation inducer (lactose in this case). The promoter and operator
A bacterial cell may exhibit different phenotypic regions lie between lac I and the structural lac genes.
appearances in different environmental conditions. The RNA polymerase has to attach to promoter region
Phenotypic variations are reversible. Some examples of and travel along the structural genes in sequence, for
environmental influence on bacteria are: transcription of RNA for enzyme synthesis. In the resting
l. Synthesis offlagella : Salmonella typhi (typhoid bacillus) stage, the repressor molecule is bound to the operator
is generally flagellated, but when grown in phenol region, preventing the travel of RNA polymerase from the
agar, the flagella are not formed . This effect is reversed promoter region to the operon. The repressor molecule
when subcultured from phenol agar into broth. also has affinity for lactose, in the presence of which, it
2. Sy nthesis of enzyme: E. coli possesses the genetic leaves the operator free so that transcription can take
information for the synthesis of the enzyme, beta- place. When the lactose is completely metabolised and
galactosidase, but the actual synthesis takes place only exhausted, the repressor again attaches to the operator,
when grown in a medium containing lactose. This switching off transcription. Lactose acts both as the
enzyme is necessary for lactose fermentation. Such inducer and the substrate for enzyme (Fig. 9.4).
enzymes which are synthesised only when induced by
the substrate (lactose) are called inducible enzymes. B. Genotypic Variation
The enzymes which are synthesised irrespective of These variations occur in the genetic material. These are
the presence or absence of the substrate are called stable, heritable and not influenced by the environment.
constitutive enzymes. Genotypic variations may occur in bacteria by mutation
or gene transfer.
Gene Regulation
('Operon' concept by Jacob and Monod) Mutation
Lactose fermentation of Esch. coli requires three Mutation is a random, undirected heritable vanation
enzymes, beta-galactosidase, galactoside permease and caused by change in nucleotide sequence of the DNA of
transacetylase. These enzymes are coded by the structural the cell. It can involve any of the numerous genes present
genes lac Z, lac Y and lac A respectively. These genes are in bacterial chromosome. The frequency of mutation
arranged in sequence, forming a functional unit, lac operon. ranges from 10- 2 to 10- 10 per bacterium per division.
- - - -- - - - - - - - - - - - Repressor molecule
Galactoside permease
• •- - Transacetylase
Lactose--
Fig. 9.4 Lac operon

Ch 9: Bacterial Genetics,_ _ _ _ _ __ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __ 65
Mutation occurs spontaneously but its frequency may

be enhanced by mutagens such as UV rays, alkylating
Point mutation
It ma occur by
r
J\
-
\ VJ
,..l
agents, 5-bromouracil and acridine dyes. ~ase pair substitution '\.____ l YOJ\.J y ttY\ /On
Mutation is a natural event, taking place all the time, ,,.(.iirFrame shift mutations I
in all dividing cells. Most mutants go unrecognised as
the mutation may be lethal or may involve some minor Base pair substitution
function that may not be expressed. Mutation is best A single base pair has been substituted for another pair.
appreciated when it involves a function which can be It can be subdivided into
readily observed by experimental methods. For example ~ransition
Esch. coli mutant that loses its ability to ferment lactose (bJTransversion
can be readily detected on MacConkey's agar. (a) Transition
Mutation can affect any gene and hence may modify This happens by replacement of one pyrimidine by
any characteristic of the bacterium e.g. sensitivity to another pyrimidine, and one purine by another purine
bacteriophages, loss of ability to produce capsule or ie. AT replaced by GC. Transition is the most frequently
flagella, loss of virulence, alteration in colony morphology, occurring mutation.
pigment production, drug susceptibility, biochemical (b) Tran~sv~e~rs~i~on~ - - - - - --:----
reactions, antigenic structure. The practical importance of When purine is replaced by pyrimidine and vice
bacterial mutation is mainly in the field of drug resistance versa, it is a,1,~,u transversion, e.g. GC changes to CG.
and the development of live vaccines.
Some mutations involve vital functions producing Frame shift mutations
nonviable mutants (lethal mutation). An important type Sometimes, during DNA replication, one or a few base
of lethal mutation is 'conditional mutation'. A conditional pairs are inserted into or deleted from the DNA. This shifts
lethal mutant may be able to live under certain conditions the normal 'reading frame' of the coded message forming
(permissive conditions) but not under restrictive or non- new set of triplet codon. The coded message is read
permissive conditions. One example of such type of correctly up to the point of addition or deletion, but the
mutant is the temperature-sensitive (ts) mutant, which subsequent codons will specify the incorrect aminoacids
is able to live at the permissive temperature (say, 35°C), (Fig. 9.5).
but not at the restrictive temperature (say, 39°C).
Multisite mutations
~ S OF MUTATION® La~number of base pairs are altered in D A (Fig.
-tli;r; are broadly two types of mutations: 9.6) . This may be of four types such as
1. Point mutation .-- 1--Vi,0 (i) Addition (gain) (ii) Deletion (loss)
2. Multisite mutations -- i"-~
Q'J? (iii) Duplication
DNA Sequence 3'---- Q)CA GGT AGT GAA TTA------------ 5'
RNA Sequence 5'----

I AIU I lc l A I I UIA I lcl u l Ij u 1--------3'
Polypeptide Ser- Pro- Ser- Leu- Asn

(Wild-Type)
deleted base
~~~~e:n~~~------------.i- 1Gr I Icj u I l cjc I 1u r 1 ~------3'
Polypeptide Vol His His Leu

(mRNA triplet codon reading frame has shifted one nucleotide rightward)
Inserted base
Restored RNA
Sequence 5'-------
Polypeptide
IGI C I
Vol- +Alo-
I UI A I
Ser-
1
lc u I
Leu-
(Second mutation results in insertion of base restores original reading order)
IAj u I -------3'
Asn
Fig. 9.5 Frame shift mutation

66 - -- -- - - -- -- - -- - -- - - - - -- - - - - Unit I: General Microbiology
Wild type DEMONSTRATION OF MUTATION

1N_or_m_gll __ I A I B I C I DI E I F I G I H I IN LABORATORY
Mutation can be recognised by both gene sequencing as
.M_d!!iq_n_ - _ I AI B I X I C I D I E I FI G I H I - - - well as by observing phenotypic changes.
Fluctuation test and replica plating methods are
.P~le_!iq_n___ I A I B I F I G I H I ____________ _ useful for demonstration of large number of mutant
colonies.
.Pl!JlliC_gl!91}_ _ I AI B I C I D I C I D I E I FI G I H I -
Fluctuation test
lnyersiQ'l __ I AI B I C I G I FI E I D I H I _____ _ Luria and Delbruck (1943) demonstrated that bacteria
undergo spontaneous mutation independent of
.StJ.bstiM[on_lAI sl x I ol El FI GI HI _____ _ environment. They studied bacteriophage sensitive
Esch. coli. Samples from small volume cultures and a
Fig. 9.6 Examples of types of mutations
single large volume culture were plated on solid media
containing bacteriophage. The mutant bacteria (resistant)
formed colonies. Colony count was compared. There was
OTljER TYPES OF MUTATIONS wide fluctuations in the number of bacteriophage resistant
J>ttfuced mutation colonies in small volume cultures, as compared to a
Although mutation occurs spontaneously, its frequency single large volume culture. On statistical analysis it was
is greatly enhanced by certain agents called mutagens. observed that mutation occurred randomly, some early
These may be physical or chemical agents. and some late, which lead to wide fluctuation. However,
in large volume cultures, fluctuations were within limits
~ ical agents / (Fig 9.7). This experiment was not widely appreciated,
(i) UV lighr / probably due to the complicated statistical evaluation.
(ii) Ionising radiatipr( e.g. X-rays It was the simple but elegant 'replica-plating' technique
(iii) Visible light that proved the point beyond doubt.
(iv) Heat
Replica plating method
/ 4emical agents (Lederberg and Lederberg, 1952)
(i) 5-Bromouracil Colonies of bacteria were transferred from master plate,
(ii) 2-Aminopurine on to a number of other plates, using a velvet template.
(iii) Nitrous acid_____........ Relative position of all the colonies was retained. By
(iv) Acridine dyes replica plating on culture plates with and without
bacteriophages, they were able to demonstrate that
Nonsense mutatio/ bacteriophage resistant mutants appeared without ever
When a non-sense codon (UAG, UAA or UGA) is having had contact with the selective agent (Fig. 9.8).
formed within a gene by mutation, the protein synthesis
is terminated prematurely and a partial polypeptide is V. GENE TRANSFER ~)
produced during translation. This is called a nonsense Various methods of gene transfer are as follows:fJ r\,
mutation. Transformation (uptake of naked DNA) '7
@ Transduction (through bacteriophage)
Missense mutation / C. Lysogenic conversion ,~
Both substitution and frame shift mutation give rise to Conjugation (plasmid-mediated)
an altered codon which specifies a different aminoacid
from that normally located at a particular position in A. Transformation
the protein. This is called missense mutation. Transformation is the transfer of gepetic info~n
through free or naked DNA. In 1 9 2 ~ found
Suppression mutation/ that mice died--wherr'"mjeted with of l.;.-i----..
A mutation (second) that restores the function of a gene noncapsulated _(rough: no capsule) ~eumoco=c...· __
altered by previous mutation (first) is called suppression derived from capsular type II and heat ki e smooth:
mutation. capsulated) strain from type I, neithe~separately

Ch 9: Bacterial Genetics _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ 67
---------n
~--U-- - - -. g-
Stock culture
Siegle la,ge vol,me '"'""'
Small volume cultures -o• ot ot ot ot ot ot ot ~---D

t ·---.
+ + + ++ + + + + + ' '
Wide fluctuation in number of Fluctuation within limits

resistance colonies
Fh!. Q.7 Fluctuation test
10 Colonies bacteria and multiply in it. During the .assembly of

bacteriophage progeny insioe infected bacte~ia, er:_rors
I may happen occasio_nally. Besides its own nucleic acid,
host DNA may accidentally be inc9rporated into the
bacteriophage. This is known as 'packaging error'. Hence,
when this bacteriophage infects another bacterium: host
DNA is transferred and the recipient cell acquires new
Master plate characters coded by donor DNA.
Types of Transduction
1. Generalised
2. Restrictec;i
10 Colonies on the 7 Colonies (bacteriophage 1. Generalised transduction

culture plate without resistant) on the culture plate Transduction may be 'generalised', when it involves any
bacteriophage with bacteriophage
segment of donor DNA at random.
Fig. 9.8 Replica plating method
2. Restricted transduction
proved fatal. The live type I capsu ated pneumococcus Transduction may be 'restricted', when a specific
could be isolated from mice that died. It showed that bacteriophage transduces only a particular genetic trait.
rough forms (noncapsulated) were transformed into It has been studied intensively in the 'lambda' phage of
the smooth forms ( "i:. 9.9). It demonstrated that Esch. coli.
some factor in heat-killed type I pnemp.ococcus had ,-
transferred the information for capsule synthesis in the Role of Transduction
noncapsulated_(raugh) strain. Later on, this transforming Transduction is not only confined to transfer of
factor (agent) was identified as free DNA by Avery, chromosomal DN ~,...,......,,isomes and plasmids o
Macleod and McCarty in 1944. Transformation has be transduced. P nicillin res1sta m staphylococci i
been studied mainly in bacteria like pneumococci, due to the plasmi s transferred from one bacterium to
H. influenzae and Bacillus species. another by transduction.
~M ~ o v i d e s an excellent tool for the genetic
B. Transduction Any bacteria for which bacteriophage
Transmission of a_portion of DNA from one bacterium exists can be subject to transduction. Transduction has
to another by a bacteriophage is known as transduction also been proposed as a method of genetic engineering
(Fig. 9.10). Bacteriophages are viruses that parasitise, in the treatment of some i n ~

68 - - - - - -- - - -- - - - -- - - -- -- - - -- - - Unit I: General Microbiology
• •••
•••
••• •• ••• •
••••
••••• •••••
• •••
Live non-capsulated Live-capsulated Heat killed
• •
Live non-capsulated Heat killed capsulated
y
(rough) type II (smooth) type I capsulated type I type II pneumococci type I pneumococci
pneumococci pneumococci pneumococci
i i t
Survives; no bacteria Dies, Live capsulated Survives; Dies, live capsulated type I
recovered type I pneumococci no bacteria pneumococci recovered
recovered recovered
II
Fig. 9.9 Transformation experiment of Griffith
•••
Bacteriophage
0 @3---•
Bacterial
DNA
Fig. 9.10 Transduction

L - - -- - - - - - - - --
Bacterial
cell
- - - - --
Portion of DNA acquired
through b a c ; ~ ~
----,----i-,=7-+-_77""~-1j'"-L..>o '-ff - - - - - - - - '

y -~
C. Lysogenic Conversion and t~ria harbourin propha es are known as lysogenic

Bacteriophages have two types of life cycle inside the bacteria._In~nic conversion the phage DN~elf
host bacteriu is the new genetic element in contrast to transduction
(i) Virulent . lyr!el where the phage acts only as a vehicle carrying bacterial
(ii) Temperate ,K,•----'H'<7'f1e eye e _ gen~s A
Example of lysogenic conversion: Diphtheria bacill~
/ %ii. -
(i) Virulent or lytic eye e produces toxin (and therefore virulence) only when lyso-
Large number of _p_ro_geny phages are formed and sub- genisation with the phage beta occurs. Elimination of the
sequently, these progeny ph-ag_e_s_a-re_r_e...,.le_a__s-ed-,--c-ausing death phage from a toxigenic strain renders it nontoxigenic.
and lysis of the host cell. -',J2.._ ~::,I.*
<t-
~ - Conjugation ::::::----'0
(ii) Temperate or nonlytic cycle The transfer of "genetic material rom one bacterium
In contrast to virulent cycle, the host bacterium is (donor or male) to another (recipient or female) by
unharmed. The phage DNA remains integrated with the ~ c o n t a ~ i s called conjugation (Fig. 9.11) .
bacterial chromosome as the prophage, which multiplies Donors are those bacteria that contain F plasmid (F+ J
synchronously with bacterial DNA. The prophage acts or male cells) while cells lacking F plasmid (F- or female
as an add~hromosomal element which encodes for cells) act as recipients. F plasmid is conjugative plasmid
new characters and is transferred to the daughter cells. which encodes for sex pilus (in p+ cells) necessary for
This process is known as lysogeny or lysogenic conversion conjugation. This plasmid is also named as 'sex factor'

Ch 9: Bacterial
69
D A from adjacent site of its attachment. Such an F

factor carrying some chromosomal DNA is named as F'
factor (F prime factor). When F' cell conjugate with a
recipient (F-), it transfers, along with the F factor, the
host DNA incorporated with it. This process is called
sexduction (Fig. 9.12).
--nf'.L + F- - - - - Sexduction
Colicogenic (Col factor and resistance transfer factor
are two medically important factors which can be
)f _ ferred by conjug\tion. (!) G,1 "t
Colicogenic (Col) Fg,ttor Q) p_,f' J
Some strains of coliform bacteria produce colicins which
are antibiotic like substances lethal to other enterobacteria.
Fig. 9.11 The process of conjugation Bacteria other than coliforms also produce similar kind
of substances e.g. pyocin by Pseudomonas pyocyanea,
or 'fertility' (F) factor. When other similar plasmids diphthericin by Corynebacterium diphtheriae, hence,
were also discovered, a name of 'transfer factor' was a general term bacteriocin has been given to these
given to all such plasmids which conferred on their substances. These bacteriocins are useful for intraspecies
host cells the ability to act as donors or male cells in classification of certain bacteria (e.g. Shigella sonnei).
conjugation. Bacterial conjugation was first discovered by Colicin production is encoded by a plasmid called the
Lederberg and Tatum in 1946 in Esch. coli K12 strains. col factor, which may be transferred by conjugation.
During conjugation , the plasmid DNA replicates
and ~~f? of it passes from d ~ p i e n t cell
probaly along the sex pilus (conjugation tube). As a
Resistance Transfer Factor (RTF)
This plasmid is responsible for the spread of multiple
result, recirfe'nt (F-) becomes donor (F+) and can in turn drug resistance among bacteria. It was first reported
conjugate with other female cells (F-). This character of by Japanese workers (1959) in shigella strains, resistant
maleness (F+) in bacteria is transmissible or infectious. simultaneously to four drugs. They observed that
patients excreting such shigella strains also shed Esch.
_Hfr ceJl and F' cell coli strains resistant to same drugs. Transfer of multiple
F factor or plasmid has the ability to drug resistance from Esch. coli to shigella strains was
exist in some cells in the i n ~ with the host demonstrated. The resistance is plasmid mediated and
chromosome (same as episome). These cells a r ~ is transferred by conjugation. This mechanism of drug
traa~r-1.,;.hromosomal to recipient -cells w i ~ resistance is known as transferable or infectious drug
fre uency, therefore, named as Hf,r cells. Conjugation resistance.
ith an r cell, an F- rarely becomes F+ but it receives Resistance transfer factor (RTF) and resistance
chromosomal DNA from the donor. determinant (r) are two components of the plasmid
The conversion of an F+ cell i n t o ~ n ell is known as an R factor (RTF + r determinants). The
reversible. "' RTF is responsible f-Or conjugational transfer while each
,__ F+ Hfr .,.. . r determinant carrie~ resistance for one of the several
When the F factor reverts from the 4!!.egrated to fr3 drugs. An R factor can have several r determinants
state, it may sometimes carry with it some chromosomal and resistance to many drugs can be transferred
Hfr F' Conjugation of F' F' F'

and F- cells
Fig. 9.12 Sexduction y

70 - - - - - - - - - -- - - - -- -- - - - - - -- -- - Unit I: General Microbiology
simultaneously (Fig. 9.13). Sometimes RTF dissociates This is the rationale of combined treatment in tuberculosis.
from the r determinants and both exist as separate However, in spite of this knowledge, inappropriate or
plasmids. In such cases, the resistance is not transferable inadequate treatment has caused extensive resistance in M
though the host cell remains drug resistant. The RTF tuberculosis, leading to 'multidrug resistant tuberculosis'
can also attach to its determinants other than those for (MDR TB) cases.
drug resistance. Enterotoxin and haemolysin production Transferable drug resistance mediated by the R factor
in some enteropathogenic Esch. coli are shown to be is an important method of drug resistance. Acquisition
transmitted by this transfer factor (RTF). of an R factor simultaneously confers resistance to
several drugs, therefore, there is no use of combined
R+ therapy. Table 9.1 shows the differences of mutational
rDeter~
[jJ and transferable drug resistance.
Resistance transfer by transformation can also be
demonstrated experimentally, but its significance in nature
is unknown. The plasmid carrying the gene for beta-
-I
lactamase production can be transferred from a penicillin
____..,
..__
o~:r------,; resistant Staphy lococcus to a susceptible Staphylococcus
by transduction .
Conjugation
Table 9.1 Differences between Mutational and
Transferable Drug Resistance
Mutational drug resistance Transferable drug resistance
1. Mutational resistance Transferable resistance
Fig. 9.13 Resistance transfer factor and drug resistance
2. Resistance to one drug at Multiple drug resistance
a time
Transferable drug resistance is seen in various
3. Low degree resistance High degree resistance
pathogenic and commensal bacteria of man and animals
such as Esch. coli, Klebsie/la, Proteus, Vibrio, Pseudomonas. 4. Resistance is not Resistance is transfe rable
Transfer of drug resistance occurs readily in vitro but in transferable to other to other organisms
organisms
the normal intestines, it is inhibited by several factors
such as anaerobic conditions, bile salts, alkaline pH and 5. Mutants may be defective Not defective
abundance of anaerobic bacteria. But in the intestines of 6. Virulence of resistant Virulence not decreased
persons on oral antibiotic therapy, transfer occurs readily mutants may be lowered
due to the selection pressure produced by the drug. 7. Resistance can be Cannot be prevented
Transferable drug resistance involves all antibiotics prevented by treatment by treatment with
in common use. Bacteria carrying R factors can be with combination of drugs. combination of drugs.
transmitted from animals to man. Hence indiscriminate
use of antibiotics in animals or in animal feeds can Transposable Genetic Elements
lead to an increase of multiple drug resistance in the Transposable genetic elements are specific sequences of DNA
community. segments that have the ability to move from one plasmid
to another plasmid or from plasmid to chromosome and
vice versa and also within the chromosome. Because of
VI. GENETIC MECHANISMS OF their ability to insert into many sites both on plasmid
DRUG RESISTANCE and chromosome, they have assumed the popular name
Bacteria may acquire drug resistance by mutation or by of jumping genes.
one of the methods of gene transfer. In clinical practice, The transfer of genetic material from one DNA
mutational resistance is very important in tuberculosis. molecule to another is called transposition . It does not
If a patient is treated with streptomycin alone, initially require any DNA homology between transposable element
organisms die in large numbers but soon resistant mutants and the site of insertion. It is, therefore, different from
appear and multiply unchecked. By use of two or more recombination.
drugs, 'a mutant resistant' to one drug will be killed by Transposons are larger (4--25 Kb) transposable genetic
the other drug. The possibility of a mutant developing elements and contain additional genes that encode for
resistance to more than one drug at one time is remote. at least one function (often conferment of antibiotic

Ch 9: Bacterial Genetics_ _ _ _ _ _ _ _____.!_ _ _ _ _ _ _ _ _ _ _ __ _ ________ 71
Transposon
I inverted repeat sequence Gene Inverted repeat sequence I

G T A C G -,----.--,----.----.--,-~ C G T A C
IIIIIIIIIIIIII IIl G C A T C
'---v-----'
0 C-
A-
G
T
Single stranded loop _ _ _( \
Double stranded stem

formed by bonding of
\ C_ G)
G-
T-
C
A
terminal inverted sequences
T- A A- T
G- C C- G
Fig. 9.14 Structure of transposon
resistance). They may also encode for toxin production. The DNA can be cleaved by enzymes called restriction
Transposon is defined as a segment of DNA with one endonucleases and the fragments containing the desired
or more genes in the centre and the two ends carrying genes are isolated. These desired DNA fragments are
inverted repeat sequences of nucleotides- nucleotide covalently bound to vectors or carriers, such as plasmids
sequences complementary to each other but in the or temperate bacteriophages. The recombinant DNA
reverse order. Because of this feature, each strand of molecule is sealed by an enzyme DNA ligase and is then
the transposon can form a single-stranded loop carrying introduced into a bacterial host, usually Esch. coli, by
the gene or genes, and a double stranded stem formed by transformation. Many other bacteria and yeasts have also
hydrogen bonding between the terminal inverted repeat been employed for introduction of recombinant DNA
sequences (Fig. 9.14). molecule. Esch. coli containing recombinant molecule
Small transposons (1-2 kb) are known as 'insertion is grown on a suitable medium and bacterial colonies
sequences' or IS elements. Transposons attach at certain carrying specified genes are produced (Fig. 9.15). Thus
regions of chromosomal, plasmid or phage DNA. Bacteria desired protein is obtained in large quantities by growing
acquire new characters by the insertion of transposable these Esch. coli.
elements. Unlike plasmids, transposons are not self
replicating and are dependent on chromosomal or plasmid Application of Genetic Engineering
DNA for replication. 1. Production of vaccines: Preparation of certain vaccines
It has been suggested that R plasmids may have is done by producing specific antigen against which
evolved as collections of transposons each carrying a antibody is required e.g. hepatitis B, rabies and B.
gene that confers resistance to one or several antibiotics. pertusis vaccines.
Transposition is a mechanism of amplifying genetic 2. Production of proteins: Genetic engineering has
transfers in nature. also been used for the production of proteins of
therapeutic interest. These include human growth
hormone, insulin, interferons, interleukin-2, tumour
VII. GENETIC ENGINEERING necrosis factor and factor VIII.
By genetic engineering or recombinant DNA technology, 3. Gene therapy: Genetic diseases can be cured by
it is now possible to isolate the genes coding for any introducing normal genes into the patient.
desired protein from microorganisms or from cells of
higher form of life including man, and to insert them Restriction Endonculeases
into suitable microorganisms in such a way so that it These are microbial enzymes which split DNA into
can be expressed in the formation of specific (desired) fragments of varying lengths. They cleave double stranded
protein. Such cloning of genes in microorganisms enables DNA at specific oligonucleotide sequences. Many such
the preparation of the desired protein in pure form and enzymes (for example, Eco RI, Hind III) which act at
in large quantities at a reasonable cost. different nucleotide sequences have been recognised. The

the detection of homologous DNA by hybridisation.

Hybridisation is the technique in which two single-strands
of nucleic acid come together to form a stable double-
+ DNA fragment to be stranded molecule.
cloned All microorganisms contain some unique sequences
Plasmid vector of nucleic acid within their genome that distinguish them
from all other organisms. This unique sequence of nucleic
! Insert DNA into plasmid

vector
acid can be recognised by hybridisation with a DNA probe
(Fig. 9.16) . Various diagnostic DNA probes have been
developed for identification of different microorganisms.
Probes containing sequences unique to the microbe can
be added to microbial cultures, body fluids or other
specimens suspected to contain the microbe or its DNA.
The DNA probe hybridises with the complementary
Recombinant plasmid sequences on the microbe's DNA. The advantages of
DNA probes are their high degree of specificity and
l lntroduce recombinant
plasmid into Esch . coli ability to detect minute quantities of DNA. They are
also very useful in diagnosis of microbes that are either
difficult or impossible to culture. DNA probes which bind
Bacterial
chromosome
@ Esch. coli
D to RNA can also be designed and has been particularly
used to locate ribosomal RNA.
t
Bacterial cell multiplication
Application of DNA Probes
Applications of DNA probe technology in microbiology
are shown in Table 9.2.
!
Colony of cells containing copies of the same recombinant plasmid
Table 9.2 Applications of DNA Probe Technology
• In clinical microbiology:
Fig. 9.15 Genetic engineering - Direct detection of microbes in specimens.
- To detect microbes which are either difficult or impossible
natural function of these enzymes in bacteria may be to culture.
• Identification of culture isolates
the dest~f ion of foreign DNA which may enter the
• Strain identification
bacteriaycell. • To identify toxins, virulence factors
• Identification of resistant markers
VIII. DNA PROBES
DNA probes are radiolabelled or chromogenically labelled Diagnostic DNA probes to recognise Legionella
pieces of single1 stranded DNA which can be used for pneumophila, Campylobacter jejuni, M. tuberculosis,
A- T
A T A A- T
C- G C G C C- G
Denature
G- C G C G G- C
Add DNA probe Labelled probe Hybridise
C- G C G G
T- A
T A A
T- A
T A A
Double stranded DNA

Fig. 9.16 Hybridisation technique

Ch 9: Bacterial Genetics, _ _ _ _ _ _ _ _ _ __ _ _ _ _ __ _ _ _ _ _ _ _ _ _ _ _ __ _ 73
Helicobacter pylori, Esch. coli (LT and ST toxins), (iii) DNA Synthesis
hepatitis B virus, Plasmodium falciparum, rotavirus and Polymerase enzyme derived from Thermus aquaticus (Taq)
human immunodeficiency virus have been developed. triggers the formation of new DNA strand from the free
nucleotides. Taq polymerase and nucleotides are added
Blotting Techniques in the tube for formation of new strands of DNA.
DNA fragments are obtained by restnct10n enzyme These three steps are repeated again and again,
digestion and separated by gel electrophoresis. These a process that is automated by the thermocycler or
fragments can be transferred from the gel by blotting PCR machine. The products of first cycle become the
to nitrocellulose or nylon membranes that bind the template for the next cycle. After 20-30 cycles or more,
DNA. The DNA bound to the membrane is first an exponential increase in the amount of DNA occurs
denatured (converted to single stranded DNA) and (Fig. 9.17).
then hybridised with radioactive single stranded DNA
- - - - - - - - - Double stranded
probes. Hybridisation results in formation of radioactive - - -- - - - ~ - template DNA
double-stranded segments, which can be detected on
X-ray film. This technique of identifying DNA fragments
by DNA:DNA hybridisation is called Southern blotting,
after E.M.Southern who developed it. This is a highly
sensitive technique.
--
- - - - - - - - - Single stranded DNA
- 0ligonucleotide primers -
- - -- - - - - - are added and
annealing occurs.
A similar procedure for analysis of RNA has been - - - -- -- - - - Extension

called northern blotting. Here the RNA is separated by
gel electrophoresis, blotted and identified using labelled One copy of DNA has
become two.
probes.
A technique for identification of proteins is called
immunoblotting. The Western blot test is one example,
which has been used as confirmatory test for the diagnosis
- _
_ __ _ _ _ _ __
PCR cycle is repeated.
DNA is separated into
two single strands;
primers are added and
--
_ _ _ _ _ _ _ _ _ annealing occurs.
-
of HIV antibody in sera.
IX. POLYMERASE CHAIN REACTION (PCR)

Polymerase chain reaction (PCR) was first developed
in 1983 at Cetus Corporation, USA. It has made a
revolutionary impact in molecular biology. In 1993, Kary
Mullis was awarded the Nobel prize for this work.
A. Principle
It is a DNA amplification system that produces a large
amount of DNA in vitro from small amounts of starting
material. It amplifies a specific DNA sequence (or gene) Two copies have
become four
of interest.
B. Procedure
It involves four main stages which are as follows:
(i) Denaturation Fig. 9.17 Polymerase chain reaction

The double stranded DNA is dissociated to a single
stranded DNA at 94°C (denaturing temperature). (iv) Detection of Amplified Product
Amplified DNA can be detected by Gel electrophoresis.
(ii) Primer Annealing
The temperature is reduced to 50-60°C, then oligonucleotide C. Application of PCR
primers attach to target DNA. This temperature is called The PCR provides extremely rapid analysis (one day).
annealing temperature and the process is known as PCR is a versatile tool useful in infectious, genetic or
annealing of primers. neoplastic diseases, in forensic investigations and in the

examination of phylogenetic relationships in evolution. this therapy is to permanently cure the physiological
It has been applied in clinical laboratory for diagnosis dysfunction by repairing the genetic defect.
of various infectious agents (Table 9.3). A specific DNA
sequence of a particular infectious agent is amplified with 1. Types
the specific primers. i. Ex vivo gene therapy
ii. In vivo gene therapy
D. Types of PCR
Besides originally described PCR, other types of PCR Ex vivo gene therapy
include reverse transcriptase PCR (RT-PCR), nested In this type of therapy, the normal gene is cloned in
PCR, multiplex PCR and real time PCR. These have vectors such as retroviruses (mouse leukaemia virus) or
been described in chapter 79. adenoviruses which are infectious but relatively harmless.
Tissues removed from the patient are incubated with these
Table 9.3 PCR in Diagnosis of Infectious Agents genetically modified viruses to transfect them with the
Bacteria normal gene. The transfected cells are then reintroduced
into the patient by transfusion.
M. tuberculosis, Legionella pneumophila, Helicobacter pylori,
Chlamydia trachomatis, Mycoplasma pneumoniae
In vivo gene therapy
Viruses
Cloning of vector is similar to that of ex vivo gene therapy.
Cytomegalovirus, Herpes simplex virus, Hepatitis B virus, However, the step of incubating the excised patient tissue
Hepatitis C virus, Coxsackie virus, Measles virus, Human
with genetically modified viruses is absent in this type
immunodeficiency virus ( HIV-I and HIV-2 ) , Human papilloma
virus, Rota virus, Rubella virus, Human herpes virus-6 ( HHV-6 ),
of therapy. Thus the naked DNA or a virus vector is
rhinovirus, parvovirus, adenovirus. directly introduced into the patient's tissues.
Fungi
2. Applications
Candida albicans, Cryptococcus neoformans, The first gene therapy experiment in humans was initiated
Pneumocystis jiroveci.
in 1990 at the National Institutes of Health (NIH), USA.
Protozoa This was done on a four year old girl suffering from a
Toxoplasma gondii, Trypanosoma cruzi, Plasmodium sp. severe immunodeficiency disease caused by the lack of
enzyme adenosine deaminase (ADA). She was transfused
with her own blood cells which had been engineered to
X. GENETICALLY MODIFIED ORGANISMS contain a functional ADA gene. Later, other children
The process of artificially introducing foreign DNA were given similar treatment. So far, the children have
into organisms is called transfection. The recombinant shown marked improvement and continue to be healthy.
organisms produced in this way are named transgenic or However, the treatment is not permanent and must be
genetically modified organisms. Foreign DNA have been repeated. Single gene defects such as haemophilia and
inserted into a variety of microbes, plants and animals sickle cell anaemia are possible candidates for therapy.
through recombinant DNA technology. Transgenic Many other clinical trials with gene therapy are also in
organisms are available for a variety of biotechnological progress.
applications.
3. Gene line therapy
XI. GENE THERAPY It is the ultimate sort of gene therapy, in which gene are
Gene therapy is a technique by which a faulty gene is inserted into an egg, sperm or early embryo. Although
replaced with a normal gene in persons with fatal or tests in animals seems it to be effective, its use on humans
extremely debilitating genetic diseases. The benefit of has been rather controversial.
KEY POINTS
1. Genetics is the study of heredity and variation.
2. All hereditary characteristics are encoded in DNA.
3. Genetic information is stored in the DNA as a code. Codon consists of a sequence of three nucleotide bases, i.e.,
the code is triplet.
Ch 9: Bacterial G e n e t i c s ' - - - - - - - - ~ - - - - - - - - - - - - - - - - - - - - - - 75
4. Besides the chromosomal DNA, some bacteria may also possess extrachromosomal DNA such as plasmids. These
plasmids are circular DNA molecules and can replicate autonomously. They may carry properties of drug resistance,
toxigenicity, conjugation and others.
5. Some plasmids have an ability to transfer themselves to other bacteria of the same and also of different species.
These are called self transmissible plasmids. This transfer occurs usually by method of conjugation.
6. Mutation is a random, undirected heritable variation caused by change in nucleotide sequence of the DNA of the
cell. The frequency of mutation ranges from 10- 2 to 10- 10 per bacterium per division.
7. Mutation occurs spontaneously but its frequency may be enhanced by mutagens such as UV rays, alkylating agents,
5-bromouracil and acridine dyes.
8. Gene transfer can occur in bacteria. Various methods of gene transfer include transformation, transduction, lysogenic
conversion and conjugation.
9. R. factor is a plasmid responsible for the spread of drug resistance among bacteria. This plasmid is transferred from
one bacterium to other by conjugation. R factor has two components, resistance transfer factor (RTF) and resistance
determinant (r). This mechanism of drug resistance is known as transferable drug resistance.
10. Some bacteria may acquire drug resistance by mutation. It is known as mutational drug resistance. This type of
reistance occurs in M. tuberculosis.
11. Transposable genetic elements are specific sequences of DNA segments that have the ability to move from one plasmid
to another plasmid or from plasmid to chromosome and vice versa and also within the chromosome. They are also
named as jumping genes.
12. By genetic engineering or recombinant DNA technology, it is possible to isolate the genes coding for any desired protein
from microorganisms, and to insert them into suitable microorganisms in such a way so that it can be expressed in
the formation of specific (desired) protein. This technique has been used for production of vaccines, insulin, interferons
and interleukin-2. Gene therapy is another application based on genetic engineering.
13. DNA probes are radiolabelled or chromogenically labelled pieces of single stranded DNA which can be used for
the detection of homologous DNA by hybridisation. Various diagnostic DNA probes have been developed for
identification of different microorganisms.
14. Polymerase chain reaction (PCR) is a DNA amplification system that produces a large amount of DNA in vitro from
small amounts of starting material. The PCR provides extremely rapid method for diagnosis of various infectious agents.
YOU MUST KNOW

1. Functioning and importance of Lac operon.
2. Types of mutation.
3. Principles and uses of various methods of gene transfer.
4. Resistance transfer factor and its role in drug resistance.
5. Differences between mutational drug resistance and transferable drug resistance.
6. Transposable genetic elements and their importance.
7. Principles and applications of genetic engineering, DNA probes, polymerase chain reaction (PCR).
8. Gene therapy and its importance.
STUDY QUESTIONS
(a) Transformation (b) Transduction (c) Conjugation (d) F factor
(e) R factor (f) Extrachromosomal genetic elements (g) Lac operon .
(a) Transposable genetic elements (b) Genetic engineering
(c) Restriction endonucleases (d) DNA probes and their clinical application
(e) PCR and its application in clinical microbiology (f) Gene therapy

76 - - - - -- - - - - - - - - -- - - - - - - - - - - - Unit I: General Microbiology
3. Define mutation. Describe various types of mutation.

4. Name the various methods of gene transfer. Discuss any one of these in detail.
5. Tabulate the differences between mutational drug resistance and transferable drug resistance.
MULTIPLE CHOICE UESTIONS (MCQs)

1. All the following are non-sense codons except:
(a) UAG (b) UGA (c) UAA (d) AAG
2. The process of transfer of genetic information from DNA to RNA is known as:
(a) Transformation (b) Transduction (c) Transcription (d) Translation
3. Which of the following properties may be plasmid mediated?
(a) Resistance to drugs (b) Enterotoxin production
(c) Lactose fermentation (d) All of the above
4. Which of the following enzymes are required for lactose fermentation?
(a) Beta-galactosidase (b) Galactoside permease
(c) Transacetylase (d) All of the above
5. During mutation, when a purine is replaced by pyrimidine and vice-versa, it is named:
(a) Transversion (b) Transition (c) Induced mutation (d) None of the above
6. During mutation, when one pyrimidine is replaced by another pyrimidine and one purine by another purine, it is
named:
(a) Transversion (b) Transition (c) Induced mutation (d) None of the above
7. Transfer of genetic information through free or naked DNA is called:
(a) Transformation (b) Transduction (c) Conjugation (d) Lysogenic conversion
8. Transmission of a portion of DNA from one bacterium to another by a bacteriophage is known:
9. The transfer of genetic material from one bacterium to another by mating or contact is called:
10. F factor carrying some chromosomal DNA is named:
(a) F factor (b) F prime factor
(c) Hfr (d) Resistance transfer factor
11. Which of the following factors is responsible for transferable drug resistance in bacteria?
(a) Resistance transfer factor (b) F factor
(c) Colicogenic factor (d) All of the above
12. Resistance transfer factor can be transferred from one bacterium to another by:
(a) Transformation (b) Transduction
(c) Conjugation (d) None of the above
13. Drug resistance in Mycobacterium tuberculosis is due to:
(c) Conjugation (d) Mutation
14. Penicillin resistance in Staphy lococcus aureus may be acquired by:
(c) Conjugation (d) Mutation
15. The technique of identifying DNA fragments by DNA: DNA hybridisation is called:
(a) Southern blotting (b) Northern blotting
(c) Western blotting (d) None of the above
Answers (MCQs):
1. (d) 2. (c) 3. (d) 4. (d) 5. (a) 6. (b) 7. (a) 8. (b) 9. (c) 10. (b)
11. (a) 12. (c) 13. (d) 14. (b) 15. (a)
- - - •++---

Chapter 10
MICROBIAL PATHOGENICITY
I. Microbes and Related Terms

A. Microbes B. Related Terms
II. Infection
A. Types of Infection B. Transmission of Infection
C. Factors Predisposing to Microbial Pathogenicity
Ill. Types of Infectious Diseases
A. Localised B. Generalised
The host-parasite relationship is determined by the 2. Pathogen: Microorganism capable of producing

interaction between host factors and the infecting disease in the host is called pathogen (pathos, suffering
microorganism. Health or disease depends on the and gen, disease).
equilibrium between the two. 3. Opportunistic pathogens: Some commensals or
saprophytes can produce disease when host resistance
I. MICROBES AND RELATED TERMS is lowered.
A. Microbes 4. Pathogenicity: It refers to the ability of a class of
Microbes can be divided into saprophytes and microbes to produce disease.
parasites, based on their relationship to their hosts. 5. Virulence: It is the degree of pathogenicity of a
1. Saprophytes: These are free-living microbes that live microbe.
on dead or decaying organic matter. They are found
in soil and water. They are generally unable to invade II. INFECTION
the living body. However, when host resistance is Infection: The l~dgement and @ultiplication of a parasite
lowered , some saprophytes like Bacillus subtilis may ~ e body is known as infection. All infections do not
cause infection. invariably result in disease.
2. Parasite: A parasite is a microorganism which lives
on a living host and derives nutrition from the host, A. Types of Infection
without any benefit to the host. 1. Primary infection: Initial infection with a parasite in
a host is termed primary infection.
B. Related Terms 2. Reinfection: Subsequent infection with the same
1. Commensals: Commensals live in complete harmony parasite in the same host is called reinfection.
with the host without causing any harm to it. They 3. Secondary infection: When the body resistance is
constitute the normal flora of the body such as lowered by a preexisting infectious disease, a new
Staphylococcus epidermidis of skin and Escherichia parasite sets up an infection, this is termed secondary
coli of gastrointestinal tract. infection.
77
78 - - - - -- - - - - - - -- -- - - - -- - -- -- - - Unit I: General Microbiology
4. Cross infection: When a patient already suffering (c) Insects

from a disease acquires a new infection from another (d) Soil and Water
host or another external source, it is known as cross (e) Food.
infect· . (a) Human cases and carriers- The commonest source
osocomial infection: ross infection acquired in of human infection is man himself. The parasite may
s 1s ca e ospital-acquired or hospital originate from a patient or a carrier.
associated or nosocomial infection. • Carrier: A carrier is a person who harbours the
6. Iatrogenic infection: An iatrogenic infection is defined pathogenic microorganism without suffering
as physician induced infection resulting from drug from its ill effects. Several types of carrier are
therapy or investigative procedures. known.
7. Subclinical infection: When clinical symptoms of an • Healthy carrier: One who harbours the pathogen
infection are not apparent, it is known as subclinical but has never suffered from the disease caused by
infection. that particular pathogen.
8. Latent infection: Following infection, some parasites • Convalescent carrier: One who has recovered from
may remain in a latent or hidden form in host tissues and the disease but continues to harbour the pathogen
they proliferate and produce clinical disease when the in his body.
host resistance is lowered. It is known as latent infection. • Temporary carrier: It depends on the duration of
9. Atypical infection: In this type of infection, the carriage. The temporary carrier state lasts for less
typical or characteristic clinical manifestations of than six months.
the particular infectious disease are not present. • Chronic carrier: When carrier state lasts for several
years and sometimes even for the rest of one's life,
B. Transmission of Infection it is called chronic carrier.
There are three links in the chain of transmission of • Paradoxical carrier: It refers to a carrier who
communicable diseases. These links include the reservoir, acquires the pathogen from another carrier.
mode of transmission and the susceptible host. • Contact carrier: A persoµ who acquires the
pathogen from a patient.
1. Reservoir Carriers are very important source of infection to
It refers to any human being, animal, plant, soil or spread the disease in a community.
inanimate matter in which parasite normally lives, (b) Animals- Many pathogens are capable of causing
multiplies and depends primarily for its survival. The infection in both man and animal. Animals may, therefore,
reservoir and source of infection are one and the same act as source of human infection. The infection in animals
in most instances, sometimes they may be different e.g. may be asymptomatic and these animals act as the
reservoir of Cl. botulinum is the soil but the source of reservoir of human infections. These are called reservoir
infection is contaminated food. hosts. Infectious diseases trare·d from animals to
man are calle ses. ,
Sources of infection Zoonof disease may be
These may be endogenous sources or exogenous . . ~ubercul;!'sis,
S;dj 1!!Jlella food
sources. po1sonmg. - 1
• Viral: Rabies from dogs. '

(i) Endogenous sources • Protozoa!: Leishmaniasis.
Organisms of normal flora are usually non-pathogenic • Helminthic: Hydatid disease from dogs.
but occasionally they behave as pathogens outside their • Fungal: Dermatophytes from cats and dogs.
habitat. Esch. coli which are the normal flora of the
intestines may cause urinary tract infection. Another (c) Insects- Blood sucking insects such as mosquitoes,
example is viridans streptococci which are the normal ticks, mites, flies, fleas and lice may transmit a number
flora of the mouth may cause infective endocarditis. of pathogens to man. The diseases so caused are called
arthropod borne diseases. Insects transmitting these
(ii) Exogenous sources pathogens are known as vectors. They are of two types:
Most of the infections are exogenous in origin. Exogenous mechanical vectors and biological vectors.
sources include: Mechanical vectors: These carry the organisms on
(a) Human cases and carriers their legs, wings and body and transmit them to the
(b) Animals eatables e.g. transmission of dysentery or typhoid bacilli

Ch 10: Microbial Path ogenicity - - - -- - ' - - - - - - - - - - -- - - - - - - - - - - - - 79
to man, through food , by domestic fly. by patients in secretions from the nose or throat during
Biological vectors: The pathogen multiplies in the body sneezing, coughing or speaking. Large drops of such
of the vector, often undergoing part of a development secretions fall to the ground and dry there. Small droplets,
cycle in it. Such vectors are named biological vectors (e.g. less than 0.1 mm in diameter, evaporate immediately to
f emale Anopheles mosquito in malarial parasite; Cu/ex become minute particles or droplet nuclei (1-10 µm in
mosquito in filarial parasite). diameter) which remain suspended in air for long periods,
Ex trinsic incubation period: After the entry of acting as sources of infection.
pathogen into the vector, the time required for the
vector to become infective is called extrinsic incubation (iii) btgestion V
period. Intestinal infections like cholera, dysentery, food
Besides acting as vectors, some insects may also act poisoning and most of the parasitic infections are
as reservoir hosts (e.g. ticks in relapsing fever) . Infection acquired 10n of foo r drink contaminated by
is maintained in such insects by transovarial passage. pathogen . ood borne infections occur mostly through
(d) Soil and water- Some pathogens may survive in carriers engaged in handling or preparation of food and
the soil for very long periods. Spores of tetanus and contaminating the food stuffs. The water supply may get
gas gangrene bacilli remain viable in the soil for several contaminated with the faeces of the patients or carriers.
decades and serve as the source of infection. Fungi causing All these may transmit infection.
histoplasmosis and parasites such as roundworm and
hookworm also survive in soil and cause human infection.
Contamination of water with pathogenic 'atin'r!?CI1rs:,:--:·ifi7some instances, may be inoculated directly
microorganisms (e.g. Vibrio cholerae, hepatitis virus) may into the tissues of the host, for example,Crabies virgs)
act as the source of infection. is inoculated directl(!?y bite of a rabid animJ!> Spores
(e) Food- Contaminated food acts as a source of of Cl. tetani are present in the soil. They get deposited
infection in cases of food poisoning, gastroenteritis, into the host tissue following severe wounds leading to
diarrhoea and dysentery. tetanus.
Infection by inoculation may be iatrogenic when
P
. Modes of Transmissio ~ .
· orga ·
7
n spread from one host to
unsterile syringes and equipments are employed. Hepatitis
B and Human Immunodeficiency Virus (HIV) may be
another by a variety of mechanisms. transmitted by use of contaminated syringes and through
transfusion of infected blood.
(i) Contact
Contact transmission may be direct or indirect. (v) Vectors
(a) Direct contact- Sexually transmitted diseases (STD) V, re arthropods or other invertebrate hosts e.g.
1 · gonorrhoea, herpex simplex type 2 os u ·toes, ies, fleas, ticks, mites and lice. Transmission
x'f'........:1.-a-re--acquired by direct contact. by vector may be either mechanical or biological (Refer
(b) ~ t contact- It may be through the agency of Chapter 81).
which are inanimate objects such as clothing,
toys etc. which may be contaminated by a pathogen (vi) Transplacental
~ a c t as a vehicle for its transmission"'____Face towels Some pathogens can cross the placental barrier and infect
shared by various persons may lead to spread of the foetus in utero. This is known as vertical transmission.
trachoma. This may lead to abortion, miscarriage or stillbirth. In
Contagious disease is term used for the disease case of congenital syphilis, live infants may be D·v_l:,J~'i-1.J.J
acquired by direct contact. manifestations of the disease. Infection with ella vir ,
Infectious disease is the disease acquired by other especially in the first trimester of pregnancy, a to
modes such as inanimate objects. The distinction between congenital malformations. Such infections are known as
contagious and infectious disease is now not generally teratogenic infections. Other organisms which may cause
employed. congenital in1ID~~;.-.a.r,e toxoplasma, cytomegalovirus
pes simplex virus.
(ii) Inhalation
Respiratory infections such as common cold, influenza, (vii) Iatrogenic and laboratory infections
whooping cough and tuberculosis are acquired by If meticulous care in asepsis is lacking, certain infections
inhalation. These organisms are shed into the environment like AIDS and hepatitisB may sometimes be transmitted

80 - - - - - - - - - - - - - - - - - - - -- - - - - - Unit I: General Microbiology
during procedures such as injections, lumbar puncture, (b) Close adherence of the bacterium assures that
blood transfusion, dialysis and surgery. These are known the toxin will be delivered in high concentrations
as iatrogenic infections. Laboratory personnel handling directly to the host cells.
infectious material are particularly at risk. Special care (c) If bacterium is invasive in nature, adherence helps
should be taken to prevent laboratory infection. in penetrating host cells.
Besides above mentioned adhesins, other structures
3. Susceptible Host which play role in adherence are outer membrane protein,
The infectious agent enters the body by one of the following flagella, lipopolysaccharide (LPS) and glycocalyx. Most
routes: oral, respiratory, genitourinary, conjunctiva! or of the bacteria make use of not just one but several of
cutaneous. This is known as portal of entry. these factors.
Incubation period: It is the time interval between Loss of adhesins often renders the strain avirulent.
the entry of infective agent and the onset of clinical Adhesins are antigenic in nature. Specific immunisation
features of the disease. The infective agent, after reaching with adhesins has been attempted for prevention of
the selective tissue, undergoes multiplication during this gonorrhoea in human beings.
period.
The outcome of an infection depends on interaction
between microbial factors which predispose to (ii) Invasiveness
pathogenicity and various host factors which contribute This refers to the ability of an organism to spread within
to resistance. the host tissues after establishing infection. Highly invasive
pathogens produce, spreading or generalised lesions (e.g.
C. Factors Predisposing to Microbial streptococcal infections), while less invasive pathogens
Pathogenicity cause localised lesions (e.g. staphylococcal abscess). Some
Pathogenicity refers to the ability of a microbial species pathogens though capable of causing fatal diseases lack
to produce disease. Virulence is the ability of a strain of invasiveness but remain confined to site of entry and
a microorganism to cause disease. The pathogenic species produce the disease by elaborating a potent toxin e.g.
M. tuberculosis contains strains of varying degrees of tetanus bacillus.
virulence including those which are avirulent, such as the
vaccine strains. The virulence of a strain is not constant
and may undergo variation. (iii) Antiphagocytic factors
Exaltation: Enhancement of virulence of a strain is Macrophages and polymorphs play role in phagocytosis
known as exaltation. This can be induced by serial passage of microbes. There are certain factors which oppose
in an experimental animal. phagocytosis for survival of bacteria. These factors are
Attenuation: Reduction of virulence of a strain is as follows:
known as attenuation. This can be achieved by repeated (a) Capsule- Cell wall in many bacteria is enclosed by
passages through unfavourable hosts, repeated cultures gelatinous layer known as capsule. Most capsules are
in artificial media, growth under high temperature or in polysaccharide in nature except that of Bacillus which
the resence o -Q.A---i..i..;septics. is polypeptide. Capsule contributes to the virulence of
bacteria by preventing phagocytosis. Non-capsulated
t --------- 5?i~'\ w
The initial event in the pathogenesis is the attachment of
variants usually exhibit low virulence.
(b) Streptococcal M protein-The M protein present on the
surface of group A streptococci binds both fibrinogen
the bacteria to body surfaces. The attachment is a specific and fibrin to the bacterial cell wall thus masking the
reaction between surface receptors on the epithelial cells bacterial receptors from complement.
and adhesive structures on the surface of bacteria. These (c) Cy totoxin- Cytotoxins produced by certain bacteria
adhesive structures are named adhesins. Adhesins usually interfere with chemotaxis or kill the phagocyte. Staph.
occur as fimbriae or fibrillae and pili or as colonisation aureus produces haemolysins (can damage both
factors. RB Cs and WBCs) and leukocidin (only WBCs are
Advantages of adherence are as follows: damaged).
(a) It prevents the microbes from being flushed away (d) Bacterial surface antigens-Vi antigen of S. typhi and
in mucus secretions, urine and by peristalsis in the K antigen of Esch. coli enable these bacteria to resist
gut. phagocytosis and lytic activity of complement.

Ch 10: Microbial Pathogen icity _ _ _ _ _ _ _ _ _ _ _ _ __ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ 81
(iv) Survival within the phagocyte (a) Endotoxins- They are lipopolysaccharide (LPS)
Ingestion of a microbe by a phagocyte results in the in nature and form an integral part of the cell
formation of a phagolysosome by the fusion of phagosome wall of Gram negative bacteria. They are released
with the lysosome. Any organism which interferes with the from the bacterial surface by natural lysis of the
formation of phagolysosome can survive intracellularly. bacteria or by the disintegration of the cell wall.
(a) Interference with the oxidative burst-Superoxide and They are heat stable. The toxicity of endotoxin
H 2O 2 produced are lethal to bacteria. Staph. aureus depends on the lipid component (lipid A). Lipopo-
produces an enzyme catalase which breaks down lysaccharide binds to neutrophils via lipid A and
H 20 2 and enables the bacteria to survive. Listeria alter neutrophil functions as follows:
monocytogens produces another enzyme superoxide • it decreases chemotactic activity.
dismutase which neutralises the oxygen radicals • random migration of neutrophils is decreased.
leading to survival of the bacteria. • alters the metabolic and bactericidal properties
(b) Prevention of fusion and degranulation- Cell walls of of neutrophils.
some organisms modify the phagosomal membrane The endotoxins are not destroyed by autoclaving,
in such a manner that fusion of lysosome with hence infusion of a sterile solution containing
phagosome is prevented e.g. Chlamydia. endotoxin can cause serious illness. They cannot
B. pertussis produces adenylate cyclase which is be toxoided. They are poor antigens and the
inhibitor of degranulation and thus enhances toxicity is not completely neutralised by the
intracellular survival. homologous antibodies. Massive Gram negative
(c) Resistance to lysosomal enzymes-This may be due septicaemia may cause the syndrome of endotoxic
to the presence of capsular polysaccharide (M. shock characterised by fever, leucopenia,
lepraemurium) and mycoside (M. tuberculosis). thrombocytopenia, profound fall of blood pressure
(d) Escape from phagosomes-Some microbes escape and circulatory collapse leading to death.
from the phagosome into the cytoplasm of the host (b) Exotoxins- They are heat labile proteins which
cell before the fusion of phagosome with the lysosome are secreted by certain species of bacteria. They
e.g. Rickettsiae are believed to escape by an effect of diffuse readily into the surrounding medium. They
phospholipase A on the phagosomal membrane. are highly potent even in minute amounts and
constitute some of the most poisonous substances
(v) Bacterial toxins known. Botulinum toxin is the most poisonous, it
Bacteria produce two types of toxins, endotoxins and has been estimated that 3.0 kg of Botulinum toxin
exotoxins (Table 10.1 ). can kill all the inhabitants of the world. Exotoxins
~1JM betw/xi.ns and Endotoxins

/ +r
Tab~
I.
~ xotoxms
Protein (polypeptides) M .W. 10,000 to 900,000.
Endo toxins
Lipopolysaccharide in nature.
2. Heat labile (>60°C). Heat stable.
3. Actively secreted by living cells into medium. Form integral part of the cell wall; released only on
disruption of bacterial cell.
4. Highly antigenic; stimulates formation of antitoxin which Weakly antigenic; antitoxin is not formed but antibodies
neutralises toxin. against polysaccharide are raised.
5. Converted into toxoid by formaldehyde. Cannot be toxoided.
6. Enzymic in action. No enzymic action.
7. Specific pharmacological effect for each exotoxin. Non-specific action of all endotoxins.
8. Very high potency. Low potency.
9. Highly specific for particular tissue e.g. tetanus toxin for CNS. on-specific in action.
10. Don't produce fever in host. Usually produce fever.
11. Produced mainly by Gram-positive bacteria and also by some Produced by Gram-negative bacteria.
Gram-negative bacteria.

can be converted into toxoids by treatment with (ix) Infecting dose

formaldehyde. Toxoids lack toxicity but retain Adequate number of bacteria is required for successful
antigenicity and thus induce protective immunity infection. The dose of infection depends on virulence of
when used as vaccines. bacteria. The dosage may be estimated as the minimum
Exotoxins are highly antigenic and stimulate infecting dose (MID) or minimum lethal dose (MLD).
formation of antitoxin which neutralises toxin. They are Minimum number of bacteria required to produce clinical
highly specific for a particular tissue e.g. tetanus toxin for evidence of infection in a susceptible animal under
CNS. They have specific pharmacological activities and standard conditions is called minimum infective dose
do not produce fever in host. They are mainly produced (MID). MLD is a minimum number of bacteria that
by Gram positive bacteria but may also be produced by produce death in the animal under standard conditions.
certain Gram negative bacteria such as Vibrio cholerae These doses are more correctly estimated as ID 50 and
and enterotoxigenic Esch. coli. LD 50 as the dose required to infect or kill 50 per cent
of the animals tested under standard conditions.
(vi) Enzymes
Some bacteria produce enzymes which directly damage (x) Route of infection
host tissue. Certain bacteria are infective when introduced through
(a) Proteases- These enzymes cleave immuno-globulin optimal route, for example, cholera vibrios can produce
IgA which protects at mucosa! surfaces. lesion only when administered by oral route, but unable to
(b) Kinase- It enhances the spread of bacteria by cause infection when introduced subcutaneously. However,
dissolving fibrin clots e.g. streptococcus and Staphylococcus aureus can cause lesion whatever may be
staphylococcus. the portal of entry.
(c) Hyaluronidase- It breaks down hyaluronic acid and
helps in spread of infection e.g. streptococcus. (xi) Communicability
(d) Coagulase- It causes deposition of fibrin around The ability of a microbe to spread from one host to another
the bacteria and thus prevents phagocytosis e.g. is known as communicability. This property determines
staphylococcus. the survival and distribution of a microbe in a community
(e) Collagenase- It breaks down collagen in connective and not necessarily produce a disease in an individual
tissue and contributes in spread of infection e.g. host. A correlation need not exist between virulence
Clostridium perfringens. and communicability. Infections in which the pathogen
is shed in secretions such as in respiratory or intestinal
(vii) Siderophores and iron acquisition diseases, are highly communicable. In other instances, as in
Bacteria require iron for their metabolism. Many hydrophobia, human infection represents a dead end. Even
bacteria produce low molecular weight compounds called a highly virulent microbe may not exhibit a high degree of
siderophores that can acquire iron from the host's iron communicability due to its rapidly lethal effect on host.
binding proteins. This property enhances the virulence. Occurrence of epidemic and pandemic diseases
requires that the pathogen should possess high degree
(viii) Genetic factors of virulence and communicability.
Plasmids are extrachromosomal DNA segments that carry
genes for antibiotic resistance known as R-factors. These
factors are readily transferable by conjugation to the
various bacteria and confer resistance to many antibiotics. III. TYPES OF INFECTIOUS DISEASES
This also indirectly contributes in survival of bacteria Infectious diseases may be localised or generalised.
due to escape from action of antibiotics.
Plasmids may also code for enterotoxin production, A. Localised
colonisation factors and siderophore synthesis and thus There is an acute inflammatory reaction at the site of
enhances the virulence of the organism. All the strains invasion to arrest infection. When this process fails, the
of C. diphtheriae produce exotoxin only when they are organisms spread via the lymph channels (lymphangitis)
lysogenised with a bacteriophage called betaphage. The to regional lymph nodes (lymphadenitis) and then to
elimination of this phage abolishes the toxigenicity of blood stream (bacteraemia) which leads to dissemination
the bacillus. of organisms.

Ch 10: Microbial Pathogenicity _ _ _ _ __ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ 83
B. Generalised community, they may be classified as endemic, epidemic,

1. Bacteraemia: It is the circulation of bacteria in blood. and pandemic.
Transient bacteraemia is frequent even in healthy
persons and may occur during chewing, brushing Endemic
of teeth and straining at stools. The bacteria are The disease which is constantly present in a particular
immediately killed by phagocytes and are unable to area, e.g. enteric fever.
initiate infection. Bacteriaemia of a longer duration
is seen during generalised infections as in typhoid Epidemic
fever. The disease that spreads rapidly, involving many persons
2. Septicaemia: It is the condition where bacteria circulate in a particular area at the same time, is called epidemic
and multiply in the blood, form toxic products and disease e.g. meningococcal meningitis.
cause high fever.
3. Pyaemia: It is condition where pyogenic bacteria Pandemic
produce septicaemia with multiple abscesses in It is an epidemic that spreads through many areas of the
internal organs e.g. liver, spleen, kidney. world involving very large number of persons within a short
Depending on the spread of infectious diseases in period e.g. cholera, influenza and enteroviral conjunctivitis.
KEY POINTS
1. The host-parasite relationship is determined by the interaction between host factors and the infecting microorganism.
Health or disease depends on the equilibrium between the two.
2. A parasite is a microorganism which lives on a living host and derives nutrition from the host, without any benefit
to the host.
3. Commensals live in complete harmony with the host without causing any harm to it.
4. Cross infection acquired in hospitals is called nosocomial infection.
5. Bacterial toxins contribute in virulence of bacteria.
6. Endotox ins are lipopolysaccharide (LPS) in nature while exotoxins are heat labile proteins.
7. Exotoxins can be converted into toxoids e.g. tetanus toxoid.
YOU MUST KNOW

1. Definitions of important terms such as saprophytes, parasite, commensals, pathogen, pathogenicity, virulence, infection.
2. Types of infection and their definitions.
3. Types of carriers and their definitions.
4. Modes of transmission and their examples.
5. Differences between exotoxins and endotoxins.
STUDY QUESTIONS
1. Describe the factors predisposing to microbial pathogenicity.
2. Tabulate the differences between exotoxins and endotoxins.
MULTIPLE CHOICE QUFS lONS (MCQs)

1. Which of the following may cause teratogenic infections?
(a) Toxoplasma (b) Cytomegalovirus (c) Rubella virus (d) All of the above

84 - - - - - -- - - - - - - - - - - -- -- -- -- - -- - Unit I: General Microbiology
2. Which toxin can be converted into toxoid by formaldehyde?

(a) Exotoxin (b) Endotoxin (c) Both of the above (c) None of the above
3. Chemical nature of endotoxin is:
(a) Lipopolysaccharide (b) Protein (c) Carbohydrate (d) None of the above
4. Chemical nature of exotoxin is:
(a) Lipopolysaccharide (b) Protein (c) Carbohydrate (d) None of the above
Answers (MCQs}:
1. (d) 2. (a) 3. (a) 4. (b)
- - - •++---

IMMUNOLOGY
11. Immunity .............................. ...................................................................... 87
12. Antigen ............. ...... ........................... .......................................................... 93
13. Antibodies-lmmunoglobulins ......... .. ........................................................ 97
14. Antigen-Antibody Reactions .... .... .... .... .......... ........ .................................. 105
15. Complement System ................................................................................ 121
16. Structure and Functions of Immune System ............................................ 127
17. Immune Response ................... ................................................................ 136
18. Immunodeficiency Diseases ..................................................................... 147
19. Hypersensitivity .... ..... ... ........ ......................................................... ............ 152
20. Autoimmunity .......................................... .... ............... ............................... 160
21. Transplantation and Tumour Immunity ...................................................... 163
22. lmmunohaematology ................................................................................ 168

Chapter 11
IMMUNITY
I. Innate Immunity
A. Types of Innate Immunity B. Factors Influencing Innate Immunity
C. Mechanisms of Innate Immunity
II. Acquired Immunity
A. Active Immunity B. Passive Immunity
Ill. Miscellaneous
A. Combined Immunisation B. Adoptive Immunity
C. Local Immunity D. Herd Immunity
The term 'immunity' is defined as resistance exhibited 2. Racial Immunity

by the host against any foreign antigen including Within one species, different races may exhibit differences
microorganisms. This resistance plays a major role in in susceptibility or resistance to infections. This is termed
prevention of infectious diseases. Immunity may be innate as racial immunity. Algerian sheep is highly resistant to
or acquired. anthrax which is a common disease of other races of
sheep. In human beings, it is reported that American
I. INNATE IMMUNITY negroes are more susceptible than the white race to
It is the resistance which individual possesses by birth. tuberculosis. Such differences in humans could be due to
It is by virtue of his genetic and constitutional make- socioeconomic status. However, in a few instances genetic
up. It does not depend on prior contact with foreign basis of racial immunity has been proved.
antigen. It may be nonspecific, when there is resistance Races with sickle cell anaemia prevalent in
to infections in general, or specific when resistance to a Mediterranean coast are immune to P falciparum
particular pathogen is concerned. infection. A genetic abnormality of erythrocytes
(sickling) confers immunity to P falciparum because
A. Types of Innate Immunity such red blood cells cannot be parasitized by these
1. Species Immunity parasites. Persons with hereditary deficiency of glucose-
It refers to the resistance to a pathogen, shown by all 6-phosphate dehydrogenase (G-6-PD) are less susceptible
members of a particular species e.g. B. anthracis infects to P falciparum.
human beings but not chickens. The mechanism of
such type of immunity is not clearly understood. The 3. Individual Immunity
physiological and biochemical differences between tissues Resistance to infection varies with different individuals
of different host species may be responsible for species of same race and species. This is known as individual
specific resistance. immunity. The genetic basis of individual immunity is
87
88 - - - - - - - - - - - - - - - - -- - - - - - - - - - - - - - - Unit II: Immunology
evident from the observation that homozygous twins normal resident flora may lead to invasion by extraneous
exhibit similar degrees of susceptibility or resistance to microbes and thus causing serious diseases. Clostridial
tuberculosis. In contrast, such correlation is not seen in enterocolitis following oral antibiotics is one such
heterozygous twins. example.
B. Factors Influencing Innate Immunity (ii) Respiratory tract

I.Age The inhaled particles are arrested in the nasal passages
The two extremes of life (foetus and old persons) carry on the moist mucous membrane surfaces. The mucous
higher susceptibility to various infections. In foetus, the secretions of respiratory tract act as a trapping mechanism
immune system is immature whereas in old age there is and hair like cilia propels the particles towards the pharynx
gradual waning of immune responses. The foetus in-utero where it is swallowed or coughed out. The cough reflex
is generally protected from maternal infections through acts as an important defence mechanism. Some particles
the placental barrier. However, some pathogens (rubella, which manage to reach alveoli are ingested by phagocytes
cytomegalovirus and Toxoplasma gondii) cross this barrier present there.
and lead to congenital malformations.
In some diseases such as chickenpox and poliomyelitis, (iii) Intestinal tract
the clinical illness is more severe in adults than in young The mouth possesses saliva which has an inhibitory
children. This may be due to more active immune response effect on many microorganisms. Some bacteria may be
which causes greater tissue damage. swallowed and are destroyed by acidic pH of gastric
juices. The normal bacterial flora of intestine further
2. Hormones exert a protective effect by preventing colonisation of
Certain hormonal disorders such as diabetes mellitus, pathogenic bacteria.
adrenal dysfunction and hypothyroidism enhance
susceptibility to infections. Staphylococcal sepsis is (iv)Conjunctiva
more common in diabetes which may be associated with Tears have a major role by flushing away bacteria and
increased level of carbohydrates in tissues. Corticosteroids other dust particles. In addition, lysozyme present in tears
depress the host resistance by its antiinflammatory, has a bactericidal action.
antiphagocytic effects and by inhibiting antibody
formation. The elevated steroid level during pregnancy (v) The Genitourinary tract
may be related to higher susceptibility of pregnant women The flushing action of urine eliminates bacteria from the
to many infections. urethra. The acidic pH of vaginal secretions in female,
due to the fermentation of glycogen by lactobacillus
3. Nutrition (normal flora) , renders vagina free of many pathogens.
Malnutrition predisposes to bacterial infections. Both In males, semen is believed to contain some antibacterial
humoral and cell mediated immune responses are reduced substances.
in malnutrition.
2. Antibacterial Substances
Besides specific antibody formation, there are number
C. Mechanisms of Innate Immunity of nonspecific antibacterial substances present in blood
l. Epithelial Surfaces and tissues. These substances are properdin, complement,
(i) Skin lysozyme, betalysin, basic polypeptides (leukins from
It not only acts as a mechanical barrier to microorganisms leucocytes, plakins from platelets) and interferons which
but also provides bactericidal secretions. The high possess antiviral activity. The complement system plays
concentration of salt in drying sweat, the sebaceous an important role in the destruction of pathogenic
secretions and long chain fatty acids contribute to microorganisms that invade the blood and tissues (see
bactericidal activity. The skin may be freed of bacteria Chapter 15).
deposited on it (transient flora) but the bacterial flora
normally present on skin (resident flora) is not easily 3. Cellular Factors
removed even by washing and application of disinfectants. Once the infective agent has crossed the barrier of
The resident bacterial flora of skin and mucous surfaces epithelial surfaces, the tissue factors come into play for
help to prevent colonisation by pathogens. Alteration of defence.

Ch 11 : Immunity_ _ __ _ _ _ __ _ _ _ _ _ __ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ 89
When an infective agent invades tissue, an exudative Fever also stimulates the production of interferon which
inflammatory reaction occurs by accumulation of helps in recovery from viral infections.
phagocytes at the site of infection and deposition of
fibrin which entangles the organisms, to act as a barrier 6. Acute phase proteins
to spread of infection . Phagocytic cells ingest these Following infection or injury, there is a sudden increase
organisms and destroy them. These phagocytic cells are in plasma concentrations of certain proteins, collectively
classified as called acute phase proteins. These include C reactive
(i) Microphages e.g. polymorphonuclear leucocytes protein (CRP), mannose binding proteins and many
(neu trophils) others. CRP and some other acute phase proteins
(ii) M acrophages e.g. Mononuclear phagocytic activate the alternative pathway of complement. They
cells are believed to prevent tissue injury and promote repair
Phagocytic action can be divided into four stages: of inflammatory lesions.
(i) Chemotaxis II. ACQUIRED IMMUNITY

Phagocytes reach the site of infection attracted by The resistance acquired by an individual during life is
chemotactic substances. known as acquired immunity. It is of two types, active
and passive.
(ii) Attachment
The infective agent gets attached to phagocytic A. Active Immunity
membrane. Active immunity is subdivided into two types : Natural
and Artificial.
(iii) Ingestion Natural- Through clinical or subclinical infection
Phagocyte engulfs the infective material into a vacuole Artificial- Induced by vaccination
(phagosome). The membrane of phagosome fuses with It is the resistance developed by an individual as a
a lysosome to form a phagolysosome. result of contact with an antigen. This contact may be in
the form of natural infection or by vaccination. It leads
(iv) Intracellular killing to stimulate the immune apparatus to form antibodies
Most bacteria are destroyed in the phagolysosome by and/or the production of immunologically active cells.
hydrolytic enzymes of lysosomes. However, some bacteria Active immunity develops after a latent period which is
(M. tuberculosis, M. leprae) resist this type of killing and required for immune system to act but once developed,
can multiply within phagolysosome and thus produce the active immunity is long lasting.
disease. Phagocytosis in such instances may actually help
to disseminate infection to other parts of the body. Mechanism
A class of lymphocytes called Natural Killer (NK) Active immune response stimulates both humoral and
cells play an important role in non-specific defence against cell mediated immunity usually in parallel.
viral infections and tumour.
(i) Humoral immunity
4. Inflammation It is antibody mediated immunity. It depends on the
Inflammation occurs as a result of tissue injury or synthesis of antibodies by plasma cells. These cells
irritation, initiated by the entry of pathogens or other produce specific circulating antibody which combines
irritants. It is an important non-specific defence mechanism. specifically with the antigens and modify their activity.
Inflammation leads to vascidilation, increased vascular This modified activity may be in the form of lysis of
permeability and cellular infiltration. Microorganisms are antigen molecules; their toxin may be neutralised, or in
phagocytosed and destroyed. Due to increased vascular the form of removal of antigen by phagocytosis.
permeability, there is an outpouring of plasma which
helps to dilute the toxic products present. A fibrin barrier (ii)Cell mediated immunity (CMn
is laid to wall off the site of infection. It depends on T-lymphocytes developed against certain
antigens. Antibody synthesis also occurs in response to
5. Fever these antigens, but their role is limited. The cell mediated
A rise in temperature following infection is a natural immunity by sensitised T-lymphocytes is important
defence mechanism. It destroys the infecting organisms. in resistance to chronic bacterial infections. In these

90 Unit II: Immunology
chronic infections, organisms can multiply and survive in (c) Bacterial products
phagolysosome i.e. intracellular parasitism (tuberculosis, Tetanus toxoid for tetanus
leprosy) and in viral infections (Herpes simplex). Diphtheria toxoid for diphtheria
Types B. Passive Immunity

(i) Natural active immunity Passive immunity is subdivided into two types : Natural
It is acquired by natural subclinical or clinical infections. and Artificial.
Such immunity is long lasting. Persons recovering from Natural- Through transplacental maternal IgG
smallpox infection develop natural active immunity. antibodies.
Artijzcial-Through antiserum injection.
(ii) Artificial active immunity Passive immunity is induced in an individual by
It ,is produced by vaccination. The vaccines are prepared preformed antibodies (generally in the form of antiserum)
from live, attenuated or killed microorganisms, or their against infective agent or toxin. This antiserum is prepared
antigens or toxoids. In killed vaccines the organisms are by injecting infective agent or toxin in another host. The
killed by heat, formalin, phenol and alcohol. These are immune system has no active role in passive immunity.
preserved in phenol, N-merthiolate and alcohol. Toxoids Protection starts immediately after transfer of immune
are prepared from bacterial exotoxins inactivated by serum. There is no latent period as present in active
formalin (formol toxoid) or by alum (alum precipitated immunity (Table 11. l ). Passive immunity is short lasting
toxoid- APT). Toxoids are immunogenic but not toxigenic. but is useful when immunity is required immediately.
VACCINES. Natural
(a) Live vaccines It is transferred from the mother to foetus or infant.
BCG for tuberculosis Transfer of maternal antibodies to foetus transplacentally
Ty 21a for typhoid and to infant through milk (colostrum) protects them till
Sabin vaccine for poliomyelitis their own immune system matures to function.
MMR vaccine for measles, mumps, rubella
17D vaccine for yellow fever Artificial
(b) Killed vaccines It is through parenteral administration of antibodies.
TAB for enteric fever The agents used for artificial passive immunity
Killed cholera vaccine are hyperimmune sera of animal or human origin,
Salk vaccine for poliomyelitis convalescent sera and pooled human gammaglobulin.
Neural and non-neural vaccines for rabies The oldest method is to employ horse hyperimmune
Hepatitis B vaccine sera. Antitetanus serum (ATS) is prepared by injecting a
Table 11.1 Differentiating Features of Active and Passive Immunity

Active Immunity Passive Immunity
1. Produced actively by the immune system. Received passively by the host. The host's immune system
does not participate.
2. Induced by infection or by contact with immunogens. Conferred by administration of ready-made antibodies.
3. Long-lasting and effective protection. Protection short lived and less effective.
4. Immunity is effective only after a lag period i.e. time required Immunity is effective immediately.
r , fo r generation of antibodies.
5. Immunological memory present, therefore, subsequent No immunological memory. Subsequent administration of
challenge is more effective (booster effect). antibody is less effective due to immune elimination.
6. egative phase may occur. This is due to antigen combining No negative phase.
with already existing antibody and lowering its level.
7. ot applicable in immunodeficient persons. Applicable in immunodeficient persons.
8. Used for prophylaxis to increase body resistance e.g. BCG Used for treatment of acute infections.
vaccine.

Ch 11: Immunity_ _ _ _ _ _ _ _ _ _ _---,-_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ 91
series of doses of tetanus toxoid to horses, and bleeding IJI. MISCELLANEOUS

them to separate the serum. As ATS is a foreign protein, A. Combined Immunisation
it is liable to cause hypersensitivity reactions. To eliminate A combination of active and passive immunisation is
these complications, human ATS is employed. This is employed simultaneously which is known as combined
prepared by hyperimmunisation of human volunteers immunisation. Passive immunity provides the protection
with tetanus toxoid. Protection with human ATS lasts necessary till the active immunity becomes effective.
longer as there is no immune elimination of the human
globulins. B. Adoptive Immunity
Convalescent sera (sera of patients recovering from Injection of immunologically competent lymphocytes
infectious diseases) contain high levels of specific antibody is known as adoptive immunity. Instead of whole
and therefore is employed for passive immunisation against lymphocytes, an extract of immunologically competent
measles and rubella. Sera of healthy adults can be pooled lymphocytes can be used. This is being attempted in
and is used for passive immunisation against common therapy of lepromatous leprosy. Such lymphocytes are
infectious diseases prevalent in the region. Pooled human collectively known as transfer factor.
gammaglobulin are used for passive immunisation against
some viral infections e.g. hepatitis A. It has to be ensured C. Local Immunity
that all preparations from human sera should be free Natural infection or the live viral vaccine administered
from the risk of infections with hepatitis B, hepatitis C, orally or intranasally provides local immunity at the
hepatitis D, HIV and other viruses. site of entry such as gut mucosa and nasal mucosa
respectively. A special class of immunoglobulins (IgA)
plays an important role in local immunity. One type of
. ''
Uses of Passive Immunisation IgA called secretory IgA is produced locally by plasma
(i) To provide immediate short term protection in a cells.
nonimmune host, faced with the threat of a serious
infection. D. Herd Immunity
(ii) For suppression of active immunity which may be It refers to the overall resistance in a community. When
injurious. Example is to use Rh immunoglobulins herd immunity is low, chances of epidemics increase
during delivery to prevent immune response to Rh on introduction of a suitable pathogen. Eradication of
factor in Rh negative mothers with Rh positive any communicable disease depends on development of
babies. a high level of herd immunity rather than of immunity
(iii) For treatment of serious infections. in individuals.
KEY POINTS
1. The Immunity is defined as resistance exhibited by the host against any foreign antigen including microorganisms.
2. Immunity may be innate or acquired.
3. Acquired immunity is of two types, active and passive.
4. Active immunity is subdivided into two types, natural and artificial.
5. Artificial active immunity may be produced by vaccination.
6. Passive immunity is also subdivided into two types, natural and artificial.
7. Artificial passive immunity is through parenteral administration of antibodies (antiserum).
8. Herd immunity refers to the overall resistance in the community.
YOU MUST KNOW

1. Innate immunity, acquired immunity, herd immunity and their role.
2. Differences between active and passive immunity.

92 - - - - -- - - - - - - - - - - - - -- - - - - - - - - -- - - - Unit II: Immunology
STUDY QUESTI ONS

1. Discuss the mechanisms of innate immunity.
2. Tabulate the differences between active and passive immunity.
(a) Innate immunity (b) Active immunity (c) Passive immunity (d) Herd immunity.

I. Clinical or subclinical infections lead to
(a) Active natural immunity (b) Passive natural immunity
(c) Active artificial immunity (d) Passive artificial immunity
2. Vaccination induces:
(a) Active natural immunity (b) Passive natural immunity
(c) Active artificial immunity (d) Passive artificial immunity
3. Artificial passive immunity may be induced by:
(a) Antiserum injection (b) Vaccination
(c) Maternal antibodies (d) All of the above
4. All of the following are live vaccines except:
(a) BCG (b) Sabin vaccine
(c) MMR (d) TAB vaccine
5. All of the following are killed vaccines except:
(a) Salk vaccine (b) Hepatitis B vaccine
(c) Non-neural vaccines for rabies (d) MMR
Answers (MCQs):
1. (a) 2. (c) 3. (a) 4. (d) 5. (d)
____ ... ____

Chapter 12
ANTIGEN
I. Types of Antigen
A. Complete Antigen B. Haptens
II. Factors of Antigenicity
An antigen is a substance which, when introduced into character of simple haptens, whereas complex haptens
a body evokes immune response to produce a specific are polyvalent. It is assumed that precipitation occurs
antibody with which it reacts in an observable manner. when antigen has two or more antibody combining
sites.
Proantigens are low molecular weight substances
I. TYPES OF ANTIGEN which do not induce antibody formation but can cause
They may be classified as delayed hyper-sensitivity reaction e.g. dinitrochloro-
A. Complete Antigen benzene (DNCB).
B. Haptens/Incomplete Antigen: Complex haptens; Simple Antigenic determinant (epitope) is the smallest unit
haptens. of antigenicity. Antigen possesses a number of these
determinants. Each type of determinant induces a specific
A. Complete Antigen antibody formation . The size of antigenic determinant is
These are substances which can induce antibody around 25-34A O and a molecular weight of about 400-
formation by themselves and can react specifically with 1000. The combining site on the antibody molecule,
these antibodies. corresponding to the epitope, is called the paratope.
B. Haptens
Haptens are substances unable to induce antibody II. FACTORS OF ANTIGENICITY
formation on its own but can become immunogenic The exact basis of antigenicity is not clear but a number
(capable of inducing antibodies) when covalently linked of factors have been implicated which make a substance
to proteins, called carrier proteins. However, haptens antigenic. These factors are:
can react specifically with induced antibodies. These
antibodies are produced not only against the hapten but 1. Foreignness
also against the carrier protein. 2. Size
1. Complex haptens can combine with specific antibodies 3. Chemical nature
to form precipitates e.g. capsular polysaccharide of 4. Susceptibility to tissue enzymes
pneumococc1. 5. Antigenic specificity
2. Simple haptens combine with specific antibodies but 6. Species specificity
no precipitate is produced. This is due to univalent 7. Isospecificity
93
94 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Unit II: Immu nology
8. Autospecificity 5. Antigenic Specificity

9. Organ specificity Antigenic specificity depends on antigenic determinants.
10. Heterophile specificity. The position of antigenic determinant group in the antigen
molecule is important for specificity. The differences in
specificity are determined in compounds with the group
1. Foreignness attached at ortho, meta or para positions. Another factor
An antigen must be foreign to the individual (non-self) is spatial configuration of the determinant group which
to induce an immune response. The immune system does makes differences in antigenic specificity of dextro, laevo
not normally mount an immune response against his own and mesa isomers of substances.
antigens (self). This tolerance to self antigens is due to Antigenic specificity is not absolute. Cross reactions
continuous contact with them during the developmental may occur between antigens which bear stereochemical
stages of immune system. However, breakdown of this similarities.
mechanism results in autoimmune disease.
The antigenicity of a substance is related to degree 6. Species Specificity
of its nonself nature. The more foreign a substance, it Tissues of all individuals in a species possess species
is likely to be more powerful antigen. Antigens from specific antigens. Some degree of cross reaction exists
other individuals of the same species are less antigenic between antigens from related species. The species
as compared to those from other species. specificity has got phylogenetic relationship and has been
of assistance in (i) tracing of evolutionary relationship
2. Size (ii) forensic applications in the identification of species
Larger molecules are highly antigenic whereas low of blood and of seminal fluid.
molecular weight (less than 10,000) are either nonantigenic
or weakly antigenic. Substance with low molecular weight 7. Isospecificity
may be rendered antigenic by adsorbing these on inert Isospecificity depends on isoantigens which may be found
particles like bentonite or kaolin. in some but not all members of a species. A species may be
grouped according to the presence of different isoantigens
3. Chemical Nature in its members. These are genetically determined. Blood
Most naturally occurring antigens are either proteins grouping is one example depending on human erythrocyte
or polysaccharides. Proteins are more effective antigens. antigens (isoantigens). Blood groups are clinically very
An exception is gelatin which is non-antigenic protein important in blood transfusion, in isoimmunisation
due to its low tyrosine (aromatic radical) content. The during pregnancy and to provide valuable evidence in
presence of aromatic radical is suggested as essential for disputed paternity cases.
antigenicity. Lipids and nucleic acids are less antigenic on Histocompatibility antigens are associated with the
their own but do so when combined with proteins. plasma membrane of tissue cells. These are specific for
each individual of a species. These are very important
when some tissue is transplanted from one individual
4. Susceptibility to Tissue Enzymes to another. Human leucocyte antigen (HLA) is the
Substances which can be metabolised and are susceptible major histocompatibility antigen determining homograft
to the action of tissue enzymes behave as antigens. rejection.
The antigen introduced into the body is degraded by
the enzymes of the phagocytic cells into fragments of 8. Autospecificity
appropriate size containing antigenic determinants. The Self antigens are generally nonantigenic, but there
hidden antigenic determinants can be identified only when are exceptions. These exceptions are lens protein and
molecule is broken down . Bovine serum albumin (BSA) sperm. These are not recognised as self antigens. Lens
has more than 18 determinants although only six of these protein normally confined within its capsule doesn't
are exposed in the intact (unbroken) molecule. come in contact with immune system, therefore, it is not
Substances unsusceptible to tissue enzymes such as recognised. Similarly, antigens (sperm) that are absent
polystyrene latex are not antigenic. Substances which during the embryonic life and develop later, are also
cannot be metabolised in body are not antigenic. not recognised by immune system. When these antigens
Synthetic polypeptides composed of D-aminoacids is are released into tissues following injury, antibodies are
one example. produced against them. Mechanism in pathogenesis

Ch 12: Antigen _ _ _ __ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ 95
of autoimmune diseases is also similar. The antigenic (ii) Weil-Felix Reaction

specificity of self antigens may be modified as a result Serum of patient suffering from typhus fever (containing
of infection or irradiation and may thus act as nonself. heterophile antibody) agglutinates strains of Proteus
The immune response against these modified antigens species (OX 19, OX 2 and OX K) . This is due to presence
may lead to autoimmune disease. of a hetrophile antigen on Rickettsiae causing typhus
fever, shared by these proteus strains.
9. Organ Specificity
Organ specific antigens are confined to a particular organ.
Some organs like brain, kidney and lens protein of one (iii) Paul-Bunnell Test
species share specificity with that of another species. As In infectious mononucleosis, heterophile antibodies appear
brain specific antigens are shared by man and sheep, in the serum of the patient. These antibodies agglutinate
antirabies vaccines (sheep brain vaccines) may cause sheep erythrocytes because Epstein Barr virus (causative
neuroparalytic complications in man. Antirabies vaccine agent of infectious mononucleosis) shares antigen with
(developed in sheep brain) contains large amount of sheep erythrocytes.
partially denatured brain tissue. In man, the sheep brain
tissue induces immune response causing damage to the
recipient's nervous tissue. SUPERANTIG
Superantigens are molecules that can interact with
10. Heterophile Specificity antigen-presenting cells (APCs) and T-lymphocytes in
The same or closely related antigens present in different a non-specific manner. These antigens do not involve
tissues of more than one species are known as heterophile the endocytic processing as required in typical antigen
antigens. Antibodies to these antigens produced by presentation. Interaction of superantigens with MHC
one species cross react with antigens of other species. class II molecules of the APC and the V~ domain of
Examples include the following: the T-lymphocytes receptor activates a large number of
T-lymphocytes than conventional antigens. This leads to
(i) Forssman Antigen massive cytokine expression and immuno-modulation.
It is a lipoprotein-polysaccharide complex widely present Staphylococcal enterotoxins, exfoliative toxin and some
in man, animals, birds and bacteria. viral proteins are examples of superantigens.
KEY POINTS
1. An antigen is a substance which, when introduced into a body evokes immune response to produce a specific antibody
with which it reacts in an observable manner.
2. Antigen may be classified as complete antigen and hapten/incomplete antigen.
3. Heterophile antigens are closely related antigens present in different tissues of more than one species. Antibodies to
these antigens produced by one species cross react with antigens of other species.
4. Weil-Felix reaction and Paul-Bunnell test are based on the presence of heterophile antibodies.
YOU MUST KNOW

1. Types of antigen.
2. Forssmam antigen.
3. Superantigens.
STUDY QUESTIONS
1. What is an antigen? Discuss briefly about various factors of antigenicity.
2. Write short notes on: (a) Haptens (b) Heterophile antigens.

96 - - - - - - - - - - -- - - - - - - - -- -- - - - - - - - - - - Unit II: Immunology

1. Weil-Felix reaction is a serological test based on:
(a) Heterophile antigen (b) Sheep brain antigen
(c) Specific antigen (d) None of the above
2. Paul-Bunnell test is based on sharing of antigens between:
(a) Sheep erythrocytes and Epstein-Barr virus (b) Mycoplasrna and human erythrocytes
(c) Rickettsial antigens and Proteus antigens (d) None of the above
Answers (MCQs):
1. (a) 2. (a)
- - - •++---

Chapter 13
ANTIBODIES-IMMUNOGLOBULINS
I. Antibodies
A. Properties of Antibodies B. Structure of lmmunoglobulin
C. lmmunoglobulin Antigen Determinants D. lmmunoglobulin Classes
II. Abnormal lmmunoglobulins
A. Multiple Myeloma B. Heavy Chain Disease
C. Cryoglobulinaemia
I. ANTIBODIES (lg) for antibody has been accepted internationally.

Antibodies are substances which are formed in the serum Immunoglobulins are mainly synthesised by plasma cells.
and tissue fluids in response to an antigen and react Immunoglobulin includes, besides antibody globulins, the
with that antigen specifically and in some observable abnormal myeloma proteins, the cryoglobulin and the
manner. macroglobulin. Thus, all immunoglobulins may not be
antibodies but all antibodies are immunoglobulins.
A. Properties of Antibodies
1. Chemical nature of antibodies is globulin and they are B. Structure of Immunoglobulin
named as immunoglobulins. Serum globulins could Porter, Edelman and Nisonoff (1959-64) developed a
be separated into pseudoglobulins (water soluble) and technique for cleavage of immunoglobulin molecule which
euglobulins (water insoluble). Most antibodies are has led to detailed structure of immunoglobulin.
found to be euglobulins. Immunoglobulins constitute An antibody molecule consists of two identical heavy
about 20 to 25 per cent of the total serum proteins. and two identical light chains. The 'heavy' (H) chains are
2. Based on sedimentation studies, most antibodies are longer and 'light' (L) chains are shorter. Both types of
sedimented at 7S (M.W 150,000- 180,000). Some chains are polypeptide in nature. The two heavy chains
heavier antibodies- 19S globulins (M.W about are held together by disulphide (S-S) bonds. Each light
900,000) were designated as M or macroglobulins. chain is also attached to heavy chain by disulphide bond
3. Tiselius and Kabat (1938) showed that most serum (Fig. 13.1).
antibodies on electrophoretic mobility, belong to The H chains are structurally and antigenically
gammaglobulins which is used now synonymously distinct in different classes of immunoglobulins. Five
with antibody. Later, many antibodies were found different classes namely IgG, IgM, IgA, IgD, and IgE
to migrate as beta or alpha globulins. are designated depending on presence of heavy chain,
The term 'Immunoglobulin' was proposed by expert gamma (y), mu (µ) , alpha (a), delta (o) and epsilon (E)
committee of WHO in 1964. The name immunoglobulin respectively.
97
y
98 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Unit II: Immunolog
Variable region Constant region

~----• +--------- ---------- --------- •
,jl
I
t
\
/ \
sI H-chain /
/ ''
,,/ \
s ..... ,," ',
sI COOH
Fd-=:l r=
(Carboxyterminus)
s region 1
! + , ,, , ,,
'''
NH 2
(Aminoterminus) ====1
Fab
Fe
=1 F(ab')2
Fragments of Fe
Fig. 13.l Structure of immunoglobulin Papain digestion Pepsin digestion
Fig. 13.2 Effect of enzymes on immunoglobulin

Class of Immunoglobulin Heavy Chain
IgG gamma (G) (ii) Pepsin Digestion (Fig. 13.2)
IgM mu (M) Pepsin cleaves at a different point of immunoglobulin
IgA alpha (A) molecule and gives rise to Fe portion and two Fab
fragments held together in position. This Fab fragment
IgD delta (D)
is bivalent and can still precipitate with antigen. It is
IgE epsilon (E) called F(ab') 2 • Pepsin also degrades the Fe portion into
smaller fragments.
The L chains are similar in all classes of
immunoglobulins. They are present in two forms kappa
(K) and lambda (L). Each immunoglobulin has either two 2. Aminoacid Sequences
kappa or two lambda light chains but both (K & L) are Each light chain contains 210-230 aminoacids whereas
never found together in a molecule. The light chains are heavy chain has 420-460 aminoacids. Molecular weight of
named kappa and lambda after the investigators Komgold light chain is 25,000 and that of heavy chain is 50,000.
and Lapari. Kappa and Lambda chains are present in a Both L and H chains consist of two portions each,
ratio of 2: 1 in human sera. a variable (V) region and a constant (C) region. In the
L chain the two regions are of equal length while in the
1. Effect of Enzymes H chains the variable region constitutes approximately
(i) Papain Digestion (Fig. 13.2) only a fifth of the chain. Variable (V) regions are present
Porter and colleagues split rabbit lgG antibody to egg at aminotermi nus (NH 2) and constant (C) region at
albumin, by a proteolytic enzyme papain in the presence of carboxyterminus (COOH). Antigen combining site is
cysteine. Papain can digest immunoglobulin molecule into at its aminotermi nus which consists of both H and L
three fragments one Fe fragment (Fe crystallisable) and chains (Fig. 13.1 ). The aminoacid sequence in the amino
two identical Fab (fragment antigen binding) fragments. terminal half of the chain is highly variable, variability
Two Fab fragments possess the antigen binding sites but determines the immunological specificity of the antibody
the Fe fragment lacks the ability to bind antigen. As the molecule. The infinite range of the antibody specificity of
Fe fragment can be crystallized, it was so named. immunoglobulins is due to the 'variable regions' of the
Each Fab fragment consists of a light chain and a H and L chains. There are many hypervariable regions
part of H chain. The portion of the heavy chain in Fab present in variable portions of both H and L chains
fragment is named Fd region. Fe fragment consists of parts and are involved with the formation of the antigen
of both H chains. It is composed of the carboxyterm inal binding sites. The sites on the hypervariable regions
portion of the H chains. It determines the biological which make actual contact with the epitope are called
properties of the immunoglobulin molecule such as 'complementarity determining regions' or CDRs. There
complemen t fixation , placental transfer, skin fixation are three hypervariable regions in the L and four in the
and catabolic rate. H chains.

Ch 13: Antibodies - lmmunoglobulins _ _ _ _ _ __ _ _ _ _ _ _ _ _ _ _ _ _ __ _ __ _ _ 99
3. Immunoglobulin Domains (Fig. 13.3) different classes of immunoglobulins are differentiated .

Immunoglobulins are folded to form globular variable Various H chain markers are gamma, mu, alpha, delta
and constant domains. There are four domains in each and epsilon in immunoglobulins IgG, IgM, IgA, IgD and
heavy chain, one in variable region (VH) and three in lgE respectively. The light chains are also distinguished
constant region (CHI , CH2, CH3). There is one additional through isotypic markers into kappa and lambda.
fourth domain on heavy chain (CH4) in lgM and lgE
molecule. Light chain has one domain in variable region 2. Allotypes
(VL) and one in constant region (CL) in all classes of These are individual specific determinants within a species.
immunoglobulin (lgG, IgA, IgD, IgM and IgE). Each Allotype markers are also present on the constant regions
domain has a separate function. of heavy and light chains. These markers are genetically
The variable region domains (VL and VH) are determined. The allotype markers in humans are Gm on
responsible for the formation of a specific antigen gamma heavy chains, Am on alpha heavy chains, and
binding site. The constant region domains mediate the Km (originally designated as Inv) on kappa light chains.
secondary biological functions. The area between CH 1 More than 25 Gm types, 3 Km allotypes and 2 Am on
and CH2 domain is hinge region where enzyme papain lgA have been identified. No allotypic markers have been
acts. The CH2 region in IgG binds Clq in the classical found for lambda light chains or µ, 8 or£ heavy chains.
complement pathway, and the CH3 mediates adherence Allotype markers are useful in testing paternity.
to the monocyte surface.
3. Idiotypes
Antigen binding Idiotype markers are located in hypervariable regions
of the immunoglobulin molecule. Idiotypes are specific
Monocyte for each antibody molecule. By immunisation with Fab
adherence fragments, antiidiotypic antibodies can be produced.
t D. Immunoglobulin Classes
Characteristics of five immunoglobulins present in human
sera are shown in Table 13.1.
1. Immunoglobulin G (IgG) (Fig. 13.4)

(i) lgG is the major serum immunoglobulin
CH3
(about 80% of the total amount). The normal
serum concentration is about 8- 16 mg/ml.
Heavy-
chain (ii) Molecular weight is 150,000 (7S).
(iii) Half life is about 23 days (longest amongst all
Light chain -
the immunoglobulins).
------------ ---+ (iv) It is the only immunoglobulin that is transported
Fig. 13.3 lmmunoglobulin domains (lgG molecule)
through placenta and provides natural passive
immunity to newborn.
C. Immunoglobulin Antigen Determinants
Variable region Constant region
Immunoglobulins are glycoproteins and can act as •--- - --••- ----------------------- -•
immunogens when inoculated into a foreign species.
L- chain- L I_ _ __,,li= ===..,tl
Differences in aminoacid sequences between different sI
immunoglobulin classes, subclasses and types determine s
their antigenic specificity. There are three major types of
immunoglobulin antigen determinants. H-chains / is
1. lsotypes
2. Allotypes " Ll l • a!!! !I _
sI
3. Idiotypes s
1. Isotypes Hypervariable regions

These determinants are shared by all members of the same
Fig. 13.4 IgG molecule
species. On the basis of isotypic markers on H chains,
Table 13.1 Properties of Immunoglobulin Classes

Property IgG lgA IgM l gD lgE
Molecular weight 150,000 160,000 900,000 180,000 190,000
Sedimentation coefficient(S) 7 7 19 7 8
Heavy chain Gamma Alpha Mu Delta Epsilon
Light chain Kor L Kor L Kor L Kor L Kor L
Serum concentration (mg/ml) 12 2 1.2 0.03 0.00004
Placental transport +
Half life 23 days 6-8 days 5 days 3 days 2-3 days
Intravascular distribution(%) 45 42 80 75 50
Present in milk + +
Complement fixation
Classical ++ +++
Alternative +
Selective secretion by seromucous glands +
Heat stability (56°C) + + + +
(v) It is distributed equally between the intravascular (xiii) It is protective against those microorganisms which
and extravascular compartments. are active in the blood and tissues.
( vi) IgG appears late but persists for longer period. It
appears after the initial immune response which 2. Immunoglobulin A (IgA) (Fig. 13.5)
is IgM in nature. (z) IgA is the second major serum immunoglobulin
( vii) It participates in precipitation, complement fixation (about 10-13% of serum immunoglobulins). The
and neutralisation of toxin and viruses. normal serum concentration is 0.6 - 4.2 mg/ml.
(viii) IgG binds to microorganisms and enhances the (ii) Half life is about 6 - 8 days.
process of phagocytosis. (iii) IgA occurs in two forms, serum IgA and secretory
(ix) Extracellular killing of target cells coated with IgA.
IgG is due to recognition of Fe receptor of IgG (iv) Serum IgA is a monomeric 7S molecule (MW
by killer cells bearing the appropriate receptors. 160,000), while IgA found on mucosa! surfaces and
(x) The catabolism of IgG is unique in that it in secretions (secretory IgA, MW 400,000) is a dimer
depends on the serum IgG concentration. When formed by two monomer units joined together by
level of IgG is raised, as in myeloma or kala- a glycoprotein named the J chain (J for joining).
azar, the synthesis of IgG against that particular The J chain joins the two monomer units at their
antigen is catabolised rapidly and may result in carboxyterminals. Secretory IgA and J chain are
deficiency of that particular antibody. Conversely, produced by the plasma cells situated near the mucosa!
in hypogammaglobulinaemia, IgG antibody given or glandular epithelium. J chains are also present
for therapeutic purpose will be catabolised slowly. in other polymeric immunoglobulins such as IgM.
(xt) Passively administered IgG suppresses the (v) Secretory IgA contains another glycine rich
homologous antibody synthesis by feed back polypeptide called the secretory piece or secretory
mechanism. Based on this property, isoimmuni-
sation of women is done by administration of
anti-RhD IgG during delivery.
(xii) Four subclasses of IgG (IgGl, IgG2, IgG3, IgG4) L-chain---
have been recognised. These can be identified

with specific antisera. Differences exist in the Fe
H-chain_._~~..,....."""'"---=-=-1.
fragment of the heavy chains of various subclasses.
In human serum, these subclasses are distributed
I~
Disulphide bond
J-chain
as IgG 1 (65%), IgG2 (23%), IgG3 (8%) and
Fig. 13.5 Secretory IgA molecule
IgG4 (4%).

Ch 13: Antibodies - lmmunoglobulins _ _ _ _ _ __ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __ 101
component. The S piece is not synthesised by (vi) IgM is mainly distributed intravascularly (80%).
lymphoid cells but by mucosal or glandular epithelial (vii) It is the earliest synthesised immunoglobulin by
cells. It attaches two IgA molecules at their Fe foetus in about 20 weeks of age.
portion during transport across the cells. The S ( viii) It appears early in response to infection before
piece is believed to protect IgA from denaturation IgG. IgM antibodies are short lived, and disappear
by bacterial proteases in sites such as the intestinal earlier than IgG. Hence, its presence in serum
mucosa which is rich in bacterial flora. indicates recent infection.
(vi) IgA is the principal immunoglobulin present in (ix) It can not cross the placenta, presence of IgM
secretions such as milk, saliva, tears, sweat, nasal antibody in serum of newborn indicates congenital
fluids, colostrum and in secretions of respiratory, infection. Its detection is, therefore, useful for the
intestinal and genital systems. It protects the diagnosis of congenital syphilis, HIV infection,
mucous membranes against microorganisms. It toxoplasmosis and rubella.
forms antibody paste on mucosa. IgA covers (x) The valency of IgM is 10 which is observed
the microorganisms to inhibit their adherence to on interactions with small haptens only. With
mucosal surfaces. larger antigens, the effective valency is 5.
( vii) IgA does not fix complement but can activate (x i) It is very effective antibody in agglutination,
alternative complement pathway. complement fixation , opsonisation and immune
(viii) IgA is mainly synthesised locally by plasma cells haemolysis. It is more efficient than IgG in these
and little is derived from serum. reactions. The greater efficacy is due to the
(ix) Two subclasses of IgA (IgAl and IgA2) are known. binding of complement to several Fe regions of
IgA2 is predominant (60%) in the secretions but pentameric IgM simultaneously, thus initiating
constitutes a minor part of serum IgA. IgA2 is complement cascade and target cell lysis with a
devoid of interchain disulphide bonds between H single molecule.
and L chains. (x ii) IgM provides protection against blood invasion by
microorganisms. Its deficiency is often associated
3. Immunoglobulin M (IgM) (Fig. 13.6) with septicaemia.
(i) IgM is a pentamer consisting of 5 immunoglobulin (x iii) The isohaemagglutinins (Anti-A, Anti-B),
subunits and one molecule of J chain, which joins antibodies to typhoid 'O' antigen (endotoxin),
the Fe region of the basic subunits. rheumatoid factor and WR. antibodies in syphilis
(ii) Each H chain of IgM has four CH domains rather belong to IgM.
than three as seen in H chain of IgG molecule. (xiv) IgM (monomeric) appears on the surface of
(iii) It constitutes about 5- 8 percent of total serum unstimulated B lymphocytes and acts as recognition
immunoglobulins. The normal level in serum is receptors for antigens.
0.5- 2 mg/ml. (xv) Treatment of serum with 2-mercaptoethanol
(iv) Half life is about five days. selectively destroys IgM without affecting IgG
(v) It is heavy molecule (19S) with a molecular antibodies. This method is utilised for the differential
weight 900,000 to 1,000,000 hence also called the estimation of IgG and IgM antibodies.
'millionaire molecule' . (xvi) Two subclasses (IgMl and IgM2) of IgM are
described. These are differentiated by characteristic
H chains i.e. µ 1 and µ2 H chains.
4. Immunoglobulin D (IgD)
(i) IgD resembles IgG structurally.
(it) IgD is present in a concentration of 3 mg per
-L-chain 100 ml in serum. It is mostly intravascular in
distribution.
(iii) Molecular weight is 1,80,000 (7 S monomer) .
(iv) Half life is about three days.
(v) Like IgM, it is also present on the surface of
unstimulated B lymphocytes in blood and acts as
Fig. 13.6 1gM mo1ecu1e
recognition receptors for antigens. Combination of
cell membrane bound IgD with the corresponding
102 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Unit II: Immunology
antigen leads to specific stimulation of these II. ABNORMAL IMMUNOGL OBULINS

B lymphocytes- either activation and cloning to Apart from antibodies, other structurally similar proteins
form antibody, or suppression. may be found in serum in following pathological
(vi) Two subclasses (IgDl and IgD2) of IgD are conditions.
known. A. Multiple myeloma
B. Heavy chain disease
C. Cryoglobulina emia
5. Immunoglobu lin E (IgE)
(i) IgE is mainly produced in the linings of respiratory A. Multiple Myeloma
and intestinal tracts. Serum contains only traces It is a plasma cell dyscrasia in which unchecked proliferation
(a few nanograms per ml). It is mostly distributed of one clone of plasma cells occur, resulting in the excessive
extravascularly. production of the particular immunoglobulin synthesised
(ii) It is also referred to as reagins. by the clone. Such immunoglobulins synthesised from one
(iii) Molecular weight is 1,90,000 (8S molecule). clone of cells are called monoclonal. Multiple myeloma
(iv) Half life is 2-3 days. may involve plasma cells synthesising any of the five
(v) It resembles IgG in structure. classes of immunoglobulins, IgG, IgA, IgD, IgM or IgE.
(vi) It is heat labile (inactivated at 56°C in one hour) About 50- 60% cases are of IgG type. Myeloma involving
whereas other immunoglobulins are heat stable. IgM producing plasma cells is named as Waldenstrom's
( vii) It has affinity for surface of tissue cells, macroglobulinaemia.
particularly mast cells of the same species Apart from excessive production of respective myeloma
(homocytotrop ism). proteins (M proteins), light chains of immunoglobulins
(viil) IgE mediates type I hypersensitivity (atopic) reaction. may occur as kappa or lambda chains in the urine of
This is responsible for asthma, hay fever, eczema 50% of myeloma patients. In any one particular patient,
and Prausnitz-Kus tner (PK) reaction. light chain is either kappa or lambda only and never both.
(ix) High level of IgE in serum is also seen in children These light chains are called Bence-Jones (BJ) proteins,
with a high load of intestinal parasitism. described by Bence-Jones in 1847. BJ proteins in urine
(x) It cannot cross the placental barrier or fix the have a peculiar property of coagulating when heated to
complement. 60°C but redissolves at 80°C.
(xi) IgE is responsible for anaphylactic type of
reaction. B. Heavy Chain Disease
(xii) It may play some unidentified role in defence against Abnormal heavy chains are produced in excess. This is
intestinal parasitic infections. due to lymphoid neoplasia.
C. Cryoglobulin aemia
Role of Different lmn u•;sh t ulm (. [.1sses It is a condition in which there is a formation of precipitate
IgG - protects the body fluids on cooling the serum, the precipitate redissolves on
IgA - protects the body surfaces warming. This is due to presence of cryoglobulins in blood.
IgM - protects the blood stream Cryoglobulinaemia is often found in macroglobulinaemia,
IgE - mediates reaginic hypersensitivity systemic lupus erythematosus (SLE) and myelomas. Most
IgD - recognition molecule on the surface of B cryoglobulins consist of either IgG, IgM or their mixed
lymphocytes precipitates.
KEY POINTS
1. Antibodies are substances which are formed in the serum and tissue fluids in response to an antigen and react with
that antigen specifically and in some observable manner.
2. Chemical nature of antibodies is globulin and they are named as immunoglobulins.
3. An antibody molecule consists of two identical heavy and two identical light chains.
4. There are five different classes of immunoglobulins namely lgG, lgM, lgA , lgD and lgE.

Ch 13: Antibodies - lmmunoglobulins _ _ _ _ _ _ _ _ _ __ _ _ _ _ _ _ _ _ _ _ _ _ _ _ 103
5. IgG is the major serum immunoglobulin (about 80% of the total amount). It is the only immunoglobulin that is
transported through placenta.
6. IgA is the second major serum immunoglobulin (about 10-13% of serum immunoglobulins). It occurs in two forms,
serum JgA and secretory lgA.
7. Serum IgA is a monomeric while secretory IgA (found in mucosa! surfaces and in secretions) is a dimer formed by
two monomer units joined together by a glycoprotein named the J chain (J for joining).
8. IgA is the principal immunoglobulin present in secretions such as milk, saliva, tears, sweat, nasal fluids, colostrum
and in secretions of respiratory, intestinal and genital systems.
9. IgM is a pentamer consisting of 5 immunoglobilin subunits and one molecule of J chain, which joins the basic
subunits. It constitutes about 5-8 per cent of total serum immunoglobulins. It is heavy molecule with a molecular
weight 900,000 to 1,000,000 hence aslo called the 'millionaire molecule'.
10. IgM is mainly distributed intravascularly (80%). It appears early in response to infection before IgG. It can not cross
the placenta, presence of IgM antibody in serum of newborn indicates congenital infection .
11 . IgE is mainly produced in the linings of respirtory and intestinal tracts. Serum contains only traces (a few nanograms
per ml). It is mostly distributed ex travascularly. It is heat labile whereas other immunoglobulins are heat stable.
12. IgE mediates type I hypersensitivity reactions.
YOU MUST KNOW

1. Definition of antibody and structure of immunoglobulin.
2. Properties of immunoglobulin classes, i.e., IgG, IgA, lgM, lgD, IgE.
3. Diagrams of structure of lgM molecule and secretory lgA molecule.
STUDY QUESTIONS
1. Name the various classes of immunoglobulins. Describe the structure and properties of IgG, IgA and IgM.
(a) IgM (b) Secretory IgA (c) lgE
MULTI LE CHOICE QUESTIONS (MCQs)

1. Which immunoglobulin class can pass through placenta?
(a) IgG (b) lgM (c) IgA (d) lgE
2. Which of the following immunoglobulins is heat labile?
(a) IgG (b) IgM (c) IgA (d) IgE
3. Which immunoglobulin class is distributed maximum intravascularly?
(a) IgG (b) IgM (c) IgA (d) IgE
4. J chain is present in:
(a) lgG (b) lgM (c) lgD (d) IgE
5. Which is the first immunoglobulin to appear in response to an antigen?
(a) IgG (b) IgM (c) IgA (d) lgE
6. Which irnmunoglobulin class has maximum concentration in the human body?
(a) IgG (b) IgM (c) IgA (d) lgE
7. Presence of which immunoglobulin class in serum is a useful indicator for diagnosis of congenital infections?
(a) IgG (b) lgM (c) IgA (d) IgE
8. Which is the earliest immunoglobulin to be synthesised by the foetus?

104 _ _ __ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __ _ _ _ __ _ _ _ Unit II: Immunology
9. Which immunoglobulin class is involved in type I hypesensitivity reaction?

(a) IgG (b) IgM (c) lgA (d) IgE
10. Which immunoglobulin class can bind to mast cells?
(a) IgG (b) IgM (c) lgA (d) IgE
Answers (MCQs):
1. (a) 2. (d) 3. (b) 4. (b) 5. (b)
6. (a) 7. (b) 8. (b) 9. (d) 10. (d)
- - - ++• ---

Chapter 14
ANTIGEN-ANTIBODY REACTIONS
I. Antigen-Antibody Reactions-General Features

A. Uses B. Characteristics
C. Stages D. Measurement
II. Types of Antigen-Antibody Reactions
A. Precipitation Reactions B. Agglutination
C. Complement-Fixation Test (CFT) D. Conglutination
E. Neutralisation Test F. Opsonisation
G. lmmunofluorescence H. Radioimmunoassay (RIA)
I. Enzyme Linked lmmunosorbent Assay (ELISA) J. lmmunochromatography
K. Chemiluminescence Assay L. lmmunoelectronmicroscopic Tests
M. lmmunoblotting
I. ANTIGEN-ANTIBODY REACTIONS (iv) Detection and quantitation of either antigens or

-GENERAL FEATURES antibodies.
Antigen combines with its specific antibody in an
observable manner and the reaction between antigen B. Characteristics
and antibody is specific. These antigen-antibody reactions 1. Reaction is specific; an antigen combines only with its
in vitro are known as serological tests. homologous antibody and vice-versa. However, cross
reactions may occur due to antigenic similarity.
A. Uses 2. Entire molecules of antigen and antibody react and
1. In the Body or in Vivo not the fragments.
(i) It forms the basis of immunity against infectious 3. Only the surface antigens participate in the antigen-
diseases. antibody reaction.
(ii) It may lead to tissue injury in some hypersensitivity 4. The reaction is firm, but reversible. The firmness
reactions and autoimmune diseases. of combination depends on the affinity and avidity.
Affinity is defined as the intensity of attraction
2. In the Laboratory or in Vitro between antigen and antibody molecules. Avidity is
(i) For diagnosis of infections the binding strength after the formation of antigen-
(ii) Helpful in epidemiological studies antibody complexes.
(iii) For identification of non-infectious agents such as 5. Antigens and antibodies can combine in varying
enzymes proportions. Antibodies are generally bivalent,
105
although IgM may have five or more combining sites. is the highest dilution of the serum which shows an
Antigens may have valencies upto hundreds. observable reaction in the particular antigen-antibody
reaction. Antigens may also be titrated against sera.
C. Stages
The antigen-antibody reaction occurs in two stages: II. TYPES OF ANTIGEN-ANTIBODY
REACTIONS
1. The Primary Stage A. Precipitation Reactions
The initial interaction between antigen-antibody is rapid Precipitation
but without any visible effect. This reaction is reversible. When a soluble antigen reacts with its antibody in the
The binding between antigen and antibody occurs by presence of electrolytes (NaCl) at an optimal temperature
the weaker intermolecular forces such as Vander Waal's and pH, the antigen-antibody complex forms an insoluble
forces, hydrogen bonds and ionic bonds, rather than by precipitate and it is called precipitation. The precipitate
firmer covalent binding. This primary interaction can be usually sediments at the bottom of the tube. Precipitation
detected by estimating free and bound antigen or antibody may occur in liquid media or in gels such as agar, agarose
separately in the reaction by a number of methods. or polyacrylamide.
2. The Secondary Stage Flocculation

The primary stage in most of the instances, but not all, When instead of sedimenting, the precipitate is suspended
is followed by secondary stage which has visible effects as floccules, the reaction is called flocculation. This is
such as precipitation, agglutination, complement fixation, a modified form of precipitation. A serological test
neutralisation and immobilisation of motile organisms. (e.g. VDRL) routinely used for diagnosis of syphilis is
When these reactions were discovered, it was believed one example of flocculation test.VDRL slide is used to
that different type of antibody was involved for each perform 'Venereal disease research laboratory' (VDRL)
type of reaction. On this basis, the antibody involved in test (Fig. 14.1 ).
agglutination was called agglutinin and those involved in
precipitation as precipitin and the corresponding antigens
were called agglutinogen and precipitinogen respectively.
This concept was replaced by Zinsser's unitarian hypothesis I
(1920) which held that an antigen would induce synthesis
of only one antibody which produces different types of
visible (observable) effects. Both of these extreme views
are not true. It is now an established fact that a single
antibody can cause different types of antigen-antibody
reactions and a single antigen can stimulate production
of different classes of immunoglobulins which differ in
their reaction capacities as well as in other properties
(Table 14.1). Fig. 14.1 VDRL slide
Table 14.1 Role of lmmunoglobulin Classes in Serological Prozone Phenomenon

Reactions When same amount of antiserum is mixed with increasing
Serological reaction Immunoglobulin class quantities of antigen in different tubes, the precipitation
IgG lgM IgA
takes place in one of the middle tubes, in which antigen
and the antibody are present in optimal or equivalent
Precipitation Strong Weak Variable
proportion (Zone of equivalence). In the preceding tubes,
Aggiu tination Weak Strong Moderate antibody is in excess and in later tubes, in which antigen
Complement fixation Strong Weak Negative is in excess, the precipitation is weak or even absent
(Fig. 14.2) . Absence of precipitation (false negative) in
the presence of excess antibodies is known as prozone
D. Measurement phenomenon. This is relevant clinically as these sera
Measurement may be in terms of mass (e.g. mg Nitrogen) should be diluted further to get the proper results of
or more commonly as titre of units. The antibody titre precipitation.

Ch 14: Antigen-Antibody R e a c t i o n s - - - - - - ' - - - - - - - - - - - - - - - - -- -
-- - 107
• ••
Antibody excess Optimal proportion of
antigen and antibody Antigen excess
(zone of equivalence)
Fig. 14.2 Prozone phenomenon
(i) Mechanis m of precipitat ion 4. Testing for food adulterant s.

Marrack (1934) proposed the lattice hypothesis which is 5. To standardis e toxins and antitoxins.
widely accepted. According to this concept, multivalen t
antigens combine with bivalent antibodies, precipitati on (iii) Types of precipita tion reaction
occurs only when a large lattice is formed . This occurs in (a) Ring test
the zone of equivalence. In the zone of antigen excess, Antigen solution is layered over an antiserum in a narrow
the valencies of the antibody are fully satisfied which tube. A precipitate ring appears at the junction of the
results in failure to form a large lattice. Similarly, in the two liquids. Examples of ring tests are C-reactive protein
zone of antibody excess, the valencies of the antigen are (CRP) test and streptococ cal grouping by the Lancefield
taken up with antibody and results in failure to form a technique.
large lattice (Fig. 14.3). This lattice hypothesis holds true
for agglutinat ion also. (b) Flocculation test
It can be done in slide and in tube.
Slide test: A drop of antigen solution is added to
a drop of inactivated patient's serum on a slide and
• mixed by shaking, floccules appear. The VDRL test for

detection of antibodies against syphilis is an example of
• slide flocculation test.
Tube test: The Kahn test (tube flocculation) was
done previously for diagnosis of syphilis. Standardi sation
of toxins and toxoids is also done by tube flocculation
Antibody test.
Antibody in Zone of equivalence Antigen in
excess (Lattice formation) excess
(c) Immunodiffusion test
Fig. 14.3 Lattice hypothesis These are precipitati on tests in gel. Immunod iffusion
is generally done in 1% agar gel. There are following
(ii) Applicati ons of precipitat ion reaction advantage s of precipitati on in gel rather than in a liquid
Precipitat ion reaction may be used either as qualitative or medium.
quantitati ve test. It is very sensitive test and can detect • The reacting band is easily visible and can be stained
as little as 1 µg of protein antigen. Precipitat ion test has for preservation. This band is stable.
a considerable value in: • The number of different antigens in the reaction can
1. Identification of bacteria e.g. Lancefield 's grouping be observed. As each antigen-antibody reaction gives
of Streptococcus. rise to a line of precipitation, therefore, it helps to
2. Detection of antibody for diagnostic purposes e.g. identify different antigens.
VDRL in syphilis. • Identity, cross reaction and non identity between
3. Forensic application in identification of human blood different antigens can also be observed by
and seminal stains. immunodi ffusion.
Various Immunodif fusion Tests Uses:

1. Single diffusion in one dimension (a) It has been widely employed for estimation of
(Oudin procedure) immunoglobulin classes i.e. IgG, IgM, IgA, in sera.
The antibody is mixed in agar gel in a test tube and (b) It has also been used for screening sera for antibodies
antigen solution is poured over it. The antigen diffuses to influenza viruses.
downward through the agar gel and a line of precipitation
is formed (Fig. 14.4). The number of precipitate bands
will indicate the number of different antigens present. It 4. Double diffusion in two dimensions
is single diffusion of antigen only and in one dimension (Ouchterlon y procedure)
i.e. towards antibody in agar gel. Agar gel is poured on a slide. Wells are cut using a template.
The antiserum (antibody) is placed in the central well and
2. Double diffusion in one dimension (Oakley- surrounding wells are filled with different antigens. If two
Fultlwrpe procedure) adjacent antigens are identical, the lines of precipitate
The antibody is incorporated in agar gel, above which is formed by them will fuse with each other. If they are
placed a column of plain agar. The antigen is poured on unrelated, the lines cross each other. Spur formation
top of this plain agar. The antigen and antibody move indicates cross reaction or partial identity (Fig. 14.5b).
towards each other through intervening column of plain A special variety of double diffusion in two dimensions
agar and a precipitate band is formed where they meet is the Elek's test for toxigenicity in C. diphtheriae.
in optimum concentration (Fig. 14.4).
5. Immunoelectrophoresis
Immunoelectrophoresis combines electrophoresis and
immunodiffusion. This is done on a glass slide layered
Antigen
with semisolid agar. A well is cut and antigen is filled.
Plain agar The first step is electrophoresis of antigen for about an
Precipitin
band
hour. Rectangular trough is cut in the agar parallel to the
direction of migration of antigen and filled with antibody
Antibody in
agar gel (antiserum). Diffusion is allowed to proceed for 18-24 hours.
Precipitation lines develop with each separated component
Single diffusion Double diffusion
of the antigen. By this technique, a number of antigens
Fig. 14.4 Single and double diffusion in one dimension can be identified in human serum. It is particularly useful
for detection of normal and abnormal serum proteins like
3. Single diffusion itt two dimensions myeloma proteins (Fig. 14.6).
(Radial immunodiffusion)
The antiserum (antibody) is incorporated in agar gel on
a slide or petridish. The wells are cut on the surface of 6. Electroimmunodiffusion
gel. Antigen is added to the wells. The antigen diffuses Immunodiffusion can be speeded up if antigen and
radially and ring-shaped bands of precipitation (halos) antibody are driven by electricity. It is combination
are formed around the well (Fig. 14.5a). The diameter of electrophoresis and diffusion. Of these methods,
of the ring is directly proportiona l to the concentration counterimmunoelectrophoresis (CIE or CIEP) and rocket
of the antigen. electrophoresis are used frequently.
- j
r- (o)(O @r::_
i) to 0 1.)
0
Anti:~:~:;ell ----1---~....,-c > o~
(\.::::
I
precipitation
0 0
'-..:.,
A ntibody in agar ---------

gel
t5 ,,,. -.
0
0
ig. 14.Sa Single diffusion in two dimensions

109
Ch 14: Antigen-Antibody Reactions---- - ' - - - - - - - - - - - - - - - - - - - - - - -
Electric current
Agar gel
on a slide
0 +
Reaction of---t-- ·""'

partial identity
----+- Reaction of
identity
l--..:§=--=-----t-- ii~:cipitation
Agarose
- - Slide
Antigen in well
Wells containing antigen and antibody
Fig. 14.7 Counterimmunoelectrophoresis

0 0
(ii) Rocket electrophoresis
Fig. 14.Sb Double diffusion in two dimensions
It is one dimensional single electro-immunodiffusion.
This technique is mainly applied for quantitation of
antigens. The antiserum to the antigen to be quantitated
c================'
0
=I= Trough cut in agar
Agar covered is mixed in agarose and gelled on the glass slide. The
~-----+!-----~ microscopic slide wells are punched in the set gel and filled with increasing
Well cut in agar concentrations of the antigen. It is then electrophoresed.
Precipitation is formed in the shape of cone like structures
O (appearance of a rocket). The length of these rocket like
I I Antigen placed
in well structures corresponds to the concentration of the antigen
(Fig. 14.8).
I~ Antigen components
separated by
electrophoresis
Antiserum
(antibody) in
~-------------,+
+I
agarose gel
Trough filled with Precipitin arcs

(rockets)
antibody
I~-----------'
I\
,H _. ++ 1 Antibody diffuses
1 't' 1 't' towards separated
antigen components
(\
~-----------'
Precipitin bands
Antigen wells
• • • •
form where antibody Increasing concentration of antigen
and antigen meet at
I
~ - - - - - - - - - ~ optimal proportions Fig. 14.8 Rocket electrophoresis
Fig. 14.6 Immunoelectrophoresis

(iii) Laurell's two dimensional electrophoresis
(i) Counterimmunoelectrophoresis (Counter-current- It is a variant of rocket electrophoresis. By this method,
immunoelectrophoresis, CIE or CIEP) several antigens in a mixture can be quantitated. In
It is one dimensional double electro-immunodiffusion. the first- stage of the technique, the antigen mixture
This test is based on movement of antigen and antibody is electrophoretically separated and in second stage,
in opposite direction. This is performed on a glass slide electrophoresis is done perpendicular to that of first stage
layered with agarose and wells are cut on the surface. to get rocket like precipitation (Fig. 14.9).
One well is filled with antigen and other with antibody.
+
Electric current is passed through the gel. The antigen - - - - - + - Precipitin
and antibody move towards each other resulting in a Antibody in arc
gel
precipitation line at a point between them (Fig. 14.7).
It takes about thirty minutes and is more sensitive
than standard immunodiffusion technique. It is applied
clinically for detecting hepatitis B antigens and antibodies, Antigen (Ag) ~ Second stage
First stage in well
antigens of cryptococcus in cerebrospinal fluid and in
Fig. 14.9 Laurell's two dimensional electrophoresis
number of other diseases.

110 - - - - - - - - -- - - - -- - - - - - - - - - - - - - - -- - Unit II: Immunology
B. Agglutination Uses of tube agglutination test

It is an antigen-antibody reaction, in which a particulate (i) Serological diagnosis of
antigen combines with its antibody in the presence of (a) enteric fever (Widal test)
electrolytes at an optimal temperature and pH, resulting in (b) typhus fever (Weil-Felix reaction)
visible clumping of particles. It differs from precipitation in (c) infectious mononucleosis (Paul-Bunnel test)
which soluble antigen is present in contrast to particulate (d) brucellosis
antigen of agglutination. The agglutination is more (ii) For diagnosis of primary atypical pneumonia
sensitive than precipitation for the detection of antibodies. (Streptococcus MG agglutination test)
The agglutination reaction takes place better with IgM
antibody than with IgG antibody. Principles governing Complications related to tube agglutination
agglutination are the same as that of precipitation. Two main complications may be
Agglutination occurs when antigen and antibody are (i) Prozone phenomenon
present in optimal proportions. Lattice formation (ii) Blocking antibody
hypothesis holds good for agglutination too. The zone
phenomenon may occur when either an antigen or an (i) Prozone phenomenon
antibody is in excess. Occasionally incomplete antibodies Serum with high concentration of antibody may not be
(e.g. anti-Rh and anti-Bruce/la) are formed that combine able to react with antigen and gives a false negative result.
with the antigen but do not cause agglutination. They Several dilutions of the serum should be tested to prevent
act as 'blocking' antibodies, inhibiting the agglutination such false negative results due to prozone.
by the complete antibody added subsequently.
(ii) Blocking antibody
TYPES OF AGGLUTINATION REACTION Blocking or incomplete antibodies may be detected by
1. Slide Agglutination Test performing the test in hypertonic (5%) saline or albumin
A uniform suspension of antigen is made in a drop of saline. Antiglobulin (Coombs) test is more reliable for
saline on a slide or tile and a drop of the appropriate detecting these antibodies.
antiserum is added. The agglutination reaction is facilitated
by mixing the antigen and the antiserum with a wire loop 3. The Antiglobulin (Coombs) Test
or by gently rocking the slide. Clumping occurs instantly This test was devised originally by Coombs, Mourant
or within seconds when agglutination test is positive. and Race (1945) for the detection of incomplete anti-
Clumping after a minute may be due to drying of the Rh antibodies. When sera containing incomplete anti-
fluid and should be disregarded. A control consisting of Rh antibodies are mixed with corresponding Rh-positive
antigen suspension in saline, without adding antiserum erythrocytes, the incomplete antibody globulin coats the
must be included on the same slide. It is to ensure that erythrocytes but no agglutination occurs. When such
antigen is not autoagglutinable. coated erythrocytes are treated with antiglobulin or
Coombs serum (rabbit antiserum against human gamma
Uses: globulin), the cells are agglutinated. This is the principle
(i) It is a routine procedure to identify the bacterial of Coombs test (Fig. 14.10).
strains isolated from clinical specimens. One example
is to identify Salmonella species. Anti red cell antibody (incomplete)
(ii) It is also used for blood grouping and cross
matching.
2. TI,be Agglutination Test

This is a standard quantitative method for determination
Red cell -
t
Antiglobulin - -Y
of antibodies. Serum is diluted serially by doubling
dilution in test tubes. An equal volume of a particulate
antigen is added to all tubes. The highest dilution of
serum at which agglutination occurs is antibody titre.
Tube agglutination is routinely employed for antibody
detection in diagnosis of typhoid (Wida] test), brucellosis
Fig. 14.10 Coombs test
and typhus fevers (Weil-Felix reaction).

Ch 14: Antigen-Anti body Reactions _ _ _ _ _ _ _ __ _ _ _ __ __ _ _ _ __ _ _ _ __ 111
There are two types of Coombs test:

(i) Direct Coombs test
(ii) Indirect Coombs test
The only difference between the two is that the
sensitisation of the erythrocytes with incomplete Antibody
Latex particles /
antibodies takes place in vivo in direct type whereas it
occurs in vitro in indirect type.
Uses of Coombs test
(a) For detection of anti Rh antibodies
(b) For demonstration of any type of incomplete
antibody eg. brucellosis
+
/
lb
Antigen
4. Heterophile Agglutination Test

Heterophile antibodies have a property to react with
microorganisms or cells of unrelated species due to
common antigenic sharing.
Fig. 14.11 Latex agglutinati on test
(i) Weil-Felix reaction
Some Proteus (OX19, OX2, OXK) strains are agglutinated
by sera of patients with rickettsial infections. This is due
to antigenic sharing between these Proteus strains and
Rickettsial species. i
(ii) Paul-Runnel test

Sheep erythrocytes are agglutinated by sera of infectious
mononucleosis.
\ \ \
(iii) Streptococcus MG agglutination test
It is positive in primary atypical pneumonia.
5. Passive Agglutination Test

t. ·,
( ~:-
(
A precipitation reaction can be converted into agglutination
test by attaching soluble antigens to the surface of Fig. 14.12 Latex agglutination tile
carrier particles such as latex particles, bentonite and

red blood cells. Such tests are called passive agglutination (ii) Haemagglutination test
tests. This conversion is done because agglutination tests Erythrocytes sensitised with antigen are used for detection
are more sensitive for detection of antibodies. Passive of antibodies. In rheumatoid arthritis, an autoantibody
agglutination tests are very sensitive. When instead of (RA factor) appears in the serum. This RA factor acts
antigen, the antibody is adsorbed on the carrier particles as an antibody to gammaglobulin. Thus, RA factor
for estimation of antigens, it is known as reversed passive can agglutinate red cells coated with garnmaglobulins.
agglutination. The antigen used for the test is sheep red blood cells
sensitised with rabbit antisheep erythrocyte antibody
(i) Latex agglutination test (amboceptor). The amboceptor is antibody, therefore,
Polystyrene latex particles (0.8 - 1 µm in diameter) are garnmaglobulin in nature. This is the principle of Rose-
widely employed to adsorb several types of antigens. Waaler test employed for detection of RA factor in cases
Latex particles can also be coated with antibody for of rheumatoid arthritis.
detection of antigen (Fig. 14.11 ). These tests are very
convenient, rapid and specific. These are used for (iii) Coagglutination
detection of hepatitis B antigen, ASO, CRP, RA factor, Principle
HCG, bacterial typing (N meningitidis) and many other It is based on the presence of protein A on the surface of
antigens. Latex agglutination tile is used to perform latex some strains of Staph. aureus (especially Cowan 1 strain).
agglutination test (Fig. 14.12). Specific IgG immunoglobulin is coated on these Cowan

112 - - - -- - - - -- - -- - - - - - -- -- - - - - - - - - - - Unit II: Immunology
1 strains of Staphylococcus aureus. Fe portion of IgG the fixation of complement, an indicator system consisting
molecule binds to proteinA whereas antigen combining of sheep erythrocytes coated with amboceptor (rabbit
Fab terminal remains free. When the corresponding antibody to sheep erythrocytes) is used. Complement
antigen is mixed with these coated cells, Fab terminal can lyse these erythrocytes coated with antibodies. If
binds to antigen resulting in agglutination (Fig. 14.13). complement is fixed and utilized in the antigen-antibody
This test is used for detection of bacterial antigens in reaction, there is no free complement to act on the
blood, urine and CSF. N gonorrhoeae, Streptococcus indicator system and hence no lysis of erythrocytes, which
pyogenes and H. infiuenzae antigens can be detected by indicates the positive complement fixation test. Lysis of
this method. erythrocytes indicates that complement was not fixed in
the first step and therefore, the serum is negative for
antibodies (negative CFT).
+ 2. Procedure
The serum (to be tested) should be inactivated by heating
Cowan 1 strain of lmmunoglobulin
Staph. aureus at 56°C for 30 minutes to destroy any complement activity
the serum may have and to remove some non-specific
inhibitors of complement. The antigen may be used as
soluble or particulate. Fresh guinea pig serum is used
as source of complement. Complement activity is heat
labile so it is used as fresh. Alternatively, the serum is
preserved either in the lyophilised or frozen state or with
addition of special preservatives.
Controls should be included in the test. Antigen
and serum controls are included to ensure that they are
not anti-complementary. Complement control is used
Agglutination to ensure that the desired amount has been added, and
cell control to make sure that sensitised erythrocytes
Fig. 14.13 Coagglutination
(erythrocytes coated with amboceptor) do not undergo
lysis in the absence of complement.
C. Complement Fixation Test (CFT)
1. Principle (Fig. 14.14) 3. Standardisation of Complement
The antigen-antibody complexes have ability to 'fix ' The guinea pig serum is titrated for complement activity.
complement. This reaction has no visible effect. To detect One unit of minimum haemolytic dose (MHD) of
I Negative CFT I
+
Complement Sheep erythrocytes coated with Lysis
amboceptor (Indicator system)
I Positive CFT J
•
T +
Antigen Antibody
+f -i::+
Complement Compement is fixed
in Ag-Ab reaction
Indicator system
No lysis
(As complement is not free to act on indicator system)
Fig. 14.14 Complement fixation test

113
Ch 14: Antigen-Antibody Reactions _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __
complement is defined as the highest dilution (smallest cause agglutination of sensitised sheep erythrocytes if
quantity) of guineapig serum which lyses one unit volume these are combined with complement, this process is called
of washed sheep red blood cells in the presence of excess conglutination.
haemolysin (amboceptor) within a fixed time (30 to 60 First step : Antigen + Antiserum (positive)
minutes) and at a fixed temperature (37°C). + Horse complement - complement is fixed
Second step : Sheep erythrocytes with conglutinin -
4. Titration of Amboceptor No agglutination occurs because complement is not free
The amboceptor is titrated for haemolysin activity. to act with sensitised erythrocytes.
One unit of minimum haemolytic dose (MHD) of
No agglutination- Positive result
haemolysin is defined as the highest dilution (smallest
quantity) of the inactivated amboceptor which lyses Agglutination -Negative result
one unit volume of washed sheep red blood cells in
the presence of excess complement within a fixed time OTHER COMPLEMENT DEPENDENT
(30 to 60 minutes) and at a fixed temperature (37°C). SEROLOGICAL TESTS
Normal saline with added calcium and magnesium is 1. Immobilisati on Test
used as diluent for titrations of complement, amboceptor When live motile Treponema pallidum is mixed with
and for CFT. patient's serum in the presence of complement, the
organism becomes non-motile. This is the principle of
INDIRECT COMPLEMENT FIXATION TEST Treponema pallidum immobilisation test.
Certain avian (e.g. duck, parrot) and mammalian (e.g.
horse, cat) sera cannot fix guinea pig complement. Indirect 2. Immune Adherence
complement fixation test is used for testing such sera. The antigen-antibody complexes in some bacteria (V.
Test is done in duplicate and after the first step, the cholerae, T. pallidum) adhere to particulate material such
standard antiserum known to fix complement is added as erythrocytes or platelets. This bacterial adherence to
in one set. cells is known as immune adherence.
First step: Antigen + test serum (negative for antibody)
+ guinea pig complement 3. Cytolytic or Cytocidal Tests
Second step: Standard antiserum will react with When a live Vibrio cholerae is mixed with its antibody
antigen and fix the free complement. in the presence of complement, the bacterium is lysed.
Indicator system: No haemolysis because This forms the basis for measurement of anticholera
complement is not free to act on indicator system. antibodies.
In a positive test, antigen would have been used up
by serum in the first step and standard antiserum would E. Neutralisation Test
not be able to fix the complement which remains free to Bacterial exotoxins are capable of producing neutralising
act on indicator system resulting in haemolysis (positive antibodies (antitoxins) which play a role in protection
result). against diseases such as diphtheria and tetanus. The
First step: Antigen + test serum (positive for tqxicity of bacterial endotoxins is not neutralised
antibody) + Guinea pig complement by antisera. Viruses may also be neutralised by their
Second step: Standard antiserum cannot react with antibodies and these are named as virus neutralisation
antigen becaµse antigen has been used up by antibody in tests.
the first step but complement is free as it is not fixed. Toxin-antitoxin neutralisation can be measured in
Indicator system: Haemolysis occurs because vivo and in vitro.
complement is free to act on the system.
IN VIVO TESTS
D. Conglutination 1. Toxigenicity Test
This is an alternative method for systems which do not Toxigenicity test is done for detection of toxin of C.
fix guinea pig complement. Horse complement (non- diphtheriae. It is an intradermal test to inject bacterial
haemolytic) is used. The indicator system is sheep toxin in animal previously protected by antitoxin of
erythrocytes sensitised with bovine serum. Bovine serum C. diphtheriae and a test animal without antitoxin. No
contains a beta globulin component named conglutinin, biological effects of toxin are observed in the control
which acts as antibody to complement. Conglutinin can animal but test animal (unprotected) dies.

114 - -- - - - - - - - - - - - --
- - - - - - - -- - - - - - - - Unit II: Immunology
2. Shick Test 1. DIRECT IMMUNOFLUORESCENCE TEST

This is similar kind of test in humans. Diphtheria toxin Principle
is injected intradermally in man, there is no reaction at The specific antibodies tagged with fluorescent dye (i.e.
the site of injection if person is immune to diphtheria ie. labelled antibodies) are used for detection of unknown
antitoxin is present in blood. Injected toxin is neutralised antigen in a specimen. If antigen is present, it reacts
by circulating antitoxin. with labelled antibodies and fluorescence can be observed
under ultraviolet light of fluorescent microscope (Fig.
IN VITRO TESTS 14.15). This is depicted in flow diagram as follows:
1. Antistreptoly sin 'O' (ASO) Test Specimen (Positive for antigen)
Serum of patients suffering from Streptococcus pyogenes +
infection contains antistreptolysin 'O' (antitoxin) which Labelled antibodies
neutralizes the haemolytic activity of streptococcal 'O' ...
haemolysin (toxin). Fluorescence observed
...
2. Virus Neutralisatio n Tests Antigen is present in specimen
Neutralisation of viruses can be demonstrated in cell
cultures, eggs and animals It is used mostly in typing Uses
viral isolates. I. It is commonly employed for detection of bacteria,
viruses or other antigens in blood, CSF, urine, faeces,
3. Nagler Reaction tissues and other specimens.
Clostridium welchii toxin (alphatoxin) is neutralised by 2. It is a sensitive method to diagnose rabies, by
antitoxin when the bacteria is grown in egg yolk medium detection of the rabies virus antigens in brain smears.
containing antitoxin. It is useful for rapid detection of
C. welchii in clinical specimens. Disadvantage
Separate specific fluorescent labelled antibody has to be
F. Opsonisation prepared against each antigen to be tested.
Opsonisation is the process by which a particulate antigen
becomes more susceptible to phagocytosis. This occurs 2. INDIRECT IMMUNOFLUORESCENCE TEST
by help of opsonin which combines with an antigen and The indirect method is employed for detection of
facilitates phagocytosis. Opsonin may be an antibody-like antibodies in serum or other body fluids.
substance or other component present in serum. Opsonic
index is defined as ratio of phagocytic activity of the Principle
patient's blood for a given bacterium, to that of a normal A known antigen is fixed on a slide. The unknown antibody
individual. Phagocytic index is the average number of (serum) is applied to the slide. If antibody (globulin) is
phagocytosed bacteria per polymorphonu clear leucocyte present in the serum, it attaches to known antigen on
from stained blood films.Phagocytic index denotes the the slide. For detection of this antigen-antibody reaction,
phagocytic activity of the blood and thus helps in fluorescin-tagged antibody to human globulin is added.
measuring opsonic index. In positive test, fluorescence occurs under ultraviolet light
(Fig. 14. I 5). One specific example of detecting antibodies
G. Immunofluorescence in serum of syphilis patient is also included.
Fluorescence is the property of certain dyes which absorb
rays of one particular wavelength (ultraviolet light) and Advantages
emit rays with a different wavelength (visible light). Coons A single antihuman globulin fluorescent conjugate can be
and his colleagues (1942) showed that fluorescent dyes can employed for detection of antibody to any antigen. All
be conjugated to antibodies and these 'labelled' antibodies antibodies are globulin in nature, therefore, antihuman
can be used to detect antigens in tissues. The commonly globulin attaches to all antibodies. This has overcome the
used fluorescent dyes are fiuorescin isothiocyanate and disadvantage of direct immunofluorescence test.
lissamine rhodamine, exhibiting blue green and orange
red fluorescence respectively. Immunofluorescence test is SANDWICH' TECHNIQUE OF
of two types: IMMUNOFLUORESCENCE
1. Direct immunofluorescence test Antigen being in the middle with labelled and unlabelled
2. Indirect immunofluorescence test antibody on either side, forms a sandwich. This is used

Ch 14: Antigen-Antibody Reactions _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __ 115
Direct immunofluorescence test
Unknown Antigen
I
E3 +
Slide
Fluorescein labelled Fluorescence under UV

specific antibody light (positive test)
Indirect immunofluorescence test
Known Antigen
+ r1i _~. . . . . . .
Patient serum antigen Flourescein Fluorescence under
containing antibody + labelled UV light
antibody antiglobulin (positive test)
Example :
Treponema pallidum + Serum of syphilis patient + Fluorescin Fluorescence
(containing anti-treponemal labelled (Positive)
antibodies which is globulin antiglobulin
in nature)
Fig. 14.15 Direct and indirect immunofluorescence tests
for detection of antibodies. was awarded the Nobel Prize for the discovery of RIA
Antibody test. This test can detect antigens up to picogram (l0- 12g)
+ quantities.
RIA is based on competition for a fixed amounts of
Antigen specific antibody between a known radiolabelled antigen
+ and unknown unlabelled (test) antigen (Fig. 14.16). This
Labelled antibody competition is determined by the level of the test antigen
present in the reacting system. After antigen-antibo dy
H. Radioimmun oassay (RIA) reaction, the antigen is separated into the 'free' and
Berson and Yallow (1959) first described the test 'bound' fractions and their radioactivity is measured. The
radioimmunoa ssay (RIA) and since then it has been concentration of test (unlabelled) antigen is calculated
utilised for quantitation of hormones, drugs, hepatitis from the ratio of the bound and total antigen labels,
B surface antigen, IgE and viral antigens. In 1977, Yallow using a reference curve (Fig. 14.17).
Unlabelled antigen (test sample) - - - + -

,._,,,~~-=-< Free fraction
Mix 0 0
Known radiolabelled antigen - - - + -
Incubate
0
y Bound fraction
a_ 0
Q_
Fig. 14.16 Radioimmunoassa y

116 - - - - - - - - -- - - - - - - - - - - -- - - - - - - - - - - Unit II: Immunology
SANDWICH ELISA
C:
Cl)
For antigen detection in a specimen, the wells of microtitre
Ol
Eca
plate are coated with specific antibody against the antigen
(/)
to be detected. Specimens to be tested are added in
Q)
.0 coated wells. If antigen is present in specimen, it binds to
.!Q
coated antibody. To detect this antigen-antibody reaction,
antiserum (antibody) conjugated with an enzyme is added.
i::i
C:
This conjugated antiserum binds to an antigen already
0
.0 attached to coated antibody. A substrate is added to know
0
0 the binding of conjugated antiserum to antigen-antibody
a: complex. In case of binding (positive result), an enzyme
acts on substrate to produce ~olour, intensity of which
Concentrtion of unlabelled antigen
can be read by spectrophotometer or ELISA reader.
Fig. 14.17 Reference curve Colour detection can also be seen by naked eye. This
type of ELISA test is also known as sandwich ELISA
The standard dose response or reference curve has (Fig. 14.19). Positive and negative controls should always
to be prepared for calculations. This is done by running be included in the test. At every step of ELISA test,
the reaction with fixed amounts of antibody and labelled incubation and washing is done to wash off unbound
antigen but with varying known amounts of unlabelled reagents.
antigen. The ratio of bound: total labels (B:T ratio)
plotted against the unlabelled antigen concentrations INDIRECT ELISA
gives the standard reference curve. The concentration For antibody detection, the wells of microtitre plate are
of antigen in the test sample is calculated with the help coated with antigen. Sera to be tested are added in these
of B:T ratio-of the test by using standard dose response coated wells. If antibody is present in specimen, it binds to
or reference curve. coated antigen. To detect this antigen-antibody reaction,
a goat antihuman immunoglobulin antibody conjugated
I. Enzyme Linked Immunosorbent Assay (ELISA) with an enzyme is added. Enzyme conjugated antihuman
ELISA has been applied widely for detection of a variety iromunoglobulin binds to antibody (immunoglobulin in
of antibodies and antigens. It is simple and nearly as nature). To detect this binding, a substrate is added and
sensitive as radioimmunoassay. It requires only microlitre enzyme acts on substrate to produce colour .in a positive
reaction. This procedure is also named as indirect ELISA
0
quantities of test reagents. The principle ·of ELISA is

almost same as tliat of immunofluorescence, the only (Fig. 14.19) . Reading of the test is same as described in
difference being, an enzyme is used instead of fluorescent . sandwich ELISA. Positive and negative controls are always
dye. The enzyme acts on substrate to produce a colour in put up alongwith test sera. Incubation and washing is done
a po_sitive test. ELISA can be used for detection of antigen at every step to wash off unbound reagents. Substrates
or antibody. Tests for specific immunoglobulin classes (e.g. are specific for each enzyme. The enzyme (horseradish
IgM ELISA) are also available. It is done on a solid phase. peroxidase, alkaline phosphatase) gives rise to a colour
The test can be done in polystyrene tubes (macro-ELISA) change by adding specific substrate (o-phenyl-diamine
or polyvinyl microtitre plates (micro-ELISA) (Fig. 14.18) . dihydrochloride for peroxidase, p-nitrophenyl phosphate
for alkaline phosphatase). Alkaline phosphatase with this
substrate produces a yellow colour.
COMPETITIV E ELISA
It has been used for detection of HIV antibodies. Positive
result shows no colour whereas appearance of colour
indicates a negative test. Like radioimmunoassay, there
are two specific antibodies, one conjugated with enzyme
and other present in serum (if serum is positive for
antibodies). Competition occurs between two antibodies
for same antigen. The microtitre plate wells are coated
with HIV antigen. Sera to be tested is added to these wells
Fig. 14.18 ELISA test and incubated at 37°C and then washed. If antibodies

Ch 14: Antigen-Antibody Reactions _ _ _ _ _ _ _ _ _ _ _ __ _ _ _ __ _ _ __ _ _ __ 117
Sandwich ELISA Indirect ELISA Competitive ELISA
LW Conjugate is washed out

as antigen is not free to
bind the conjugate
Colour (Positive)
lilJ
Colour (Positive) No Colour (Positive)
Enzyme (tagged to conjugate)
is not there to act on substrate
A Antigen Conjugate [antibody to antigen (& )]

Antibody Colour product
Conjugate [Antibody to antibody( ~ )]
Fig. 14.19 ELISA
are present, antigen-antibody reaction occurs. To detect Human immunoglobulin immobilised at a spot on the
this reaction, enzyme labelled specific HIV antibodies are membrane serves as a control, as shown by development
added. There is no antigen left for these antibodies to of colour at that site. ·
act. These antibodies remain free and washed off during
washing. Substrate is added but there is no enzyme to Uses of ELISA
act on it. Therefore, positive results show no colour (Fig. It has been used for detection of antigens and antibodies
14.19). If serum to be tested is negative for antibodies, of various microorganisms. Some examples are
antigen is there to combine with enzyme conjugated (i) Detection of HIV antibodies in serum
antibodies and enzyme acts on substrate to produce (ii) Detection of mycobacterial antibodies m
colour. tuberculosis
(iii) Detection of rotavirus in faeces
CASSETTE OR CYLINDER ELISA (iv) Detection of hepatitis B markers in serum.
It is a simple modification of ELISA for testing one or ( v) Detection of enterotoxin of Escherichia coli m
few samples of sera at a time. The test is rapid (about 10 faeces.
minutes) as compared with the 2-4 hours taken for micro-
ELISA The result is read visually. J. Immunochromatography
Cassette ELISA is being used for detection of HIV Immunochromatography or lateral flow immunoassay is
type 1 and 2 antibodies. Specific type 1 and 2 antigens one of the most popular form of rapid immunoassays. It
are immobilised on the nitrocellulose membrane in the can detect both antigens and antibodies. It has advantage
cassette. Test serum is added on the membrane. In positive of being a one step test. It can be completed within
serum, antibody will bind to the appropriate antigen. After 30 minute. It is a strip based test. The strip contains
washing to remove unbound antibody, conjugate (enzyme a chromatographic pad with three zones: sample pad,
labelled antihuman immunoglobulin antibody) is added. conjugate pad and capture line (Fig 14.20). The conjugate
It is washed again to remove unbound conjugate and a pad may be having colloidal gold, dye, or latex beads
substrate is added. A positive result shows a coloured spot. as conjugate which produces signal. The specimen is
118 - - - - - - - - - - - - - - - -
- - - - - - - - - - - - - - - - - - Unit II: Immunology
Sample
i TT
POSITIVE
SAMPLE
Sample pad Conjugate pad

I
Test line positive Capture line Control line
Sample
i 'in 'i ~
!···
H ~ 'i'i'i'i~ lr
zone
I 7
NEGATIVE
SAMPLE
Sample pad Conjugate pad Test line negative Capture line

I
Control line
zone
• Antigen Conjugate [antibody to antigen (A )] J Antibody to antigen (A ) Antibody to antibody( ~ )
Fig. 14.20 lmmunochromatography strip for antigen detection in sample
applied to the sample pad and flows laterally by capillary as clumps. This method is applied in some viruses such
action. Upon reaching the conjugate pad, it may bind to as hepatitis A virus and viruses causing diarrhoea.
conjugate if antigen or antibody is present in the specimen
and forms antigen antibody complex. This complex then 3 lmmunoenzym e Test
flow laterally to reach capture line. Here it is captured Some enzymes such as peroxidase can be conjugated with
by antigen or second antibody present in the capture antibodies. Tissue sections are treated with peroxidase
line. The presence of colour line is a positive test. There labelled antisera to detect corresponding antigen. The
is a positive control line also to check that the test was peroxidase bound to the antigen is visualised under the
properly performed. electron microscope.
K. Chemilumine scence Assay

Chemiluminescence refers to a chemical reaction in which M. Immunoblott ing
energy is emitted in the form of light. On the basis of In immunoblots, antibodies can detect proteins (antigens)
this property chemiluminescence compounds are used in mixtures. The mixture of proteins (antigens) is
in this method to provide the signal (i.e. light) during electrophoretically separated in a gel. The separated
the antigen-antibody reaction. The emitted light can be proteins are then transferred from gel to a nitrocellulose
measured and the concentration of the analyte calculated. paper. These nitrocellulose paper strips are reacted with
The method has been fully automated. This property test sera and subsequently with enzyme-conjugated anti-
of chemiluminescence has also been applied for drug human immunoglobulin. A suitable substrate is added,
sensitivity testing of M. tuberculosis (refer chapter 42). colour is produced by enzyme where specific antibody
in test sera has reacted with separated proteins on strip.
L. Immunoelectronmicroscopic Tests This test has been widely used to confirm the ELISA
1. lmmunoferrit in Test positive HIV antibody cases. This is known as Western
Ferritin (electron dense substance) conjugated antibody Blot test. It detects antibodies against various protein
is used to react with an antigen. This antigen-antibody (antigen) fractions in test sera.
reaction can be visualised under electron microscope. The above procedure may also be applied to analyse
DNA or RNA. When DNA is transferred on nitrocellulose
2. lmmunoelectronmicroscopy strips from gel, this test is referred to as Southern Blot
Viral particles are mixed with specific antisera and are test. Similarly, if RNA is transferred, it is named as
observed under the electron microscope. These are seen Northern Blot test.

Ch 14: Antigen-Antibody Reactions _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __ _ _ _ __ __ 119
KEY POINTS
1. Antigen combines with its specific antibody in an observable manner and the reaction between antigen and antibody
is specific. These antigen-antibody reactions in vitro are known as serological tests.
2. There are various types of antigen-antibody reactions. Some of these include precipitation, agglutination, complement
fixation test, immunoftuorescence and enzyme linked immunosorbent assay (ELISA).
3. When a soluble antigen reacts with its antibody in the presence of electrolytes (NaCl) at an optimal temperature
and pH, the antigen-antibody complex forms an insoluble precipitate and it is called precipitation. When instead of
sedimenting, the precipitate is suspended as floccules, the reaction is called flocculation. This is a modified form of
precipitation.
4. Flocculation test, radial immunodiffusion, immunoelectrophoresis, counterimmunoelectrophoresis and rocket electrophoresis
are different types of precipitation reactions.
5. Agglutination is an antigen-antibody reaction, in which a particulate antigen combines with its antibody in the
presence of electrolytes at an optimal temperature and pH, resulting in visible clumping of particles. It differs from
precipitation in which soluble antigen is present in contrast to particulate antigen of agglutination.
6. Slide agglutination test, tube agglutination test, heterophile agglutination test and passive agglutination test are different
types of agglutination reaction.
7. Passive agglutination reaction is a precipitation reaction which has been converted into agglutination test by
attaching soluble antigens to the surface of carrier particles such as latex particles, bentonite and red blood cells.
8. Latex agglutination test, haemagglutination test and coagglutination are examples of passive agglutination reaction.
9. Immunofluorescence test is of two types: direct immunoftuorescence test and indirect immunoftuorescence test. The
antibodies tagged with flourescent dye (i.e. , labelled antibodies) are used in these tests.
10. Enzyme linked immunosorbent assay (ELISA) has been applied widely for detection of a variety of antibodies and
antigens.
11. Different types of ELISA include sandwich ELISA , indirect ELISA and competitive ELISA.
12. Detection of HIV antibodies, mycobacterial antibodies, rotavirus in faeces and hepatitis B markers in serum are some
examples where ELISA is commonly used.
YOU MUST KNOW

1. Difference between precipitation and agglutination.
2. Prozone phenomenon.
3. Mechanism and applications of precipitation reaction.
4. Types of precipitation reaction.
5. Principles and applications of radial immunodiffusion, immunoelectrophoresis, counter current immunoelectropheresis
(CIEP).
6. Types of agglutination reactions and their uses.
7. Principles and uses of latex agglutination test and coagglutination.
8. Principle of complement fixation test.
9. Principles and uses of immunofluorescence tests and enzyme linked immunosorbent assay (ELISA).
STUDY QUESTIONS
1. Name various antigen-antibody reactions and describe the principle and application of precipitation reactions.
2. Define agglutination reaction? Discuss the principle and application of agglutination reactions.

120 - - - - - -- - - - -- - - - -- - - - -- - - - -- - - - - - Unit II: Immunology

(a) Immunoelectro phoresis (b) CIEP (c) Rocket electrophoresis
(d) Prozone phenomenon (e) Latex agglutination test (f) Coagglutination
(g) CFT (h) Conglutination (i) Neutralisation test.
4. Discuss briefly about:
(a) Irnmunofluorescence tests (b) RIA
(c) ELISA (d) Chemiluminescence assay.
U TIP E C OICE QUESTIONS (MCQs)

1. Which immunoglobuli n class is the most efficient to produce agglutination reaction?
2. Which immunoglobuli n class is the most efficient to produce precipitation reaction?
(a) IgG (b) lgM (c) lgA (d) lgE
3. Ring test is used for:
(a) C-reactive protein test (b) Streptococcal grouping by Lancefield technique
(c) Both of the above (d) None of the above
4. VDRL test is an example of:
(a) Agglutination test (b) Flocculation test (c) Immunofluorescence (d) All of the above
5. Radial immunodiffusio n can be used to estimate the following immunoglobuli n classes:
(a) IgG (b) IgM (c) IgA (d) All of the above
6. Counterimmun oelectrophoresi s is used for detecting:
(a) Hepatitis B antigens (b) Cryptococcal antigens (c) Neisseria meningitidis (d) All of the above
7. Tube agglutination test is used for serological diagnosis of:
(a) Enteric fever (b) Infectious mononucleosis
(c) Typhus fever (d) All of the above
8. Which of the following is/are example/s of heterophile agglutination test?
(a) Weil-Felix reaction (b) Paul-Bunnel test
(c) Streptococcus MG agglutination test (d) All of the above
9. Which of the following is/are example/s of passive agglutination test?
(a) Latex agglutination test (b) Haemagglutina tion test
(c) Coagglutination (d) All of the above
10. Which of the following is/are example/s of neutralisation test?
(a) Schick test (b) Antistreptolysin 'O' test
(c) Nagler reaction (d) All of the above
11. Direct immunofluorescence test may be used for detection of:
(a) Rabies virus antigens (b) Antibodies in syphilis
12. Indirect immunofluorescence test may be used for detection of:
(a) Rabies virus antigens (b) Antibodies in syphilis
13. ELISA can be used for detection of antigens andbr antibodies in:
(a) HIV (b) Rotavirus (c) Hepatitis B virus (d) All of the above
14. The technique of immunoblotting to analyse RNA is named:
(a) Southern blot (b) Nort~ern blot (c) Western blot (d) None of the above
Answers (MCQs):
1. (b) 2. (a) 3. (c) 4. (b) 5. (d)
6. (d) 7. (d) 8. (d) 9. (d) 10. (d)
11. (a) 12. (b) 13. (d) 14. (b)
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Chapter 15
COMPLEMENT SYSTEM
I. Complement
A. Components of Complement B. Classical Pathway
C. Alternative Pathway of Complement Activation D. Biological effects of Complement
E. Regulation of Complement Activation F. Biosynthesis of Complement
G. Quantitation of Complement
II. Deficiencies of the Complement
I. COMPLEMENT Complement is normally present in circulation

The 'complement' (C) refers to a system of some non- in inactive form, but when its activity is induced by
specific proteins present in normal human and animal antigen-antibody reaction or other stimuli, complement
serum, which are activated characteristically by antigen- components react in a specific sequence as a cascade
antibody reaction and subsequently lead to a number either through the classical or alternative pathway. Both
of biologically significant consequences. The term the pathways have same result i.e. lysis or damage of
complement was coined by Paul Ehrlich, because it target cell. Classical pathway is triggered by specific
augmented (complemented) the action of antibody. The antigen-antibody complex; the alternative pathway can
complement constitutes 10-15 percent of total human be initiated by endotoxin, lipopolysaccharides or zymosan
serum globulins. C as a whole is heat labile (inactivated (yeast cell wall).
at 56°C for 30 minutes) though some of its components
are heat stable. C ordinarily does not combine with either B. Classical Pathway
free antigen or antibody, but only with antigen-antibody Complement components react in a specific sequence,
complex. Only lgM, IgG 3, IgG 1 and IgG 2 in that order following activation by antigen-antibody complex, and
fix complement. This property is due to the presence of C results in immune cytolysis. This is known as the classical
binding site on the Fe portion of these immunoglobulins. pathway of complement (Fig. 15.1) .
The complement system consists of about 20 proteins The traditional model used to explain C activity for
which include the complement components, the properdin immune cytolysis is the lysis of erythrocytes sensitised by
system and the control proteins. its antibodies. The erythrocyte (E)-antibody (A) complex
is named EA and later on, when C components are
A. Components of Complement attached to EA, it is called EAC.
There are nine components of complement called Cl Immune cytolysis is initiated by the binding of
to C9. The component Cl is made up of three protein component Cl to EA. Clq is the recognition unit of Cl,
subunits named Clq, Clr and Cls. In normal serum hence, Clq reacts with the Fe piece of bound antibody
C3 is present in the highest concentration (1.2 mg/ml) molecule (IgM or IgG) in EA. The binding of Clq in
whereas C2 in the lowest concentration (0.015 mg/ml). the presence of calcium ions leads to activation of Clr
121
E+A
Ca++
Cl --------;• EA
+
EAC1
+
C4
C4a C4b
+
EAC14b
Mg++
+
C2
C2a C2b It has kinin like activity

and increases vascular
+ permeability.
C14b2a (C3 convertase)
+
C3
Chemotatic and /'-..__
anaphylatoxic /
properties -+- C3a C3b
C14b2a3b (C5 convertase)
i
C5
Anaphylatoxic and -+- c a C5b

5
chemotactic activity
-+- C6
-+- C7
It also sensitises C567 -+- binds to cell membrane and prepares
bystander cells to make
l
the cells for lysis by CB and C9
them susceptible to lysis
-+- ca
-+- C9
C14b2a
3b5b6789
Cell damage or lysis
Fig. 15.1 The classical pathway of complement

Ch 15: Complement System _ _ _ _ _ _ __;___ _ _ __ __ _ __ _ _ _ __ _ _ __ _ 123
and Cls. The activated Cls is an esterase which splits range of substances (activators) are known to activate
C4 into C4a and C4b, of which C4b joins the cascade. alternative pathway. These include bacterial endotoxins,
C14b in presence of magnesium ions act on C2 and forms yeast cell walls, IgA and D , the cobra venom factor
C2a and C2b. The larger fragment C2a attaches to C4 and the nephritic factor (a protein present in the serum
to form C42 which has enzymatic activity and is called of glomerulonephritis patient). The binding of C3b to
CJ convertase. The other C2 fragments possess kinin like an activator is the first step in the alternative pathway.
activity and increase vascular permeability. C42 splits C3 Although C3b is present in the circulation but in the
into C3a and C3b, of which, C3b joins the cascade. C3a free state it is rapidly inactivated by the serum protein
has chemotactic and anaphylatoxic properties. C14b2a3b factors H and I. However, bound C3b is protected from
has enzymatic activity and is referred to as C5 convertase. such inactivation.
It acts on CS, to split it into C5a (anaphylatoxic and The bound C3b, in the presence of Mg++, interacts
chemotactic) and C5b which joins the cascade. C6 and C7 with plasma protein factor B forming C3bB. The factor
join to form C567, some of which binds to cell membrane B portion of C3bB complex is split by factor D into
and prepares the cell for lysis by C8 and C9. Most of Ba and Bb. Bb fragment binds to C3b forming C3bBb.
C567 absorb to unsensitised 'bystander' cells making them This C3bBb is C3 convertase of alternative pathway (Fig.
susceptible to lysis by C8 and C9. In this way, C567 serves 15.2). This enzyme is extremely labile. The properdin or
to amplify the reaction. The unbound C567 complex has factor P helps to stabilise the enzyme C3 convertase. C3
chemotactic and leucocyte activating properties. The lysis convertase splits more C3 to C3a and C3b. The newly
is done by producing 'holes' approximately 100° A in formed C3b binds more factor B. The alternative pathway
diameter on cell membrane. This disrupts the osmotic then proceeds from C3 to C9 in the similar way as that
integrity of the cell membrane which results into release occurs in the classical pathway.
of the contents of the cell.
D. Biological Effects of Complement
C. Alternative Pathway of Complement 1. Bacteriolysis and Cytolysis
Activation Complement mediates immunological membrane
The activation of C3 has the major role in the damage. This results in bacteriolysis and cytolysis.
complement cascade. In the classical pathway, C3 Neutralisation of certain viruses requires the participation
activation occurs by classical C3 convertase (C142). This of complement.
activation of C3 without help of Cl42, is known as the
'alternative pathway'. 2. Amplification of Inflammatory Response
The alternative pathway contributes to antimicrobial C3a and C5a are anaphylatoxic by degranulation of
defence without requiring specific antibodies. A wide mast cells to release histamine and other mediators.
C3b in circulation Free C3b

+ inactivated by
Activator e.g. endotoxin factors H and I
.j,
C3b (Bound)
Mg++ .J., f-- factor B
C3bB
Ba .J., f-- Factor D
C3bBb (C3 convertase)
.j,
C3
Chemotactic f-- C3a C3b

and anaphylatoxic .J.,
Cascade
After this, remaining reaction (C3 onward) is the same as that in the classical pathway (Fig. 15.1 ).
Fig. 15.2 Alternative pathway of complement

124 - -- - - -- - - - - - - - - - - - -- - - - -- - - - - - - - Unit II: Immunology
They cause increased vascular permeability and are also inhibitors which bind to complement components and
chemotactic. C567 is chemotactic and also brings about halt their further function , and inactivators which destroy
reactive lysis. complement proteins.
3. Hypersensitivity Reactions 1. Inhibitors

Complement participates in Type II (cytotoxic) and Type Normal serum contains natural inhibitors of complement
III (immune complex) hypersensitivity reactions. which regulate the complement activity. A heat labile
alpha neuraminoglycoprotein in serum inhibits Cl
4. Endotoxic Shock esterase. The S protein binds to C567 and modulates
Endotoxin can activate the alternative pathway of the cytolytic action of the membrane attack complex.
the complement cascade. In endotoxic shock there is
excessive C3 activation and platelet adherence. Platelet 2. Inactivators
lysis and release of large amount of platelet factor lead A serum betaglobulin, factor I provides control of C3
to disseminated intravascular coagulation (DIC) and activation. It cleaves C3b and C4b. Another betaglobulin
thrombocytopenia. Gram negative septicaemias and factor H regulates alternative pathway by binding to C3b.
dengue haemorrhagic syndrome may have a similar C4 binding protein is a normal serum protein, which
pathogenesis. Schwartzman reaction is a good model of controls the activity of cell bound C4b. An enzyme named
excessive C3 activation and depletion of complement anaphylatoxin inactivator degrades C3a, C4a and C5a
protects against this reaction. which are anaphylatoxins released during the complement
cascade.
5. Immune Adherence
Complement bound to antigen-antibody complexes F. Biosynthesis of Complement
adheres to erythrocytes or to platelets. This reaction is Complement components are synthesised in various sites
called immune adherence. Adherent particles are rapidly as follows:
phagocytosed and thus help in eliminating the pathogenic Site Complement Component
microorganisms. C3 and C4 are necessary for immune Intestinal epithelium Cl
adherence. Macrophages C2, C4
Spleen C5, CS
6. Opsonisation Liver C3 , C6, C9
An important function of complement is to facilitate the Not known C7
destruction of pathogens by phagocytic cells. Phagocytic
cells (macrophages, monocytes, neutrophils and others) Rise in complement levels (C4, C3, CS and C6) occurs
possess surface receptors for C3b. If immune complexes in response to acute inflammation. Complement alongwith
have activated the complement system, the C3b bound some other plasma proteins (CRP) are collectively known
to them stimulate phagocytosis and removal of immune as 'acute phase substances' which show a rise in acute
complexes. This facilitated phagocytosis is referred to inflammation.
as opsonisation.
G. Quantitation of Complement
7. Autoimmune diseases Complement activity of serum is measured by estimating
Serum complement levels are decreased in many the highest dilution of the serum that lyses sheep
autoimmune diseases such as systemic lupus erythematosus erythrocytes sensitised by antierythrocytic antibody.
and rheumatoid arthritis. They may, therefore, be involved Complement components can be measured by radial
in a pathogenesis of autoimmune diseases. immunodiffusion method but this does not differentiate
between active and inactive fractions.
E. Regulation of Complement Activation
The destruction of tissues by excessive activity of II. DEFICIENCIES OF THE COMPLEMENT
complement is prevented by regulation of complement Deficiency of complement leads to poor host resistance
activation. Apart from damage to tissues, unchecked against infections and results in recurrent bacterial and
complement activity can also cause exhaustion of fungal infections and collagen disorders. Some deficiency
the complement system. Control of the complement states of various complement components are listed in
cascade is exerted by two types of regulatory proteins: Table 15.1.

Ch 15: Complement System _ _ _ _ __ _ _ _ _ _ _ _ _ __ __ _ _ _ _ _ __ __ _ 125
Table 15.1 Complement Deficiency and Associated Syndromes

Deficiency Syndrome
Cl inhibitor Hereditary angioneurotic oedema

Cl , C2, C4 components Systemic lupus erythematosus (SLE) and other collagen vascular diseases
C3 and its regulatory protein C3b inactivator Recurrent pyogenic infections
CS , C6, C7, C8 , C9 components Bacteraemia, mainly with Gram negative diplococci; toxoplasmosis
C9 No particular disease
KEY POINTS
1. The complement refers to a system of some non-specific proteins present in normal human and animal serum,
which are activated characteristically by antigen-antibody reaction and subsequently lead to a number of biologically
significant consequences.
2. There are nine components of complement Cl to C9.
3. Complement components react in a specific sequence as a cascade either through the classical or alternative
pathway .
4. Classical pathway is triggered by specific antigen-antibody complex; the alternative pathway can be initiated by
endotoxin, lipopolysaccharides or zymosan (yeast cell wall).
5. Deficiency of complement leads to poor host resistance against bacterial and fungal infections and collagen
disorders.
YOU MUST KNOW

1. Components of complement.
2. Steps in activation of classical pathway and alternative pathway of complement.
3. Biological effects of complement.
4. Complement deficiency and associated syndromes.
ST JDY QUESTIONS
1. Discuss briefly the classical pathway of complement activation.
(a) Alternative pathway of complement (b) Biological effects of complement.
ULT PLE CHO CE QUESTIONS (MCQs)

1. Classical pathway of the complement is activated by:
(a) Antigen (b) Antibody
(c) Antigen-Antibody complex (d) None of the above
2. The alternative pathway of the complement is initiated by:
(a) Endotoxins (b) Lipopolysaccharides
(c) Yeast cell walls (d) All of the above
3. What is the chemical nature of components of complement?
(a) Protein (b) Lipopolysaccharide
(c) Lipid (d) None of the above

126 - - - - - -- - - - - - - -- -- - - -- -- -- - -- - -- - Unit II: Immunology
4. First component of complement which binds to antigen-antibody complex in classical pathway is:
(a) C lq (b) Clr
(c) Cls (d) C3
5. Which component of complement is present in the highest concentration in the serum?
(a) C l (b) C2
~O ~G
6. Factor B and Factor D are important components of:
(a) Classical pathway of complement (b) Alternative pathway of complement
Answers (MCQs):
1. (c) 2. (d) 3. (a) 4. (a) 5. (c) 6. (b)
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Chapter 16
STRUCTURE AND FUNCTIONS OF IMMUNE SYSTEM
I. The Lymphoid System

A. Central Lymphoid Organs B. Peripheral Lymphoid Organs
C. Cells of Lymphoreticular System
II. Major Histocompatibility Complex (MHC)
A. The HLA Complex B. HLA Typing
The lymphoreticular system is responsible for immune Types of Immune Response

response and it consists of lymphoid and reticuloendothelial The specific immune response to an antigen can be of
components. The lymphoid cells (lymphocytes and plasma two types:
cells) are responsible for specific immunity. The phagocytic 1. The humoral or antibody mediated immunity (HMI
cells (polymorphonuclear leucocytes and macrophages) or AMI)
which are part of reticuloendothelial system, are mainly It is mediated by antibodies which are produced by
concerned with scavenger functions, of eliminating plasma cells.
microorganisms and other foreign particles from blood 2. The cell-mediated immunity (CMI)
and tissue, thus contributing to non-specific immunity. It is mediated by sensitised lymphocytes.
The central lymphoid organs are structures in
which precursor lymphocytes proliferate, develop and
I. THE LYMPHOID SYSTEM become immunologically competent. After acquiring
Lymphoid cells immunological capabilities, the lymphocytes migrate to
e.g. Lymphocytes peripheral lymphoid organs, where appropriate immune
Plasma cells response occurs when exposed to an antigen.
Lymphoid organs
A. Central (Primary) lymphoid organs A. Central Lymphoid Organs
e.g. Thymus 1. Thymus
Bursa of Fabricius in birds The primary function of the thymus is to produce thymic
Bone marrow in mammals lymphocytes. It is a major site for lymphocyte proliferation
B. Peripheral (Secondary) lymphoid organs in the body. During maturation, the lymphocytes acquire
e.g. Spleen new surface antigens (Thy antigens) and are called
Lymph nodes T-lymphocytes or T-cells (thymus dependent) . Unlike in
Mucosa associated lymphoid tissue (MALT) the peripheral lymphoid organs, lymphocyte proliferation
Lymphoid tissues in gut, lungs, liver, bone in the thymus is not dependent on antigenic stimulation.
marrow The thymus confers immunological competence on these
127
cells during their stay in the organ. Majority of these 2. Spleen

proliferative lymphocytes die in the thymus and only about It is the largest lymphoid organ. It contains two distinct
one percent migrate to the secondary lymphoid organs. areas- White and red pulps. Spleen consists of a cortex
These T-cells circulate through blood and lymphatics containing densly packed T and B lymphocytes and a
in man. The T-lymphocytes are responsible for cell loosely structured medulla. The periarterial lymphoid
mediated immunity (CMI). The importance of thymus collections in the white pulp are called Malphigian
in CMI is evident from, the lymphopaenia, deficient graft corpuscles or follicles. Germinal centres develop within
rejection and so called 'runting disease' seen in neonatally white pulp following antigenic stimulation. Surrounding
thymectomised mice. Congenital aplasia of the thymus the germinal centre is a mantle layer of lymphocytes.
in man (Di-George syndrome) and in mice ('nude mice') Like in lymph node, T and B lymphocyte areas are
are other examples of deficient CMI. segregated.
(i) B-lymphocyte area: Perifollicular region, germinal
2. Bone Marrow centre and mantle layer.
Some lymphoid cells develop and mature within the (ii) T-lymphocyte area: The lymphatic sheath immediately
bone marrow and are called B-cells (B for Bursa or Bone surrounding the central arteriole.
marrow). In birds, these B lymphocytes mature in Bursa of The spleen serves as the graveyard for blood cells
Fabricius. Bone marrow in mammals is equivalent to bursa. and as a systemic filter for trapping circulating blood
Bone marrow is the site for stem cell proliferation, the borne foreign particles.
origin of pre-B cells and their maturation into functional
B-lymphocytes. Competence for IgM production by B-cells 3. Mucosa Associated Lymphoid Tissue (MALT)
is acquired early (14th day of embryonation) and for IgG The subepithelial accumulation of lymphoid tissue
late (21st day). Mature B-lymphocytes also acquire C3 protects the mucosa lining the alimentary, respiratory
and Fe receptor on their surface. B-lymphocytes bear and genitourinary tracts which is exposed to numerous
either surface IgM alone or in combination with IgG antigens. The lymphoid tissue may be present as diffuse
or IgA depending upon the production of a particular collection of lymphocytes or as aggregates with well
immunoglobulin class. Following appropriate antigenic formed follicles (pharyngeal tonsils; small intestinal
stimulation, B-lymphocytes transform into plasma cells Peyer's patches). These are collectively named as mucosa-
and secrete antibodies. associated lymphoid tissues (MALT).
Such lymphoid tissues in the gut are called the gut
B. Peripheral Lymphoid Organs associated lymphoid tissue (GALT) and those in the
1. Lymph Nodes respiratory tract, the bronchus associated lymphoid
The lymph nodes are round bodies and lie along the tissue (BALT). MALT contains both lymphoid cells (B
course of lymphatic vessels. The mature node has an and T cells) as well as phagocytes. The predominant
outer cortex and an inner medulla. A paracortical area immunoglobulin in the mucosa is secretory IgA, other
lies between cortical follicles and base of medullary immunoglobulin classes are also formed locally.
cords. The cortex contains primary lymphoid follicles
(accumulation of lymphocytes) within which secondary C. Cells of Lymphoreticular System
follicles (germinal centres) develop during antigenic These consist of structural cells (reticulum cells, endothelial
stimulation. Besides these lymphocytes, the lymphoid cells and fibroblasts) and immunologically competent
follicles also contain dendritic macrophages which capture cells (lymphocytes, plasma cells and macrophages).
and process the antigen.The medulla is predominantly In the circulating blood, about 70% lymphocytes are
composed of medullary cords which are elongated T-lymphocytes, 20% B-lymphocytes and 10% Null Cells.
branching bands of the lymphocytes, plasma cells and
macrophages. The cortical follicles and medullary cords 1. Lymphocytes
contain B-lymphocytes whereas paracortical area (a These are small, round cells present in blood, lymph,
zone between the cortical follicles and medullary cords) lymphoid organs and many other tissues. Lymphocytes
contains T-lymphocytes. constitute 20-45% of leucocyte population in peripheral
Lymph nodes are filter for the lymph. They phagocytose blood, while they are predominant cell types in lymph and
foreign particles including microorganisms. They help in lymphoid organs. Lymphocytes may be small (5- 8 µm),
the proliferation and circulation of T and B lymphocytes. medium (8- 12 µm) and large (12- 15 µm). Among these, the
They enlarge following antigenic stimulation. small lymphocytes are the most numerous. According to

Ch 16: Structure and Functions of Immune System _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ 129
their life span, the lymphocytes are classified as short lived produce certain activation products (lymphokines) and
(10-12 days) and long lived (three years or more, or even induce CMI, while stimulated B-cells divide and transform
for life). The short lived lymphocytes act as effector cells into plasma cells which synthesise antibodies and induce
in immune response, while the long-lived cells are store HMI.
houses of immunological memory and act as memory
cells. The long-lived lymphocytes are mostly thymus DIFFERENCES BETWEEN T AND B CELLS
derived. 1. T cells bind to sheep erythrocytes to form rosettes
(E rosette or SRBC rosette) while B cells do not. T
LYMPHOCYTE RECIRCULATION cells bind to sheep erythrocytes by CD 2 antigen.
Lymphopoiesis occurs at three places- the bone marrow, 2. B cells also form rosette (EAC rosette or Erythrocyte
central lymphoid organs and the peripheral lymphoid amboceptor complement rosette) but with sheep
organs. The lymphocytes of these three sites mix together erythrocytes coated with antibody (amboceptor) and
in the process known as lymphocy te recirculation. There is complement. T cells are unable to form EAC rosette.
a constant circulation of lymphocytes through the blood, B-cell surface has C3 receptor which binds to
lymph, lymphatic organs and tissues. This recirculation complement of EAC.
of lymphocytes from one site to another ensures that 3. T cells have thymus specific antigens on surface, which
lymphocytes of appropriate specificity would reach the site are lacking on B-cells.
during their careless wandering, following introduction of 4. All T cells have CD3 receptor on their surface.
antigen. Most of the recirculating lymphocytes are T-cells. 5. B-cells have immunoglobulins on surface and surface
B-lymphocytes tend to be more sessile. receptors for the Fe fragement of IgG. These are
Lymphocytes which are 'educated' by the central absent on T-cells. Instead T cells have T cell receptors
lymphoid organs become immunologically competent (TCR) composed of two chains of polypeptides,
cells (ICCs). They are fully equipped to deal against an linked to CD3.
antigen. They serve the following functions: 6. Blast transformation of T-cells occur with mitogens
1. recognition of antigens ('R' for recognition). such as phytohaemagglutinin (PHA) or concanavalin
2. storage of immunological memory ('R' for A (ConA) while B cells have similar transformation
rememberance or memory). with bacterial endotoxins.
3. immune response to antigens ('R' for response). 7. Viewed under the scanning microscope, B-cells possess
The nature of immune response depends on the numerous microvilli on their surface while T-cells are
type of cells stimulated by an antigen. Stimulated T-cells free of these projections (Table 16.1).
Table 16.1 Characteristic Features of T Cells and B Cells

Feature T Cell B Cell
A. Location
I. Peripheral blood 65-85% 15-25%
2. Lymph node 60-75% 30-35%
3. Spleen 25-45% 55-60%
4. Thoracic duct 80-90% 10-20%
5. Thymus 96% egligible
B. Thymus specific antigens +
C. CD3 receptor +
D. Surface immunoglobulins +
E. Receptor for Fe fragment of IgG +
F. SRBC rosette (E-rosette) +
G. EAC rosette (C3 receptor) +
H. Numerous microvilli on surface +
I. Blast transformation with
I. anti-CD3 +
2. anti-lg +
3. Phytohaemagglutinin (PHA) +
4. Concanavalin A +
5. Endotoxins +

130 - - - - - - -- -- -- - - - - - - - - - - - - - - - - - -- - Unit II: Immunology
(i) T-lymphocytes Delayed type hypersensitivity (DTH) cells

T-lymphocytes can be classified into different subpopulations These cells are responsible for delayed (Type IV)
or subsets based on functional capacity and on surface hypersensitivity. These cells are not distinguishable from
antigenic determinants. Th cells on the basis of surface markers. They possess
CD4 markers as those in Th cells. It is believed that these
( a) ON THE BASIS OF FUNCTIONS cells may be one type of T helper cells (Th I).
Various subsets are as follows: These cells secrete different lymphokines (e.g.
gamma interferon) responsible for delayed type of
REGULATORY T CELLS hypersensitivity.
T helper (CD4) cells
There are two subsets of T helper (Th) cells, Thl and
Th2. Thl produce mainly the cytokines such as interferon Mixed lymphocyte reactivity (MLR) cells
gamma (IFN-y) and interleukin-2 (IL-2) which activate These are cells which undergo rapid proliferation in mixed
macrophages and T cells to promote cell mediated lymphocytic reactivity.
immunity (CMI). Th2 are the helper T cells which interact
with B-lymphocytes to develop them into plasma cells that (b) ON THE BASIS OF SURFACE ANTIGENIC
produce immunoglobulins. This interaction is mediated DETERMINANTS (Table 16.2)
by lymphokines (such as IL4, IL5, IL6) secreted by Th2
Table 16.2 Subsets of Cells in Man
cells. Thus T helper cells facilitate B cell response to
produce immunoglobulins. Type of Cell Old Term ew Term
Balanced activity of helper and suppressor T-cells Pan T marker T3 CD3
produces optimum immune response. Overactivity (present on all T-cells) Tl! CD2
of helper cells or decreased suppressor cell activity T helper/delayed T4 CD4
leads to autoimmunity, while decreased helper cell hypersensitiviiy
function or increased suppressor cell activity results in T cytotoxic/suppressor T8 CD8
immunodeficiency state. Helper cells are about 65 percent
B cells B4 CD19
and suppressor cells about 35 percent of circulating
T-lymphocytes.
The surface antigens on the T-cells can be detected
T suppressor (CDB) cells by monoclonal antibodies. These thymic antigens or
Suppressor T-cells block immune response by their action markers were previously designated by the letters Tl to
on T- helper or B cells. Tl 1. These markers are now designated as CD (clusters
of differentiation). More than fifty T-cell antigens are
EFFECTOR CELLS identified, relevant CD molecules are given below.
Cytotoxic T (Tc) cells CD2 (Tll)
These cells can lyse specific target cells whose surface It is an early antigen which acts as a receptor
antigens are recognised by them e.g. virally infected for sheep red blood cells. It is present in all mature
cells, tumour cells. Cytotoxic T cells possess membrane T-lymphocytes.
glycoprotein CD8. CD3 (T3)
It is present on all T-lymphocytes. It is closely
associated with T cell receptor (TCR) on cell membrane.
T cells It is supposed to be involved in transmitting signal to
the cell following antigen binding.
CD4 (T4)
Regulatory T cells Effector T cells
(t) T helper (CD4) cells (!) Cytotoxic T-cells It is present on all T-helper cells. It is also present
on macrophages and monocytes. CD4 also acts as a
(it) T suppressor (CD8) (ii) Delayed type hyper- receptor for HIV (human immunodeficiency virus). CD4
cells sensitivity (DTH) cells cells recognise MHC class II antigens.
(ii!) Mixed lymphocyte CDS (Tl)
reactivity (MLR) Its function is not known but it is found in majority
of T-cells from the stage of maturation.

CD8 (T8) can synthesise an antibody of single specificity, of a

It is present on suppressor T cells and cytotoxic single immunoglobulin class (IgM or IgG or IgA). An
T-cells. CD8 cells recognise MHC class I antigens. exception is seen in primary immune response, when a
plasma cell synthesising IgM initially, may later switch
(ii) Null Cells over to IgG production. Mature plasma cells survive
The small proportion of lymphocytes which are neither only a few weeks. However, myeloma plasma cells are
T-cells nor B-cells are known as null cells. Among the null capable of an unlimited number of cell divisions. Plasma
cells, killer cells (K cells), natural killer (NK) cells and cells are present in germinal centres of lymph nodes,
lymphokine activator killer (LAK) cells are recognised. spleen and diffuse lymphoid tissue of respiratory and
K cells are now classified with NK cells. alimentary tracts. Although plasma cell is the principle
antibody producing cell, but other cells like lymphocytes,
(a) Killer Cells (K Cells) lymphoblasts and transitional cells may also synthesise
These lymphocytes possess surface receptor for Fe portion antibody to a certain extent.
of IgG. These cells are capable of killing target cells B-lymphocytes bind to sheep RBC (SRBC) coated
sensitised with IgG antibodies. They do not require with antibody and complement (EAC) forming EAC
complement for such lysis or killing. These are responsible rosette. B-lymphocytes also have immunoglobulin on
for antibody dependent cell mediated cytotoxicity (ADCC). their surface. They possess receptor for Fe fragment of
This antibody dependent cell mediated cytotoxicity differs IgG. There are numerous microvilli on the surface of
from the action of cytotoxic T lymphocytes, which is B-lymphocytes when these cells are viewed under scanning
independent of antibody. electron microscope.
Interaction of T-lymphocytes and B-lymphocytes in
cell mediated immunity (CMI) and humoral or antibody
(b) Natural Killer Cells (NK Cells) mediated immunity (AMI) is shown in Fig. 16.1.
These are large lymphocytes which contain azurophilic
granules in the cytoplasm and for which, they are called
large granular lymphocy te (LGL). NK cells are cytolytic 2. Phagocytic Cells
for virally transformed target cells, certain tumor lines The phagocytic cells are the mononuclear macrophages
and are involved in allograft rejection. NK cells are found (of blood and tissues) and the polymorphonuclear
in spleen and peripheral blood. rnicrophages.
Action of NK cells is independent of antibody in
contrast to K cells which are dependent on antibody. (i) Macrophages
Their activity is 'natural' or 'nonimmune' as it does not The blood macrophages (monocytes) are the largest
require sensitisation by prior antigenic contact. They of the lymphoid cells present in peripheral blood.
release several cytolytic factors. One of these, perfo rin , Tissue macrophages (histiocytes) are named as alveolar
which resembles complement component C9, causes macrophages in the lungs and Kupffer cells in the liver.
transmembrane pores through which cytotoxic factors Tissue macrophages proliferate locally. Macrophages
enter the cell and destroy it by apoptosis (programmed express many surface receptors such as Ia proteins, those
cell death). NK cell activity is enhanced by interferon . for the Fe part of IgG, activated complement components
It is believed that NK cells play an important role in and various lymphokines.
antiviral and antitumour immunity.
Functions
(c) Lymphokine Activated Killer Cells (LAK Cells) (1) Phagocytosis
These are NK cells treated with interleukin-2 The primary function of macrophages is phagocytosis.
(IL-2), which are cytotoxic to tumour cells without Macrophage envelops the foreign particle by small
affecting normal cells. IL-2 also acts as a growth factor pseudopodia. The membranes of pseudopods fuse to
for NK cells. form phagosome. Lysosomes containing hydrolytic
enzymes fuse with the membrane of phagosome to form
(iii) B-Lymphocytes and Plasma Cells phagolysosome. Most bacteria are killed intracellularly by
Antigenically stimulated B-lymphocytes undergo blast lysosomal enzymes, however, some (bacilli of brucellosis
transformation to become plasmablasts, intermediate and tuberculosis) may resist and multiply intracellularly
transitional cells and then plasma cells. A plasma cell for transportation to other locations.

132 - - - - - - - - - - -- - - - - - -- - - - - - - - - - - - - - Unit II: Immunology
Memory T cells
T lymphocytes / Cytotoxic
T (Tc)cells -----,),• CMI
/
T helper (CD4) cells
lgG
B lymphocytes ----..-\----'I), • Plasma cells -----1),•

lgA
IgM
IgD
------i),• AMI
lgE
Memory B cells
Fig. 16.1 Interaction of T and B lymphocytes
(2) Specific immune response (ii) Microphages

(i) They trap and process bacterial antigens and present The microphages contain polymorphonuclear leucocytes
them in optimal concentration to the lymphocytes (neutrophils, eosinophils and basophils) of blood.
for induction of specific immune response. Too high
a concentration of antigen may be tolerogenic and (a) Neutrophils
too low a concentration may not be able to induce They have non-specific phagocytic property. They play
a immune response. It is an essential prerequisite a prominent role of phagocytosis in acute inflammation
for some antigens. The T cells accept the processed by locating a foreign particle.
antigen on macrophage only when both the cells (T
cell and macrophage) possess surface determinants (b) Eosinophils
coded for by the same major histocompatibility Eosinophils are less phagocytic than neutrophils. They are
complex (MHC) genes. This is known as MHC found in large numbers in allergic inflammation, parasitic
restriction. infections and diseases associated with antigen-antibody
complexes, but their function is not clear.
(ii) Activated macrophages secrete IL-1 which induces
T-cells to synthesise IL-2. IL-2 facilitates T-cell (c) Basophils
activation and thus initiate CMI. Activated T-cells They are present in blood and tissues (mast cells). Their
also help B-cells to form antibodies. cytoplasm contain basophilic granules which contain
heparin, histamine, serotonin and other hydrolytic
(3) Antitumour activity and graft rejection enzymes. Degranulation of mast cells release these
The functional activity of macrophages may be pharmacologically active agents resulting in anaphylaxis
increased by lymphokines, complement components or and atopic allergy.
interferon. Activated macrophages are not antigen specific.
They secrete a number of biologically active substances, 3. Dendritic Cells
including interleukin-I, tumour necrosis factor and colony Although macrophages are the major antigen presenting
stimulating factor (CSF). When stimulated by cytophilic cells (APC) but the dendritic cells also perform this
antibodies and certain lymphokines, macrophages become function. Dendritic cells are bone marrow derived cells.
'armed'. These armed macrophages are capable of antigen They possess MHC Class II antigens. They have little
specific cytotoxicity and are important in antitumour or no phagocytic activity. They are present in peripheral
activity and graft rejection. blood and in the peripheral lymphoid organs. These cells

are specially involved in the presentation of the antigens (All of these are present within HLA-D
to T cells during primary immune response. region of HLA complex.)
Class III - Complement loci encode for C2, C4 and
Factor B of complement system and
II. MAJOR HISTOCOMPAT IBILITY tumour necrosis factors (TNF) alpha
COMPLEX (MHC) and beta.
Transplants from one individual to another member of A locus is the position where a particular gene is
the same species ('allografts') are recognised as foreign located on the chromosome. HLA loci are multiallelic i.e.
and rejected. Gorer (1930) identified the antigens the gene present on the locus can be any one of several
responsible for allograft rejection in inbred mice that alternative forms (alleles). Each allele determines a distinct
led to the discovery of the major histocompatibility antigen. There are 24 alleles at HLA-A locus and 50 at
complex (MHC) . He identified two blood group antigens HLA-B. HLA system is very pleomorphic. Every
in mice. Antigen 1 was common to all strains of mice individual inherits one set of HLA-genes from each
while antigen 2 was present in certain strains only and parent.
was responsible for allograft rejection. This was named The nomenclature of the HLA-system is regulated
H2 antigen. The H2 antigen was found to be the major by official committee of WHO. They have officially
histocompatibility antigen and to be coded for by a closely recognised alleles and their corresponding antigens by
linked multiallelic clusters of genes called the major the locus and a number e.g. HLA-Al, HLA-DR7 etc.
histocompatibility complex named as H-2 complex. The
MHC in humans is known as the human leucocyte antigen 1. Class I MHC Antigens (A, B, C)
(HLA) complex. The major transplantation antigens of The MHC class I antigens are present on the surface of
man include carbohydrate antigens of erythrocytes (blood all nucleated cells. They are involved in graft rejection
groups) and glycoprotein antigens of cell membranes and cell mediated cytolysis. The cytotoxic T cells (CD8)
(HLA system). In 1980, Snell, Dausset and Benacerraf recognise MHC class I antigens for their action.
were awarded the Nobel Prize for their work on MHC
and the genetic control of immune response. 2. Class II MHC Antigens (DR, DQ and DP)
They have a very limited distribution and are principally
A. The HLA Complex found on the surface of macrophages, monocytes, activated
Histocompatibility antigens mean cell surface antigens T-lymphocytes (CD4) and B-lymphocytes. They are
that evoke immune response to an incompatible host primarily responsible for the graft-versus-host response
resulting in allograft rejection. These alloantigens are and the mixed leucocyte reaction (MLR). The immune
present on surface of leucocytes in man and are called response genes (Ir) in mice are identical to MHC class
human leucocyte antigens (HLA) and the set of genes II genes in man. The antigens coded for by the Ir genes
coding for them is named the HLA Complex. are termed the Ia (I region associated) antigens.
The HLA complex of genes is located on short arm
of chromosome 6 and is grouped in three classes (Fig. 3. Class III MHC Antigens
16.2). Class III genes encode C2, C4 complement components
Class I HLA-A, HLA-B and HLA-C of the classical pathway and properdin factor B of the
Class II HLA-DR, HLA-DQ and HLA-DP alternative pathway.
Cla:;;s II Class Ill Class I
.. -.
DR DQ DP C4a C4b BF C2 TNF B C A
Fig. 16.2 HLA complex

134 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Unit II: Immunology
B. HLA Typing 2. Paternity determination

Antisera from multiparous women are collected for HLA 3. Diseases and HLA association
typing. These multiparous women tend to have antibodies HLA-B27 - Ankylosing spondylitis
to HLA antigens of their husbands, due to sensitisation HLA-DR4 - Rheumatoid arthritis
during pregnancy. However, monoclonal antibodies are
now available. Typing is done serologically by complement MHC RESTRICTION
dependent cytotoxic reaction. Lymphocytes of donor is T cells respond to processed antigens on macrophages
typed against recipient sera in the presence of complement. only when they are presented along with the self-MHC
However, serological typing is not possible for HLA-D antigen. This is called MHC restriction. Both class I and
and HLA-DP antigens. HLA-D is detected by mixed class II MHC antigens interact in this phenomenon.
leucocytic reaction (MLR) and HLA-DP by primed Cytotoxic T cells (CD8) react to an antigen in association
lymphocyte typing (PLT). with Class I MHC antigens while helper T cells (CD4)
recognise class II MHC antigens. Cytotoxic T cells can
Indications of HLA Typing kill or lyse virally infected target cells only when T cells
1. Tissue typing prior to transplantation and target cells are of the same MHC type.
KEY POINTS
1. The lymphoreticular system is responsible for immune response and it consists of lymphoid and reticuloendothelial
components.
2. The lymphoid cells (lymphocytes and plasma cells) are responsible for specific immunity.
3. Lymphocytes which are 'educated' by the central lymphoid organs become immunologically competent cells (ICCs).
They are fully equipped to deal against an antigen.
4. The nature of immune response depends on the type of cells stimulated by an antigen. Stimulated T-cells produce
certain activation products (lymphokines) and induce cell mediated immunity (CMI), while stimulated B-cells divide
and transform into plasma cells which synthesise antibodies and induce humoral immunity.
5. The surface antigens on the T-cells can be detected by monoclonal antibodies. These thymic antigens are designated
as CD (clusters of differentiation) .
6. On the basis of functions, T cells can be classified into T helper ( CD4) cells and T suppressor ( CD8) cells.
7. Histocompatibi/ity antigens mean cell surface antigens that evoke immune response to an incompatible host resulting
in allograft rejection. These alloantigens are present on surface of leucocytes in man and are called human leucocyte
antigens (HLA) and the set of genes coding for them is named the HLA complex.
YOU MUST KNOW

1. Differences between T and B cells.
2. Clusters of differentiation (CD).
3. Killer cells and Natural killer cells.
4. Human leucocyte antigen (HLA) and the HLA complex.
STUDY QUESTIONS
1. Differentiate between T and B cells in a tabulated form .
(a) Subsets of T lymphocytes (b) B-lymphocytes
(c) Null cells (d) Mucosa associated lymphoid tissue.

Ch 16: Structure and Functions of Immune System _ _ __ _ _ _ _ _ _ _ __ _ _ _ _ __ _ 135
3. Describe briefly about:

(a) Major histocompatibility complex
(b) HLA typing
(c) MHC restriction.

1. E-rosettes are formed by
(a) T cells (b) B cells
(c) Macrophages (d) Monocytes
2. EAC rosettes are formed by
(c) Macrophages (d) None of the above
3. CD 3 receptor is present on:
(c) Macrophages (d) Monocytes
4. CD4 antigen is present on:
(a) T-helper cells (b) Macrophages
(c) Monocytes (d) All of the above
5. CDS antigen is present on:
(a) T helper cells (b) T suppressor cells
6. Natural killer cells play an important role in:
(a) Antiviral activity (b) Antitumour activity
(c) Allograft rejection (d) All of the above
7. CDS cells can recognise:
(a) MHC class I antigens (b) MHC class II antigens
(c) MHC class III antigens (d) All of the above
8. Class II MHC antigens are present on:
(a) Macrophages (b) Monocytes
(c) Activated T lymphocytes (CD4) (d) All of the above
9. Which of the following HLA types is associated with ankylosing spondylitis?
(a) HLA-B27 (b) HLA-DR4
(c) HLA-DP (d) None of the above
10. Which of the following HLA types is associated with rheumatoid arthritis?
(a) HLA-B27 (b) HLA-DR4
(c) HLA-Al (d) None of the above
Answers (MCQs):
1. (a) 2. (b) 3. (a) 4. (d) 5. (b)
6. (d) 7. (a) 8. (d) 9. (a) 10. (b)
---- • .. ----

Chapter 17
IMMUNE RESPONSE
I. Introduction
II. Humoral Immune Response
A. Primary and Secondary Humoral Responses B. Fate of Antigen in Tissues
C. Production of Antibodies D. Theories of Antibody Formation
E. Monoclonal Antibodies F. Factors Influencing Antibody Production
Ill. Cell Mediated Immune Responses
A. Induction of CMI B. Cytokines
C. Detection of CMI D. Transfer Factor
IV. Immunological Tolerance
I. INTRODUC TION II. HUMORAL IMMUNE RESPONSE

The specific reactivity induced in a host following an Antibody production follows a characteristic pattern (Fig.
antigen stimulus is known as the immune response. It 17. I) which consists of:
is of two types: I. a lag phase- The immediate stage following antigenic
1. Humoral or antibody mediated immunity stimulation when no antibody is detectable m
2. Cell-mediated Immunity circulation.
Antibody mediated immunity (AMI) provides 2. a log phase-There 1s steady nse m titre of
primary defence against most extracellular bacteria and antibodies.
helps in defence against viruses those infect through 3. a plateau-There is an equilibrium between antibody
respiratory or intestinal tracts. AMI also participates synthesis and catabolism.
in the pathogenesis of immediate (type I, II and III)
hypersensitivity and certain autoimmune diseases. Cell
mediated immunity (CMI) protects against fungi, viruses Lag Log Plateau Decline
i--+f• )I• )1-----
and intracellular bacteria like M. tuberculosis, M. leprae
and parasites such as Leishmania and trypanosomes. It
plays an important role in allograft rejection, graft-versus- ''
'
-
host reaction (GVH), and mediates the pathogenesis of
delayed (type IV) hypersensitivity and certain autoimmune
diseases. It also provides immunological surveillance and
immunity against cancer. Both AMI and CMI usually
i
Antigen
Time
develop together, though at times one or other may be Fig. 17.l Primary humoral response
the predominant type.
136
Ch 17: Immune Response _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ 137
4. the phase of dec/ine- Catabolism exceeds the phase'. It is followed by an increase in antibody level
production and the titre falls. exceeding the initial level. These effects of repeated
antigenic stimulus forms the basis of secondary humoral
A. Primary and Secondary Humoral Responses response.
The primary humoral response has a long lag phase and
low titre of antibodies that do not persist for long. In B. Fate of Antigen in Tissues
contrast, the secondary humoral response has a short or Antigens introduced intravenously are rapidly localised
negligible lag phase and a much higher level of antibodies in the spleen, liver, bone-marrow, kidneys and lungs.
that last for long periods. (Fig. 17.2). The antibody formed About 70-80% of these antigens are broken down by
in the primary humoral response is predominantly IgM reticuloendothelial cells and excreted in the urine. On
and in secondary humoral response IgG. The duration the other hand, antigens introduced subcutaneously are
of the lag phase depends upon the nature of antigen, its mainly localised in the draining lymph nodes, only small
amount, route of administration and species of animal. amount being found in the spleen.
With some antigens such as diphtheria toxoid, the lag Antigens are presented to immunocompetent cells in
phase in primary humoral response may be 2-3 weeks, two ways- by macrophages and by the dendritic cells in
while with pneumococcal polysaccharide, it may be as the lymph node follicles. With many antigens, processing
short as a few hours. by macrophages appears to be a necessary prerequisite
for antibody formation. Both macrophages and dendritic
cells present the antigen, native or processed, at the cell
surface. Macrophages modulate the dose of antigen
Secondary response
presented to lymphocytes so that it may be optimum to
induce immune response.
C. Production of Antibodies
Antigens are presented to immunocompetent cells
Primary response by antigen presenting cells (APC) (macrophages and
dendritic cells). With many antigens (T cell dependent
antigens such as proteins and erythrocytes), processing
by macrophages is essential pre-requisite for antibody
l formation. In case of T cell independent antigens, such

as polysaccharides, antibody production does not require
T cell participation.
Time
Fig. 17.2 Primary and secondary humoral response

Only when the processed antigen is presented on the
surface of APC, in association with MHC molecules, to
the T cell carrying antigen recognition receptors (TCRs),
A single injection of an antigen helps more in priming is the T cell able to recognise it. In the case of helper T
the immunocompetent cells to produce the particular cells, the antigen has to be presented with MHC Class II
antibody rather than in actual elaboration of high levels of and for cytotoxic T cells with MHC class I molecules.
antibody. Effective levels of antibody are usually induced The TH cell requires two signals for activation.
by subsequent exposures to the same antigen. Due to this The first is a combination of TCR with MHC class II-
reason, the killed vaccines are given in multiple doses for complexed antigen. The second is interleukin-I (IL-1)
immunisation. The first injection is known as the 'priming' produced by APC. The activated TH cell produces
dose and the rest as 'booster' doses. A single dose of live interleukin-2 (IL-2) and other cytokines required for
vaccine is sufficient as multiplication of the organism in B-cell stimulation. These include IL-4, IL-5 and IL-6
the body provides a continuous antigenic stimulus that which act as B-cell growth factor (BCGF) and the B cell
acts both as priming and booster doses. differentiation factor (BCDF) for activation of B cells
When the same animal is subsequently exposed to the which have combined with their respective antigen. These
same antigen, a temporary fall in the level of circulating B cells undergo clonal proliferation and differentiate into
antibody occurs due to the combination of antigen with plasma cells that synthesise and secrete antibodies. B
pre-existing antibody (antibody formed during the first cells carry surface receptors which consist of IgM or
antigen exposure). This has been called the 'negative other immunoglobulin classes. Depending upon these

138 - - -- - - - -- -- - - - - -- - - - -- - -- - - - - - - - Unit II: Immunology
receptors, a plasma cell secretes an antibody of a single the receptors. As a compensatory mechanism, there is
specificity of a single antibody class (IgM, IgG or any an over production of the same type of receptors which
other single class). However, in primary humoral response, circulate as antibodies.
plasma cells initially secrete IgM and later switching
over to form IgG. Following antigenic stimulus, not all (ii) Natural selection theory
B-lymphocytes get converted into plasma cells. A small This theory was proposed by Jerne (1955). He postulated
proportion of them develop into 'memory cells' which that about a million globulin (antibody) molecules were
have a long lifespan and can recognise the same antigen formed in embryonic life, which covered the full range
on subsequent exposure. The increased antibody response of antigens. When an antigen is introduced, it combines
during secondary antigenic stimulus is due to the memory selectively with the globulin molecule that has the nearest
cells induced by the primary contact with the antigen. complementary 'fit'. The globulin, with the combined
Antibody production by B lymphocytes is regulated antigen, stimulates antibody forming cells to produce
by T cells. Helper T cells (TH) stimulate and suppressor T same kind of antibody.
cells (TS) inhibit antibody production. Optimum antibody
response depends on the balanced activity of these TS (iii) Clonal selection theory
and TH cells. This theory was proposed by Burnet (1957). The theory
states that during foetal development a large number of
D. Theories of Antibody Formation clones of immunological competent cells (ICCs) bearing
There are two broad groups: specific antibody patterns are produced by a process of
1. Instructive theories somatic mutation of ICCs against all possible antigens.
2. Selective theories Clones of cells with immunological reactivity with self
The instructive theories postulate that an antigens are eliminated during embryonic life. Such
immunocompetent cell (ICC) is capable of synthesising clones are known as 'forbidden clones' . Persistence of
antibodies of all specificity. The antigen instructs ICC to forbidden clones or their development in later life by
produce the complementary antibody. Selective theories, somatic mutation lead to autoimmune processes. Each
on the contrary, postulate that ICCs have only a restricted ICC is capable of reacting with one antigen or a small
immunological range. The antigen selects the appropriate number of antigens. Contact with specific antigen leads
ICC to synthesise an antibody. to cellular proliferation to form clones synthesising the
antibody. The clonal selection theory is widely accepted
1. Instructive Theories nowadays.
(i) Direct template theory
According to this theory, the antigen (or the antigenic E. Monoclonal Antibodies
determinant) enters the antibody forming cell and serves 1. Principle
as a 'template' against which antibodies are synthesised Antibodies that are usually produced in response to a
so that they have combining sites complementary to the single antigen are heterogenous as they are synthesised
antigenic determinant. by several different clones of cells i.e. polyclonal. A
single antibody forming cell or clone produces antibodies
(ii) Indirect template theory directed against a single antigen or antigenic determinant
This was proposed by Burnet and Fenner (1949). According only and such antibodies are called monoclonal antibodies.
to this theory, the antigenic determinant enters into the In multiple myeloma, antibodies are produced by a
ICC so that a 'genocopy' of the antigenic determinant is single clone of plasma cells against a single antigenic
incorporated in its genome and transmitted to the progeny determinant and hence the antibodies are monoclonal.
cells (indirect template). The method for production of monoclonal antibodies
against any desired antigen was first described by Kohler
2. Selective Theories and Milstein in 1975. They were awarded Nobel Prize
(i) Side chain theory for Medicine in 1984.
According to side chain theory, ICCs have surface
receptors capable of reacting with antigens which have 2. Technique
complementary side chains. When foreign antigens are Antibody forming spleen cells are fused with myeloma
introduced into the body, they combine with those cell cells to produce hybrid cells (hybridomas). The resultant
receptors which have a complementary fit. This inactivates hybridoma retains the antibody producing capacity of the

Ch 17: Immune Response - - - - - - - - - - ' - - - - - - - - - - - - - - - - - - -- - - - 139
spleen cells and the ability of myeloma cells to multiply indefinitely while unfused myeloma cells are killed
indefinitely (Fig. 17.3). The details of technique are as by aminopterin in HAT medium.
follows: (v) Clones that secrete the desired antibody are selected
(i) Animal (usually mouse) is immunised with the for continuous cultivation. These hybridomas can
desired antigen and lymphocytes are harvested from be maintained indefinitely and will continue to
the spleen. form monoclonal antibodies. They can also be
(ii) Spleen cells (lymphocytes) are then fused with grown as tumours in the peritoneal cavity of mice
mouse myeloma cells, grown in culture, which are by intraperitoneal inoculation and monoclonal
deficient in the enzyme hypoxanthine phosphoribosyl antibodies are obtained by harvesting the ascitic fluid
transferase (HPRT). Fusion is done by incubating produced. Hybridomas may be frozen for prolonged
these cells in the presence of polyethylene glycol storage.
(PEG).
(iii) The fused cells (hybrid cells) are grown in basal culture Mouse monoclonal antibodies, however, proved
medium containing hypoxanthine, aminopterin and unsuitable for human therapeutic use because of strong
thymidine (HAT medium). antimouse immune response. Human monoclonal
(iv) Only hybrid cells having properties of both the antibodies have been developed by modification of
splenic lymphocytes (HPRT +) and myeloma cells the original technique. Genes for particular antibody
(HPRT-) can grow in culture. The enzyme HPRT is fragments have been fused to bacteriophage genes. Large
necessary for nucleic acid synthesis and is provided quantities of the desired antibody can be obtained by
by the splenic lymphocytes in hybrid cells. Splenic infecting bacteria with the appropriate bacteriophage. Such
lymphocytes alone (unfused) cannot replicate antibodies hold great promise for immunotherapy.
Polydooal aotl,e: m
___,,,;r :'Z Antibody producing spleen cells

Spleen \J 1
e~
+ - Fusion - - < (j
F",ed oell,
lcat,,doma J~4 Myeloma cells
Best antibody-producing cell cloned and expanded
Antibody produced in culture
Fig. 17.3 Production of monoclonal antibodies

140 - -- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Unit II: Immunology
3. Applications 7. Adjuvants
(i) Diagnostic Use: Many commercial diagnostic systems Any substance that enhances the immunogenicity of an
use monoclonal antibodies for identification of antigen is called adjuvant. A number of substances such
bacterial, viral and other antigens. Direct fluorescence as aluminium hydroxide or phosphate and incorporation
and enzyme-linked assays utilise monoclonal of protein antigens in the water phase of a water-in-oil
antibody conjugates. emulsion (Freund's incomplete adjuvant), delay the release
(ii) Pure antibody : Large amount of pure antibody of of antigen and prolong the antigenic stimulus.
defined class can be prepared.
(i)Types of adjuvants
F. Factors Influencing Antibody Production (a) Depot: Aluminium hydroxide or phosphate, alum and
1.Age Freund's incomplete adjuvant (water in archis oil).
The embryo is immunologically immature. Production of (b) Bacterial: Freund's complete adjuvant is the Freund's
antibodies starts after the development and differentiation incomplete adjuvant along with a suspension of killed
of lymphoid organs. During embryonic life clones of tubercle bacilli.
cells that have specificity towards self-antigens are (c) Chemical: Bentonite, calcium alginate and silica
eliminated. particles.
Immunocompetence is not complete at birth. However,
full competence is acquired only by the age of 5-7 years Action of adjuvants
(ii)
for IgG and 10-15 years for IgA. (a) Sustained release of antigen from depot
2. Genetic Factors (b) Stimulate lymphocytes non-specifically
The immune response in different individuals to same (c) Activate macrophages
antigen varies due to genetic factors. Persons capable of (d) Stimulate CMI
responding to a particular antigen are called responder Freund's complete adjuvant develops delayed
and those who do not respond are termed nonresponder. hypersensitivity in addition to enhanced antibody
The Ir (immune response) genes control this property. formation. This adjuvant cannot be used in humans
because it produces local granuloma.
3. Nutritional Status
Protein calorie malnutrition suppresses both humoral and 8. Immunosuppressive Agents
cellular immunity. These agents inhibit the immune response. They are
useful in certain situations such as transplantation
4. Route of Administration when it becomes necessary to prevent graft rejection.
There is better immune response following parenteral Immunosuppressive agents include X-irradiation,
administration of antigen than through oral or nasal radiomimetic drugs, corticosteroids, antimetabolites and
routes. antilymphocytic serum.
5. Dose of Antigen (i) X- irradiation

An antigen is immunogenic only above a minimum critical It is more cytotoxic to replicating cells and has been used
dose. Very small dose does not stimulate immune system to prolong transplant survival.
to produce antibodies while very large dose may inhibit
the antibody producing system and paralyse it. This (ii) Radiomimetic drugs
phenomenon is known as immunological paralysis. They belong to the class of alkylating agents e.g.
cyclophosphamide, nitrogen mustard. Cyclophosphamide
6. Multiple Antigens given for three days after the antigen, completely
When two or more antigens are administered suppresses the antibody response. It is much less effective
simultaneously, the effects may vary. Antibodies may be when given before the antigen. It selectively prevents B
produced against all the antigens, or antibody response cell replication.
to one or the other of the antigens may be enhanced as
in triple vaccine (diphtheria, pertussis, tetanus), or the Corticosteroids
(iii)
response to one or more of them may be diminished They cause depletion of lymphocytes from the blood and
(antigenic competition). For optimal effect, the nature lymphoid tissues. They are antiinflammatory drugs that
and relative proportions of the different antigens should diminish the responsiveness of both B and T cells. They
be carefully adjusted. impair maturation of activated cells by suppressing the
Ch 17: Immune Response - - - - - - - - - - - - ; - - - -- - - - - - - - - - - - - - - - - - - 141
production of interleukins. However, therapeutic doses A. Induction of CMI

have little effect on immune response. Foreign antigen is presented by antigen presenting cells
(APCs) to T-lymphocytes. T-lymphocytes possess antigen
(iv) Antimetabolit es recognition receptors known as T cell receptors (TCRs)
These are substances that interfere with the synthesis of that recognise foreign antigen and a self MHC molecule
DNA, RNA or both and thus inhibit cell division and on the surface of the APC. These sensitised T-lymphocytes
differentiation necessary for humoral and cellular immune undergo blast transformation , clonal proliferation and
responses. They are used clinically in the prevention of differentiation into memory cells and effector cells (Th, Tc,
graft rejection. Some examples of antimetabolites are Td and Ts). The activated lymphocytes release biologically
folic acid antagonists (methotrexate), analogues of purine active products (lymphokines) which are responsible for
(6-mercaptopurine, azathioprine), cytosine (cytosine various manifestations of CMI.
arabinoside) and uracil (5-Fluorouracil). T cells recognise antigens only when presented
with MHC molecules. CD8 + cells can recognise the
(v) Antilymphocy tic serum (ALS) combination of foreign antigen and Class I MHC antigen
Antilymphocytic serum is a heterologous antiserum and differentiate into Tc and Ts lymphocytes whereas
raised against lymphocytes. Antiserum prepared against CD4 + cells can recognise the combination of foreign
thymus cells is called antithymocyte serum (ATS). antigen with Class II MHC antigen and differentiate into
The corresponding globulin preparations are called Th and Td cells. Tc lymphocytes recognise foreign antigen
antilymphyocyte globulin (ALG) or antithymocyte globulin and Class I MHC antigen and gets attached to the target
(ATG). ALS is raised in horses. It is devoid of undesirable cell. This stimulates Tc lymphocytes to release cytolysins
effects and its action is only on circulating lymphocytes which leads to lysis of the target cell. Subsequently, the Tc
and not on lymphocytes in lymphoid organs. It is mainly cell may detach from the target cell and repeat the same
used to prevent graft rejection. Antilymphocytic serum process with another. Tc lymphocytes also synthesise and
is effective primarily against T-lymphocytes. Humoral secrete interferon-y and thus they probably also contribute
response to thymus independent antigens is unaffected to some extent to macrophage activation.
but the response to thymus dependent antigens may be
inhibited. As ALS is a foreign protein, it may lead to B. Cytokines
hypersensitivity reactions. These are biologically active substances secreted by
monocytes, lymphocytes and other cells. They are named
(vi) Cyclosporine lymphokines if they are derived from lymphocytes and
It has been widely employed as an immunosuppressant monokines if they are derived from monocytes and
in organ transplantation. It does not have any cytotoxic macrophages. Interleukins are chemical substances that
effect on lymphocytes and has no antimitotic activity. It function primarily as growth and differentiating factors.
selectively inhibits helper T-cell activity. They exert a regulatory influence on other cells. All these
biologically active substances (lymphokines, monokines,
III. CELL MEDIATED IMMUNE RESPONSES interleukins) are collectively known as cytokines. They
The term cell mediated immunity refers to specific are not specific for antigens. Various cytokines are shown
acquired immune responses mediated by sensitised T in Table 17.1.
cells. This form of immunity can be transferred from Some of the important cytokines are described
donor to recipient with intact lymphocytes, but not with below:
antisera, hence it is called cell mediated immune reaction. l. Interleukin-I: Interleukin-l(IL-1) is principally
Cell mediated immunity (CMI) plays an important role secreted by macrophages and monocytes. It occurs
in the following immunological functions: in two forms IL-1 alpha and beta. Its production
1. delayed hypersensitivity (type IV hypersensitivity) is stimulated by antigens, toxins, inflammatory
2. immunity in infectious diseases caused by intracellular processes and inhibited by corticosteroids and
orgarusms cyclosporin A. It is a stable polypeptide.
3. transplantation immunity and graft-versus-host
(GVH) reaction Immunological effects of IL-1
4. immunological surveillance and immunity against 1. Stimulation of T cells for the production of IL-2
cancer and other lymphokines
5. pathogenesis of certain autoimmune diseases e.g. 2. B cell proliferation and antibody synthesis
thyroiditis. 3. Neutrophil chemotaxis and phagocytosis

142
Unit II: Immunology
Table 17.1 Cytokines and their Functions

Cytokine Major sources Main functions
I. Interleukins:
IL-1 Macrophages and Stimulation of T cells for the production of IL-2 and other lymphokines;
monocytes B cell proliferation; neutrophil chemotaxis.
IL-2 T cells Major activator of T and B cells, cytotoxicity of T and NK cells, helps
in destruction of tumour cells.
IL-3 T cells Acts as a growth factor for bone marrow stem cells.
IL-4 TH cells Proliferation of B and cytotoxic T cells, augments lgE synthesis.
IL-5 TH cells Proliferation of activated B cells.
IL-6 T, B cells, macrophages, Induces immunoglobulin synthesis by B cells; formation of IL-2 receptors
fibroblasts on T cells.
IL-7 Spleen, bone marrow B and T cell growth factor.
stromal cells
IL-8 Macrophages Neutrophil chemotactic factor.
IL-9 T cells Proliferation of T cells.
IL-10 T, B cells, macrophages Inhibit interferon production.
IL-11 Bone marrow stromal cells Induce acute phase proteins.
IL-12 T cells Activate natural killer (NK) cells.
IL-13 T cells Inhibit the functions of mononuclear cells.
IL-14 T cells Stimulates proliferation of activated B cells, inhibits lg secretions.
IL-15 Monocytes Proliferation of T cells and activated B cells.
IL-16 Eosinophils, CD8+ T cells Chemoattraction of CD4+ cells.
IL-17 CD4+ T cells Release of IL-6, IL-8, GCF and PGE2.
IL-18 Hepatocytes Induces interferon-y production, enhances NK cells activity.
2. Colony stimulating
factors (CSF):
G-CSF Fibroblasts, endothelium Granulocyte growth stimulation.
M-CSF Fibroblasts, endothelium Mononuclear growth stimulation.
3. Tumour necrosis
factors (TNF):
TNF-a. Activated macrophages Tumour cytotoxicity, antiviral effects, endotoxic shock.
and monocytes
TNF-~ TH cells Induce other cytokines.
4. Interferons (IFN):
IFN-a. Leucocytes Antiviral activity.
IFN-~ Fibroblasts Antiviral activity.
IFN-y Activated T cells Antiviral, macrophage activation, increases the cytotoxicity of NK cells.
Together with the tumour necrosis factor (TNF), it which help to destroy NK resistant tumour cells. This
is responsible for many of the hematological changes in property has been used in the treatment of cancers.
septic shock. IL-2 was previously named as T cell growth factor
2. Interleukin-2: Interleukin-2 (IL-2) is the major (TCGF). It is produced by T cells.
activator of T and B cells and stimulates cytotoxic 3. Interleukin-3: IL-3 is a growth factor for bone marrow
T (Tc;) cells and NK cells. It converts certain null cells stem cells. It is also known as multicolony stimulating
(LGL) into lymphokine activated killer (LAK) cells factor (multi-CSF).

Ch 17: Immune Response _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _
_ _ _ _ _ _ _ 143
4. Interleukin-4: IL-4 acts as B cell differentiating factor. 2. Macrophage activating factor (MAF): It restricts
It activates resting B cells. It also acts as growth macropha ge movemen t and increases phagocytic
factor for T cells and mast cells. It augments IgE activity.
synthesis and therefore may have a role in atopic 3. Macrophage chemotact icfactor (MCF): It stimulates
hypersensitivity. It enhances the activity of cytotoxic chemotax is of macrophages.
T cells. It was formerly known as B cell growth 4. Macrophage stimulating factor (MSF): It stimulates
factor-I (BCGF-I) . macropha ge migration to the site of action.
5. Interleukin-5: IL-5 causes proliferation of activated B
cells. It was formerly known as B cell growth factor-
II (BCGF-II ). C. Detection of CMI
6. Interleukin -6: IL-6 is produced by stimulated T and CMI can be detected by following methods:
B cells. It is also produced by macropha ges and 1. Skin tests for delayed hypersensitivity
fibroblasts. It induces immunogl obulin synthesis by 2. Lymphocy te transform ation test
B cells and formation of IL-2 receptors on T cells. 3. Migration inhibiting factor (MIF) test
7. Colony stimulating factors ( CSF): They stimulate the 4. Rosette formation
growth and differentiation of pluripoten t stem cells 5. Detection of T-cells by immunofluorescence technique.
in the bone marrow. They are named after the types
of cells they induce- for example, granulocyte (G), 1. Skin Tests for Delayed Hypersen sitivity
or mononucl ear (M) CSF. IL-3 induces growth of It is useful to detect delayed hypersensitivity to commonly
all types of haematopoietic cells and thus known as encounter ed antigens. Tuberculin skin test is commonly
multi-CSP. They are responsible for adjusting the rate used. Other antigens used for skin testings are l % solution of
of production of blood cells according to requirements l-Chloro-2 , 4 dinitrochl orobenzen e (DNCB) or
e.g. massive granulocyte response is seen in pyogenic dinitrofluorobenzene (DNFB).
infections.
8. Tumour necrosis factors (TNF): The tumour necrosis 2. Lymphoc yte Transform ation Test
factor occurs as two types namely alpha and beta. This is in vitro test which detects transform ation of
TNF-alph a is produced principally by activated cultured sensitised T lymphocytes on contact with specific
macropha ges and monocyte s. It resembles IL-1 in antigen.
having a very wide spectrum of biological activities
such as its role in the manifesta tions of endotoxic 3. Migratio n Inhibitin g Factor (MIF) Test
shock. It also has immunom odulatory influences Human peripheral leucocytes are incubated in capillary
on other cytokines. TNF-beta is formed principally tubes in culture chambers containing culture fluid. In the
by T helper cells. Its actions are similar to those absence of antigen, the leucocytes migrate out to the open
of TNF-alph a. TNF-beta was formerly known as end of the tube to form a fan like pattern. When an antigen
lymphotoxin. to which the individual has CMI is introduced into the
9. Interferons (IFN): There are three classes of IFNs, culture fluid, the leucocytes are prevented from migrating .
alpha, beta and gamma. IFN-alpha is produced by By compariso n with the control (without antigen), it is
leucocytes, beta produced by fibroblasts and gamma by possible to make a semiquantitative assessment of the
activated T cells. IFN-gamm a increases the cytotoxicity migration inhibition.
of NK cells and macrophages. It has inhibitory effect
on malignant cells. Interferons also have antiviral 4. Rosette Formatio n
activity. Most T cells form rosettes when incubated with sheep
Cytokine productio n is regulated by exogenous erythrocytes. Rosette is a lymphocyte to which three or
factors such as antigens, as well as by endogenous factors. more sheep erythrocytes are attached. T cell rosette is
They also regulate each other by positive and negative called as E-rosette. T cells can be estimated by counting
feedbacks. IL-1, 2, 3, CSF and IFNs have already found E-rosettes and thus help in detection of CMI status.
therapeuti c application.
5. Detection of T cells by Immunofluorescence
Lymphok ines Technique
1. Migration inhibiting factor (MIF): It inhibits the T cells can be detected by immunofluorescence technique
migration of normal macrophages. using monoclon al antibodies.

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D. Transfer Factor induced in adults when immunocompetence is temporarily

Lawrence (1954) reported transfer of CMI in man by interrupted by immunosuppressive agents.
injecting the extracts from the leucocytes. The extract Soluble antigens and haptens are more tolerogenic
contains a soluble factor known as 'transfer factor' than particulate antigens. For example, when human
(TF). The transferred immunity is specific as CMI can gamma globulin is heat aggregated, it is immunogenic
be transferred only to those antigens to which the donor in mice, but when de-aggregated, it is tolerogenic. The
is sensitive. induction of tolerance is dose dependent. High doses
TF is a low molecular weight substance (MW 2000 to of antigen induce B cell tolerance whereas repeated
4000), resistant to trypsin but gets inactivated at 56°C in 30 minute doses induce T cell tolerance. A moderate
minutes. It remains stable for several years at - 20°C and in dose of same antigen may be immunogenic. The route
the lyophilised form at 4°C. It is not antigenic. Chemically, of administration is also important for induction of
it appears to be polypeptide-polynucleotide. It is highly tolerance. Certain haptens are immunogenic in guinea
potent. The transferred CMI is systemic and not local. pigs by the intradermal route but are tolerogenic orally
Humoral immunity is not transferred by transfer factor. or intravenously.
The mechanism of action of TF is not known. It probably Tolerance to humoral and cellular immunity is
stimulates the release of lymphokines from sensitised usually induced simultaneously. When unresponsiveness
T lymphocytes. TF has been used in: is established for one parameter (humoral or cellular)
1. T cell deficiency (Wiskott-Aldrich syndrome) of the immune response, it is called 'split tolerance'.
patients In guinea pigs, delayed hypersensitivity to tuberculin
2. treatment of disseminated infections associated can be inhibited, without affecting the production of
with deficient CMI (lepromatous leprosy and a circulating antibody, by injection of tuberculoprotein
tuberculosis). prior to vaccination with BCG.
3. treatment of malignant melanoma and other types Tolerance can be overcome spontaneously or by an
of cancer. injection of cross reacting immunogens. Tolerance to
bovine serum albumin in rabbits can be abolished by
IV. IMMUNOLO GICAL TOLERANC E immunisation with cross-reacting human serum albumin.
Immunological tolerance is defined as a state in which
contact with an antigen specifically abolishes the capacity Mechanisms of Tolerance
to mount an immune response against that particular Tolerance can arise through following mechanisms:
antigen when it is administered subsequently, the immune
reactivity to other antigens being unaffected. Immune 1. Clonal Deletion
tolerance may be of two types, natural or acquired. In embryonic life clones of B and T lymphocytes that
Natural tolerance is non-responsiveness to self recognise self antigens are selectively deleted and are no
antigens. Any antigen that comes in contact with the longer available to respond upon subsequent exposure to
immunological system during its embryonic life would that antigen. This is called clonal deletion.
be recognised as self antigen and would not provoke
an immune response in the mature animal. Burnet and 2. Clonal Anergy
Fenner (1949) postulated that tolerance could also be Clones of B and T lymphocytes that recognise self
induced against foreign antigens if they were administered antigens might be present but they cannot be activated.
during embryonic life. This is known as clonal anergy.
Acquired tolerance arises when a potential antigen
induces a state of unresponsiveness to itself For acquired 3. Suppression
tolerance to be maintained, the antigen must persist or Clones of B and T lymphocytes expressing receptors that
be repeatedly administered. recognise self antigens are preserved. Antigen recognition
The induction of tolerance depends on the species might be capable of causing activation, however, expression
and immunocompetence of the host, physical nature, of immune response might be inhibited through active
dose and route of administration of antigen. Rabbits and suppress10n.
mice can be rendered tolerant more rapidly than guinea
pigs and chickens. It is difficult to induce tolerance when 4. Other Mechanisms
the degree of irnmunocompetence of the host is higher. Access of the antigen to immunocompetent cells is
For this reason, embryos and newborns are particularly interfered with (afferent block), or antibody synthesised
susceptible for induction of tolerance. Tolerance can be is neutralised or destroyed (efferent block).

Ch 17: Immune Response _ _ _ _ _ __:__ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __ 145
KEY POINTS
1. The specific reactivity induced in a host following an antigen stimulus is known as the immune response.
2. Immune response is of two types: humoral immunity and cell mediated immunity.
3. Humoral immunity is due to antibody production while cell immunity is because of sensitised T cells.
4. There are many theories of antibody formation but the clonal selection theory is widely accepted.
5. Antibodies that are usually produced in response to a single antigen are polyclonal, i.e., synthesised by several different
clone of cells.
6. A single antibody forming cell or clone produces antibodies directed against a single antigen or antigenic determinant
only and such antibodies are called monoclonal antibodies.
7. Hybridoma technique is used to produce monoclonal antibodies.
8. Many commercial diagnostic systems use monoclonal antibodies for identification of bacterial, viral and other
antigens. Direct fluorescence and enzyme-linked assays utilise monoclonal antibody conjugates.
9. Cell mediated immunity (CMI) plays an important role in delayed hypersensitivity (type IV hypersensitivity),
transplantation immunity, immunity in infectious diseases caused by intracellular organisms, immunity against cancer
and pathogenesis of certain autoimmune diseases.
10. The biologically active substances are responsible for various manifestation of CMI. These are named cytokines.
These include lymphokines, monokines and interleukins.
11. Immunological tolerance is defined as a state in which contact with an antigen specifically abolishes the capacity
to mount an immune response against that particular antigen when it is administered subsequently, the immune
reactivity to other antigens being unaffected.
YOU MUST KNOW

1. Primary and secondary humoral immune responses.
2. Theories of antibody formation.
3. Principle, technique and applications related to monoclonal antibodies.
4. Cytokines and their functions.
5. Immunological tolerance.
STUDY QUESTIONS
1. Discuss primary and secondary humoral responses.
2. Discuss briefly about:
(a) Theories of antibody formation
(b) Production of monoclonal antibodies and their applications.
(a) Cytokines (b) Adjuvants (c) Transfer factor
(d) Immunological tolerance (e) Clonal selection theory.
MULTJPLE CHOICE QUESTIONS (MCQs)

1. Cell mediated immunity (CMI) plays an important role in:
(a) Allograft rejection (b) Type IV hypersensitivity reactions
(c) Graft-versus-host reaction (d) All of the above
2. B-lymphocytes which have a long lifespan and can recognise the same antigen on subsequent exposure are named:
(a) Memory cells (b) Killer cells
(c) Natural killer cells (d) None of the above

146 - - - - - - -- - - - - -- - - - - - - - - - - - - - - - - - - Unit II : Immunology
3. The widely accepted theory of antibody formation is:

(a) Direct template theory (b) Indirect template theory
(c) Natural selection theory (d) Clonal selection theory
4. The animal used for monoclonal antibodies production is:
(a) Mouse (b) Guinea pig
(c) Rabbit (d) None of the above
5. The technique used for monoclonal antibodies production is called:
(a) Hybridoma (b) Rosette formation
(c) Opsonisation (d) Immunofluorescence
6. Interleukin-I (IL-I) is principally secreted by:
(a) Macrophages (b) Monocytes
7. Main functions of interleukin-I (IL-I) are:
(a) Stimulation of T cells for production of IL-2 (b) B cell proliferation
(c) Neutrophil chemotaxis (d) All of the above
8. Which of the following methods can be used to detect cell mediated immunity?
(a) Skin tests for delayed hypersensitivity (b) Lymphocyte transformation test
(c) Migration inhibiting factor test (d) All of the above
9. Which of the following mechanisms can be used to induce immunological tolerance?
(a) Clonal anergy (b) Clonal deletion
(c) Suppression (d) All of the above
Answers (MCQs):
1. (d) 2. (a) 3. (d) 4. (a) 5. (a)
6. (c) 7. (d) 8. (d) 9. (d)
- - - ••• - - -

Chapter 18
IMMUNODEFICIENCY DISEASES
I. Primary Immunodeficiencies
A. Humoral Immunodeficiencies B. Cellular Immunodeficiencies
(B Cell Defects) (T Cell Defects)
C. Combined Immunodeficiencies D. Disorders of Complement
(Both B and T Cell Defects)
E. Disorders of Phagocytosis
II. Secondary Immunodeficiencies
A. Depression of Humoral Immune Responses B. Depression of Cell-Mediated Immunity
Immunodeficiency diseases are conditions where the defence A. Humoral Immunodeficiencies

mechanisms of the host are impaired, leading to repeated (B Cell Defects)
microbial infections and sometimes enhanced susceptibility 1. X-Linked Agammaglob ulinaemia
to malignancies. Such defects may be primary or secondary. This syndrome is the first immunodeficiency disease to
Primary immunodeficiencies result from abnormalities have been recognised. It was first described by Bruton
in the development of immune mechanisms. Secondary in 1952 and is also called Bruton's disease. It is seen
immunodeficiencies are due to consequences of some other only in male infants. Manifestations are not apparent
disease, malnutrition, drugs and other processes that affect till about six months of age due to passive protection by
the normal functioning of the mature immune system. maternal immunoglobulins. Patients suffer from recurrent
These immunodeficiency diseases may involve specific infections with pyogenic bacteria, particularly with
immune functions- humoral immunity, cell mediated pneumococci, streptococci, meningococci, Pseudomonas
immunity or both-or nonspecific mechanisms such as and Haemophilus infiuenzae.
phagocytosis and complement. The basic defect is a failure of pre-B cells to differentiate
into mature B cells. All classes of immunoglobulins are
I. PRIMARY IMMUNODE FIC ENCIES grossly depleted in the serum. Tonsils and adenoids are
The defects may be in T cell, B cell or both, complement atrophic. Lymph node reveals a depletion of cells of the
system or phagocytes. Some primary immunodeficiency bursa-dependent areas. Plasma cells and germinal centres
syndromes are listed in Table 18 .1. are absent even after stimulation with antigen. Affected
It must be realised that there is considerable individuals have a marked decrease in the proportion
overlapping due to intimate interaction between the B of B-cells in circulation but pre-B cells are present
cell and the T-cell. For instance, T cell defici~ncies of in normal numbers in bone marrow. T cell functions
helper or suppressor T cells will also have a profound are normal in these patients and can normally handle
effect on antibody response. viral, fungal, protozoa! diseases. Allograft rejection is
147
148 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Unit II: Immunology
Table 18.1 Primary Immunodeficiency Syndromes and unable to differentiate into plasma cells and secrete
their Classification immunoglobulins. Increased suppressor T cell and
A. Humoral Immunodeficiencies (B cell Defects) decreased helper T cell activity have been proposed as
1. X-linked agammaglobulinaemia a cause of this disorder. In this condition, the total
2. Transient hypogammaglobulinaemia of infancy immunoglobulin level is usually less than 300 mg per 100
3. Common variable immunodeficiency ml, with IgG level less than 250 mg per 100 ml. Treatment
4. Selective immunoglobulin deficiencies is by administration of gammaglobulin preparations
5. Immunodeficiencies with hyper-IgM intramuscularly or intradermally.
6. Transcobalarnin II deficiency
B. Cellular Immunodeficiencies (T Cell Defects)
4. Selective Immunoglobulin Deficiencies
1. Thymic hypoplasia (DiGeorge's syndrome) In these conditions, there occurs selective deficiency of
2. Purine nucleoside phosphorylase (PNP) deficiency one or more immunoglobulin classes, whereas the other
C. Combined Immunodeficiencies (Both Band T Cell Defects) immunoglobulin classes remain normal or elevated.
1. Cellular immunodeficiency with abnormal
immunoglobulin synthesis (Nezelof's syndrome) (a) Selective IgA deficiency
2. Ataxia telangiectasia
There is absence or near absence of serum and secretory
3. Wiskott-Aldrich syndrome
4. Immunodeficiency with thymoma IgA in this most common well-defined immunodeficiency
5. Severe combined immunodeficiency diseases disorder. These patients exhibit increased susceptibility
6. MHC Class II deficiency to respiratory and gastrointestinal infections. Atopic
D. Disorders of Complement disorders among IgA deficient individuals are high. Anti-
1. Complement component deficiencies IgA antibodies are present in many of these patients.
2. Complement inhibitor deficiencies
E. Disorders of Phagocytosis (b) Selective IgM deficiency
1. Chronic granulomatous disease (CGD) Selective IgM deficiency has been found to be associated
2. Myeloperoxidase deficiency with septicaemia due to meningococci and other Gram-
3. Chediak-Higashi syndrome negative bacteria.
4. Leucocyte G-6-PD deficiency
5. Immunodeficiencies with Hyper-IgM
There is low levels of IgA and IgG with elevated IgM.
normal and delayed hypersensitivity to tuberculin can be Some of these immunodeficiencies are X-linked and
demonstrated. Treatment consists of routine injections some inherited as autosomal recessive. Patients are
of normal immunoglobulins. vulnerable to infections and autoimmune disorders such
as thrombocytopaenia , neutropaenia and haemolytic
2. Transient Hypogammaglobu linaemia of Infancy anaemia. Some patients develop malignant infiltration
This is due to an abnormal delay in the initiation of IgG with IgM producing cells.
synthesis in some infants. It may occur in infants of both
sexes. Maternal IgG is slowly catabolised and reaches 6. Transcobalamin II Deficiency
a very low level by the second month. Ordinarily, the It is an autosomal recessive disorder. These individuals
infants start producing their own IgG by this age. When show metabolic effects of vitamin B12 deficiency
there is a delay, immunodeficiency occurs. Treatment with including megaloblastic anaemia and intestinal villous
gammaglobulin is not recommended in this condition, as atrophy. Immunological defects include plasma cells
it may contribute to prolongation of immunodeficiency depletion, diminished imrnunoglobulin levels and impaired
by a negative feedback inhibition of IgG synthesis. phagocytosis. Treatment with vitamin B 12 may restore
haematopoietic, gastrointestinal and B cell functions but
3. Common Variable Immunodeficiency not phagocytic activity.
This is also known as late onset hypogammaglobulinaemia
because it usually manifests only by 15-35 years of age. B. Cellular Immunodeficienc ies
It is characterised by recurrent pyogenic infections. This (T Cell Defects)
may also be associated with an increased incidence of Abnormalities of T cell development results in viral,
autoimmune disease. B cells may be present in circulation intracellular bacterial, fungal and protozoa! infections
in normal numbers, but they appear defective and are rather than acute bacterial infections.

Ch 18: Immunodeficiency Diseases _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ 149
1. Thymic Hypoplasia (DiGeorge's Syndrome) rejection. Transfer factor and foetal thymus transplants
This is a developmental defect affecting the third and have been tried with limited benefit.
fourth pharyngeal pouches, which leads to aplasia or
hypoplasia of the thymus and parathyroid glands. T cells 3. Wiskott-Aldrich Syndrome
are deficient or absent in the circulation. The thymus It is an X-linked recessive disease characterised by
dependent areas of spleen and lymph nodes are depleted eczema, bleeding and recurrent infections. Most of
of T lymphocytes. Delayed hypersensitivity and graft the affected children die due to complications such as
rejection are depressed. The humoral immune response bleeding, infection or lymphoreticular malignancy during
is largely unaffected. childhood. The bleeding occurs due to thrombocytopenia,
Patients show enhanced susceptibility to viral, eczema from elevated IgE level and recurrent infections
fungal and bacterial infections. Most of these infants from abnormalities in cell-mediated (thyrnic hypoplasia)
usually show other associated developmental anomalies and antibody mediated immunity. B cells are unable
such as cardiac defects particularly involving great to respond to polysaccharide antigens. Treatment with
vessels. Neonatal tetany is present. Transplantation of transfer factor and bone-marrow transplantation have
foetal thymus tissue has been reported to restore the been found to be beneficial.
immunological functions.
4. Immunodeficiency with Thymoma
2. Purine Nucleoside Phosphorylase (PNP) This syndrome consists of a benign thyrnic tumour, impaired
Deficiency cell mediated immunity and agammaglobulinaemia. It
Inherited deficiency of enzyme PNP occurs due to a occurs usually in adults. Antibody formation is poor and
gene defect in chromosome 14 which results in impaired progressive lymphopenia develops. Patients have been
metabolism of cytosine and inosine to purines. These shown to have excessive suppressor T cell activity.
patients show decreased T cell proliferation leading
to decreased cell mediated immunity and recurrent or 5. Severe Combined Immunodeficiency Diseases
chronic infections. These include many syndromes with severe combined
deficiency of both humoral and cell mediated immunity.
C. Combined Immunodeficiencies (Both B and T They are inherited in the autosomal recessive mode.
Cell Defects) Many distinct patterns of this immunodeficiency have
1. Cellular Immunodeficiency with Abnormal been described.
Immunoglobulin Synthesis (Nezelof's Syndrome)
There is depressed cell mediated immunity associated (a) Swiss type agammaglobulinaemia
with selectively elevated, decreased or normal levels of Such persons are born with lymphoid stem-cell defects
immunoglobulin. Patients are susceptible to recurrent and lack both humoral and cell-mediated immunity. They
viral, fungal, bacterial and protozoa! diseases. The thymus have agammaglobulinaemia and lymphocytopaenia. The
is small and peripheral lymphoid tissues are hypoplastic. basic defect is presumed to be at the level of the lymphoid
Autoimmune processes such as haemolytic anaemia are stem cell.
common. Bone marrow transplantation, transfer factor
and thymus transplantation have been used for treatment, (b) Reticular dysgenesis
with success in some cases. Adequate antimicrobial therapy This is the most serious form of combined
is essential for the treatment of microbial infections. immunodeficiency. Here the defect is at the level of
multipotent bone marrow stem cell. There is a total failure
2. Ataxia Telangiectasia of myelopoiesis. The individual is born with lymphopenia,
This is an autosomal recessive genetic disorder, where neutropaenia, thrombocytopaenia, anaemia and bone
combined immunodeficiency is associated with cerebellar marrow aplasia. The condition is invariably fatal in the
ataxia, telangiectasia (dilated capillaries), ovarian first week of life.
dysgenesis and chromosomal abnormalities. Death occurs
due to sinopulmonary infection early in life, or malignancy (c) Adenosine deaminase deficiency
in second or third decade. The majority of p.:!iP-nts lack Adenosine dearninase (ADA) is an enzyme which catalyses
IgA and IgE but some patients possess antibody to IgA. the conversion of adenosine to inosine in purine metabolic
Cell mediated immunity is also defective which results pathway. In these patients, ADA levels are low in all the
in an impairment of delayed hypersensitivity and graft tissues, including red blood cells. The mechanism by which

150 Unit II : Immunology
this deficiency causes immunological impairment is not as enzyme deficiency and extrinsic defects may be due
clear. It is, however, believed that deficiency of ADA to deficiency of opsonin antibody, complement or other
leads to accumulation of adenosine and deoxyadenosine factors promoting phagocytosis. Phagocytic dysfunction
triphosphate, which are toxic to lymphocytes, particularly leads to increased susceptibility to infections.
T-lymphocyte lineage. There is greater loss of T-cell
immunity than that of B-cell. The immunodeficiency 1. Chronic Granulomatous Disease (CGD)
varies from complete absence to mild abnormalities of It is a fatal genetic disorder in which there is a deficiency
B and T cell functions. Infants with these severe immune of NADPH oxidase. In these patients, polymorphonuclear
disorders are vulnerable to all forms of infections and leucocytes phagocytose invading bacteria normally but are
most die during the first year of life. unable to kill ingested microorganisms because engulfment
of bacteria is not followed by activation of oxygen-
6. MHC Class II Deficiency dependent killing mechanisms. The bacteria involved in
It is an autosomal hereditary recessive disorder in which the recurrent infections are catalase positive organisms such
failure to express MHC II molecules on the surface of as staphylococci and coliforms. Catalase negative bacteria
antigen presenting cells (macrophages and B cells) results are handled normally. Leucocytes from these patients fail to
in a deficiency of CD4+ T-cells. This is due to the fact reduce nitroblue tetrazolium (NBT) during phagocytosis.
that T-cell (CD4+) development is dependent on positive This property has been used (NBT test) for the diagnosis
selection by MHC class II molecules in thymus. Thus of chronic granulomatous disease.
MHC II deficient infants exhibit the deficiency of T
cells (CD4+ ). The lack of these helper T-cells results in 2. Myeloperoxidase Deficiency
deficient antibodies. In this rare disease, leucocytes are deficient in
myeloperoxidase. Patients are liable to develop recurrent
Candida albicans infection.
D. Disorders of Complement
1. Complement Component Deficiencies 3. Chediak-Higashi Syndrome
Genetic deficiencies of almost all complement components It is an autosomal recessive disorder. Polymorphonuclear
have been detected in man. Deficiency of Clr and C4 leucocytes in these patients possess large lysosomes. These
is associated with systemic lupus erythematosus. The abnormal lysosomes do not fuse readily with phagosome.
most severe abnormalities of host defences occur with Thus leucocytes possess diminished phagocytic activity.
C3 deficiencies which result in increased susceptibility to Patients suffer from severe pyogenic infections.
pyogenic infections. Deficiency of C6, C7, C8 is associated
with neisserial infections. 4. Leucocyte G-6-PD Deficiency
The defects of all these complement components are Leucocytes are deficient in glucose-6-phosphate
transmitted as autosomal recessive traits, except that of dehydrogenase and show diminished bactericidal activity
properdin which is X-linked recessive disease. after phagocytosis. Such patients are vulnerable to
microbial infections.
2. Complement Inhibitor Deficiencies
Hereditary angioneurotic oedema is due to a genetic II. SECONDARY IMMUNODEFICIENCIE S
deficiency of Cl inhibitor and this defect is transmitted as an These can occur secondary to a number of disease states
autosomal dominant. Normally, Cl inhibitor is involved in such as metabolic disorders, malnutrition, malignancy or
inactivation of complement system and other pathways, like as a consequence of certain therapeutic measures which
clotting, fibrinolytic and kinin. In Cl inhibitor deficiency, depress the immune system. Secondary immunodeficiencies
these systems may be activated. Activation of factor XII are more common than primary immunodeficiencies.
(Hageman factor) leads to formation of bradykinin and
C2 kinin. These kinins act on the post-capillary venules A. Depression of Humoral Immune Responses
causing contraction of endothelial cells and formation This results when B-cells are depleted as in lymphoid
of gaps that allow the plasma leakage and production malignancy, particularly in chronic lymphatic leukaemia;
of oedema. when excessive loss of serum proteins occurs as in
exfoliative skin disease and in protein losing enteropathies.
E. Disorders of Phagocytosis In multiple myeloma, there is excessive production of
Phagocytosis may be impaired either by intrinsic or extrinsic abnormal immunoglobulins but normal immunoglobulin
defects. Intrinsic defects are within phagocytic cells such level is decreased .

Ch 18: Immunodeficiency Diseases _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ 151
B. Depression of Cell-Mediated Immunity leprosy and some cases of sarcoidosis. Acquired

Cell mediated immunity is depressed in acquired immunodeficiency syndrome (AIDS) is the most important
immunodeficiency syndrome (AIDS), Hodgkin's of secondary immunodeficiency diseases (see Chapter
lymphoma, obstruction to lymph circulation, lepromatous 63).
KEY PO NTS
1. Immunodeficiency diseases are conditions where the defence mechanisms of the host are impaired leading to repeated
microbial infections and sometimes enhanced susceptibility to malignancies.
2. Primary immunodeficiencies result from abnormalities in the development of immune mechanisms.
3. Secondary immunodeficiencies are due to consequences of some other disease, malnutrition, drugs and other processes
that affect the normal functioning of the mature immune system.
4. These immunodeficiency diseases may involve specific immune functions- humoral immunity, cell mediated immunity
or both-or nonspecific mechanisms such as phagocy tosis and complement.
YOU MUST KNOW

1. List of primary immunodeficiency diseases.
2. Secondary immunodeficiency diseases.
STUDY QUEST ONS

1. What are immunodeficiency diseases? Classify pnmary immunodeficiency diseases and describe briefly about
DiGeorge's syndrome.
(a) B cell defects (b) T cell defects (c) Disorders of complement.
MULTIPLE CHOICE QUESTIONS (MCOs)

1. DiGeorge's syndrome is an immunodeficiency disease due to:
(a) T cell defects (b) B cell defects (c) Both of the above (d) None of the above
2. Which of the following defects occur in Nezelof's syndrome.
3. Wiskott-Aldrich syndrome is an immunodeficiency disease which occurs due to:
4. Chediak-Higashi syndrome is an immunodeficiency disease which occurs due to:
(a) T cell defects (b) B cell defects
(c) Complement disorder (d) Diminished phagocytic activity
5. Chronic granulomatous disease occurs due to deficiency of:
(a) NADPH Oxidase (b) G6PD enzyme (c) Both of the above (d) None of the above
Answers (MCQs):
1. (a) 2. (c) 3. (c) 4. (d) 5. (a)
- - - - . .• - - - -

r
Chapter 19
HYPERSENSITIVITY
I. Classification
A. Type I (Anaphylactic) Reaction B. Type II (Cytotoxic) Reaction
C. Type Ill (I mmune Complex) Reaction D. Type IV (Delayed or Cell Mediated) Reaction
E. Type V (Stimulatory Type) Reaction
II. Shwartzman Reaction
Immune response is generally a protective process but it hypersensitivity. The term 'allergy' means an altered state
may sometimes be injurious to the host. Hypersensitivity of reactivity to an antigen, it may include both protective
refers to a condition in which immune response results as well as injurious immune response. This term allergy
in excessive reactions leading to tissue damage, disease was originally coined by von Pirquet (1905).
or even death in the sensitised host.
Hypersensitivity occurs in individuals who have I. CLASSIFICATION
had previous contact with the antigen (allergen). The Hypersensitivity reactions are classified into two main
initial contact sensitises the immune system by priming types, 'immediate' and 'delayed' types based on the time
appropriate B or T lymphocytes. It is known as 'priming' required by sensitised host to develop clinical reactions
or 'sensitising' dose. Subsequent contact with the same upon exposure to the shocking dose of the antigen.
antigen causes hypersensitivity. It is known as 'shocking' The major differences between the two hypersensitivity
dose. These allergens may be complete antigens or reactions are shown in Table 19 .1.
haptens. Coomb and Gel (1963) classified hypersensitivity
Allergy is most commonly used as a synonym for reactions into four major types, types I to IV.
Table 19.1 Distinguishing Features of Immediate and Delayed Type of Hypersensitivity

Feature Immediate type Delayed type
I. Onset and duration Appears and recedes rapidly Appears slowly in 24-72 hours and lasts longer
2. Immune response Antibody mediated Cell mediated (T-lymphocytes)
3. Passive transfer Possible with serum Cannot be transferred with serum but possible with lymphocytes
or transfer factor
4. Desensitisation Easy but short lived Difficult but long lasting
5. Induction Antigens or haptens, by any route By antigen injected intradermally or with Freund's adjuvant or by
skin contact
152

Ch 19: Hypersensitivity _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ 153
Type I (Anaphylactic) E. Type V (Stimulatory Type)

Type II (Cytotoxic) It is a modification of Type II hypersensitivity reaction.
Type III (Immune complex) Antibodies interact with antigens on cell surface that
Type IV (Delayed or cell mediated) leads to cell proliferation and differentiation instead of
Type I, II and III depend on the interaction of inhibition or killing. Antigen-antibody reaction enhances
antigen with humoral antibodies and are known as the activity of affected cell.
immediate type reactions. Type IV reaction is mediated
by T-lymphocytes. Reaction develops in 24 to 72 hours TYPE I (ANAPHYLACTIC) REACTION
and is called delayed hypersensitivity reaction. Later on Anaphylaxis is the classical example of type I
type V hypersensitivity reaction was also described. hypersensitivity reaction. It is IgE mediated reaction.
The term anaphylaxis (ana-without, phylaxis-protection)
A. Type I (Anaphylactic) was described by Richet (1902) who observed that dogs
Antibodies (IgE type) are fixed on the surface of tissue which had survived a sublethal dose of toxic extracts
cells (mast cells and basophils) in sensitised individuals. of sea anemones, were rendered highly susceptible to
The antigen combines with the cell fixed antibody, leading minute doses of the same extract given days or weeks
to release of pharmacologically active substances which later, instead of becoming immune to it.
produce the hypersensitivity reaction. Type I reactions occur in two forms- the acute,
potentially fatal, systemic form called anaphylaxis and
B. Type II (Cytotoxic) the recurrent non-fatal localised form called atopy.
It is a cytotoxic reaction mediated by antibodies that
react with antigens present on the surface of cell or 1. Mechanism of Anaphylaxis
other tissue components resulting in damage of the Anaphylaxis occurs when a sensitised individual comes
cell. Combination with antibody may sometimes cause in contact with a shocking dose of antigen. Sensitisation
stimulation instead of cell damage. An example is 'long may occur by any route such as parenteral, inhalation
acting thyroid stimulator' (LATS), an antibody against or ingestion but the most effective route is parenteral.
thyroid cells, which stimulates the excessive secretion of Antigens as well as haptens may induce anaphylaxis.
thyroid hormone. An interval of 2-3 weeks is required between sensitising
and shocking dose, during which cytotropic antibody
C. Type III (Immune Complex) lgE (previously known as reagenic antibody) produced
The damage is caused by antigen-antibody complexes. against the antigen attaches to surface receptors of mast
These complexes may be deposited in tissues causing cells and basophils. IgE molecules bind to these receptors
immune complex disorders. by their Fe end. Once sensitised, the individual remains
so for a long period. When a shocking dose of the same
D. Type IV (Delayed or Cell Mediated) or immunologically related antigen is given, the antigen
The tissue damage is mediated by combines with cell bound IgE antibody on mast cells
T-lymphocytes and not by antibody, hence, is also called rapidly. The antigen-antibody complex stimulates mast
cell mediated hypersensitivity. The antigen activates cells and basophils to release mediators that cause clinical
specifically sensitised T-lymphocytes which lead to the manifestations of anaphylaxis (Fig. 19.1) . Shocking dose
secretion of lymphokines, with fluid and phagocyte is most effective when given intravenously, less effective
accumulation. subcutaneously, and least effective intradermally.
+ lgE +Allergen
0-0 0
t1
Allergan
Mast cell 21
lgE
Release of mediators - - . Anaphylaxis

Fig. 19.1 Type I (anaphylactic) hypersensitivity

r
2. Chemical Mediators (c) Platelet activating factor ( PAF)

The chemical mediators are of two types- primary It is released from basophils which causes
mediators which are the preformed contents of mast aggregation of platelets and release of their
cell and basophil granules and the secondary mediators vasoactive amines.
which are newly formed upon stimulation of mast cells, (d) Other mediators of anaphylaxis
basophils and other leucocytes. These are the anaphylatoxins released by
( i) Primary mediators: e.g. Histamine, serotonin, complement activation and bradykinin and other
eosinophil chemotactic factor of anaphylaxis, kinins from plasma kininogens.
neutrophil chemotactic factor of anaphylaxis, various
proteolytic enzymes.
(a) Histamine 3. Features of Anaphylaxis
This is the most important in human anaphylaxis. (i) Anaphylaxis occurs within a few seconds to few
It is formed by the decarboxylation of histidine minutes following shocking dose of antigen.
present in the granules of mast cells, basophils (ii) Cytotropic lgE antibody is responsible.
and in platelets. It causes vasodilatation, (iii) Tissues or organs which are affected in anaphylaxis
increased capillary permeability and contraction are called 'target tissues' or 'shock organs'. Lung is
of smooth muscle. the principal shock organ in humans. Bronchospasm,
(b) Serotonin laryngeal oedema, respiratory distress, shock and
It causes vasoconstriction, increased capillary death may occur.
permeability and smooth muscle contraction. It (iv) It can be induced artificially by serum of sensitised
is found in the brain tissue, intestinal mucosa individual.
and platelets. ( v) It is not related to heredity.
(c) Eosinophil chemotactic factor of anaphylaxis
( ECF-A ) 4. Types of Anaphylaxis
These are released from mast cell granules and Apart from systemic anaphylaxis, there are other types
are strongly chemotactic for eosinophils. These of anaphylaxis which are as follows:
probably contribute to the eosinophilia associated
with many hypersensitivity conditions. Another (i) Anaphylaxis in vitro
chemotactic factor which attracts neutrophils (Schultz-Dale phenomenon)
is known as neutrophil chemotactic factor of Isolated tissues (uterus or ileum) from sensitised guinea
anaphylaxis. pigs when kept in a bath of Ringer's solution, the organ
(d) Proteolytic enzymes contracts vigorously on addition of the specific antigen to
Proteases and hydrolases are also released from the bath. This is named as Schultz-Dale phenomenon.
mast cell granules.
(ii) Secondary mediators: e.g. Slow reacting substances (ii) Cutaneous anaphylaxis
(SRS), prostaglandins and thromboxane, platelet Cutaneous anaphylaxis may be induced when a small
activating factor (PAP), cytokines such as IL3 to shocking dose of an antigen is administered by intradermal
IL6, GM-CSP, ILl , INF-y, INF-a. route to a sensitized host. It is manifested by appearance
(a) Slow reacting substance of anaphylaxis of local 'wheal and flare' response. Cutaneous anaphylaxis
( SRS-A ) is useful in testing for hypersensitivity and in identifying
It is produced by leucocytes. These are much more the allergen responsible in atopy.
potent bronchoconstrictors than histamine. These
are principal mediators in bronchoconstriction (iii) Passive cutaneous anaphylaxis (PCA)
of asthma. They cause sustained contraction This is a sensitive in vivo method for detection of
of smooth muscles. These are not inhibited by antibodies. A small volume of the antibody is inoculated
an tihistaminics. intradermally into a normal animal. If the antigen along
(b) Prostaglandins and thromboxane with a dye Evans blue is injected intravenously 4-24 hours
Both of these are derived from arachidonic acid afterwards, there will be an immediate blueing at the
which is formed from disrupted cell membranes site of intradermal injection due to vasodilatation and
of mast cells and leucocytes. Prostaglandin F2a increased capillary permeability (wheal and flare reaction).
is a bronchoconstrictor. Thromboxane A2 is also PCA can be used to detect human lgG antibody which
a powerful, but transient, bronchoconstrictor. is heterocytotropic (antibody is capable of fixing to cells

Ch 19: Hypersensitivity _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __ _ _ _ _ _ _ _ _ _ _ _ _ _ 155
of other species) but not IgE which is homocytotropic site of injection within minutes. The special affinity of
(can fix to cells of homologous species only). IgE (reaginic antibody) for skin cells forms the basis of
PK reaction. As reaginic IgE is homocytotropic, the test
5. Atopy has to be performed on human skin. This test is not
The term 'atopy' (atopy meaning out of place or done nowadays due to risk of transmission of serum
strangeness) was first introduced by Coca (1923) to refer hepatitis.
to familial hypersensitivities which occur spontaneously in In-vitro methods for detection of IgE include
man. It is typified by hay fever and asthma. The antigens radioallergosorbent test (RAST), ELISA and passive
commonly involved in atopy are pollens, house dust and agglutination.
foods. These atopens induce IgE antibodies, formerly
termed as 'reagin' antibodies. Atopic sensitisation occurs
spontaneously following natural contact with atopens. 6. Anaphylactoid Reaction
Predisposition to atopy is genetically determined, probably This reaction resembles anaphylactic shock clinically and is
linked to MHC genotypes. provoked by intravenous injection of peptone, trypsin and
some others substances. It has a nonspecific mechanism
(i) Features of atopy involving the activation of complement pathway with
1. Atopy runs in families. These individuals have release of anaphylatoxins. It has no immunological basis.
tendency to produce reagin antibody in unusually The clinical resemblance is due to action of same chemical
large amounts. mediators participating in both reactions.
2. Reactions occur at the site of entry of the antigen,
inhalation of pollens affect lungs (bronchial asthma),
ingestion of fish, milk, eggs, drngs etc. (gastrointestinal TYPE II (CYTOTOXIC) REACTION
disorders or cutaneous eruptions) and contact leads Cytotoxic reaction is mediated by IgG (or rarely IgM)
to local allergy (conjunctivitis). antibodies directed against antigens on the surface of cells
3. It is IgE mediated hypersensitivity reaction. IgE is resulting in cell damage. Antibodies bind to an antigen
homocytotropic i.e. species specific. Only human IgE on the cell surface and cause (i) phagocytosis of the cell
can fix to the surface of human cells. through opsonic or immune adherence (ii) cytotoxicity
4. Induction of atopy is difficult artificially because by natural killer (NK) cells (iii) lysis through activation
atopens are poor antigens. of complement system (Fig 19.2).
(ii) Mechanism of atopy Examples

The atopen combines with the cell bound IgE antibodies 1. Autoimmune anaemias and haemolytic disease of the
fixed on the surfaces of mast cells and the basophils newborn
and this antigen-antibody complex stimulates these cells Lysis of red cells occur due to formation of
to release the mediators resulting in clinical features of antierythrocyte antibodies.
atopy. 2. Drug reactions
Sedormid purpura is a classical example. A sedative
Examples drug sedormid (not used nowadays) combines with platelets
1. Food allergy e.g. egg, mushroom, prawn, shell fish. and antibodies are formed against these sedormid coated
2. Dust allergy e.g. pollens of ragweeds, grasses or trees, platelets. On subsequent exposure to drug, antibodies
house dust. attack the platelets leading to thrombocytopenic purpura.
3. Drug allergy e.g. penicillin, sulphonamides. Other drugs such as sulphonamide, thiazide diuretics and
quinidine cause similar type of purpura. Many drugs
(iii) Prausnitz-Kustner (PK) reaction attach to the cell membranes of erythrocytes, neutrophils
This was the original method for detecting atopic antibody. or platelets and lead to antibody formation. On subsequent
Prausnitz and Kustner (1921) reported that if serum exposure to drug, antigen-antibody reaction produces
collected from Kustner, who had a gastrointestinal allergy cytotoxic or cytolytic (haemolysis) reactions.
to certain cooked fish, was injected intracutaneously into
Prausnitz, followed 24 hours later by injecting (intra- Demonstration of type II reaction
cutaneously) small amount of cooked fish extract into Coombs test (direct antiglobulin test) 1s usually
the same site, a wheal and flare reaction occurred at the positive.

Opsonic adherence
concentration of foreign serum. Antibodies to foreign
and phagocytosis Immune adherence serum reach high enough titres by 7-12 days but still
some excess antigen remains in the circulating blood.
l Immune complexes get deposited on the endothelial lining

of blood vessels in various parts of the body, causing
inflammatory infiltration. Massive complement activation
and fixation by antigen-antibody complexes leads to fall
;:\
bb
@ +
Antibody
(ab)
+
Complement
in complement concentration. Serum sickness differs from
other hypersensitivity reactions in that a single injection
Antigen on the
serves both as the sensitising and shocking dose. Immune
I
surface of a cell Complemen~1
C56789 complexes damage the tissue in the same way as that
in Arthus reaction. Serum sickness is characterised
Cytotoxic action by
NK cells
Fig. 19.2 Type II (cytotoxic) hypersensitivity

57 Lysis
by fever, urticaria, arthralgia, lymphadenopathy and
splenomegaly. Some important immune complex diseases
are poststreptococcal glomerulonephritis, endocarditis,
hepatitis B, dengue haemorrhagic fever and malaria.
TYPE III (IMMUNE COMPLEX) REACTION

It is characterised by deposition of antigen-antibody TYPE IV (DELAYED OR CELL MEDIATED)
complexes in tissues, activation of complement and REACTION
infiltration of polymorphonuclear leuococytes leading The reaction is mediated by sensitised T-lymphocytes
to tissue damage. Type III reactions differ from type II which, on contact with specific antigen, release
reactions because its antigens are not attached to the lymphokines that cause biological effects on macrophages,
surface of a cell. The interaction of these antigens with leucocytes and tissue cells (Fig. 19.3). In contrast, all
antibodies produce free floating complexes which can be others hypersensitivity reactions are induced by circulating
deposited in the tissues leading to an immune complex antibodies. Type IV or delayed type of hypersensitivity
reaction. occurs within 48-72 hours of antigen challenge. As it is
Two typical type III reactions include Arthus not antibody mediated, it cannot be passively transferred
reaction (localised) due to relative antibody excess and by serum, but can be transferred by lymphocytes or the
serum sickness (generalised) because of relative antigen transfer factor. Two types of delayed hypersensitivity
excess. reactions are well recognised, the tuberculin (infection)
type and the contact dermatitis type.
1. Arthus Reaction
Arthus (1903) observed that with repeated subcutaneous Lymphokines
injections of normal horse serum (antigen) into rabbits,
the initial injections were without any local effect, but with
later injections, intense local oedema and haemorrhagic
necrosis develop. This type of reaction is called Arthus
/'
reaction. The tissue damage is due to antigen-antibody
complexes formed at equivalence or slight antibody
excess. The antigen antibody complexes are deposited
on the walls of blood vessels. These complexes activate
complement and attract neutrophils at the local site.
Leucocyte-platelet thrombi are formed which reduce
the blood supply and cause tissue necrosis. The Arthus
reaction can be passively transferred with sera containing
high titre of antibodies (lgG, IgM).
2. Serum Sickness Cytotoxic T-cell Target cell

It is a systemic form of type III hypersensitivity
Fig. 19.3 Type IV (delayed or cell mediated) hypersensitivity
reaction. It appears following a single injection of high

Ch 19: Hypersensitivity _ _ _ _ __ _ _ _ _ _ _ _ _ _ __ _ _ _ _ _ __ _ _ _ _ _ _ 157
1. Tuberculin (Infection) Type dermis is infiltrated predominantly by lymphocytes and

When a small dose of tuberculin is injected intradermally few macrophages.
in an individual sensitised to tuberculoprotein by prior
infection or immunisation, an erythema and swelling Detection
(induration) occurs at the site of injection within 48-72 Patch test
hours. The injection site is infiltrated by large number The suspected allergen is applied to a small area of
of lymphocytes and about 10-12% macrophages. In skin under an adherent dressing. Sensitivity is indicated
unsensitised individuals, the tuberculin injection provokes by itching appearing in 4-5 hours, and local reaction
no response. Purified protein derivative (PPD) which is (erythema, vesicle or blister formation) after 24-48 hours.
the active material of tubercle bacilli, is used in tuberculin
test. 1YPE V (STIMULATORY 1YPE) REACTION
The tuberculin test is a useful indicator for delayed It is a modification of Type II hypersensitivity reaction.
hypersensitivity (cell mediated immunity) to the bacillus. Antibodies interact with antigens on cell surface that
Cell mediated hypersensitivity reaction develops in many leads to cell proliferation and differentiation instead of
infections with bacteria (M. tuberculosis, M leprae), fungi inhibition or killing. Antigen-antibody reaction enhances
and parasites. It occurs when infection is subacute or the activity of affected cell.
chronic and the pathogen is intracellular. The typical example is Grave's disease in which thyroid
Various skin tests are performed to detect delayed hormones are produced in excess quantity. Long acting
type of hypersensitivity. Positive skin test does not indicate thyroid stimulating (LATS) antibody is an autoantibody
present infection but implies that the person has been to thyroid membrane antigen. It is presumed that LATS
infected or immunised by the microorganism in the past. combines with a TSH receptor on thyroid cell surface and
Some of these skin tests include: produces the same effect as TSH resulting in excessive
(i) Lepromin test: It is positive in tuberculoid leprosy secretion of thyroid hormone.
but negative in lepromatous type of leprosy.
(ii) Frei test: This test is positive in lymphogranuloma II. SHWARTZMAN REACTION
venereum (LGV). It is not an immune response but it has superficial
(iii) Histoplasmin test: It is positive in histoplasmosis resemblance to hypersensitivity reaction. It is probably a
(a fungal infection caused by Histoplasma specialised type of intravascular coagulation precipitated
capsulatum). by endotoxin.
Shwartzman (1928) injected a culture filtrate of
2. Contact Dermatitis Type S. typhi intradermally in a rabbit and same filtrate was
Delayed hypersensitivity may sometimes develop as injected intravenously after 24 hours. A haemorrhagic
a result of skin contact with a range of sensitising necrotic lesion developed at the site of intradermal
materials- metals such as nickel and chromium, drugs inoculation. The initial intradermal (preparatory)
such as penicillin or other antibiotics in ointments, injection causes accumulation of leucocytes which by
simple chemicals like hair dyes, picryl chloride, release of lysosomal enzymes damage capillary walls.
dinitrochlorobenzene, cosmetics and soaps. Following the intravenous (provocative) injection, there
These substances can act as haptens. After occurs intravascular clotting, the thrombi leading to
absorption through skin, these molecules combine necrosis of vessel walls and haemorrhage. This is known
with skin protein to become antigenic. Cell mediated as Shwartzman reaction.
immunity is induced in skin. As most of the antigens When both preparatory and provocative doses are
involved are fat soluble, their likely portal of entry is given intravenously, the animal dies 12-24 hours after the
along the sebaceous glands. Sensitisation is particularly second dose. Autopsy reveals bilateral cortical necrosis of
liable to occur when the chemical is applied in an oily the kidneys and patchy haemorrhagic necrosis in other
base (ointment or cream) on an inflammed area of the organs such as liver, spleen etc. The reaction is also called
skin. The Langerhan's cells of skin carry these antigens the Sanarelli-Shwartzman reaction or the generalised
to regional lymph nodes where T-lymphocytes are Shwartzman reaction.
sensitised. On subsequent exposure to the offending Mechanisms similar to the Shwartzman reaction may
agent, sensitised lymphocytes release lymphokines which operate in certain clinical conditions, such as fulminating
cause superficial inflammation of skin characterised by meningococcal septicaemia (Waterhouse-Friderichsen
redness, induration, vesiculation within 24-48 hours. The syndrome).

158 - - - - - - - -- - - -- - - - - - - - -- -- - - -- - -- - Unit II: Immunology
KEY POINTS
1. Hypersensitivity refers to a condition in which immune response results in excessive reactions leading to tissue damage,
disease or even death in the sensitised host.
2. Hypersensitivity reactions are mainly of four types: type I (anaphylactic) , type II (cytotoxic), type Ill (immune complex)
and type IV (delayed or cell mediated).
3. Type I, II and III depends on the interaction of antigen with humoral antibodies and are known as immediate type
reactions. Type IV reaction is mediated by T-lymphocytes.
4. lgE is involved in type I hypersensitivity reaction. Type I reactions occur in two forms, the systemic form called
anaphylaxis and the localised form called atopy.
5. Type II reaction is mediated by antibodies that react with antigens present on the surface of cell or other tissue
components resulting in damage of the cell. Examples of this reaction are autoimmune anaemias and haemolytic
disease of the new born.
6. Type III reaction is caused by antigen-antibody complexes. These complexes may be deposited in tissues causing
immune complex disorders. Two typical type III reactions include Arthus reaction (localised) and serum sickness
(generalised).
7. Type IV reaction is mediated by T-lymphocytes. The antigen activates specifically sensitised T-lymphocytes which
lead to the secretion of lymphokines, with fluid and phagocyte accumulation. Two types of delayed hypersensitivity
reactions include the tuberculin (infection) type and the contact dermatitis type.
YOU MUST KNOW

1. Differences between immediate and delayed type of hypersensitivity reactions.
2. Mechanisms and examples of type I, type II, type III and type IV hypersensitivity reactions
STUDY QUESTIONS
1. Classify hypersensitivity reactions. Describe in detail about type I hypersensitivity reactions.
(a) Arthus reaction (b) Serum sickness (c) Delayed hypersensitivity reaction
(d) Atopy (e) Anaphylaxis (f) Type II hypersensitivity reactions.
u (' Q fSTIONS (MCQs)

1. Which of the following hypersensitivity reactions has cell mediated immune response?
(a) Type I (b) Type II
(c) Type III (d) Type IV
2. Which class of immunoglobulin mediates type I hypersensitivity reactions?
(a) IgG (b) IgM
(c) IgA (d) lgE
3. Chemical mediators released during type I hypersensitivity reactions may be:
(a) Histamine (b) Serotonin
(c) Eosinophil chemotactic factors of anaphylaxis (d) All of the above
4. Schultz-Dale phenomenon is an example of:
(a) Type I hypersensitivity reaction (b) Type II hypersensitivity reaction
(c) Type III hypersensitivity reaction (d) Type IV hypersensitivity reaction
5. Arthus reaction is an example of:

Ch 19: Hypersensitivity - -- - - - - - - - - - - ' - - - - - - - - - - - - - - - - - - - - - - 159
6. Delayed hypersensitivity reaction is mediated by:

(a) T lymphocytes (b) B lymphocytes
(c) Macrophages (d) All of the above
7. Lepromin test is an example of:
8. Excess production of thyroid hormones in Grave's disease is due to:
9. Shwartzman reaction is an example of:
(c) Type III hypersensitivity reaction (d) None of the above
Answers (MCQs):
1. (d) 2. (d) 3. (d) 4. (a) 5. (c)
6. (a) 7. (d) 8. (d) 9. (d)
---- +++ ----

Chapter 20
AUTOIMMUNITY
I. Introduction
II. Mechanisms of Autoimmunity
A. Hidden or sequestered antigens B. Antigen Alteration
C. Cross Reacting Foreign Antigens D. Forbidden Clones
E. T and B Cell defects
Ill. Classification of Autoimmune Diseases
A. Haemocytolytic Diseases B. Localised or Organ Specific Diseases
C. Systemic or Non-Organ-Specific Diseases
IV. Pathogenesis of Autoimmune Diseases
I. INTRODUCTION circulation, they may evoke an immune response. Examples

Normally an animal's immune system recognises its own are lens antigen of eye, sperm and thyroglobulin. These
tissue antigens as 'self' and therefore it doesn't produce self antigens are present in closed system and have no
antibodies against these self antigens. Autoimmunity is access to the immune apparatus. The lens protein is
a condition when the body produces autoantibodies and enclosed in its capsule and has no contact with blood
immunologically competent T-lymphocytes against its own circulation. Hence immunological tolerance against lens
tissues. This leads to structural or functional damage of protein is not developed during foetal life. When this
tissues. Autoimmunity literally means 'protection against antigen leaks out, following cataract surgery or injury, it
self', while it leads to development of 'injury to self ' . may elicit an immune response which results in damage
Self tolerance was originally described by Ehrlich to the lens of the other eye. As spermatozoa develop
(1901) as 'horror autotoxicus'. He observed that after only during puberty, the tolerance against this antigen
immunisation of a goat with erythrocytes of other goats, is not induced during foetal life. Following mumps, the
antibodies were produced against erythrocytes of other virus damages the basement membrane of seminiferous
goats but not against their own. tubules which leads to leakage of sperms and therefore
induce an immune response resulting in orchitis.
II. MECHANISMS OF AUTOIMMUNI'IY
Autoimmunisation can occur under the following
conditions: B. Antigen Alteration
Tissue antigens may be altered by physical, chemical or
A. Hidden or Sequestered Antigens biological factors and these new cell surface antigens are
Certain hidden or 'sequestered' antigens are not recognised called 'neoantigens' . These neoantigens are no longer
as self antigens. When these antigens are released into recognised as 'self' and may elicit an immune response.
160
Ch 20: Autoimmunity - - - - - - - - - - - ' - - - - - - - - - - - - - - - - - - - - - - - 161
Viruses and other intracellular pathogens may activity of T helper and T suppressor cells. Enhanced
induce alteration of cell surface antigens leading to function of T helper cell and decreased T suppressor cell
autoimmunity. function are suggested as causes of autoimmunity.
Another hypothesis is polyclonal B cell activation.
C. Cross Reacting Foreign Antigens An antigen generally activates only its corresponding B
Sharing of antigens by different organisms is the basis of cell but certain microorganisms and their products can
'cross reacting antigen' theory. Streptococcus (M proteins) cause polyclonal (i.e. antigen nonspecific) activation of
and heart muscle share antigenic characteristics. Repeated B-lymphocytes to produce antibodies.
streptococcal infection can therefore induce the immune Nonspecific antibodies are formed in some infectious
response which damages the heart. Nephritogenic strains diseases, such as antihuman erythrocyte cold antibodies
of streptococci may lead to glomerulonephritis due to in mycoplasma pneumonia.
the antigenic sharing.
Injection of Semple rabies vaccine may elicit an
III. CLASSIFICATION OF
immune response against sheep brain antigens. This may
AUTOIMMUNE DISEASES
lead to encephalitis due to cross reaction between human
and sheep brain antigens. Autoimmune diseases may be classified as
A. Haemocytolytic diseases
D. Forbidden Clones B. Localised or organ specific diseases
According to clonal selection theory an innumerable C. Systemic or non-organ-specific diseases.
number of clones of immunologically competent cells
(ICCs) bearing specific antibody patterns are produced A. Haemocytolytic Diseases
against all possible antigens. Any clone of cells carrying a These conditions involve various cells present in blood
pattern reactive against self antigens is destroyed during circulation. Haemocytolytic diseases include autoimmune
embryonic life. Such clones are known as 'forbidden haemolytic anaemias, leucopenia and thrombocytopenia.
clones'. At the time of birth only cells against nonself
persist. Persistence of forbidden clones or development in B. Localised or Organ-Specific Diseases
later life by somatic mutation could lead to autoimmune In these diseases, specific organs are targets for
diseases. autoimmune reactions.
E. T and B Cell Defects C. Systemic or Non-Organ-Specific Diseases

T helper cells facilitate B cell response to antigens while In systemic diseases, immune response occurs against a
T suppressor cells inhibit antibody production by B cells. variety of self antigens and involves damage to several
Optimal antibody response depends on the balanced organs and tissue system (Table 20.1).
Table 20.1 Autoimmune Diseases

Type Disease Autoantibody
A. Haemocytolytic 1. Autoimmune haemolytic anaemia 1. Anti-red blood cell antibodies.

diseases 2. Idiopathic thrombocytopenic 2. Antiplatelet antibodies.
purpura
B. Localised or Organ 1. Grave's disease I. Anti-TSH receptor autoantibody
specific diseases 2. Hoshimoto's thyroiditis 2. Antibodies to thyroglobulin and to microsomal antigens
3. Myasthenia gravis 3. Antiacetylcholine antibodies
4. Pernicious anaemia 4. Antibody to gastric parietal cells and to vitamin B-12 binding
site of intrinsic factor
5. Addison's disease 5. Antibodies to adrenal cells
6. Chronic active hepatitis 6. Antinuclear antibodies; antihepatocyte antibodies
7. Sympathetic ophthalmia 7. Antibody to uveal or retinal tissue
C. Systemic diseases 1. Rheumatoid arthritis 1. Antigammaglobulin antibodies

(non-organ 2. Systemic lupus erythematosus 2. Antinuclear (anti DNA)
specific) antibodies
3. Goodpasture's syndrome 3. Antibasement membrane

162 - - - - - - - - - - -
- - - - - - - - - - - - - - - - - - - - - Unit II : Immunology
IV. PATHOGENESIS OF like SLE, myasthenia gravis and Grave's disease. In SLE,
AUTOIMM UNE DISEASES
deposition of circulating immune complexes in glomerular
Autoimmun e diseases may occur either by humoral
response or by cellular immune response against self basement membrane results in tissue damage. A third
antigens. Autoantibodies are more easily detectable than mechanism in autoimmune diseases is by type IV reaction
T-cell assessment for cellular response. Antibodies may (delayed type of hypersensitivity). Humoral and cellular
cause damage by type II (cytotoxic) or type III (immune immune responses may act together in some autoimmune
complex) hypersensitivity reactions. Type II reaction diseases. Experimental orchitis can be induced only when
is important in autoimmun e haemolytic anaemias in both type of immune responses are in action.
which autoantibod ies alongwith complemen t destroy Progress of autoimmun e diseases can be arrested by
erythrocytes. Type III reaction has a role in diseases immunosuppressive therapy.
KEY POINTS
1. Autoimmuni ty is a condition when the body produces autoantibodies and immunologically competent T-lymphocytes
against its own tissues.
2. Autoimmunity can occur in certain conditions such as hidden or sequestered antigens, antigen alteration, cross reacting
foreign antigens, forbidden clones, T and B cell defects.
YOU MUST KNOW

1. Mechanisms of autoimmunity.
2. List of autoimmune diseases.
STUDY QUESTIONS
1. What is autoimmunity? Discuss the various mechanisms of autoimmunity.
2. Classify autoimmune diseases and describe their pathogenesis.
UL IPLE CHOICE QUESTIONS (MCQs)

1. Lens protein of eye is an example of:
(a) Sequestered antigen (b) Neoantigen
(c) Cross-reacting foreign antigen (d) None of the above
2. Which antibody is responsible for systemic lupus erythematosus?
(a) Antiplatelet antibodies (b) Antinuclear antibodies
(c) Antibasement membrane antibodies (d) None of the above
Answers (MCQs):
1. (a) 2. (b)
- - - - +++ - - - -

Chapter 21
TRANSPLANTATION AND TUMOUR IMMUNITY
I. Transplantation
A. Types of Transplants B. The Allograft Reaction
C. Histocompatibility Antigens D. Histocompatibility Testing
E. Prevention of Graft Rejection F. Graft-Versus-Host Reaction
II. Tumour Immunity
A. Tumour Antigens B. Immune Response in Malignancy
C. lmmunosurveillance D. lmmunotherapy of Cancer
I. TRANSPLANTATION 4. Xenograft (formerly called heterografts): These are

It is a useful procedure for replacement of diseased tissues grafts between members of different species and are
or organs which are irrepairably damaged. Transplantation called xenografts.
is necessary for restoration of function. The tissue or Autografts and isografts are usually accepted and
organ transplanted is called transplant or graft. It has survive. Allografts and xenografts are usually rejected
been known for a long time that transplants survive due to genetic and antigenic incompatibility.
only when the tissue or organ is taken from the recipient
himself, while grafts from another individual of the same B. The Allograft Reaction
species or from a different species would be rejected. The Rejection of the graft by the recipient is called the allograft
immune response induced by the transplantation (HLA) reaction. The graft becomes vascularised during the first
antigens is the reason for rejection of these exogenous two or three days and appears to be accepted initially.
grafts. By the fourth day, inflammation occurs and the graft is
invaded by lymphocytes and macrophages. Vascularity
A. Types of Transplants diminishes due to thrombosis and the graft undergoes
1. Autograft: It is a tissue or organ taken from one ischaemic necrosis. By the tenth day, graft sloughs off.
site and engrafted to another site in the same These events resulting in the rejection of allograft is
individual. known as the 'first set response'. Cell mediated reaction
2. Isograft: It is a graft taken from an individual and is responsible for the reaction. Rejection is primarily
placed on another individual of the same genetic by helper T lymphocytes, which activate cytotoxic T
constitution e.g. grafts made between identical lymphocytes, macrophages and B lymphocytes.
twins. When a second allograft from the same donor is
3. Allograft (formerly called homografts): It is graft applied on a sensitised recipient (in which previous graft
transfer between two genetically nonidentical members has been rejected by the first set response), it will be
of the same species. rejected in an accelerated fashion. Necrosis sets in early
163

164 - - - - -- - - - -- - - - - - - - - - - - - - - - - - - - - - Unit II: Immunology
and the graft sloughs off by the sixth day. This type cultured together. Donor cells are irradiated to prevent
of rejection is known as 'second set response'. In this, DNA synthesis and proliferation. A radioactive DNA
antibodies play a dominant role along with cell mediated precursor is also added to the mixture of donor and
immunity. 'Second set response' is seen only if the second recipient lymphocytes. If the class II antigens are foreign,
graft is from the same donor as the first. Application of the recipient cells will be stimulated to divide. As these
the graft from another donor will evoke only the first stimulated recipient lymphocytes replicate their DNA,
set response. they incorporate the radioactive precursor. The amount
of radioactivity incorporated can be easily measured.
C. Histocornpatibility Antigens
Histocompatibility antigens are specific for each individual E. Prevention of Graft Rejection
and are under genetic control. When the recipient 1. Immunosuppression
possesses all the antigens present in the graft, there Immunosuppression is produced in the recipient so that
will be no immune response and the transplantation is the transplanted tissue survives for a longer period.
successful. Rejection of graft depends upon the antigenic Irradiation, corticosteroids and antilymphocytic serum
differences between graft and recipient. The term 'major (ALS) are employed for immunosuppression in clinical
histocompatibility complex' (MHC) is a system of cell transplantation. Fungal metabolites such as cyclosporin
antigens that are responsible for allograft rejection or A and repamycin are also used. They show specific T cell
acceptance. The MHC in man is the 'human leucocyte inhibitory activity.
antigen (HLA) system'. A description of the HLA system
is presented in Chapter 16. 2. Transplantation in anatomically protected sites
There are certain privileged sites where allografts are
D. Histocornpatibility Testing permitted to survive. These include cornea, cartilage
Procedures used for matching donor and recipient for and testicle grafting. Lack of vascularity prevents graft
transplantation are described below. rejection in case of corneal transplants.
The foetus can be one example of an intrauterine
1. ABO Grouping allograft as it contains antigens which are foreign to the
ABO compatibility is essential in all tissue transplantation mother. The reason why the foetus is exempt from rejection
because blood group antigens are strong histocompatibility is not clear. Various explanations are as follows:
antigens. (i) The placenta produces certain hormones which
are locally immunosuppressive.
2. Tissue Typing (ii) The placenta produces mucoproteins which coat
(Detection of MHC Antigens) foetal cells, thus masking HLA-antigens and
The HLA antigens of class I type on leucocytes are prevent recognition.
identified by means of antisera obtained from multiparous (iii) The mother produces specific blocking antibodies
women, from persons who have received multiple blood to foetal antigens and thus blocking immune
transfusions, from individuals who are repeatedly skin recognition.
grafted and from volunteers who have been immunised (iv) The high concentration of alpha-fetoprotein may
with cells from another individual with a different HLA protect the foetus against immunological damage
haplotype. Monoclonal antibodies are now being used from any maternal leucocytes entering foetal
instead of these antisera. Following methods are used: circulation. This is due to immunosuppressive
properties of alpha-fetoprotein.
(i) Cytotoxic test (v) P1-glycoprotein, of foetal origin, has been shown
Lymphocytes are incubated with a panel of standard sera to inhibit maternal cellular immunity.
for HLA antigens (class I MHC antigens) in presence of These factors depress the immune system of
complement. Cells carrying antigens corresponding to the the mother and its response is localised in the
HLA antiserum are killed. These can be detected by the uterus.
addition of trypan blue which stains only dead cells.
F. Graft-Versus Host Reaction
(ii) Mixed lymphocytes culture (MLC) Graft rejection is generally due to the reaction of the host
MHC class II antigens are identified by this method. to the grafted tissue (host- versus-graft response). Contrary
Lymphocytes from both donor and recipient are to that, the graft may mount an immune response against

Ch 21: Transplantati on and Tumour Immunity _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ 165
the antigens of the host this is known as the 'graft-versus- specific. All tumours produced by one virus will possess
host (GVH) reaction'. the same antigens, even if the tumours are in different
The GVH reaction occurs when the following animal strains or species.
conditions are present:
1. The graft contains immunocompetent 2. Tumour Associated Antigens (TAAs)
T-lymphocytes. MHC antigens of recipient activate These are present on tumour cells and also on some
transplanted immunocompetent T-lymphocytes normal cells. These include:
which lead to the production of lymphokines, Tc (i) Tumour associated carbohydrate Antigens (TACAs):
cells, antibodies etc. They attack the recipient cells They represent abnormal forms of glycoproteins and
leading to manifestations of GVH reaction. glycolipids such as mucin-associated antigen detected
2. The recipient possesses HLA antigens that are absent in pancreatic and breast cancers.
in the graft. (ii) Oncofetal antigens: These are foetal antigens which
3. The recipient's immunological responsiveness is either are present in embryonic and malignant cells, but not
destroyed or so impaired (following whole body in normal adult cells. The best known examples are
irradiation) that he cannot reject a graft. alpha-fetoprotein in hepatomas and carcinoembryonic
The GVH reactions are predominantly cell antigen (CEA) in colonic cancers.
mediated. The manifestations of GVH reaction consist (iii) Differentiation antigens: CD 10, an antigen expressed
of splenomegaly, fever, rash, anaemia, weight loss and in early B-lymphocytes, is present in B-cell leukaernias.
sometimes death. Neonatally thymectornised animal Similarly, prostate specific antigen (PSA) has been
receiving an allograft of spleen or blood lymphocytes do used as a diagnostic indicator in prostatic cancer.
not grow normally but develop a fatal wasting syndrome
known as runting disease. B. Immune Response in Malignancy
Both cell-mediated and humeral responses can be
demonstrated in malignancy. The sensitised T-cells attack
II. TUMOUR IMMUNilY the 'foreign' tumour cells and tend to limit its growth.
When a cell undergoes malignant transformation, it Of all the T-cell subsets, delayed-type hypersensitivity
expresses new surface antigens and may also lose some T (Td) cells play a significant role in tumour killing by
normal antigens. The tumour associated antigens are means of lymphokines that they release. The natural killer
immunologically distinct from normal tissue antigens. (NK) cells can also kill the tumour cells in absence of
The host recognises the tumour associated antigens antibody. In addition to direct lysis of tumour cells, NK
as "nonself," therefore, tumour can be considered cells also participate in antibody dependent cytotoxicity
as an allograft and is expected to induce an immune (ADCC). Thus NK cells may provide the first line of
response. defence against many tumours.
Humoral responses may also participate in tumour
A. Tumour Antigens cell killing by activation of complement and induction
They can be classified into two groups: of ADCC by NK cells.
1. Tumour specific antigens (TSAs)
2. Tumour associated antigens (TAAs). C. Immunosurveillance
It is believed that malignant cells arise by mutation of
1. Tumour Specific Antigens (TSAs) somatic cells that occur frequently. It is postulated that
Tumour specific antigens are present on the membrane the immune system keeps a constant vigilance on these
of cancer cells and not on the normal cells. They induce malignant mutation of somatic cells and destroy them
an immune response when the tumour is transplanted in on the spot. Inefficiency of the immunosurveillance,
syngenic animals. Such tumour specific antigens reject either as a result of ageing or in congenital or iatrogenic
tumour transplants in immunised hosts, these are termed immunodeficiencies, leads to an increased incidence of
as 'tumour specific transplantation antigens' (TSTA) or cancer. The development of tumours represents an escape
'tumour associated transplantation antigens' (TATA). from this surveillance. The mechanisms of such escape
In chemically induced tumours, these antigens are are not clear but various possibilities have been suggested.
tumour specific. Different tumours possess different These are as follows:
antigens, even if they are induced by the same carcinogen. 1. Modulation of Surface Antigens: Certain tumour cells
In contrast, the TSAs of virus induced tumours are virus may shed or stop expressing the surface antigens thus

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making the tumour cells immunologically invisible. injected directly into certain tumours may lead to tumour
2. Masking Tumour Antigens: Some cancers produce a regression. Antitumour effect of BCG is believed to be
mucoprotein called sialomucin. It binds to the surface due to activation of macrophages and NK cells. BCG
of the tumour cells. Since sialomucin is a normal has been reported to be useful in malignant melanomas,
component, the tumour cells are not recognised as bladder cancer, lung cancer and certain leukaemias.
foreign by the immune system. Corynebacterium parvum has been reported to be
3. Production of Blocking Antibodies: Certain tumour beneficial in various types of lung cancers and metastatic
cells invoke immune system to produce blocking breast cancer. Dinitrochlorobenzene (DNCB) has been
antibodies which can not fix and activate complement tried in squamous and basal cell carcinoma of the skin.
resulting in prevention of tumour cell lysis. Levamisole, originally introduced as an antihelminthic,
4. Fast Rate of Proliferation of Malignant Cells: Due has been tried for stimulating cell mediated immunity
to the fast rate of proliferation of malignant cells, and macrophage functions.
tumour cells may be able to 'sneak through' before Other nonspecific immune modulators include thymic
the development of an effective immune response hormones to restore T-cell function, interferon to stimulate
and once they reach a certain mass, the tumour load NK cell function and IL-2 to stimulate killing of cancers
may be too great for the host's immune system to by Tc cells, NK cells and macrophages.
control.
5. Suppression of Cell Mediated Immunity (CMI): Some 2. Specific Immunotherapy
tumours may form cytokines like Transforming This has been attempted by vaccination with tumour
Growth Factor (TGF-~) which suppresses CMI. antigens, treatment with 'immune RNA' and transfer
factor. Monoclonal antibody to tumour antigens may
D. Immunotherapy of Cancer be administered, either alone or tagged with a cytotoxic
Immunotherapy of cancer is of two types: drug, will bind to and specifically destroy only cancer
cells. Lymphokine activated killer (LAK) cells obtained by
1. Non-Specific Active Immunotherapy treatment of natural killer (NK) cells with interleukin-2
This activates the immune system in a generalised manner (IL-2) have been tried in the treatment of certain
which destroys the tumour cells. BCG vaccine when malignancies, such as renal carcinomas.
KEY POINTS
I. Transplantation is a useful procedure for replacement of diseased tissues or organs which are irrepairably damaged.
The tissue or organ transplanted is called transplant or graft.
2. There are four types of transplant namely autograft, isograft, allograft and xenograft.
3. Rejection of graft depends upon the antigenic differences between graft and recipient. The transplantation (HLA)
antigens are responsible for graft rejection or acceptance.
4. Graft rejection is generally due to the reaction of the host to the grafted tissue (host-versus-graft response). Contrary
to that, the graft may mount an immune response against the antigens of the host this is known as the 'graft-versus-
host (GVH) reaction'.
5. It is believed that malignant cells arise by mutation of somatic cells that occur frequently. It is postulated that the
immune system keeps a constant vigilance on these malignant mutation of somatic cells and destroy them on the
spot. This is known as immunosurveillance.
6. The development of tumour represents an escape from this immunosurveillance.
YOU MUST KNOW

1. Types of transplants.
2. Histocompatibility antigens.
3. Graft-versus-host (GVH) reaction.
4. Immunosurveillance.

Ch 21: Transplantation and Tumour Immunity _ _ _ _ _ __ _ _ _ _ _ _ _ _ _
_ _ _ _ _ _ _ 167
STUDY QUEST IONS

1. Define various types of transplant s. Describe briefly about the allograft reaction.
(a) Histocomp atibility antigens (b) Graft-vers us-host (GVH) reaction.
3. Discuss briefly about role of immunity in tumours.
(a) Tumour antigens (b) Immunosu rveillance.
MULTIPLE CHOICE QUEST IONS (MCQs )

1. Grafts between two genetically non-identi cal members of the same species are known as:
(a) Autografts (b) Isografts
(c) Allografts (d) Xenograft s
2. Which of the following cells may provide the first line of defence against many tumours?
(a) Eosinophils (b) atural killer cells
(c) Monocyte s (d) one of the above
Answers (MCQs):
1. (c) 2. (b)
____ ... ____

Chapter 22
IMMUNOHAEMATOLOGY
I. ABO Blood Group System

II. Rh Blood Group System
Ill. Haemolytic Disease in Newborn
IV. Complications of Blood Transfusion
The ABO system is the most important of all the blood Table 22.1 Distribution of ABO Antigens on the Red
group systems known and its discovery made blood Cells and Isoantibodies in the Serum.
transfusion possible. The other blood group systems Blood group Antigen on red cells Isoantibodies in serum
include Rh, MN, P, Lutheran, Lewis, Kell, Duffy, Kidd,
A A Anti-B
Diego, Yt, Kg, Dombrock and Colton. The ABO and
Rh systems are the major blood group antigens. B B Anti-A
AB AB None
I. ABO BLOOD GROUP SYSTEM 0 None Anti-A and Anti-B
The ABO blood group system was originally described by
Landsteiner (1900) and now contains four blood groups. H Antigen
In 1930, Landsteiner was awarded the Nobel Prize for The H antigen (H substance) is present on red cells of
the discovery of human blood groups. The blood group all ABO blood groups. It is the precursor substance
is determined by presence or absence of two antigens A on which A-B-0 genes operate to produce A and B
and B on the surface of the red cell membrane. Red cells antigens. Due to universal distribution of H antigen, it
of blood group A carry antigen A, cells of blood group is not ordinarily important in blood grouping or blood
B antigen B and cells of blood group AB have both A transfusion. However, Bhende et al (1952) from Bombay
and B antigens, while blood group O cells have neither reported a rare instance in which A and B antigens as
A nor B antigens. These groups are also distinguished well as H antigens were absent from red cells. This is
by the presence or absence of two distinct isoantibodies called as OH blood group or Bombay type. Sera of these
in the serum. The serum contains the isoantibodies for persons contain anti-A, anti-B and anti-H antibodies,
the antigen that is absent on the red cell. The serum of therefore, they can accept the blood only from their own
blood group A individual possesses anti-B antibodies, group (OH or Bombay type).
blood group B has anti-A antibodies, and blood group A, B and H antigens are glycoproteins. H antigen
0 both anti-A and anti-B, while in blood group AB both is structurally L-fucose, A and B antigens are formed
anti-A and anti-B antibodies are absent (Table 22.1). by addition of N-acetylgalactosamine and galactose
Anti-A and anti-B isoantibodies are called natural respectively to H antigen (L-fucose) . In addition to
antibodies because they are seen to arise without any erythrocytes, these antigens are also present in almost
apparent antigenic stimulation. all tissues and fluids of the body.
168
Ch 22: lmmunohaematology _ _ _ _ _ _ __ __ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __ _ _ _ 169
II. RH BLOOD GROUP SYSTEM 2. ABO incompatibility between foetus and mother:
Landsteiner and Wiener produced an anti-Rhesus serum In faeto-maternal ABO incompatibility, the foetal
by injecting rabbits with red cells from Rhesus monkey. cells entering th.e maternal circulation are destroyed
Anti-Rhesus serum agglutinated about 83% human red rapidly by the mother's incompatible anti-A or anti-B
cells. This type of antigen was then named Rhesus antigen antibodies before they can induce Rh antibodies.
or Rh factor. 3. Number of pregnancies: The risk increases with each
Of all the Rh antigens, antigen D (Rho) is the most successive pregnancy of mother with Rh-positive
potent antigen. Rh positive or Rh-negative blood depends foetuses. The first child usually escapes damage
on the presence or absence of D-antigen on the surface because sensitisation occurs only during its delivery.
of red cells respectively. Among Indians, approximately Rh isoimmunisation can be prevented by administration
93% are Rh positive and about 7% negative. The Rh of anti-Rh IgG immediately after delivery. To be effective,
factor can be detected by testing the blood with anti-D this should be employed from first delivery onwards.
(anti-Rh) serum. The passively administered lgG antibody may prevent
In contrast to ABO system, there are no natural isoimmunisation by a negative feedback mechanism.
antibodies against Rh antigen. Rh antibodies arise in
serum only in consequence of Rh incompatible pregnancy ABO Haemolytic Disease
or transfusion. Most of these antibodies belong to the Due to maternofoetal ABO incompatibility haemolytic
IgG class, and being 'incomplete antibodies', they do not disease may occur in the newborn. Persons of blood
agglutinate Rh positive red cells in saline. A minority are group A or B possess IgM natural antibodies in blood,
complete (saline agglutinating) antibodies of lgM class. which cannot cross the placenta to harm the foetus. But
These are not relevant in the pathogenesis of haemolytic persons with blood group O contain predominantly lgG
disease of newborn as they do not cross the placenta. antibodies in blood. Hence ABO haemolytic disease is
The IgG class incomplete antibodies can be detected in seen more frequently in O group mothers having A or B
the maternal blood by indirect Coombs test and in cord group foetus. As ABO haemolytic disease is because of
blood of newborn by direct Coombs test. naturally occurring maternal isoantibodies, it may occur
even in first birth, without prior immunisation. ABO
III. HAEMOLYTIC DISEASE haemolytic disease is more common than Rh haemolytic
IN NEWBORN disease but is much milder. The direct Coombs test with
When an Rh negative person gets transfusion with Rh foetal erythrocytes is often negative but indirect Coombs
positive blood or when an Rh negative mother carries an test with neonatal serum (cord blood) is more commonly
Rh-positive foetus, antibodies to Rh are produced. positive.
The haemolytic disease of the newborn occurs most
commonly when the mother is Rh negative while the IV. COMPLICATIONS OF BLOOD
father and foetus are Rh positive. The mother is usually TRANSFUSION
immunised at first delivery and consequently the first child The complications of blood transfusion may be of two
escapes damage (except when the woman has already been types, immunological and non-immunological.
transfused earlier with Rh positive blood). During first
delivery, foetal cells enter the maternal circulation in large 1. Immunological Complications
numbers and induce Rh antibodies. During subsequent Following an incompatible blood transfusion, the red cells
pregnancy, the Rh antibodies (lgG class) pass from the undergo intravascular haemolysis or they may be coated
mother to the foetus and damage its erythrocytes. The by antibodies and engulfed by phagocytes, removed from
clinical features of haemolytic disease of newborn may circulation and subjected to extravascular lysis.
vary from erythroblastosis foetalis to intrauterine death Some transfusion reactions may be due to processes
due to hydrops foetalis. other than blood group incompatibility. These are due to
However, the incidence of haemolytic disease of the recipient being hypersensitive to some allergen present
newborn is much less than the expected figures which is in the donor blood. Patient develops rigor, urticaria and
probably due to the following: other manifestations.
1. Immunological unresponsiveness to Rh antigens: Some
Rh-negative individuals do not form Rh antibodies 2. Non-immunological Complications
even after repeated injection of Rh-positive cells. The most important is transmission of infectious agents
They are called 'nonresponders'. The reason for this during blood transfusion. These may include viruses,
unresponsiveness is not known. bacteria and protozoa. HIV and hepatitis viruses are of

170 - - - - - - - - - - - - - - - - - - - - - - - - - - - Unit II: Im munology
great concern among all the infectious agents associated 2. Bacteria

with blood transfusion. List of various infectious agents Treponema pallidum
transmitted via blood transfusion is as follows: L eptospira interrogans
I.Viruses
Human immunodeficiency viruses (HIV-1 and HIV-2) 3. Protozoa
Hepatitis B virus (HBV) Plasmodia
Hepatitis C and D viruses Leishmania donovani
Cytomegalovirus (CMV) Tox oplasma gondii
KEY POINTS
1. The A BO system is the most important of all the blood group systems known. Another important system is Rh blood
group system .
2. Transmission of infectious agents is an important complication of blood transfusion. HIV and hepatitis viruses are
of great concern among all the infectious agents associated with blood transfusion.
YOU MUST KNOW

1. Rh blood group system.
2. List of infectious agents transmitted via blood transfusion .
STUDY QUESTIONS
1. Name various blood group systems and describe Rh blood group system.
(a) Haemolytic disease in newborn (b) Complications of blood transfusion.

1. Red cells of individual with group A carry:
(a) Antigen A (b) Antigen B (c) Both of the above (d) None of the above
2. Erythrocytes of individual with blood group B carry:
3. Red cells of individual with blood group AB carry:
4. Erythrocytes of individual with blood group O carry:
5. Haemolytic disease in newborn may occur when:
(a) An Rh negative mother carries an Rh positive foetus
(b) An Rh positive mother carries an Rh negative foetus
(c) Both of the above
(d) None of the above
6. The following infectious agents may be transmitted via blood transfusion:
(a) Human immunodeficiency virus (b) Hepatitis C virus
(c) Treponema pallidum (d) All of the above
An swers (MCQs):
1. (a) 2. (b) 3. (c) 4. (d) 5. (a) 6. (d)
- - - - ++• ----
SYSTEMIC BACTERIOLOGY
23. Staphylococcus ......................................................................................... 173
24. Streptococcus and Enterococcus ............................................................. 184
25. Pneumococcus ......................................................................................... 196
26. Neisseria and Moraxella ........................................................................... 201
27. Corynebacterium ...................................................................................... 210
28. Bacillus ..................................................................................................... 219
29. Clostridium ................................................................................................ 226
30. Nonsporing Anaerobes ............................................................................. 237
31. Enterobacteriaceae ................................................................................... 243
32. Shigella ..................................................................................................... 257
33. Salmonella ................................................................................................ 262
34. Vibrio ........................................................................................................ 276
35. Campylobacter, Helicobacter, Mobiluncus ................................................ 287
36. Pseudomonas, Stenotrophomonas, Burkholderia .................................... 292

37. Yersinia, Pasteurella, Francisella .............................................................. 300
38. Legionella ................................................................................................. 308
39. Haemophilus and Gardnerella ................................................................. 311
40. Bordetella .................................................................................................. 319
41. Brucella ..................................................................................................... 324
42. Mycobacterium Tuberculosis ..................................................................... 330
43. Non-Tuberculous Mycobacteria ............................................................... 346
44. Mycobacterium Leprae ............................................................................. 352
45. Spirochaetes ............................................................................................. 360
46. Mycoplasma and Ureaplasma .................................................................. 375
47. Actinomycetes ........................................................................................... 381
48. Miscellaneous Bacteria ............................................................................ 386
49. Rickettsia, Orientia, Coxiella, Ehrlichia, Bartonella ................................. 393
50. Chlamydia and Chlamydophila ................................................................. 402

Chapter 23
STAPHYLOCOCCUS
I. Species
A. Staphylococcus aureus B. Staphylococcus epidermidis
C. Staphylococcus saprophyticus
~~loco ccus aureus
_k'Morphol ogy ~re
._.....G:-13iochemical Reactions .rr.-Resistance
E. Antigenic Structure F. Toxins and Enzymes
G. Pathogenesis H. Typing Methods
I. Antibiotic Sensitivity J. Epidemiology
K. Laboratory Diagnosis L. Treatment
M. Control
Ill. Other Coagulase Positive Staphylococci
IV. Coagulase Negative Staphylococci (CONS)
A. Staphylococcus epidermidis B. Staphylococcus saprophyticus
C. Other coagulase negative staphylococci
V. Micrococci
Staphylococci are Gram positive cocci arranged in of Staphylococcus, that produces lemon yellow colour
,grape-like clusters. They are the commonest cause of and named it as Staph. citreus. Since pigment production
suppuration. Sir Alexander Ogston (1880) a Scottish is not a constant character, this classification is now
surgeon, established the pathogenic role of coccus in obsolete.
abscesses and other suppurative lesions. He gave the name
Staphy lococcus (Staphyle, meaning a bunch of grapes,
kokos, meaning a berry) from the typical arrangement
of the cocci in grape like clusters. He also observed that The genus Staphy lococcus c ains various species but
staphylococcal strains from pyogenic lesions produce the medically important pecies are:
golden yellow colonies while those from the normal ~)J-<\C } ~
skin produce white colonies on solid culture media. A. Staphyloco ccus aureus '1J ,..--
Rosenbach (1884) named them Staph. aureus and Staph. Staphylococcus aureus is a pathogenic staphylococcus that
a/bus respectively. Passel (1885) described a third strain causes pyogenic infections in man.
173 ty,,. ~()

174 - -- - - - - - - - - ~ I - ' - - - ~ __;_
:___
_ _ _ _ _ _ _ _ _ _ _ Unit Ill: Systemic Bacteriology
B. Staphylococcus epiderm' s 1. Nutrient Agar

Staphylococcus epidermidis is a skin commensal. After overnight or 24 hours incubation, the colonies are
2-4 mm in diameter, circul<U, smooth, convex, opaque and
C. Staphylococcus saprophyticus easily pi1ulsifiab)e. Most of the strains produce golden
Staphylococcus saprophyticus acts as an opportunistic yellow pigment. The pigment is not diffusable into the
pathogen. medium. Pigment production is best seen at 22- 25°C
Pathogenic strains (e.g. Staph. aureus) have certain and only in aerobic cultures. Pigment production is
characteristics like production of enzymes coagulase, enhanced by incorporation of 1% glycerol monoacetate
phosphatase, deoxyribonuclease and their ability to or milk in the medium. Thep1gment IS believed to be
ferment mannitol. Based on production of enzyme a carotenoid.
coagulase, staphylococci are classified as coagulase positive
and coagulase negative. Most of the coagulase positive
strains produce golden yellow colonies and are known
2. Blood Agar ( ft -- ~ s1'j)
Colonies are similar ta tbose on nutrient agar and in
as Staph. aureus (also named as Staph. pyogenes). These addition~et~ type of haemolysis is seejfH'aemoly sis
strains are toxigenic. The coagulase negative strains are is best observed with sheep or rabbit blood.
generally non-pathogenic, non-toxigenic and form white
colonies, these are called Staph. epidermidis (formerly 3. MacConkey's Agar l. ~ f y
known as Staph. a/bus). Colonies are very small and pink due to lactose
II. STAPHYLOCOCCUS AUREUS ~-
A. Morphology 4. Selective Media . ' \.}.>i,~
They are Gram positive cocci arranged i9 grape-like These media are used forf so latin~ Staph. aureus from
clusters, non-motile, non-spormg, approximately I µm in , samples..,__ such as faeces containing other bacteria.
diameter {F'1g. 23.1) . Cfuster formation is due to sequential Staphylococci can tolerate 8- 10% NaCl, lithium chloride,
division of bacteria in three perpendicular planes with tellurite and polymyxin. Incorporation of these reagents
daughter cells remaining in close proximity. They may "1nto media make them selective. Examples of selective
also be present singly, in pairs, tetrads or short chains of media include salt milk agar (containing 8-10% Nacl)
three or four cells especially when examined from liquid and Ludlam's medium (containing lithium chloride and
culture. A few strains may possess microscopically visible tellurite). c----
capsules, especially in young cultures. Many apparently
non-capsulated strains also have some amount of capsular 5. Mannitol Salt A ar
material on the surface. This is both a selective and an lliiilicator medi~. It
contains nutrient agar with 1% mannitol, 7.5% sodium
chloride and phenol red as indicator. Yellow coloured
colonies are seen on this medium due to fero?eotation
of mannito) by most strains of Staph. aureus. Mannitol
""""" ...
fermentation leads to production of acid and lowers the
pH of medium (phenol red produces yellow colour in
acidic pH).
6. Milk Agar
This medium is prepared by mixing sterile n u t ~ r and
s t e r i ~ . Colonies of Staph. than
those on nutriept agar and pigment production is enhan~ ---
is produced in peptone water or

B. Culture nutrient broth.
They grow readily on ordinary culture media within a
temperature range of I 0-42°C, the optimal temperature C. Biochemical Reactions
being 37°C and pH 7.4-7.6. They are aerobes and Staph. aureus is catalase positive (unlike streptococci)
facultative anaerobes. _;, and oxidase negat0e. It breaks aown carbohydrates by

Ch 23: Staphylococcus _ _ _ __ __ - ' - - - - - - -- -- -
- - - - - - - -- - 115
fermentation whereas micrococci break down sugars Most strains grow well in the presence of 10% NaCl.
aerobically. This property can be tested on Hugh-Leifson These features are significant in food preservation.
(0/F) medium. They are killed by crystal violet at a concentration of
It ferments a number of sugars producing acid without one in five hundred thousand and by brilliant green, at a
gas. These fermentation reactions are of no diagnostic concentration of one in ten million. This is the reason that
value except mannitol fermentation which may be useful crystal violet agar is used as selective medium for isolation
to differentiate Staph. aureus (maunitol positive) from of streptococci. Staphylococci are resistant to 1% phenol
Staph. epidermidis (roaooito) oegatiw). 3/ for 15 minutes while mercury perchloride (1 %) solution
_.The following characteristics help to distinguish a kills them in 10 minutes. They are resistant to lysozyme
pathogenic strain of staphylococcus (Staph. aureus) from but some micrococci are sensitive to it. Staphylococci
other non-pathogenic strains (e.g. Staph. epidermjdisJ.:__, are generally sensitive to lysostaphin, which is a mixture
~ e t a type of haemolysis on blood agarv of enzymes produced by a particula r strain of Staph.
_2:-"Production of a golden yellow pigment epidermidis.
3. Coagulase production
(\
..,.k'Man nitol fermentation
5. Gelatin liquefaction
E. Antigen ic Structure (Fig. 23.2) \_
1. Capsule
lJ)·~ ~i \
-)
osphatase production "f..) Some strains of Staph. aureus possess capsule and inhibit
oduction of enzyme deoxyribonuclease
llurite reduction P};pJpLA.~
(1/~" ph~gocytosis. The capsule is composed of polysaccharide.
~sula ted strains tend to be more virulent.
'P. Sdt c.f::-J Sp~~ =-". --·- - - - - - - - - - - - - - - ,
osphatase test LV,fl 'N
Most strains of Staph. aurclus produce enzyme phosphatase.
For detection of this enzyme, Staph. aureus is grown on
nutrient agar containing phenolphthalein diphosphate.
Enzyme phosphatase acts on phenolphthalein salt to
release free phenolphthalein. The colonies tum pink when
exposed to ammoni a vapour due to the presence of free
phenolphthalein. lmembrane
Cytoplasmic I
Cytoplasm
ce'J:11
Peptidoglycan teichoic acid complex

/i>eoxyribonuclease test
~z~:e~J:jces enzyme deoxyribonuclease which
Fig. 23.2 Antigenic structure of Staphylococcus aureus
2. Peptido glycan
Tellurite reduction C~ ,-:~
~Cl_V-L)
__~-~'" 'i Peptidoglycan is a polysaccharide polymer that provides
r=,,;~;.:;_.=....,,.._~reduces tellurite to tellurium producing rigidity to the cell wall. It activates complement and
black colonies, when grown in a medium containing evokes production of inflammatory cytokines.
potassium te urite (e.g. Potassium tellurite blood agar
used for C. d!!!f;Jheriae). 3. Teichoic Acid ( /U lo~ lt0)
It is a major antigenic determinant of all strains of Staph.
Other biochemical tests aureus. It is the group specific libitol teichoic acid of the
Staphylococci hydrolyse urea, reduce nitrates to nitrites, cell wall. It facilitates adhesion of the cocci to the host
are indole negative and are MR and VP positive. Most cell surface and prot~cts them from complement medjated
strains are lipolytic and produce a dense opacity when opsonisation. It is associated with the peptidoglycan in
grown on egg yolk medium. These tests are of lesser an insoluble form. It is absent in Staph. epidermidis. The
importance. latter contains glycerol teichoic acid. l
D. Resistan ce
Staphylococci are !,ru)Te. resistant among the non-sporing
4. Protein A l
\'7~
It is a cell wall compon ent of most strains of Staph.
~ - They survive in dried pus for 2- 3 months. Most aureus (especially <;::owan I strain). It js chemot acticy-
of the staphylococci are killed at 62°C for 30 minutes ~nti~ocytic, anticomplementary and elicits platelet
but some may require 80°C for one hour. Heat resistant Protein A has ability to bind the Fe portton of
strains may even grow at a higher temperature of 45°C. immunoglobulin G (IgG). Binding IgG in this manner
_ _ _ __ _ _ _ Unit Ill: Systemic Bacteriology
176 _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _
can block p ~ Cowan I strains coated with

any IgG antiserum will be agglutinated if mixed with e toxin 1s responsible for staphylococcal food
its corresponding antigen. This procedure is known as ,!?oisoning- nausea, vomiting and diarrho ea occurring
Coagg lut7n (refer Chapte r 14). within 2- 6 hours of consuming contaminated food.
Nine (A, B, Cl , C2, C3, D, E, H and I) antigenically
distinct enterotoxins have been identified. Some strains
F. Toxins and Enzym es may form toxin of more than one type. Type A toxin is
Staph. aureus forms a number of to~n d etlzymes. responsible for the most cases. The enterotoxin is resistant
They are importa nt virulence factors of the organism to gut enzymes. It also resists boiling for 30 minutes, a
for producing a disease m the host. temperature that kills Staph. aureus. Toxin is produced
when Staph. aureus grows in carbohydrate and protein
1. Toxins . L ,.,(_ 1 I>, X- · ()) foods, usually cooked food. Milk and milk products have
also been implicated. The food may contain preformed
' f ~distinct types called alpha, beta, gamma toxin but not the bacteria . The source of infection is
and delta haemolysins are produced by staphylococci. usually a staphylococcal lesion in skin of fingers of food
These are exotoxins. handler. Ingestion of micrograms of toxin can lead to
food poisoning. The toxin is believed to act directly
(a) Alpha lysin (a-lysin ) on the autonom ic nervous system to cause the illness.
Entero xi lso a su eran · and thus stimulates
Alpha lysin (also known as alpha toxin) is the most
important in athogenicity. In cultur~ it is produced only - -kins
~lymphocytes to release interleu -- , , necrosis
and-tumour
factor Th€1Se factors contrib ute in product ion of clinical
under erobi conditions. ~tis a prot~i and is inactivated
illness. Enterotoxins have also been implica ted in some
at 60° . The toxin regains activity if 1t is further heated
to 80°C- 100°C. This is due to toxin combining with a cases of post-antibiotic diarrhoea. The toxin can be
inhibitor at 60°C. At higher temperature, the inhibitor detected by serological tests such as ELISA and latex
is inactivated resulting in setting the toxin free. Alpha- agglutination.
lysin is lytic to rabbit erythrocytes but less active against
StAf Ut,M\W)
(iv) Toxic shock syndrom e toxin (TSST)r u;
sheep or human red cells. It is al~ cytotoxic (platelet),~
Jisucocidal and dermonecrotic. It causes injury to the Toxic shock syndrome (TSS) is characterised by fever,
c i r ~ system and muscle tissue. h~io n, vomiting, diarili,Q.ea and ~eryth ematou s
rash with subsequent desquamation and weraem ia
(b) Beta lysin ([}--lysin) of mucous membranes. Most TSST producing strains
Beta lysin is haemolytic for sheep cells, but not for human belong to bacteriophage group-I. TSST type-1 (formerly
or ra~r ocyt es. Lysis is initiated at 37 C 6~'t it is
6
also known as enterotoxin F) is most often responsible.
evident only on cold temperature. It is named as hot-cold The TSST-1 toxin producing strains colonise vagina
phenomenon. It is produced both aerobically as well as more frequently. TSST is a superantigen and thus a
anaerobically. It has been identified as sphingomyelinase potent activator of T-lymphocytes resulting in release
C. It acts on sphingomyelin in the plasma membrane of cl interleukins and tumour necrosis factor. This results
in clinical condition of TSS. J:_ L , ' ~ .
erythrocytes and is also called the hot-cold lysis.
(c) Gamma lysin (y-lysin ) (v) Exfolia tive (epidermolytic) toxin

y t s on human, sheep and rabbit erythrocytes. Two types of exfoliative toxin, A and B have been
described. Toxin A is heat stable while toxin B is heat
(d) Delta lysin (&-lysin) labile. Production of toxin A is under chromosomal
The delta lysin is lytic to human, sheep and rabbit red control whereas toxin B is plasmid mediated. These toxins
blood cells. are produced by some strains of Staph. aureus (phage
group II). They cause epidermal splitting resulting in
~listering dise~~s. It may kad to generalised desquamation
It is composed of two components, S (slow) and F (fast). producing staphylo coccal scalded skin syndrome ( SSSS) .
These components damage polymorphonuclear leucocytes The severe form oL.S~S is known as Ritter's disease in
and macrophages. This toxin is also called the l'itnton - the ~ew born. Milder forms are pemphigus neonato rum
V a l ~ after its discoverers. and bullous impetigo.

Ch 23: Staphylococcus _ _ _ _ _ __ __ _ _ _ _ _ _ _ _ __ _ _ _ _ _ _ _ _ _ _ __ 177
2. Enzymes False positive

Staph. aureus produces a number of enzymes, coag~, Citrated plasma should not be used because some
phospha~nd deoxyri~ease which are related to contaminated (e.g. Pseudomonas) bacteria may utilize
virulence of the bacteria. the citrate and produce false positive reaction. Oxalate,
Staph. aureus produces an enzyme coagulase. There EDTA or heparin are more suitable anticoagulants.
are eight (A-H) aQtigenic types of coagulase. Most human
strains produce coagulase type A. It ha~ a property to clot ~ ogenesis lit-
human or rabbit plasma. Coagulase is secreted free into Staph. aureus is /cul important pyogenic organism and
=
the culture medium. It is heat labile. It requires a plasma lesions ar{Iocalise<ljin nature in contrast to streptococcal
factor (coagulase reacting factor, CRF) for its clotting lesions which are snreading in nature. Coagulase enhances
action. CRF is present in rabbit and human plasma. virulence 9J' Staph. aureus by , inhibitin&__phagocytosis.
Clotting does not take place with guinea pig plasma It forms a ...vall of fibrin clot around the les!.Q.!1 . .Thick
because it lacks CRF. S:-2.agulase converts fibrinogen creamy pus is formed in staphylococcal infections.
jnto fibcin. , · - Staphylococcal diseases may be classified as cutaneous
Clumping factor (also called bound coagulase) is heat and deep infections; food poisoning, nosocornial infections,
stable constituent of the cell wall which reacts directly skin exfoliative diseases and toxic shock syndrome.
with the fibrinogen and causes clumping of cocci due to
the precipitation of fibrin on the cell surface.
The test for coagulase is done by the slide and the
<'1.Cutaneous Infections tj
Superficial infections include pustules, boils, carbuncles,
tube method. It is done to differentiate pathogenic (Staph. abscesses, styes, impeti10emphigus neonatorum, wound
aureus) strain from non-pathogenic strains. and burn infections. trocJi ~ } - 'o'ec(
~ p Infections
(i) Slide coagulase test These include QSteomyeljtjs, tonsillitis, pharyngitis,
Slide coagulase test detects bound coagulase. A few sinusitis, p~eumo'ititis.,__empyema, endocarditis, meningitis.,
colonies of bacteria are emulsified in a drop of normal bacteriaemia septicaemia and aernia. ,,-- \.JT[_
saline on a clean glass slide and mixed with a drop of They may caus~rina tract infecti specially in
undiluted rabbit or human plasma. Prompt clumping of association with local instrumentation or diabetes.
the suspension occurs with coagulase positive strains.
False positive results may occur in cases of citrate utilising ,,..J-Food Poisoning
bacteria (Enterococcus and Pseudomonas). Some strains Staphylococcal food poisoning may follow 2- 6 hours
which are negative for bound coagulase but positive after the ingestion of contaminated food which contains
for free coagulase gives false negative results by 'slide preformed ei1terotaxin af Staph aure.us.
coagulase test'. Hence, these should be confirmed by 'tube
coagulase test'. --CNosocomial Infections ...-----'
They are important cause of hospital acquired infections.
(ii) Tube coagulase test
Tube coagulase test is done for detection of extracellular ~ in Exfoliative Diseases
free coagulase. 0.1ml of an overnight broth culture or an These diseases are produced by the strains of Staph.
agar culture suspension of the organism is mixed with aureus that produce epidermolytic toxin. Stripping of the
0.5 ml of a 1 in 5 dilution of human or rabbit plasma. superficial layers of the skin from the underlying tissue
Diluted plasma alone in a second tube serves as a control. occurs in the various exfoliative syndromes caused by
The tubes are incubated in a water bath at 37°C for three staphylococci (bullous impetigo, pemphigus neonatorum,
to six hours. In case of a positive test, the plasma clots f[itter's disease). )staphylococcal scalded skin syndrome ~JJ
and does not flow when the tube is inverted. If clot does (SSSS) is one example of exfoliative diseases in which
not appear it is left overnight at room temperature and toxin spreads systemically. It is seen, but not exclusively,
is re-examined. On continued incubation, the clot may in small children.
be lysed by production of fibrinolysin by some strains.
Controls with known coagulase-positive and coagulase- 6. Toxic Shock Syndrome (TSS)
negative cultures must be included with each batch of It is caused by toxin shock syndrome toxin (TSST-1)
tests. and characterised by high fever, hypotension, vomiting,

178 _ _ _ __ __ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Unit Ill: Systemic Bacteriology
diarrhoea and scarlatiniform rash. Although TSS became 2. Other Typing Methods
widely known in association with the use of tampons by Molecular typing methods such as DNA fingerprinting,
menstruating women but it occurs in other situations also. ribotyping and PCR are used. Pulse field gel electrophoresis
Subsequently non-menstrual associated TSS has also been (PFGE) and sequence-based typing methods are now used
reported in both males and females as a complication in investigating of an staphylococcal outbreak.
of staphylococcal abscesses, osteomyelitis, post-surgical
wound infections etc. I. Antibiotic Sensitivity
Most of the strains of Staph. aureus were sensitive to
H. Typing Methods penicillin originally. Soon after penicillin came to be used
1. Bacteriophage Typing clinically, resistant strains began to emerge._Penicilliq_
Strains of Staph. aureus may be distinguished by resistance in"- staphylococci is_.,due to the following:
their susceptibility to different bacteriophages. An 1. Production of beta lactamase (penicillinase) which is
internationally accepted set of phages is used. A set of plasmid coded and transmitted by transduction or
23 bacteriophages is employed. The strain to be typed conjugation. The same plasmid may also carry genes
is grown on nutrient agar to produce a lawn culture. for resistance to other antibiotics (erythromycins,
After drying, the phages of basic set are applied in a tetracyclines, aminoglycosides etc.) and heavy metals.
fixed dose (routine test dose, RTD). After overnight J3eta Jactamase inactivates peni9illin by splitting the
incubation, the culture will be observed for lysis by the bet_a )actaru ring. Four types (A to D) of penicillinases
phages. The phage type is designated according to the are produced by staphylococci.
phages capable of lysing the bacterial strain (Fig. 23 .3). 2. Methicillin resistant staphylococci: There is reduction
Thus a strain of phage type 52/80/94 is the one that in affinity of penicillin binding proteins of the
is lysed only by phages 52, 80 and 94. Phage typing is staphylococcal cell wall for P-lactam antibiotics. This
important in epidemiological studies of staphylococcal change is normally chromosomal in nature. This type
infections. of resistance also occurs in beta lactamase resistant
penicillins such as methicillin, nafcillin and oxacillin.
They may cause outbreaks of hospital infection.
These strains are called methicillin resistant Staph.
aureus ( MRSA ) . These are i~portant cause.-of-
pos_!9perative wound infections and other hospital
acquired infections. They may also cause epidemics of
hospital cross infections. {yancomycin]or Jeicoplanin
is used for treatment of infections with MRSA.
Methicillin resistant strains of Staph. epidermidis
(MRSE) have also been described. As methicillin is an
unstable drug, oxacillin is used for sensitivity testing.
These strains are resistant to all P-lactam agents and
often to other agents such as aminoglycosides and
Fig. 23.3 Bacteriophage typing of staphylococci
fluoroquinolones. Methicillin resistance correlates
with the presence of the resistance gene Mee A that
The reference centre for staphylococcal phage typing codes for a unique penicillin binding protein PBP 2a
in India is located in the Department of Microbiology, (or PBP2') not affected by beta lactamase resistant
Maulana Azad Medical College, New Delhi. penicillins. This gene is transmitted chromosomally.
Typing set of staphylococcal phages Although predominantly a hospital pathogen,
MRSA is becoming more common in community.
Group I 29, 52, 52A 79, 80
These MRSA strains are named community acquired
Group II 3A, 3C, 55, 71 MRSA strains (CA-MRSA). MRSA strains restricted
Group III 6,42E,47, 53, 54, 75, 77, 83A, 84, 85 to hospital settings are named hospital acquired
Group IV MRSA strains (HA-MRSA) . Hospital personnel
harboring MRSA have been implicated as the chief
Group V 94, 96
source of nosocomial infection. These strains can
Not allocated 81, 95, cause minor and systemic staphylococcal infections.

Ch 23: Staphylococcus _ _ _ __ _ _ _ _ __ __ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __ 179
MRSE has been recognised as important agents asal and perinea! swabs are collected for detection
of nosocomial infections in hospitalised patients of carriers
who have undergone prosthetic heart valve surgery.
Eradication of colonised sites (e.g. nose or skin) with 2. Collection and Transport
topical agents is often indicated for reducing the risks Specimens should be collected in sterile containers
of cross infection in hospital. under all aseptic conditions. Sterile swabs should be
The following measures may be adopted to control used for collection of specimens from sites such as nose
staphylococcal infections in hospitals. or perineum. In case of urine, midstream urine should
• To isolate the payents with open staphylococcal be collected. Blood should be collected in blood culture
lesiorls. bottles comprising of glucose broth and taurocholate
• Detection of staphylococcal lesions among hospital broth.
staff and keeping them away from work till they . Specimens should be transported immediately to the
are treated for their lesions. laboratory and processed.
• Strict aseptic techniques to be followed.
• Hand washing, the most effective method to control 3. Direct Microscopy
hospital infections. Direct microscopy with Gram-stained smears of pus or
• In case of out-break of staphylococcal sepsis, a wound exudate is useful, where Gram positive cocci in
search may be made for carriers among hospital clusters may be seen (Fig. 23.4). This is of no value
staff Carriers detected should be treated with local for specimens where mixed bacterial flora are normally
application of muprocin or chlorhexidine. present e.g. sputum.
3. Bacterium may also develop tolerance to penicillin,
by which it is only inhibited and not killed.
_J_.Epidemiology
' Human patients and carriers are the commonest source
of infection. About 10-30% of healthy persons carry
the bacteria in the nose and less often in skin, axilla,
perineum and throat. In hospital, more than 50% of
nursing staff are carriers of Staph. aureus. Staphylococcal
disease may be exogenous or endogenous. It spreads by
contact, either direct or through fomites or by other
vehicles. Staphylococci are the commonest cause of
hospital cross infections. These bacteria are often antibiotic
resistant strains. These hospital strains spread from the Fig. 23.4 Staphylococci in stained smear of pus
h~tal staff or other patients. The postoperative wards,
operating rooms, intensive care units, newborn nursery 4. Culture
and cancer chemotherapy wards are at the highest risk The specimens are inoculated on following media:
of these infections. !-Blood agar
-Peptone Water
K. Laboratory Diagnosis Specimens where staphylococci are expected to be
1. Specimens scanty or outnumbered by other bacteria (e.g. faeces,
These are collected according to the nature of lesion as swabs from carriers), are inoculated on selective media
follows : such as:
Specimen Lesion • Salt agar
Pus Suppurative lesions • Salt milk agar
Sputum Respiratory infections c;-,Robertson's cooked meat medium (RCM)
Blood Septicaemia or PUO containing 10% NaCl.
Urine Urinary tract infection The inoculated media are incubated at 37°C for
CSF Meningitis 18- 24 hours. Next day, culture plates are examined for
Faeces Food poisoning morphology of bacterial colonies and other characters.
Food or vomit Food poisoning
Uniform turbidity is produced in liquid medium such

180
----- ------x-r-:1/"::P-' ><---------- ----- Unit Ill: Systemic Bacteriology
as peptone water. Gram staining~_ colony on blood in the community. Other typing methods include
agar and hanging drop preparation from peptone w?tter antibiogram pattern, plasmid typing, ribotyping and
are done to further charactense the DNA fingerprinting.
5. Colony Morphology and Gram Staining 8. Antibiotic Susceptibility

On blood agar, colonies are 2-4 mm in diameter, circular, This is important as staphylococci develop resistance
raised, opaque and produce golden~ pigrnent.~a to drug readily. Antibiotic sens1ttv1ty can be
haemolysis is seen around these colonies. The pigment is determined by Stokes metb,od or Kirby-Bauer method.
not diffusable into the medium (Fig. 23.5). L. Treatment
On Gram staining, they are Gram positive cocci Benzyl penicillin is the most effective antibiotic in sensitive
(1 µm in diameter) arranged in grape like clusters. strains. Cloxacillins are used against beta-lactamase
~-motile cocci in clusters are seen in hanging dro,P producing strains. Vancomycin is used in treatment
preparation. r of infections with MRS strains. Strains resistant to
vancomycin and teicoplanin have appeared in hospitals.
For mild s"'uperficja) lesions, topic~! applications of
bacitracin or chlorhexidine may be sufficient.
Some s t r ~ tolerance. These strains will
be found susceptible in the disc diffusion method but
their minimum bactericidal concentration (MBC) will be
very much higher than minimum inhibitory concentration
(MIC). They are not killed by antibiotics in the routine
doses.
The carriers are treated by local application of
antibiotics such as bacitracin and antiseptics such as
chlorhexidine. In resistant cases,trifampicin along with
Fig. 23.5 Staphylococcus aureus on blood agar another oral antibiotic may be useful.
6. Biochemical Reactions M. Control

(i) Catalase test: All staphylococci (pathogenic and 1. Source: Isolation and treatment.
--
non-pathogenic) are catalase nositive. This test
distinguishes Staphylococcus from Streptococgis
W C
2. Detection of carriers among hospital staff, their
isolation and treatment.
3. Sterilisation of instruments.
(ii) oagulase s : It is positive in Staph. aureus 4. Detection of staphylococcal lesions and their
but ative ·n other staphylococci. It helps in treatment.
differentiating a pa g ic strain (Staph. aureus) 5. Stop misuse of antibiotics.
,r
from non-path--~,. ., strains. This test has been
described in detail on page no. 181 of this chapter.
III. omER COAGULASE
Other tests which are positive in pathogenic strain
POSITIVE STAPHYLOCOCCI
(Staph. aureus) and distinguish it from non-
pathogenic staphylococci are described below. Certain strains of coagulase positive staphylococci do
(iii) Mannitol fermentation: Acid production witho~gas not possess all the characteristics of Staph. aureus. These
(iv) Gelatin liquefaction: Positive ,,,,_ organisms are called Staphylococcus intermedius. They
(v) Tellurite reduction: \._..,/"' are mostly of animal origin. Staph. hyicus is another
( vi) Production of enzymes phosphatase and coagulase positive staphylococcus but does not infect
deoxyribo ~e: Both are positive. humans.
Besides mannitol fermentation, other carbohydrates
IV. COAGULASE NEGATIVE
may also be fermented but are not helpful in diagnosis.
STAPHYLOCOCCI (CoNS)
·· 7. Bacteriophage Typing Coagulase negative staphylococci (CoNS) form the part
It is done for epidemiological purposes to trace the of the normal flora of the skin. They are opportunistic
source of Staph. aureus jnfections. It is especially useful pathogens, which cause infection in debilitated or
in food poisoning in large number of persons affected immunocompromised patients.

Ch 23: Staphylococcus _ __ _ _ __ _ _ __ _ _ _ __ _ _ _ __ _ _ __ _ _ _ __ 181
A. Staphylococcus epidermidis Other coagulase negative staphylococci include Staph.

It is a skin commensal and acts as opportumstlc lugdunensis, Staph. schleiferi and Staph. simulans.
pathogen in prosthetic devices e.g. prosthetic heart valves,
intraperitoneal catheters, orthopaedic prostheses and
vascular grafts. It may cause septicaemia and subacute
V. MICROCOCCI
bacterial endocarditis. It may produce stitch abscess. Micrococci are free living in the environment. These are
It mainly acts as pathogen in immunocompromised Gram positive cocci, catalase positive, coagulase negative,
individual. Their aetiological role may be proved by arranged in clusters which differ from staphylococci
repeated isolation. in attacking sugars oxidatively. They may appear in
irregular clusters, groups of four or of eight. They are
B. Staphylococcus saprophyticus often larger than staphylococci. Colonies are generally
It acts as an opportunistic pathogen. It is an important white in colour. Their staining is often not uniform. They
cause of urinary tract infection in young, sexually are saprophytes and commensals. They may rarely cause
active females. When present in urine cultures, Staph. opportunistic infection. Micrococci can be differentiated
saprophy ticus may be found in low numbers (<104/mL) from staphylococci by Hugh-Leifson's oxidation-
and still be considered as significant. Staph. saprophyticus fermentation (0 /F) test in which micrococci show
can also cause septicaemia and endocarditis in patients oxidative and staphylococci show fermentative breakdown
with cardiac surgery. The aetiological role is again by of carbohydrate. They are also resistant to lysostaphin
repeated isolation. Staph. saprophy ticus is novobiocin while Staphylococcus aureus is sensitive (Table 23.2).
resistant which distinguishes it from Staph. epidermidis.
Table 23.2 Distinguishing Characters of Staphylococcus
Novobiocin resistance can be determined by using
aureus and Micrococcus
novobiocin disc (5 µg) over the lawn culture of a test
Character Staphylococcus Micrococcus
strain and measuring the zone of inhibition. A zone of
aureus
inhibition less than 12 mm indicates resistance.
Differentiating features of Staph. aureus, Staph. Gram staining Gram positive Gram positive,
darkly stained
epidermidis and Staph. saprophyticus are shown in Table
23.1. Grape like Present in groups of
clusters four (tetrad) or eight
Table 23.1 Differentiating Features of Three Species of Uniform Often staining is not
staining uniform
Staphylococcus
I µmin size Larger than
Feature Staph. Staph. Staph. staphylococcus
a11re11s epidermidis saprophy ticus
Colony characters Colonies are Colonies are
l./4gulase test + golden yellow in generally white in
colour colour
~ ol +
fermentation Coagulase test Positive Negative
Progi.wtion of Breakdown of Fermentatively Oxidatively
L------'D N Aase + carbohydrates (O/F
Phosphatase + - /weak+ test)
a - lysin + Negative
Aerobic acid Positive
Protein A in the + production from
cell wall glycerol in the
presence of
Sensitivity to +
erythromycin
lysostaphin
Sensitivity to Sensitive Resistant
Novobiocin +
lysostaphin (200 µg
resistance
disc)
Pathogenicity Primary Opportunistic Opportunistic
Furazolidine Sensitive Resistant
susceptibility (JOO µg
of furazolidone disc)
C. Other coagulase negative staphylococci
Bacitracin (0 .04 unit Resistant Sensitive
Staphy lococcus haemoly ticus may cause wound infection, disc) susceptibility
bacteraemia, urinary tract infection and endocarditis.

182 _ _ _ _ _ _ _ _ _ __ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Unit Ill : Systemic Bacteriology
KEY POINTS
1. Staphylococci are Gram positive coci arranged in grape-like clusters. They are the commonest cause of suppuration.
2. Medically important species of staphylococus include Staphylococcus aureus, Staphylococcus epidermidis and
Staphylococcus saprophyticus.
3. Staphylococcus aureus is a pathogenic staphylococcus.
4. Pathogenic strains (e.g. Staph. aureus) have certain characteristics like production of enzymes coagulase, phosphatase,
deoxyribonuclease and their ability to ferment mannitol.
5. Based on production of enzyme coagulase staphylococci are classified as coagulase positive (e.g. Staph. aureus) and
coagulase negative (e.g. Staph. epidermidis, Staph. saprophyticus).
6. Mannitol salt agar is a selective medium for isolation of Staph. aureus.
7. Haemolysins, leucocidin, enterotoxin, toxic shock syndrome toxin (TSTT), exfoliative toxin are some toxins produced
by Staph. aureus.
8. Staph. aureus also produces a number of enzymes, coagulase, phosphatase and deoxyribonuclease.
9. The test for coagulase is done by the slide and the tube method. It is done to differentiate pathogenic (Staph. aureus)
strain from non-pathogenic strains.
10. Staph. aureus is an important pyogenic organism and lesions are localised in nature. They may also cause food
poisoning, nosocomial infections, scalded skin syndrome and toxic shock syndrome.
11. Strains of Staph. aureus may be distinguished by bacteriophage typing.
12. Penicillin resistance in staphylococci may be due to production of beta lactamase (penicillinase) which is plasmid
coded and transmitted by transduction.
13. Methicillin resistant Staph. aureus (MRSA) may cause outbreaks of hospital ~on.
14. Coagulase negative staphylococci form the part of the normal flora of the skin. They are opportunistic pathogens,
which cause infection in debilitated or immunocompromised patients.
15. Staph. saprophyticus is novobiocin resistant which distinguishes it from Staph. epidermidis and Staph. aureus.
16. Micrococci are saprophytes and commensals. They may rarely cause opportunistic infections.
17. Micrococci differ from staphylococci in attacking sugars oxidatively.
YOU MUST KNOW

1. Morphology and culture characteristics of Staph. aureus.
2. Toxins and enzymes of Staph. aureus.
3. Diseases caused by Staph. aureus.
4. Methicillin resistant Staph. aureus (MRSA).
5. Laboratory diagnosis of infections caused by Staph. aureus.
6. Differences between Staph. aureus, Staph. epidermidis and Staph. saprophyticus.
7. Coagulase negative staphylococci (CNS).
8. Micrococci.
STUDY QUESTIONS
1. Describe the laboratory diagnosis of infections caused by Staph. aureus.
(a) Toxins and enzymes produced by Staph. aureus.
(b) Toxic shock syndrome
(c) Staphylococcal food poisoning

Ch 23 : Staphylococc us_ _ _ _ __ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __ 183
(d)Coagulase test
(e)Clumping factor
(f)Bacteriophage typing
(g)Staphylococcal scalded skin syndrome.
(a) MRSA
(b) Coagulase negative staphylococci
(c) Micrococci
(d) Antigenic structure of Staph. aureus.
MULTIPLE CHO CE QUESTION S (MCQs)

1. Which of the following characteristics help to distinguish Staphylococcus aureus from non-pathogenic strains of
Staphylococcus ?
(a) Beta type of haemolysis on blood agar (b) Production of a golden yellow-pigment
(c) Coagulase production (d) All of the above
2. Protein A is a cell wall component of:
(a) Staphylococcus aureus (b) Coagulase negative staphylococci
(c) Micrococci (d) None of the above
3. Which of the following toxins is responsible for Staphylococcal scalded skin syndrome?
(a) Enterotoxin (b) Leucocidin (c) Haemolysin (d) Exfoliative toxin
4. Slide coagulase test detects:
(a) Free coagulase (b) Bound coagulase (c) Both of the above (d) None of the above
5. Tube coagulase test detects:
(a) Free coagulase (b) Bound coagulase (c) Both of the above (d) None of the above
6. Which of the following bacteria produce coagulase enzyme?
(a) Staphylococcus aureus (b) Staph. epidermidis (c) Staph. saprophyticus (d) None of the above
7. Which of the following staphylococci is/are novobiocin resistant?
(a) Staphylococcus aureus (b) Staph. epidermidis (c) Staph. saprophyticus (d) None of the above
8. All of the following are coagulase negative staphylococci except:
(a) Staphylococcus epidermidis (b) Staph. saprophyticus
(c) Staph. aureus (d) Staph. haemolyticus
Answers (MCQs):
1. (d) 2. (d) 3. (d) 4. (b) 5. (a)
6. (a) 7. (c) 8. (c)
____ ... ____

Chapter 24
STREPTOCOCCUS AND ENTEROCOCCUS
I. Classification
A. Alpha Haemolytic Streptococci B. Beta Haemolytic Streptococci
C. Gamma or Non-Haemolytic Streptococci
II. Streptococcus pyogenes
A. Morphology B. Culture
C. Biochemical Reactions D. Resistance ..-- ' \ (Uf
E. Antigenic Structure F. Toxins a n d Q
G. Pathogenesis H. Epidemiology
I. Laboratory Diagnosis J. Treatment
K. Prophylaxis
Ill. Other Haemolytic Streptococci
A. Group B Streptococci B. Group C and G Streptococci
C. Group D Streptococci
IV. Enterococcus
V. Viridans Streptococci
Streptococci are Gram positive cocci which are arranged anaerobic streptococci are described in chapter on
in c;hains. They are part o""f the normal human flora. 'Nonsporing anaerobes' (Chapter 30). Aerobic and
So~ hem are important human pathogens causing facultative anaerobic streptococci are classified on the
..,pyogenic infections. They grow poorly in simple media but basis of their haemolytic properties. Three types of
their growth is enhanced by the addition of fermentable haemolytic reactions (a, and y) are observed on blood
carbohydrate (e.g. glucose), blood or serum. agar medium.
The name streptococci (streptos meaning twisted or
coiled) was given by Bi/froth (1874). Rosenbach (1884) A. Alpha (a) Haemolytic Streptococci
coined the name Streptococcus pyogenes for the organisms They produce , a greenish discolouration around the
which he isolated from human suppurative lesions. colonies. This is due to partial haemolY.Sis. The zone of
lysis is small (1 or 2mm -;ide) with presence of unlysed
I. CLASSIFICATIO N (FIG. 24.1) erythrocytes which are detectable microscopically. Alpha
The streptococci are divided into aerobic streptococci, haemolysis is seen with viridans group of streptococci
obligate anaerobes and facultative anaerobes. Obligate and pneumococcus.
184
Ch 24: Streptococcus and Enterococcus _ _ _ _ _ _ _ _ __ _ __ __ _ _ _ __ _ __ __ _ 185
Streptococci ._.,,A
I 0 2 requirement
I
Obligate anaerobes
e.g. Peptostreptococci
(A_.)\ ·- <:.. Cdo r-0~ t

1. Alpha- molytic 2. Beta-haemolytic 3. Gamma-haemolytic
(The viridans group) (_ ~'L,._,b..,) (The enterococcus group)
These are classified into Serological grouping These are classified into
species by physiological (Carbohydrate C) species by physiological and
and biochemical biochemical properties.
20 Lancefield groups
properties.
~KLMNOPQRSTUV)
Streptococcus pyogenes
Serological typing
(M Protein)
Griffith types (1,2,3, etc. up to 80)
Fig. 24.1 Classification of streptococci
B. Beta (~) Haemolytic Streptococci 1, 2 etc.) based on their surface proteins (M, T and R).
These streptococci produce a clear, colourless zone of Serotyping of isolated strains is useful in epidemiological
complete haemolysis (2-4 mm wide) around the colonies, studies.
within wfocfi erythrocytes are completely lysed. The
lysis of erythrocytes is due to the production of two II. STREPTOCOCCUS PYOGENES [ ~'f-
types of streptolysin by the organisms: streptolysin O A. Morphology
and strfl}tolysin S. The former is destroyed by oxygen The individual cocci are spherical or oval, 0.5 to 1.0 µm
and the latter is oxygen stable (S for stable). Streptolysin in diameter and are arranged in chains. Chain formation
S is responsible for surface colony haemolysis. Since is due to successive cell divisions occurring in one plane
most strains produce both types of haemolysin, they only and daughter cells failing to separate completely
can usually be recognised by haemolysis of surface (Figs. 24.2 and 24.3) .
colonies. Most of the pathogenic streptococci fall into this
group. Streptococcus pyogenes is the most important and
is responsible for many important human infections. r-.N
... /"
C. Gamma (y) or Non-Haemolytic Streptococci ...\.. .. I. " .," .. \
......\ ..... ..
fl' ....
They do not produce haemolysis and Str. faecalis is a •-:
I :
typical example.
r
.y···: / , .........
The beta haemolytic streptococci were classified by
Lancefield (1933) serologically into a number of broad
,. . \I.. \....... =•• :
groups based on the nature of a carbohydrate (C) antigen
on the cell wall. These are known as Lance.field groups, 20
of which have been identified, named A-V (without I and Fig. 24.2 Streptococci
J) by precipitation reaction performed with appropriate
sera. Majority of streptococci that produce human
infections belong to group A named as Streptococcus Larger chains are formed in liquid than in solid
pyogenes. These are further subdivided by type specific media. The length of bacterial chain has no relevance
antisera into approximately 80 Griffith serotypes (type to virulence or other properties. In fact, non-pathoge~

186 - - - - - - - - - - - - - - - - - - - - - - - - - - - - Unit Ill: Systemic Bacteriology
In liquid media, such as glucose broth, growth occurs

as a granular turbidity with a powdery deposit. Bacterial
chains being heavier settle down as deposit.
C. Biochemical Reactions
Streptococci are catalase negative, unlike staphylococci
which are catalase positive. They are not soluble in 10
per cent bile, unlike Str. pneumoniae. Hydrolysis of
pyrrolidonyl naphthylamide (PYR test) and failure to
ferment ribose are useful to differentiate Str. pyogenes
from other streptococci. Str. pyogenes produces the
Fig. 24.3 Streptococcus pyogenes enzyme L-pyrrolidonyl-~-naphthylamidase (Pyrase).
This enzyme hydrolyses L-pyrrolidonyl-~-naphthylamide
strains such as Streptococcus salivarius forms the longest to p-naphthylamine which produces red colour in the
chain. .,.,,,,--- _.,,.- presence of ~-dimethylamino cinnamaldehyde. PYR-
Streptococci are Gram positive, non-motile and non- positive bacteria produce a deep red colour whereas
sporing. Some strains of Streptococcus pyogenes (Group A) PYR-negative organisms produce a slight orange colour
and group C have capsules composed ofJiyaluronic acid. or no colour change.
while members of groups B and D show polysaccharide Several sugars are fermented by streptococci
"
These capsules are best noticeable in very young producing acid but no gas. These are of little value in
cultures. laboratory identification.
B. Culture D. Resistance
They are aerobes and facultative anaerobes, growing Streptococcus pyogenes is a delicate organism which is
best at a temperature of 37°C (range 22-42°C). These inactivated by heat at 56°C for 30 minutes. It dies in a
are most exacting in nutritive requirements, growth few days in cultures but can be stored in Robertson's
occurring only in media containing blood, serum or cooked meat medium at 4°C. It is rapidly inactivated by
'sugars (fermentable carbohydrates). On blood agar, after antiseptics. It is more resistant to crystal violet than many
overnight incubation, the colonies are small (0.5- 1.0 other bacteria, therefore this dye is used to prepare selective
mm, pin point), circular, semitransparent, low convex media for the isolation of streptococci. It is susceptible to
with a wide zone of j)-haemolysis around them (Fig. sulphonamide and many antibiotics. Unlike Staph. aureus,
24.4) . Growth and haemolysis are promoted by presence it does not develop drug resistance. Sensitivity to bacitracin
of 10% CO2 in the environment. Mucoid colones are is employed for differentiating Str. pyogenes (Group A)
formed by strains which produce large capsules. Selective from other haemolytic streptococci. Str. pyogenes 1s
media containing 1:500,000 crystal violet (crystal violet resistant to SXT (trimethoprim-suphamethoxazole)
blood agar) permit growth of streptococci but inhibit
other bacteria especially staphylococci. Another selective
medium is PNF medium (horse blood agar containing E. Antigenic Structure
polymyxin B sulphate, neomycin sulphate and fusidic 1. Capsular Hyaluronic Acid
acid) used for isolation of streptococci. Capsule may be present on groups A and C streptococci.
The capsule when present inhibits phagocytosis.
2. Group Specific Polysaccharide Antigen

The cell wall is composed of an outer layer of protein
(fimbria containing protein) and lipoteichoic acid, a
middle layer of group-specific C carbohydrate and
inner layer of peptidoglycan (mucoprotein) (Fig. 24.5) .
The peptidoglycan is responsible for cell wall rigidity.
It has also some biological activities e.g. pyrogenic and
thrombolytic activity. Lancefield grouping is done on
the basis of group specific C carbohydrate. Str. pyogenes
Fig. 24.4 Streptococcus pyogenes
belongs to group A.

Ch 24: Streptococcus and Enterococcus _ _ _ _ _ _ _ _ _ _ __ _ _ _ _ _ _ _ _ _ __ _ _ 187
F. Toxins and Enzymes

Str. pyogenes produces several exotoxins and enzymes
which contribute to its virulence.
Protein and
lipoteichoic acid
Group specific 1. Toxins
carbohydrate
(i) Haemolysins
-Peptidoglycan
Streptococci produce two type of haemolysins, streptolysin
Cytoplasm 'O' and 'S'. Streptolysin 'O' is so named because it is
- - Cytoplasmic
oxygen labile. It is inactivated in the presence of oxygen,
membrane but may be reactivated by treatment with reducing
substances. It is heat labile. It lyses red cells and is also
- - - - Cell wall
cytotoxic for neutrophils, platelets and cardiac tissue.
Fimbria
(Pilus) It is demonstrable only in deep colonies and not in
surface cultures. It is antigenic and antistreptolysin 'O'
Fig. 24.5 Antigenic structure of Streptococcus pyogenes (ASO) regularly appears in sera following streptococcal
infection. An ASO titre in serum in excess of 200 units
3. Type Specific Antigen suggests either recent or recurrent streptococcal infection.
The outer part of the cell wall contains protein antigens. Streptolysin 'O' and 'S' are produced by groups A, C
Str. pyogenes is further subdivided on the basis of their and G.
surface proteins M, T and R. Streptolysin 'S' is an oxygen stable haemolysin and
is responsible for the haemolysis seen around colonies
(i) M protein of streptococci on the surface of blood agar plates. In
It is the most important antigen and acts as a virulence addition to haemolytic property, it has got leucocidal
factor by inhibiting phagocytosis. It is antigenic in nature. action. It is protein in nature but is not antigenic.
The antibody to M protein is protective. The M protein Convalescent sera do not neutralise streptolysin 'S' activity.
is heat and acid stable but susceptible to trypsin. About
80 M-protein types have been recognised. After extraction (ii) Pyrogenic exotoxin (erythrogenic toxin)
of M protein, typing is done with type specific sera. This toxin is responsible for the rash of scarlet fever.
M-protein along with other surface antigens probably It is only produced by lysogenic strains of group A
have an important role in pathogenesis of rheumatic streptococci. Three antigenically distinct forms of
fever. pyrogenic exotoxin, A, B and C have been described.
Types A and C are coded for by bacteriophage genes
(ii) T and R proteins but type B gene is chromosomal. These exotoxins are
The T protein is an acid labile, trypsin resistant antigen. It 'superantigens'- T cell mitogens which induce massive
may be specific, but many different M types may have the release of inflammatory cytokines. The toxin is antigenic
same T antigen. It is usually differentiated by agglutination and is neutralised by antibodies present in the convalescent
with specific antisera. Another surface protein R has been sera. It causes pyrogenicity, cytotoxicity and enhancement
identified in some types of Str. pyogenes (type 2,3, 28 and of susceptibility to endotoxin. This toxin was renamed
48) and some strains of group B, C and G. The T and R Streptococcal pyrogenic exotoxin (Spe) because induction
proteins have no relation to virulence of streptococci. of fever is the primary effect of the toxin. There are four
distinct types of these exotoxins namely Spe A, Spe B,
(iii) Other associated factors Spe C and Spe F. These toxins act as superantigens.
A non-type-specific protein associated with the M This toxin is responsible for Dick test and
protein is identified. This is known as M associated Schultz Charlton reaction. When the toxin is injected
protein (MAP). Some M types of Str. pyogenes produce intradermally into the skin of a susceptible child, a
a lipoproteinase which is known as serum opacity localised erythematous reaction appears. This is called the
factor (SOF). These strains produce opacity when Dick test. The test becomes negative during convalescence
applied to agar gel containing horse or swine serum. due to neutralisation of toxin by antibody. In scarlet fever,
Hair-like pili (fimbria) which consist partly of M when homologous antitoxin is injected locally into the
protein and are covered with lipoteichoic acid are present rash, blanching of the rash occurs, it is named as Schultz
in group A streptococci. They are important in the Charlton reaction. These tests are now only of historical
attachment of streptococci to epithelial cells. importance as scarlet fever is now rarely seen.
188 ~\,cy><-~ ~ nit Ill: Systemic Bacteriology
2. E'3Jfmes .---, produce serum opacity factor (SOP) which has been
J:irStreptokinase (fibrinolysin) described earlier in this chapter.
It promotes the lysis of h~man fibrin doL by catalysing
the conversion of plasminogen (plasma precursor) into G. Pathogenesis (Table 24.1)
..12lasmin ...!t is protein. in nature and antigenic. Neutralising Str. pyogenes produces pyogenic infections with a
antibodies (antistreptokinase) appear in convalescent tendency to spread locally. Non-suppurative seguelae
sera which may be estimated for retrospective study of !,ocal infectious include acute g)omern)onephritis and
of streptococcal infection. Streptokinase facilitates the [ rheumatic fever'._)
s read of infection b f e fibrin mer Table 24.1 Streptococcal Diseases
Streptococcus Lesions
\ JiiJ-- f ! f Q ~ es (streptodornase) Str. pyogenes

Group A streptococci elaborate~ur antigenically
distinct deoxyribonucleases (DNAases), A,B,C, and D,
Q
Pyogenic infections
of which, type B is tbe m.m,t aJ:1.tigeoic in man. These ~ a t o ry tract Acute tonsillitis or pharyngitis (sQ.re
-----
enzymes liquefy the highly viscous DNA that accumulate
in thick pus. DNA present in pus is derived from the ~ i n infections
t1;-;'-oat), scarlet feve;-
Infections of wounds, burns and skin.

nuclei of necrotic cells. This explains the thin serous lesions (eczema), erysipelas, impetigo,
character of streptococcal exudate. Enzyme preparation necrotizing fascitis ·-
containing b o t ~ e and streptodornase have __,Genital infections Puerperal sepsis
been applied clinically in liquefying localised collections -=--
Streptococcal toxic Bacteraemia, necrotizing fascitis
of thick exudates, as in empyema. shock syndrome
Antibodies against deoxyribonuclease B is useful
in retrospective diagnosis of Str. pyog5nes infections, Deep infections Bone and joint infections,
lyr'iiphadenitis, septicaemia, @_SCess in
particularly in skin infections, where _ASO titre may
internal organs
be low. Apart from group A, deoxyribonuclease is also --=----=
elaborated by group C and G streptococci. ® Non-suppurative R,heumatic fever, ,acute
..._/ com lications glomerulonephritis
(iii) Nicotinamide adenine dinucleotidase (NADase)
NADase acts on the ~enzyme NAD and liberates
nicotinamide from the molecule. It is produced by groups
\ Neonatal infections (septicaemia and
meningitis)
Urinary tract infection, endocarditis

A, C and G streptococci and is antigenic. It is believed faecalis
to be leucotoxic.
Viridans Endocarditis, dental caries
Streptococci
(iv) Hyaluronidase
It is produced by strains of group A,B,C, and G
streptococci. It breaks down hyaluronic acid of the 1. Pyogenic Infections
tissues and favours spread of s ~ e ~ o n along (i) Respiratory infections
1ntercellular spaces. The enzyme is antigenic and specific Sore throat (acute tonsillitis and/or pharyngitis) is the
antibodies appear in patient's sera. most common of streptococcal diseases. Tonsillitis
Streptococci possess a hyaluronic acid capsule and is more common in older children and adults. The
also produce an enzyme hyaluronidase- a seemingly self organisms may spread to surrounding tissues, leading
destructive process. But it is found that those strains which to complications such as cervical adenitis, otitis media,
form hyaluronidase in large quantities are non-capsulated. quinsy, Ludwig's angina and mastoiditis. It may rarely
There is also evidence that hyaluronidase produced m lead to meningitis.
tissues is more in amounts than in cultures.
Scarlet fever
(v) Other extracellular products .._/ / It consists of a combination of sore throat and a
Many strains produce neuraminidase, esterases, phos- generalised erythematous rash. It is caused by a strain
phatase, amy~).ir,a,se, N-acetyl glucosaminidase and producing the erythrogenic toxin. It is now rarely seen
beta-glucuronidase. Contribution of these products in in U.K. and other colder countries. It does not occur
pathogenesis is not known. Some M types of Str. pyogenes in India.
\- 4
Ch 24: Streptococcus and Enterococcus _ _---'---- - - - -- - - - - - - - - - - - -- - - - 189
l!)l0 jS( ""~~
(ii) Skin infections occur one to four weeks after the acute infection. Str.
Str. pyogenes causes suppurative infections of the pyogenes is no longer detectable when these complications
skin with a predilection to produce lymphangitis and set in. The la -t period suggests an immune response.
cellulitis. Infection of minor abrasions may sometimes umatic fever is often preceded m:
fu ore throaI\vhile
lead to fatal septicaemia. Str. pyogenes has been associated acute glomeru onephritis by the skin infection. These
with necrotizing fascitis (NF), an invasive, infection sequelae or complications are believed to be the result
characterised by inflammation and necrosis of the skin, of hypersensitivity to some streptococcal components.
subcutaneous fat and fascia. Although uncommon, NF Rheumatic fever may follow infection with any serotype
is a life-threatening infection. The strains causing NF of Str. pyogenes while acute glomerulonephritis is caused
have been named is 'flesh eating bacteria'. by only a few nephritogenic types (Table 24.2).
The two typical streptococcal skin infections are
erysipelas and impetigo. Erysipelas is an acute spreading Table 24.2 Differentiating Features of Acute Rheumatic
lesion. Skin shows massive brawny oedema with erythema. Fever and Acute Glomerulonephritis
Elderly persons are usually affected. Impetigo is found Feature Acute Acute
mainly in young children. It is caused by Str. pyogenes Rheumatic Fever Glomerulonephritis
belonging to certain limited number of serotypes. These Primary site of Throat Throat or skin
skin infections are the main cause leading to acute infection
glomerulonephritis in children in the tropics. Prior sensitisation Essential Not necessary
In skin infections, antibody response to streptolysin Pyoderma types 49,
Serotypes of Str. Any
0 is not high, therefore ASO titre does not have as pyogenes responsible 52- 55, 57- 61 and
much clinical significance as in pharyngeal infections. throat infection types
Antibodies to DNAase Bare more useful in retrospective 12, I , 25, 4 and 3
diagnosis of skin infections antecendent to acute Immune response Marked Moderate
glomerulonephritis.
Complement level Unaffected Lowered
(iii) Streptococcal toxic shock syndrome Repeated attacks Common

,.---- Absent
Streptococcal toxic shock syndrom (TSS) is a condition in Course Progressive or Spontaneous
which the entire organ system collapses, leading to death. static resolution
Group A streptococci associated with streptococcal TSS Prognosis Variable Good
produce a streptococcal pyrogenic exotoxin notably Spe Not known
Hereditary tendency Present
A. These toxins play a major role in pathogenesis and
P~icillin 2ro 12hv,laxis Not indicated
act as superantigens leading to overstimulation of the Ess~
immune response. Other virulence factors, such as SLO
and various cell wall antigens, can also contribute to The mechanism by which streptococci produce
toxic shock. Streptococcal TSS resembles Staphylococcal rheumatic fever is still not clear. A common cross-reacting
TSS. Patients are often bacteraemic and have necrotizing anti en may exist in some grou p A streptococci and
fascitis. Young children with chickenpox (vericella), and ..heart, therefore, antibo 1es pro uce m response to the
elderly adults seem to be at greater risk. streptococcal infection could cross react with myocardial
and h ~ e , causing cell~lar destruction.
(iv) Other pyogenic infections • Acute glomerulonephritis probably develops because
(a) Puerperal sepsj.s: Sh: pyogenes was an important cause some components of glomerular basement membrane are
<of puerperal sepsis. It used to take a heavy toll of antigenically similar to the cell membranes of nephritogenic
life before antibiotics became available. streptococci. Antibodies formed against streptococci cross
(b) S~psis· Ipfections of skin lesions (eczema, psoriasis, react with glomerular basement membrane and result
scabies), wounds and burns. in damage. Alternatively, streptococcal antigen-antibody
(c) P~aemia, septicaemia, abscess in internal organs complexes may deposit in the glomeruli. In either case,
(brain, lung, liver and kidney). the activation of the complement components C3 and
C5 leads to tissue destruction .
2. Non-Suppurative Complications
Str. pyogenes infections are sometimes followed by two H. Epidemiology
important non-suppurative sequelae, acute rheumatic The major sources of infection are patients and carriers.
fever and acute glomerulonephritis. Thesecomplicauon s Symptomless infection is common and maintains the

190
organism m the community. Transmission is either (vi) Biochemical Reactions

by direct contact or fomites. Streptococcal infections Streptococci are catalase negative which is an important
of respiratory tract are more frequent in children 5- 8 test to differentiate streptococci from staphylococci.
years of age. Outbreaks of infection occur in closed They are not soluble in 10% bile unlike Streptococcus
communities such as army camps or boarding schools. pneumoniae.
Control measures include early detection of patients and Streptococci ferment sugars producing acid but no
carriers and their treatment. gas. PYR test and failure to ferment ribose are useful in
identification of Str. pyogenes from other streptococci.
I. Laboratory Diagnosis
Diagnosis of acute suppurative infections is made by culture, (vii) Identification of Various Groups of Streptococci
while in the non-suppurative complications, d1agn6ts Str. pyogenes (group A) is sensitive to bacitracin (0.04
is mainly based on the demonstration of antibodies. unit/disc). Group '.N streptococci can be rapidly identified
by fluorescent antibody technique. PYR test may be
1. Acute Suppurative Infections done to differentiate Streptococcus pyogenes from other
(i) Specimens streptococci. PYR test is positive for Streptococcus
Specimen is collected according to the site of lesion, such pyogenes. Group 'B' streptococci may be identified
as swab, pus, blood or CSF. by the CAMP reaction (Christie, Atkins and Munch-
Peterson). Both group A and group B are resistant to
(i1) Collection and Transport SXT (trimethoprim-sulphamethox azole) while all other
Specimens should be collected in sterile containers under streptococci are sensitive to it. However enterococci
all aseptic conditions. These should be plated immediately are also resistant to SXT. Group 'D' can be isolated
or sent to the laboratory in Pike's transport medium by performing heat resistance test. Details of these tests
(blood agar containing 1 in 1,000,000 crystal violet and are described in respective streptococci group.
1 in 16,000 sodium azide).
(viii) Lancefield Grouping
(iu) Gram Stainittg of Smears Haemolytic streptococci are grouped serologically by the
Gram positive cocci in chains is indicative of streptococcal Lancefield technique. It is done for definite classification and
infection. Smears are of no value where streptococci are epidemiological studies. Lancefield grouping depends on
present as part of the resident flora such as in infections of C carbohydrate, which is specific for each group.
the throat and genitalia. Smear examination is important C Carbohydrate is extracted from the cell wall of
in specimens such as pus and CSF. streptococi and grouping done by a precipitation test
with group antisera. Streptococci are grown in Todd-
(iv) Culture Hewitt broth and the extraction of C carbohydrate
The specimen is inoculated on blood agar medium and may be done with hydrochloric acid (Lancefield's acid
incubated at 37°C for 18-24 hours. Haemolysis develops extraction method), or formamide (Fuller's method)
better under anaerobic conditions or in the presence of or by autoclaving (Rantz and Randall's method) or by
5- 10% carbon dioxide. Sheep blood agar is preferred as an enzyme produced by Streptomyces a/bus (Maxted's
human blood may contain certain inhibitors. method). The extract and the specific antisera are allowed
to react in capillary tubes. Precipitation occurs within five
(v Colony Morphology and Staining minutes. Besides capillary precipitation, groupmg may
The colonies of streptococci are small 0.5- 1.0 mm (size of also be done by agar gel precipitation.
pinpoint as compared to pinhead size of staphylococci),
circular, low convex with a zone of ~-haemolysis around (ix) Antigen Detection Tests
them. ELISA and agglutination tests are used to demonstrate
In liquid medium, such as glucose broth, growth
occurs as a granular turbidity with a powdery deposit.
group A streptococcal antigen from throat swabs.K" '1 /\ /
Streptococci chains being heavier settle down as a powdery 2. Non-Suppurative Complications \ , . Y ~v
deposit. In rheumatic fever and glomeruloneph\i'?G;3;erological
Gram staining from colonies show Gram positive tests provide retrospective evidence of streptoco 1
cocci in chains. Hanging drop preparation from liquid
medium shows non-motile cocci. (ASO) titration. A titre of 200 units or mots
infection. The routine test done is antistre tolysin 0
significant
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Ch 24: Streptococcus and Enterococcus._ _ _ _ _ _ _ _ _ __ _ _ _ __ _ _ __ _ _ _ _ _ 191
in rheumatic fever and is indicative of prior streptococcal hippuricase (also called hippurate hydrolase), which
infection. hydrolyses sodium hippurate to form sodium benzoate and
ASO test is a ~utralisation reactim;k,here antibodies glycine. This reaction can be detected by adding ninhydrin,
to streptolysin 'O' (ASO) are neutralised with streptolysin which reacts with glycine to form a purple colour. Another
'O' antigen. ASO titre is usually found in .b igb levels test to identify group B is the CAMP reaction (Christie, '
in rheumatic fever but in glomerulonephritis, titres tend Atkins and Munch-Peterson), which can be demonstrated
to be low, therefore, anti-deoxyribonuclease B (anti- by the production of accentuated zone of haemolysis
DNAase B) estimation is more reliable. Titres higher (as butterfly appearance) when Group B streptococci are
than 300 or 350 are significant. This test is very useful inoculated perpendicular to a streak of Staph. aureus grown
for retrospective diagnosis of s~~.ca,1 J)lfoder.rpa., on blood agar (Fig. 24.6). Group B streptococci produce a
for which ASO is of less vallie. Antihyaluro1ildase is CAMP factor that enhances the lysis of sheep erythrocytes
another useful test for pyoderma infection of streptococci. by staphylococcal ~-lysin. Other two methods to perform
The streptozyme test, a passive slide hemagglutination CAMP test include disc method and rapid CAMP test
test is a sensitive and specific screening test. It becomes (or spot CAMP test). In disc method, a disc impregnated
positive after nearly all types of streptococcal infection, with the ~-lysin is used on the plate containing growth of
whether of the throat or the skin. Group B streptococci. There is an enhanced haemolysis
around the disc but it is not of butterfly appearance as
J. Treatment disc is round. A third method, the rapid CAMP test,
Pen jci]]iu G is the drug of choice. In patients allergic to a drop of extracted ~-lysin is placed on the growth of
penicillin, erythromycin or cephalexin is_ used. Antibiotics suspected Group B streptococci in the culture plate. It is
,bave no efThcton established glomerulonephritis and then incubated at 35°C for at least 20 minutes, enhanced
rheumatic fever. haemolysis is observed.
K. Prophylaxis
Prophylaxis is indicated only in the prevention of
rheumatic fever. This prevents streptococcal reinfection Butterfly
Staph . aureus
appearance
and further damage to the heart. Penicillin is administered streak
for a long period in children who have developed early
signs of rheumatic fever. ~-haemolytic
streptococci
III. OTHER HAEMOLYTIC STREPTOCOCCI
e
Besides Str. pyogenes, haemolytic streptococci belonging Fig. 24.6 CAMP reaction
to other groups like B, C, D, F, G and rarely H, K, 0
and R may also lead to human infections. Besides hippurate hydrolysis and CAMP test,
/ ~r/v resistance to sulphamethoxazole (SXT) is another test
A. Group B Streptococci (GBS) /I · in identification of Group B streptococci. This test can
Str. CfJalactige is an important pathogen of bovine be performed by applying SXT disc on the growth of
mastitis in cattle. In recent years, it has been recognised Group B streptococci. Resistance to SXT can be observed
as the single most important pathogen in neonates causing in case of Group B streptococci.
neonatal septicaemia and meningitis. It may also cause Nine capsular serotypes of Group B streptococci have
septic abortion and puerperal sepsis. Str. agalactiae is a been identified.
commensal of female genital tract from where bacterial
infection in neonates occur. Other Group B infections in B. Group C and G Streptococci
neonates include osteomyelitis, arthritis, conjunctivitis, The species Str. dysgalactiae subsp. equisimilis is usually
respiratory infections, endocarditis and peritonitis. isolated from human sources. The clinical spectrum of
The presumptive identification depends on their ability disease caused by this species resembles infections caused
to hydrolyse hippurate. Hippurate positive bacteria produce by Str. pyogenes. It may cause human infections like
a deep purple colour whereas hippurate negative organisms pharyngitis, skin infections, endocarditis, septicaemia and
produce a slightly yellow pink colour or fail to produce meningitis. Str. dysgalactiae subsp equisimilis resembles Str.
any colour. Group B streptococci possess the enzyme pyogenes in fermenting trehalose but differs in fermenting

192 _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Unit Ill: Systemic Bacteriology
ribose. Most isolates of this species possess either the mannitol and sorbose, and arginine hydrolysis. Enterococcus
Lancefield group C or group G antigen. However some faecalis and E. faecium belong to Group II.
strains containing Lancefield group L or A antigen On MacConkey's medium they grow as tiny deep pink
have been reported. Similar to St,: pyogenes cases of colonies. On Gram's staining, enterococci appear as pairs
glomerulonephritis and acute rheumatic fever have been of oval cocci and short chains. The identification of the
reported following Str. dysgalactiae subsp. equisimilis species is based on biochemical reactions. Enterococcus
infections. St,: equi subsp. zooepidemicus may also cause faecalis is the most commonly isolated enterococcus from
human infections. It possesses Lancefield group C antigen. human sources. Other enterococci are E. faecium and E.
Str. dysgalactiae subsp. dysgalactiae and Str. equi subsp. durans. E. faecalis can be identified by fermentation of
equi are animal pathogens. Both species possess Lancefield mannitol, sucrose, aesculin and sorbitol, and by producing
group C antigen. Another group is Str. anginosus group black colonies when grown on tellurite blood agar.
which can harbour the Lancefield group antigen A, C, E. faecalis is frequently isolated from cases of urinary
F, G or none at all. tract infection and wound infection. They may also cause
other infections like subacute bacterial endocarditis,
C. Group D Streptococci septicaemia, peritonitis and infection of biliary tract.
Streptococci belonging to group 'D' are classified as Strains resistant to penicillin and other antibiotics occur
enterococci (faecal streptococci) and non-enterococci frequently. Vancomycin is the primary alternative drug to
(non-faecal streptococci). They are usually non-haemolytic penicillin for treating enterococcal infections. Vancomycin
while some strains may be alpha or beta haemolytic. resistant enterococci (VRE) have also been isolated.
The enterococci group has been reclassified as a separate Resistance is most common in E.faecalis and E. faecium.
genus called Enterococcus. Non-enterococci of group D Seven types of resistance to vancomycin in enterococci
(Str. bovis group) are inhibited by 6.5 per cent sodium have been described. These seven types of resistance are
chloride. These are now named as Group D streptococci. encoded by seven genes namely Van A, Van B, Van C,
Both Group D streptococci and enterococci are positive Van D, Van E, Van G and Van L.
for bile-aesculin hydrolysis test. They can grow in Enterococcus can be differentiated from Group 'D'
40% bile and hydrolyse aesculin to form aesculetin streptococcus by a number of tests as shown in Table 24.3.
which combines with ferric citrate to give a black
complex. Aesculin and ferric citrate are present in the Table 24.3 Differences between enterococcus and Group
bile aesculin agar slant which is used to perform this D streptococcus
test. The test is named as bile-aesculin hydrolysis test. Test Enterococcus Group 'D'
Group D streptococci may cause genitourinary streptococcus
infection or endocarditis rarely. They are susceptible to .....L..-i'Jlle-aesculin + +
penicillin. "I...-' hydrolysis test
IV. ENTROCOCCUS . A o w t h in the presence +

of o aCI
Enterococci also belong to group 'D' as they contain
the same antigen as that of group 'D' streptococci. +
They are normal inhabitants of human intestinal tract ~ ensitivity to SXT s
and possess some distinctive properties as follows:
1. They are positive for bile-aesculin hyrolysis test. 5. Ability to grow at +
45°C
2. They are relatively heat resistant and can withstand
heat at 60° C for 30 minutes ( heat test or heat
resistance test). V. VIRIDANS STREPTOCOCCI
3. Their ability to grow in the presence of 6.5 per cent
sodium chloride. This group of streptococci produce alpha haemolysis on
4. Their ability to grow at 45°C and at pH 9.6. blood agar. They are known as viridans streptococci (from
5. They are PYR test positive. viridis meaning green) due to greenish discolouration
6. They are resistant to SXT (trimethoprim- (a-haemolysis) on blood agar. They are commensals of
sulphamethoxazole). mouth and upper respiratory tract. Str. viridans includes
Enterococcal species have been divided into five groups St,: mitis group, Su: anginosus group, Str. salivarius
(Group I to Group V) based on acid formation from group, St,: mutans group and Str. bovis group. Each

Ch 24: Streptococcus and Enterococcus _ _ ---'---- -- -- - - - - -- - - - - - -- - - - 193
group contains many species. Str. milleri and Group F and gastrointestinal tract. They cause endocarditis
streptococci are now included in Str. anginosus group. in persons with predisposing factor such as valvular
This group can possess Lancefield group antigen A, C, disease of the heart, congenital heart disease and cardiac
F, G or none at all. surgery. Following some dental procedures such as tooth
They are ordinarily non-pathogenic but on occasions extraction, they may cause transient bacteraemia and
cause disease. The viridans streptococci are associated get implanted on damaged or prosthetic valves or in a
with dental caries and subacute bacterial endocarditis. congenitally diseased heart, and grow to form vegetations.
Dental caries is caused by Str. mutans. It breaks down Prophylactic antibiotic should be given in such persons
dietary sucrose, producing acid and a tough adhesive before tooth extraction or other procedures. Viridans
dextran. The acid damages dentine while the dextran streptococci are generally penicillin sensitive but some
binds together food debris, mucus, epithelial cells and strains may be resistant. Hence, antibiotic sensitivity
bacteria to form dental plaques. These plaques lead to of these bacteria must be determined for appropriate
dental caries.About 40% of subacute bacterial endocarditis treatment of endocarditis.
is caused by viridans group of streptococci.Members of Important laboratory tests for diagnosis of different
St,: mitis group are the predominant agent in urogenital groups of streptococci are shown in Table 24.4.
Table 24.4 Some characteristic features of different groups of streptococci

Lancefield S pecies or common name Diagnostic tests Diseases caused
group
A Str. pyogenes Bacitracin sensitive; PYR positive; ribose Upper respiratory tract infections; Uf .C.
not fermented; resistant to SXT skin infections; acute rheumat~
--- fever;acute glomerulonephritis

----
r B
C or G
SIi: agalactiae
Str. dysgalactiae subsp.

equisimilis
CAMP test positive; hippurate hydrolysis
positive; resista~ SXT
Ribose fermented; trehalose fermented
Neonata1se,12_ticaemia and
m~itis
Pharyngitis; endocarditis
D (i) Enterococcus sp. (E. fae calis -----

Bile aesculin test positive; heat test positive; Urinary tract infections; wound
---
and other enterococci) growth in the presence of 6.5% NaCl; PYR infections
positive; resistant to SXT
(ii) Non-enterococci (Str. bovis) Bile aesculin test positive; no growth in Genitourinary infections
6.5% NaCl; PYR negative; sensitive to SXT
Not typed Viridans streptococci (Str. Optochin resistant; species diffe rentiation on Endocarditis; dental caries
salivarius group, Str. mutans biochemical tests
group and many others)
KEY POINTS
I. Streptococci are Gram positive cocci which are arranged in chains. They grow poorly in simple media but their growth
is enhanced by the addition of fermentable carbohydrate (e.g. glucose), blood or serum.
2. Three types of haemolytic reactions (a, and y) are observed on blood agar medium.
3. The beta haemolytic streptococci were classified by Lancefield serologically into 20 Lancefield groups named A-V
(without I and J). These groups are based on the nature of a carbohydrate (C) antigen on the cell wall.
4. Streptococcus pyogenes belongs to Lancefield group A.
5. Crystal violet blood agar is a selective medium for isolation of streptococci.
6. Haemolysins and pyrogenic exo tox ins are the toxins produced by Str. pyogenes.
7. Streptokinase, deoxyribonucleases, nicotinamide adenine dinucleotidase (NADase) and hyaluronidase are important
enzymes produced by Str. pyogenes.

194 _ _ _ __ __ _ _ _ _ __ _ _ __ _ _ _ _ __ _ _ __ _ Unit Ill: Systemic Bacteriology
8. Str. pyogenes produces pyogenic infections with a tendency to spread locally. Sore throat (acute tonsilitis and/or
pharyngitis) is the most common of streptococcal diseases. Non-suppurative sequelae of local infections include acute
glomerulonephritis and rheumatic fever.
9. Group B streptococci are important pathogens in neonates causing neonatal septicaemia and meningitis. CAMP
reaction is used to identify Group B streptococci.
10. Enterococcus faecalis is frequently isolated from cases of urinary tract infection and wound infection. Heat resistance
test is positive for E. faecalis.
11. Viridans streptococci produce alpha haemolysis on blood agar. They are commensals of mouth and upper respiratory
tract. They are ordinarily non-pathogenic but on occasions cause disease. They are associated with dental caries and
subacute bacterial endocarditis.
YOU MUST KNOW

1. Classification of streptococci.
2. Morphology and culture characteristics of Streptococcus pyogenes.
3. Toxins and enzymes of Str. pyogenes.
4. Infections caused by different streptococci.
5. Non-suppurative complications of Str. pyogenes infections.
6. Laboratory diagnosis of infections caused by Str. pyogenes.
7. Group B streptococci, Group D streptococci and viridans streptococci.
STUDY QUESTIONS
1. Classify streptococci. Describe the laboratory diagnosis of streptococcal sore throat.
(a) Antigenic structure of Str. pyogenes
(b) Toxins and enzymes of Streptococcus pyogenes
(c) Non-suppurative complications of Str. pyogenes infections
(d) Lancefield grouping.
(a) Group B streptococci
(b) Group D streptococci
(c) Enterococci
(d) Viridans streptococci
(e) CAMP reaction
(f) Heat test.

1. On blood agar which type of haemolysis is produced by Streptococcus pyogenes?
(a) Alpha haemolysis (b) Beta haemolysis
(c) Gamma haemolysis (d) None of the above
2. Erythrogenic toxin is responsible for:
(a) Dick test (b) Schultz-Charlton reaction
(c) Both of the above (c) None of the above
3. Which of the following is selective medium for Streptococcus pyogenes?
(a) Blood agar (b) Crystal violet blood agar
(c) Potassium tellurite blood agar (d) Chocolate agar

Ch 24: Streptococcus and Enterococcus _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __ _ _ __ 195
4. Sensitivity to bacitracin can be used to identify:

(a) Streptococcus pyogenes (b) Str. agalactiae
(c) Str. equisimilis (d) Str. mitis
5. CAMP reaction can be used to identify:
(a) Streptococcus pyogenes (b) Str. agalactiae
(c) Str. equisimilis (d) Str. mitis
6. The most important bacterial cause of sore throat is:
(a) Streptococcus pyogenes (b) Staphylococcus aureus
(c) Staphylococcus epidermidis (d) Neisseria gonorrhoeae
7. Which of the following are non-suppurative complications of Streptococcus pyogenes infections?
(a) Acute rheumatic fever (b) Acute glomerulonephritis
8. Which of the following tests may be used for diagnosis of non-suppurative complications of Streptococcus pyogenes
infections?
(a) ASO test (b) Anti ONAase B test
(c) Streptozyme test (d) All of the above
9. Enterococci contain the same antigen as that of:
(a) Group A streptococci (b) Group B streptococci
(c) Group C streptococci (d) Group D streptococci
10. Which of the following can cause dental caries?
(a) Streptococcus mutans (b) Staphy lococcus aureus
(c) Enterococcus (d) Streptococcus agalactiae
Answers (MCQs):
1. (b) 2. (c) 3. (b) 4. (a) 5. (b)
6. (a) 7. (c) 8. (d) 9. (d) 10. (a)
----+++----

Chapter 25
PNEUMOCOCCUS
Pneumococcus
C. Biochemical Reactions D. Resistance
E. Antigenic Structure F. Variation
G. Toxins and Other Viru lence Factors H. Pathogenesis
I. Epidemiology J. Laboratory Diagnosis
K. Treatment L. Prophylaxis
Pneumococci are normal commensals of the upper slightly elongated cocci arranged in pairs (diplococci) with
respiratory tract. They are important pathogens of the broad ends in apposition. Each coccus has one end
pneumonia and otitis media in children. They are broad or rounded and other pointed (flame shaped or
reclassified as Streptococcus pneumoniae because of lanceolate appearance) (Fig. 25.1) . They are capsulated
its genetic relatedness to streptococcus. They differ and the capsule encloses each pair. The capsule may be
from streptococci in their morphology (diplococci), demonstrated as a clear halo in India ink preparation
bile solubility, optochin sensitivity and by a specific (Fig. 25.2) . In old cultures, the capsule is usually lost.
polysaccharide capsule. They are non-motile and non-sporing.
PNEUMOCOCCUS B. Culture
A. Morphology Pneumococci have complex nutritional requirement
Pneumococci are Gram positive, small (1 µm diameter), and therefore grow only in enriched media especially
-· - c9 0
0 a(S)
@
Fig. 25.1 Str. pneumoniae in pus Fig. 25.2 India ink preparation to show pneumococcus capsule
196

Ch 25: Pneumococcus _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ 197
supplemented with blood. They are aerobes and facultative due to production of beta lactamase, but alteration in
anaerobes and their growth is improved by 5- 10 per cent the penicillin binding proteins on the bacterial surface.
CO 2. The optimum temperature for growth is 37°C (range These strains are also resistant to multiple drugs.
25°C to 42°C) and pH 7.8 (range 6.5- 8.3). Pneumococci are sensitive to optochin (ethyl-
On blood agar, after incubation for 18 hours, the hydrocuprein hydrochloride) in a concentration of
colonies are usually small (0.5- 1 mm), dome shaped, with 1/500,000. When a 6 mm optochin disc (5 µg) is applied on
an area of greenish discolouration (alpha haemolysis) a blood agar plate inoculated with pneumococci, a wide
around them. On prolonged incubation, the colonies zone (14 mm or more) of inhibition occurs on incubation.
become flat, with raised edges and central umbonation This is very useful test to differentiate pneumococci from
(due to autolysis occuring at centre) which creates a other streptococci which do not show zone of inhibition
draughtsman appearance (concentric rings are seen when by optochin disc.
viewed from above). Some strains such as those of serotype
3 and 7 form large mucoid colonies due to production E. Antigenic Structure
of abundant capsular polysaccharides. Pneumococci are The most important antigen of the pneumococcus is
typically alpha haemolytic but under anaerobic conditions capsular polysaccharide. Other antigens are somatic
colonies show beta haemolysis due to liberation of oxygen M protein and a group specific cell wall carbohydrate.
labile pneumolysin O by these bacteria.
In liquid medium such as glucose broth, pneumococci 1. Capsular Polysaccharide
produce uniform turbidity. These cocci readily undergo Capsular polysaccharide is type specific. Since this
autolysis in cultures due to the action of intracellular polysaccharide is soluble in tissue and culture fluid, it diffuses
enzymes. Autolysis is enhanced by bile salts and into the culture medium or tissues and infective exudates,
other surface active agents. This property is helpful to hence, it is also called the soluble specific substance (SSS).
differentiate it from other streptococci. Pneumococci are classified into types based on the nature
of the capsular polysaccharide. More than 90 serotypes
C. Biochemical Reactions are recognised. These are named 1, 2, 3, and so on.
Pneumococci ferment several sugars with production of Serological typing of pneumococcus is carried out
acid only. Fermentation is tested in Hiss's serum water. by three methods.
Fermentation of inulin by pneumococci is of great value (i) Agglutination of organisms with type specific
to differentiate them from streptococci. antiserum.
Pneumococci are soluble in bile. When 2% sodium (ii) Precipitation of capsular polysaccharide with
deoxycholate solution is added to a broth culture at type specific antiserum.
neutral pH, the culture clears due to the lysis of the cocci. (iii) Quellung reaction or capsule swelling reaction
Alternatively, if a loopful of 10% sodium deoxycholate was described by N eufeld (1902). In this reaction,
solution is placed on a pneumococcus colony, lysis of a suspension of pneumococci is mixed on a slide
colony occurs within a few minutes. Bile solubility test is with a drop of specific antiserum and a loopful
an important diagnostic test to differentiate pneumococcus of methylene blue solution . In presence of the
from other streptococci. This test is based on the presence homologous antiserum, the capsule around
of an autolytic enzyme amidase in the pneumococci. This pneumococci reveals an apparent swelling,
enzyme cleaves the bond between alanine and muramic sharply delineated and refractile under the
acid in the peptidoglycan. The amidase is activated by microscope. The Quellung test can be done either
bile salts, resulting in lysis of the bacteria. in sputum or in culture, and used to be a routine
Pneumococci are catalase and oxidase negative. bedside procedure in olden days.
D. Resistance 2. M Protein
Pneumococci are delicate organisms and are destroyed M protein is characteristic for each type of pneumococcus.
at 52°C for 15 minutes. They are more sensitive to usual It is not associated with virulence and antibody to M
antiseptics. protein is non-protective.
They are sensitive to a wide range of antibiotics including
penicillins, erythromycin, tetracycline, chloramphenicol, 3. Cell Wall Carbohydrate (C-Substance)
vancomycin and clindamycin. Penicillin resistant strains Pneumococci contain a species specific carbohydrate
first appeared in 1967. It may be due to mutation or antigen which is named as C-substance. It is present in
gene transfer. The drug resistance to penicillin is not all pneumococci. The C-substance is precipitated by an

198 _ _ __ _ _ _ _ __ __ _ _ _ _ __ __ _ _ __ _ __ _ Unit Ill: Systemic Bacteriology
abnormal protein (~-globulin), that appears in the acute infections of the respiratory tract. Any serotype of
phase sera of cases of pneumonia but disappears during pneumococcus can produce bronchopneumonia. Other
convalescence. It is also detected in blood of patients causative agents responsible for bronchopneumonia
with some other illnesses. This is known as the C-reactive include Staph. aureus, K. pneumoniae, Str. pyogenes,
protein (CRP). It is not an antibody of C substance. It H. influenzae, Fusobacterium species and Bacteroides.
is an 'acute phase' substance, produced in hepatocytes.
Its production is stimulated by bacterial infections, 3. Meningitis
malignancies and tissue destruction. CRP is used as an It is the most serious of pneumococcal infections. Str.
index of activity in rheumatic fever and certain other pneumoniae is the second most important cause of
conditions. CRP is routinely detected in serum by latex pyogenic meningitis after Nmeningitidis. This disease is
agglutination test. commoner in children. Pneumococcus spreads from the
pharynx to the meninges via blood stream. Other bacterial
F. Variation agents of pyogenic meningitis include N meningitidis,
Pneumococcus is virulent in capsulated (smooth) form but H. influenzae, Str. agalactiae (group-B) and Listeria
non-capsulated (rough) forms are avirulent. On repeated monocytogenes.
subculture, a smooth to rough (S-.R) variation occurs.
R forms arise as spontaneous mutants and outgrow the 4. Other Infections
parental S forms in artificial culture. Such R mutants are Pneumococcus may also produce empyema, pericarditis,
eliminated in tissues by phagocytosis. otitis media, sinusitis, conjunctivitis, peritonitis and
Rough pneumococci of one serotype may be made suppurative arthritis, usually as complications of
to produce capsules of the same or different serotypes, pneumoma.
on treatment with DNA from the respective serotypes
of pneumococci. This transformation was discovered by I. Epidemiology
Griffith (1928) as the first instance of genetic exchange The source of human infection is carrier and less often
of information in bacteria. patient. Pneumococci are transmitted by inhalation of
contaminated dust, droplets or droplet nuclei. Infection
G. Toxins and Other Virulence Factors usually leads to pharyngeal carriage. Disease results only
Pneumococci produce an oxygen labile intracellular when the host resistance is lowered by factors such as
haemolysin and a leucocidin. The virulence of pneumococci respiratory viral infections, malnutrition etc.
is dependant upon its capsule which prevents or inhibits Lobar pneumonia is usually a sporadic disease but
phagocytosis. Non-capsulated strains are avirulent. The epidemics may occur. The incidence of bronchopneumonia
antibody to the capsular polysaccharide protects against is more when an epidemic of influenza or other viral
infection. Due to the abdundance of capsular material, infections of the respiratory tract occurs.
the virulence of type-3 pneumococcus is enhanced.
Pneumolysin, a toxin produced by penumococci, is J. Laboratory Diagnosis
another virulence factor. It has cytotoxic and complement 1. Specimens
activating properties. It is immunogenic. Clinical samples, such as sputum, cerebrospinal fluid
(CSF), pleural exudate or blood are collected according to
H. Pathogenesis the site of lesion. Blood culture is useful in pneumococcal
Str. pneumoniae is one of the most common bacteria septicaemia.
causing pneumonia, both lobar and bronchopneumonia.
It is also responsible for acute tracheobronchitis and 2. Collection and Transport
empyema. All the specimens should be collected in sterile containers
under all aseptic conditions. They should be processed
1. Lobar Pneumonia immediately. In case of delay, CSF specimen should never
Pneumonia results only when the general resistance is be refrigerated but kept at 37°C (H. influenzae, another
lowered. Common infective types of Str. pneumoniae causative agent of pyogenic meningitis may die at cold
include types 1- 12 in adults and types 6, 14, 19 and 23 temperature).
are responsible in children.
3. Direct Microscopy and Antigen Detection
2. Bronchopneumonia Gram staining of smear reveals a large number of
It is almost always a secondary infection following viral polymorphs and typical organism. In case of meningitis,

Ch 25 : Pneumococcus _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ 199
presumptive diagnosis may be made by finding Gram Inoculated mice die in 24-48 hours. Heart blood and
positive diplococci which may be intracellular as well peritoneal exudate of the animal shows pneumococci.
as extracellular in CSP smear. Capsule swelling may be The test may be negative with occasional strains which
observed under microscope, when pneumococci are mixed are avirulent for mice (type 14 strains).
with type specific antisera.
Capsular polysaccharide antigen can be demonstrated 8. Antibiotic Sensitivity Test
by counterimmunoelectrophoresis. This has been employed It is especially useful in strains which are resistant.
in blood, urine and cerebrospinal fluid. Antigen may
also be detected by immunochromatographic assay, latex K. Treatment
agglutination or coagglutination. The antibiotic of choice is parenteral penicillin.
Cephalosporin is indicated in case of penicillin resistant
4. Culture strains.
Specimen is inoculated on blood agar and incubated
at 37°C for 24 hours in the presence of 5- 10% CO 2. L. Prophylaxis
Typical colonies develop with a - haemolysis. Organisms Immunity is type specific and is associated with antibody
from the isolated colony are identified by Gram staining to the capsular polysaccharide. The existence of more
and biochemical reactions. than 90 serotypes makes prophylactic immunisation
impracticable. But, as a limited number of serotypes cause
5. Colony Morphology and Staining most of the pneumonia cases, vaccination is possible.
Colonies are usually small (0.5- 1 mm), with alpha Three pneumococcal vaccines are available.
haemolysis around them. On prolonged incubation, A polysaccharide vaccine containing prevalent
colonies have draughtsman appearance. serotypes (23 serotypes) is administered by a single dose
On Gram staining pneumococci are Gram positive, injection. Such vaccines are used only in those persons
small (1 µm diameter), diplococci, They are flame who are at enhanced risk of pneumococcal infection.
shaped or lanceolate in appearance. The capsule may These include persons with absent or dysfunctional
be demonstrated as a clear halo in India ink preparation spleen, nephrotic syndrome, sickle cell anaemia,
(Fig. 25.2) . multiple myeloma, hepatic cirrhosis, diabetes mellitus and
immunodeficiencies including HIV infection. Vaccination
6. Biochemical Reactions is contraindicated in lymphomas.
Important biochemical tests are inulin fermentation and The heptavalent pneumococcal conjugate vaccine (7
bile solubility tests. Another test which has a great value is serotypes) is composed of purified polysaccharides of
optochin sensitivity test. Details of these tests have already seven serotypes conjugated to a diphtheria protein is now
been described. Str. pneumoniae can be differentiated from available for use in children from two months to two years.
Str. viridans by various characteristics (Table 25 .1). More recently, a new 13-valent vaccine that contain six
additional serotypes was approved for young children.
7. Animal Pathogenicity Test However, protection would depend on the principle that
From specimens where organisms are expected to be serotypes prevalent in the community and serotypes used
scanty, intraperitoneal inoculation in mice may be used. in the vaccine are the same.
Table 25.1 Differential Characteristics of Str. pneumoniae and Str. viridans

Characteristic Str. pneumoniae Str. viridans
Morphology Capsulated, lanceolate diplococci (in pairs) Non-capsulated, oval or round cocci in chains
Colonies on blood agar Initially dome shaped with a - haemolysis, Dome shaped with a - haemolysis
later 'draughtsman' colonies
Colonies in liquid medium Uniform turbidity Granula r turbidity, powdery deposit
Bile solubility Positive Negative
Inulin fermentation Positive Negative
Optochin sensitivity Positive Negative
Animal pathogenicity Fatal infection Non-pathogenic
(lntraperitoneal inoculation in mice)

200 _ _ _ _ _ _ __ __ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Unit Ill: Systemic Bacteriology
KEY POINTS
I. Pneumococci are Gram positive, small, slightly elongated cocci arranged in pairs (diplococci) with broad ends in
apposition. Each coccus has one end broad or rounded and other pointed (flame shaped or lanceolate appearance).
They are capsulated.
2. They have complex nutritional requirement and grow only in enriched media especially supplemented with blood.
Their growth is improved by 5-10 per cent CO2 .
3. On blood agar, the colonies are usually small, dome shaped with an area of greenish discolouration (alpha haemolysis)
around them. On prolonged incubation, the colonies become flat , with raised edges and central umbonation which
creates a draughtsman appearance (concentric rings are seen when viewed from above).
4. Pneumococci differ from streptococci in their morphology (diplococci), bile solubility and optochin sensitivity.
5. They may cause pneumonia, meningitis, empyema, otitis media etc.
YOU MUST KNOW

I. Morphology and culture characteristics of Pneumococcus.
2. Quellung reaction.
3. Laboratory diagnosis of pneumococcal infections.
4. Differences between Str. pneumoniae and Str. viridans.
STUDY QUESTIONS
I. Describe the laboratory diagnosis of pneumococcal infections.
2. Differentiate between Str. pneumoniae and Str. viridans in a tabulated form .
1ULTIPLE CHOICE QUESTIONS (MCQs)

1. Draughtsman colony is a characteristic feature of:
(a) Streptococcus pyogenes (b) Strep. pneumoniae
(c) Enterococcus facecalis (d) Viridans streptococci
2. Capsule of Streptococcus pneumoniae can be demonstrated by:
(a) India ink staining (b) Quellung reaction (c) Both of the above (d) None of the above
3. Streptococcus pneumoniae may cause:
(a) Lobar pneumonia (b) Bronchopneumonia (c) Meningitis (d) All of the above
4. Pneumococcal antigen in CSF can be demonstrated by:
(a) Counterimmunoelec trophoresis (b) Latex agglutination
(c) Coagglutination (d) All of the above
5. Which of the fo ll owing tests can be useful to identify Streptococcus pneumoniae?
(a) Bile solubility test (b) Inulin fermentation
(c) Optochin sensitivity test (d) All of the above
6. Which of the following bacteria produce alpha haemolysis on blood agar?
(a) Staphylococcus aureus (b) Streptococcus pyogenes
(c) Streptococcus pneumoniae (d) All of the above
...
Answers (MCQs):
1. (a) 2. (c) 3. (d) 4. (d) 5. (d) 6. (c)
____ ____

Chapter 26
NEISSERIA AND MORAXELLA
I. Neisseria meningitidis
C. Biochemical Reactions D. Antigenic Structure
E. Resistance F. Pathogenesis
G. Epidemiology H. Laboratory Diagnosis
I. Treatment J. Prophylaxis
II. Neisseria gonorrhoeae
E. Resistance F. Pathogenesis
I. Treatment J. Prophylaxis
Ill. Non-Gonococcal (Non-specific) Urethritis
A. Causative Agents B. Treatment
IV. Commensal Neisseriae
V. Moraxella catarrhalis
C. Biochemical Reactions D. Pathogenesis
VI. Moraxella lacunata
C. Biochemical reactions D. Pathogenesis
E. Treatment
The genus N eisseria consists of Gram negative, aerobic, other species. N meningitidis and N gonorrhoeae are two
oxidase positive, non-motile diplococci (arranged in important species causing human infections.
pairs). They may be classified into pathogenic and non-
pathogenic (commensals). The two pathogenic species I. NEISSERIA MENINGITIDIS
are N meningitidis (causes pyogenic meningitis) and N (MENINGOCOCCUS)
gonorrhoeae (causes gonorrhoea). The non-pathogenic Meningococcus was first isolated by Weichselbaum m
species include N fiavescens, N sicca, N subfiava and 1887, from the spinal fluid of a patient.
201
202 _ _ _ _ _ _ _ _ _ _ _ _ __ _ _ __ _ _ _ _ _ __ _ _ _ Unit Ill: Systemic Bacteriology
A. Morphology and maltose are fermented with acid production but no

They are Gram negative, spherical or oval cocci, 0.6- gas (gonococci ferment glucose but not maltose). They
0.8 µm in size, arranged in pairs with the adjacent sides do not ferment lactose or sucrose. Indole and H 2S are
flattened (Fig. 26.1) . They are non-motile. The cocci are not produced and nitrates are not reduced.
generally intracellular when isolated from lesions.
Oxidase Test
When freshly prepared oxidase reagent (1 % tetramethyl
paraphenylene diamine hydrochloride) is poured on the
culture plate, neisseria colonies become deep purple. These
colonies can be picked up for subcultures immediately, as
the organism dies on prolonged exposure to the reagent.
The test may also be performed by rubbing bacterial
growth with a loop on a filter paper strip moistened
with the oxidase reagent (Kovac's method). A deep purple
colour is a positive reaction.
Fig. 26.1 N meningitidis in cerebrospinal fluid. Inset-enlarged

view showing flat adjacent sides of cocci.
D. Antigenic Structure
N meningitidis has been divided into 12 serogroups on
the basis of immunological specificity of the capsular
B. Culture polysaccharide. These serogroups are A , B, C, X, Y, Z,
They do not grow on ordinary media but have exacting W-135, 29E, H , I, K and L. Groups A, B and C are
growth requirements. Growth occurs on media enriched the most important. Groups 29E, W-135 and Y also
with blood or serum. These substances promote growth frequently cause meningitis. These six groups (A, B, C,
by neutralising certain inhibitory substances found 29E, W-135 and Y) are responsible for the large majority
in culture media rather than by providing additional of meningitis.
nutrients. Blood agar, chocolate agar and Mueller-
Hinton agar are commonly used media. Thayer-Martin E. Resistance
(TM) medium with antibiotics (vancomycin, colistin, These are very delicate organisms, being highly susceptible
and nystatin), modified Thayer-Martin (MTM) medium to heat, desiccation and to disinfectants. They are
with antibiotics (vancomycin, colistin, nystatin and susceptible to penicillin, ampicillin, chloramphenicol,
trimethoprim), Martin-Lewis (ML) medium and New- macrolides and ciprofloxacin. Strains resistant to penicillin
York City (NYC) medium are selective media used for have now been reported from several countries.
its isolation. Thayer-Martin medium and Martin-Lewis
(ML) medium are chocolate agar based media, while F. Pathogenesis
New-York City medium is a clear peptone-comstarch agar N meningitidis causes pyogenic meningitis in all ages,
based medium. All the three media contain antibiotics but is most common in children and young adults. The
to inhibit bacteria, yeasts and moulds. These media can other common bacterial agents responsible for pyogenic
also be used to grow N gonorrhoeae. meningitis include H. influenzae and Str. pneumoniae.
They are strict aerobes and cannot grow anaerobically. Meningococci are strict human pathogens. The
The optimum temperature and pH for growth are 35- infection is acquired by droplet spread via the carriers
360C and 7.4-7.6 respectively. A moist environment with and the cases. Human nasopharynx is the reservoir of N
5- 10% CO 2 is must for growth to occur. meningitidis. The incubation period of the disease is about
On solid media, the colonies are small (1 mm in 3 days. The spread of cocci from the nasopharynx to the
diameter), round, convex, grey, translucent and with meninges is controversial. The organism may spread along
entire edges. The colonies are butyrous in consistency the perineural sheath of the olfactory nerve, through the
and easily emulsifiable. In liquid media, it produces a cribriform plate to the subarachnoid space or through
granular turbidity. the blood stream.
Meningococcaemia presents as acute fever with
C. Biochemical Reactions petechial rash. A few develop fulminant meningococcaemia
N meningitidis is catalase and oxidase pos1t1ve. The ( Waterhouse-Friderichsen syndrome) characterised by
prompt oxidase reaction helps to identify neisseria (both shock, disseminated intravascular coagulation and
meningococci and gonococci) in mixed cultures. G lucose multisystem failure. It is usually a fatal condition.

Ch 26: Neisseria and Moraxella _ _ _ _ _ __ _ _ _ _ _ _ _ __ _ __ _ _ _ _ _ _ _ _ _ 203
Deficiency of complement components (C5- C9) favours as countercurrent immunoelectrophoresis (CIEP),

the meningococcal infections. coagglutination or latex agglutination test. Antigen
Bacterial endotoxin appears to play the pathogenic detection is useful when organisms are scanty.
role. The vascular endothelium is sensitive to the The second portion of the CSP is used for direct
endotoxin. All major inflammatory cascade systems as culture while the third portion of the CSP is incubated
well as cytokines are triggered. overnight after adding an equal volume of glucose broth
and then subcultured on blood agar or chocolate agar.
G. Epidemiology
The humans are the only reservoir of the meningococcus.
Asymptomatic nasopharyngeal carriers serve to infect 4. Culture
their contacts. Transmission is by airborne droplets, (i) CSF
or less often by fomites. The carrier rate is about 5-10 Centrifuged deposit is inoculated on blood agar or
per cent which rises during epidemics. Meningitis is chocolate agar. The plate is incubated at 35-36°C under
more common in children below the age of five years 5- 10% CO 2 . Colonies appear after 18- 24 hours. These
and in males. Epidemic usually occurs in overcrowded are identified by colony morphology, Gram staining and
area. Serogroup A is usually associated with epidemics. biochemical reactions.
Serogroup B causes both epidemics and outbreaks, while
serogroup C is associated mostly with localised outbreaks. (ti) Blood
Group A meningococcus affected Delhi during epidemic Blood culture is often positive in meningococcaernia and
in early 1985. in early cases of meningitis. Blood culture bottles (glucose
broth and sodium taurocholate broth) are incubated at
H. Laboratory Diagnosis 35-36°C for 24 hours. Subcultures are made from these
1. Specimens broths on to blood agar and chocolate agar. These plates
• CSP are again incubated at 35-36°C under 5-10% CO 2 for
• Blood 18- 24 hours. Identification of organism is done by colony
• Petechial lesions specimen morphology, Gram staining and biochemical reactions.
• N asopharyngeal swab-especially to detect Blood culture should be incubated for 4-7 days, with
carriers. daily subcultures.
2. Collection and Transport (iu) Otltt,. spt<.,lll ns

Collection of specimen is done under sterile conditions Other specimens (petechial lesions, nasopharyngeal
by lumbar puncture for CSP and by venepuncture swab, autopsy specimens) are inoculated on blood agar
for blood. Blood is injected into blood culture bottles and chocolate agar and are processed in similar way as
(glucose broth and sodium taurocholate broth) through described for CSP. Specimens may be collected from
the hole in the bottle cap. Nasopharyngeal specimen the meninges, lateral ventricles or brain and spinal cord
is collected by using a sterile swab. All the specimens in case of autopsy. Meningococci may die if specimens
must be transported immediately. CSP should never be are not collected within 12 hours of the death of the
refrigerated as H. in.fiuenzae (another common causative patient.
agent of meningitis) may die at the cold temperature.
The nasopharyngeal swab should be held in a transport 5. Colony Morphologtj and Gram Staining
medium (e.g. Stuart's) until it is inoculated on a culture On solid media, colonies are small (1 mm in diameter),
medium. round, convex, grey, translucent and with entire edges.
Smear is prepared from the suspected colony and Gram
3. Direct Microscopy and Antigen Detection staining is done. On Gram staining, they are Gram negative,
CSF is divided into three portions. One portion is spherical or oval cocci, 0.6----0.8 µmin size, arranged in pairs
centrifuged and smear is prepared from the deposit (diplococci) with the adjacent sides flattened (Fig. 26.1 ).
for Gram staining. Meningococci are seen as Gram
negative diplococci present mainly inside polymorphs 6. Biochemical Reactions
(intracellular), but may also be present extracellularly. A N meningitidis is catalase and oxidase positive. Glucose
large number of pus cells are also seen. The supernatant and maltose are catabolised with acid production but no
fluid may be used for detection of meningococcal gas. This breakdown of glucose and maltose occurs by
antigen (capsular polysaccharide) by rapid tests, such oxidation and not by fermentation.
204 _ _ _ __ __ _ _ _ _ _ _ __ _ _ _ _ _ _ _ _ _ _ _ _ _ Unit Ill: Systemic Bacteriology
7. Slide Agglutination
Direct slide agglutination of the organism may be done
with specific antisera.
8. Serological Diagnosis
This may be attempted in chronic meningococcal
septicaemia where no organism has been isolated.
Specific antibodies to capsular polysaccharide may be
demonstrated by haemagglutination test.
9. Polymerase Chain Reaction

Meningococcal DNA in CSF or blood can be amplified Fig. 26.2 N. gonorrhoeae in urethral pus. Inset - enlarged
and then detected. It is a rapid method. view showing diplococci with adjacent surfaces concave
I. Treatment
Meningococci are uniformly sens1t1ve to penicillin.
Chloramphenicol is used in persons allergic to penicillin.
Penicillin G in high doses is given intravenously or
intrathecally if necessary. Cefotaxime or ceftriaxone are
as effective as chloramphenicol and the possibility of
blood dyscrasia may be avoided.
J. Prophylaxis
1. Chemoprophylaxis Fig. 26.3 Gram-negative intracellular diplococci
It is indicated for close contacts of patients for (N gonorrhoeae)
eliminating the bacteria from nasopharynx. Rifampicin

or ciprofloxacin are recommended. Gonococci possess pili on their surface. These pili
facilitate adhesion of cocci to host cells and promote
2. Immunoprophylaxis virulence by resisting phagocytosis. Pili of different strains
Meningococcal vaccines prepared from polysaccharides of are antigenically different. Piliated gonococci agglutinate
serogroups A, C, W-135 and Y are available. Single dose human erythrocytes and the haemagglutination is not
is given intramuscularly. The protection is group specific inhibited by mannose.
and lasts for at least three years but does not prevent
meningococcal carriage. No vaccine is available against B. Culture
group B meningococci because capsular polysaccharide This organism is more difficult to grow than N. meningitidis.
of this group is a poor immunogen. They are aerobic, but may grow anaerobically as well.
They grow best at a temperature of 35- 36°C in presence
of 5- 10% CO 2 and at pH 7.2- 7. 6. They require an enriched
II. NEISSERIA GONORRHOEAE medium like chocolate agar for their growth. A popular
(GONOCOCCUS) selective medium is the Thayer-Martin medium which
N. gonorrhoeae causes the sexually transmitted disease, contains chocolate agar with antibiotics (vancomycin,
gonorrhoea. The organism was first described by colistin and nystatin) . It inhibits most contaminants
Neisser in 1879 in gonorrheal pus. N. gonorrhoeae and including commensal neisseria. Trimethoprim may be
N. meningitidis are closely related and have 70% DNA added to Thayer-Martin medium to inhibit swarming
homology. of Proteus sp. This medium is known as modified Thayer-
A. Morphology Martin medium.
They are Gram negative oval cocci arranged in pairs
(diplococci) with adjacent sides concave (pear or bean Colony Morphology
shaped). In smear from purulent material, they are Colonies are small, round, grey, translucent, convex with
intracellular within polymorphs, some cells containing finely granular surface. They are easily emulsifiable. N.
as many as a hundred cocci (Figs. 26.2 and 26.3). gonorrhoeae produces five types of colonies, Tl, T2,

Ch 26: Neisseria and Moraxella _ _ _ _ __ _ __ _ _ __ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ 205
T3, T4 and TS. Type 1 and 2 produce small brown gonococci. There are two variants of protein I, named
colonies and possess pili. These are autoagglutinable and IA and IB. Any one strain carries either IA or IB but
virulent strains. Type 3, 4 and S form larger, granular, not both. 24 serovars of type IA and 32 serovars of type
non-pigmented colonies and are non-piliated. They form IB have been demonstrated.
smooth suspensions and are avirulent. Tl and T2 are also
known asp+ and p++ respectively, while T3, T4 and TS (ii) Protein II (opa)
are named as P-. One part of protein II is in outer membrane and the
rest is exposed on the surface of bacteria. This protein
C. Biochemical Reactions takes part in adhesion of bacterium and its attachment
N gonorrhoeae is oxidase positive. Gonococci ferment to host cell. Type II protein is present in those strains
glucose (with acid production only) and not maltose. which form opaque colonies, therefore, it is also called
Rapid carbohydrate utilisation test (RCUT): These the opacity associated (OPA) protein.
rapid tests also detect acid production from various
carbohydrates. These are based on the presence of (iii) Protein III
preformed enzymes for carbohydrate utilisation rather than It is associated with protein I in the formation of pores
on bacterial growth. They take 2-4 hours rather than 24 to on the cell surface and hence plays a role in the exchange
72 hours required for the conventional carbohydrate tests. of molecules across the outer membrane.
D. Antigenic Structure 5. Other Proteins

The antigenic structure is complex. The surface structures IgAl protease produced by gonococci as well as
of N gonorrhoeae include the following : meningococci splits and inactivates IgA which plays a
major role in mucosa! defence. Two other proteins H8
1. Capsule and iron binding protein are described but their role in
It is polyphosphate and not polysaccharide. Capsule pathogenicity is not known.
is most evident in freshly isolated gonococci. It is
loosely associated with cell surface. The capsule inhibits E. Resistance
phagocytosis. Presence of capsule and its role requires The gonococcus is a very delicate organism. It is strict
further elucidation. parasite and dies rapidly outside the human host, in 1
to 2 hours in exudate and in 3 to 4 days in culture. It
2. Pili survives at 3S°C if kept in slant cultures under sterile
These are hair like structures extending from the surface. paraffin oil. Cultures may also be stored for years if
The piliated organisms produce altered appearance of frozen quickly and kept at - 70°C.
colonies on culture. Pili enhance attachment of the Gonococci are readily killed by heat, drying and
organism to host cells and resist phagocytosis. They act antiseptics. Formerly, they were highly susceptible to
as virulence factor. They are made up of pilin proteins. sulphonamides and penicillin but have steadily developed
These pilin proteins are antigenically different in almost resistance to many antibiotics. PPNG (penicillinase
all strains and a single strain can produce several producing Neisseria gonorrhoeae) strains are resistant to
antigenically distinct pilins. penicillin due to production of ~-lactamase (penicillinase)
enzyme by these strains.
3. Lipooligosaccharide (LOS)
There is absence of long O-antigenic side chains in the F. Pathogenesis
chemical structure of lipooligosaccharide (LOS). This 1. Gonorrhoea
property differentiates it from lipopolysaccharide (LPS) It is a sexually transmitted disease involving urethra in
of Gram negative bacilli. Toxicity in gonorrhoea is largely both sexes but in females, the endocervix is the primary
due to the endotoxic effects of lipooligosaccharide (LOS). site of infection. The incubation period is 2-8 days. The
disease is an acute urethritis characterised by purulent
4. Proteins urethral discharge. Asymptomatic infection is common
The outer membrane antigens (proteins) are the porins. in women. In males, the acute urethritis may extend
to the prostate, testes, seminal vesicles and epididymis.
(i) Protein I (por) If untreated, it may lead to fibrosis and stricture. The
It forms pores on surface. Each strain of gonococcus infection may spread to the periurethral tissues, causing
expresses one type of protein I. It helps in serotyping of abscesses and multiple discharging sinuses ('watercan

perineum') In females, the primary infection may spread be examined. Centrifuged deposits of urine is examined
from urethra and cervix to Bartholin's glands, uterus, in cases where no urethral discharge is available.
fallopian tubes, ovaries and may cause pelvic inflammatory
disease resulting in sterility. In some patients, who develop 2. Transport
pelvic inflammatory disease, there is inflammation of All the specimens should be transported and processed
liver capsule (Fitz-Hugh-Curtis syndrome). Occasionally immediately. If this is not possible, specimens should be
hematogenous spread of bacteria may lead to septicaemia, collected with charcoal coated swabs and transported to
endocarditis or acute purulent arthritis. the laboratory in Stuart's transport medium. Another
transport medium is Amies medium with charcoal.
2. Ophthalmia Neonatorum Several commercial transport systems such as JAMBEC
It is a nonvenereal gonococcal conjunct1v1tJs m the plates (James E. Martin Biological Environmental
newborn through infected birth canal. Chamber), Bio-Bag, Gono-Pak, and Transgrow, contain
selective media and a carbon dioxide atmosphere to provide
3. Gonococcal Vulvovaginitis optimal conditions for the specimens during transport.
In adult women the vagina usually is resistant to gonococcal
infection because of the acidic pH of the vaginal 3. Direct Microscopy
secretions, but vulvovaginitis can occur in prepubertal girls. Gram staining of smear provides a presumptive evidence
of gonorrhoea in men. Gram negative intracellular
4. Other Infections diplococci are found in smear of at least 95% cases of
Sometimes the disease may involve rectum or oropharynx acute gonococcal urethritis in males. In females, diagnosis
following rectal intercourse or by orogenital contact of gonorrhoea by smear examination is unreliable as some
respectively. Involvement of oropharynx may lead to of the normal genital flora have similar morphology.
gonococcal pharyngitis. Fluorescent antibody tests are more sensitive and specific
methods for diagnosis by microscopy especially in females.
G. Epidemiology
Gonorrhoea is an exclusively human disease. The only 4. Detection of Antigen or Nucleic acid
source of infection is a human carrier or less often a The gonococcal antigens can be detected by ELISA in
patient. Asymptomatic infection in woman makes them clinical specimens. Nucleic acid can be directly detected
a reservoir to spread infection among their male contacts. in urethral discharge using DNA probe.
The mode of infection is almost exclusively venereal.
Ophthalmia neonatorum is a non-venereal gonococcal 5. Culture
infection. The specimens should be inoculated directly on preheated
plates immediately on collection. Chocolate agar is used
H. Laboratory Diagnosis for culture of the specimens and incubated at 35°C to
The diagnosis is readily made in acute stage, as urethral 36°C under 5- 10% CO 2 for 48 hours. In chronic cases,
discharge contains large number of gonococci. It is where mixed infection is usual and in lesions like proctitis,
difficult to detect gonococci from chronic cases or from selective medium such as Thayer-Martin medium
patients with metastatic lesions such as arthritis. or modified Thayer-Martin medium is used. Typical
translucent colonies appear on culture medium.
1. Specimens
Urethral discharge and cervical discharge (in females) are 6. Colony Morphology and Gram Staining
collected in acute urethritis. The meatus is first cleaned On solid medium, colonies are small, round, grey,
with a gauze soaked in saline. A sample of the urethral translucent, convex with finely granular surface. They
discharge is then collected with a platinum loop or by a are easily emulsifiable. Smear is made from the colony
sterile swab. Calcium alginate and some cotton swabs have and Gram staining is done. Gonococci are Gram negative
been shown to be inhibitory to gonococci, so Dacron or cocci arranged in pairs (diplococci) with adjacent sides
Rayon swabs are preferred. In women, cervical swab is concave (pear or bean shaped).
collected in addition to urethral discharge. High vaginal
swabs are not satisfactory. 7. Superoxol Test
In chronic urethritis, urethral discharge is observed Superoxol test is helpful in rapid presumptive identification
only in few cases. In these cases, some exudate obtained of N gonorrhoeae. Superoxol is 30% hydrogen peroxide
after prostatic massage or morning drop of secretion may (not the 3% solution used routinely for catalase test).

Ch 26: Neisseria and Moraxella _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ 207
N gonorrhoeae produce immediate brisk bubbling when uncomplicated gonococcal infections. Coinfection with
some colonies of this bacteria are emulsified with 30% C. trachomatis is common in patients with gonorrhoea
hydrogen peroxide reagent on a glass slide. In contrast, therefore dual therapy is frequently recommended. Dual
both N meningitidis and N lactamica, produce weak, therapy includes ceftriaxone/cefixime (for gonorrhoea)
delayed bubbling. and azithromycin/doxycycline (for C. trachomatis).
8. Biochemical Reactions
They are oxidase positive. They breakdown glucose with J. Prophylaxis
acid only by oxidation. They do not catabolise maltose Control of gonorrhoea consists of early detection of cases,
unlike meningococci. tracing of contacts, health education and other general
measures. Vaccination has no role in prophylaxis.
9. Serology
It may not be possible to isolate gonococci in culture
from some chronic cases or from some patients with III. NON-GONOCOCCAL (NON-SPECIFIC)
metastatic lesions such as arthritis. Serological tests may URETHRITIS
be of value in such cases. Complement fixation test, It is a condition of chronic urethritis where gonococci
immunofluorescence and ELISA tests have been used cannot be demonstrated. It is known as non-gonococcal
to detect antibodies. However, no serological test has urethritis (NGU).
been found useful for routine diagnostic purposes.
A. Causative agents
I. Treatment 1. Bacterial
The organism is sensitive to large doses of penicillin • Chlamydia trachomatis is the most common.
(intramuscular) or doxycycline. • Ureaplasma urealyticum
• Mycoplasma hominis
Two types of penicillin resistance have been observed: • Gardnerella vagina/is
1. Low Level Resistance • Acinetobacter lwoffi
The bacterial strains are sensitive to higher concentrations
of penicillin and are not related to beta-lactamase 2. Viral
production. The resistance is chromosomally determined. Herpes virus
These resistant gonococci are designated as 'chromosomally- Cytomegalovirus
mediated resistant Neisseria gonorrhoeae' (CMRNG).
3. Fungal
2. High Level Resistance Candida albicans
It is due to production of enzyme beta-lactamase by the
bacteria and is plasmid coded. In such cases, the strains 4. Protozoa[
are totally resistant to penicillin. It was first recognised in Trichomonas vagina/is
1976. Since then these penicillinase producing Neisseria
genorrhoeae (PPNG) strains have spread widely. B. Treatment
In penicillin-resistant gonorrhoea, cefotaxime, Tetracycline is effective for both C. trachomatis and
ceftriaxone, ciprofloxacin, tetracycline or spectinomycin Ureaplasma urealyticum infections.
are used.
In 1985, plasmid mediated high level tetracycline
resistance was recognised. These gonococci are named IV. COMMENSAL NEISSERIAE
as 'tetracycline resistant Neisseria gonorrhoeae' (TRNG). This group of bacteria inhabits the normal respiratory
Chromosome mediated spectinomycin resistance has tract. These commensals (N fiavescens, N sicca and N
also been known in gonococci. This is a high level subflava) are differentiated from the pathogenic Neisseriae
resistance due to mutation. In 1991 'quinolone-resistant by following methods.
N gonorrhoeae' (QRNG) was identified. Mechanism of 1. Commensals can grow on ordinary agar not enriched
resistance is mutation. with blood or serum at 22°C.
Currently a single dose of ceftriaxone intramuscular 2. Their primary isolation does not require presence of
or cefixime orally is recommended for treatment of CO 2 .

3. These bacteria produce pigmented (yellow to VI. MORAXELLA LACUNATA

greenish yellow) colonies and ferment a number of (MORAX-AXENFELD BACILLUS)
carbohydrates. This was formerly included in the genus Haemophilus but
as it does not require X or V factor, it has been separated
into the genus Moraxel/a. It was described by Morax
V. MORAXELLA (BRANHAMELLA) (1896) and Axenfeld independently from cases of subacute
CATARRHALIS conjunctivitis. The most important species is M. lacunata.
Neisseria catarrhalis was initially classified as Branhamel/a
catarrhalis but now reclassified as Moraxel/a catarrhalis. A. Morphology
It is part of the normal flora of the upper respiratory It is a Gram negative, short (2- 3 µm) ovoid, non-motile
tract and genital tract. bacillus arranged in pairs.
Gram negative diplococci, 0.6-lµm oval with adjacent They require blood or serum for growth and are strict
sides flattened. aerobes. On loeffier's serum slope, the colonies form pit
or lacunae (hence the name lacunata).
B. Culture
It grows on ordinary medium like nutrient agar. C. Biochemical Reactions
It does not ferment sugars and is oxidase and catalase
C. Biochemical Reactions positive.
It does not ferment any carbohydrate but hydrolyses
tributyrin-a test for its identification. It is catalase and D. Pathogenesis
oxidase positive. Moraxella are parasitic and their habitat includes mucosa
of upper respiratory tract, genital tract and conjunctiva.
D. Pathogenesis M. lacunata causes catarrhal conjunctivitis (angular
It causes lower respiratory tract infections, especially in conjunctivitis). A few species of Moraxella occasionally
adults with chronic obstructive airway disease. It has also give rise to septic arthritis, meningitis and endocarditis.
been isolated in cases of otitis media, less commonly in
meningitis, endocarditis and sinusitis. Some strains of E. Treatment
B. catarrhalis produce beta-lactamase and are resistant M. lacunata is very sensitive to zinc salts. They are
to penicillin. sensitive to penicillin and most other antibiotics.
KEY POINTS
1. The genus Neisseria consists of Gram negative, aerobic, oxidase positive, non-motile diplococci.
2. The two pathogenic species are N meningitidis (causing pyogenic meningitis) and N gonorrhoeae (causing
gonorrhoea).
3. They grow on media enriched with blood or serum. Chocolate agar is the commonly used medium. Modified Thayer-
Martin medium and New-York City medium are selective media used for their isolation.
4. N meningitidis is strict aerobe and cannot grow anaerobically. A moist environment with 5-10% CO 2 is must for
growth to occur.
5. Both N meningitidis and N gonorrhoeae are catalase and oxidase positive. N meningitidis ferments both glucose and
maltose but N gonorrhoeae only ferments glucose.
6. N meningitidis has been divided in 13 serogroups (A, B, C, D, X, Y, Z, W-135, 29E, H, I, Kand L) on the basis of
immunological specificity of the capsular polysaccharide. Serogroups A, B, C, 29E, W-135 and Y are responsible for
the large majority of meningitis.
7. M eningococcal vaccines prepared from polysaccharides of serogroups A, C, W-135 and Y are available. Single dose
is given intramuscularly.
8. N gonorrhoeae is pear or bean shaped. It is more difficult to grow than N meningitidis. It is aerobic but may grow
anaerobically as well. It can grow on all those culture media used for growth of N meningitidis.

Ch 26: Neisseria and Moraxella _ _ __ __ _ _ _ _ _ _ __ _ __ _ _ _ _ _ __ __ __ 209
9. Penicillin resistance in N.gonorrhoeae may be due to production of enzyme beta-lactamase (penicillinase). It is plasmid
coded. These strains are named penicillinase producing Neisseria gonorrhoeae (PPNG).
IO. Non-gonococcal urethritis (NGU) is a condition of chronic urethritis where gonococci cannot be demonstrated.
11. Non-pathogenic species of Neisseria include N. flavescens, N. sicca and N. subflava. They are commensals and inhabit
the normal respiratory tract.
YOU MUST KNOW

1. Morphology, culture characteristics, biochemical reactions and antigenic structure of Neisseria meningitidis.
2. Laboratory diagnosis of meningococcal meningitis.
3. Meningococcal vaccines.
4. Morphology, culture characteristics, biochemical reactions of Neisseria gonorrhoeae.
5. Laboratory diagnosis of gonorrhoea.
6. Penicillinase producing N. gonorrhoeae strains (PPNG).
7. Non-gonococcal urethritis (NGU).
8. Moraxella catarrhalis.
STUDY QUESTIONS
1. Describe the laboratory diagnosis of meningococcal meningitis,
2. Discuss the laboratory diagnosis of gonorrhoea.
(a) Non-gonococcal urethritis (NGU) (b) Antigenic structure of N.gonorrhoeae
(c) Moraxella catarrhalis.
MULT PLE CHOICE QUESTIONS (MCQs)

1. All of the following bacteria are non-pathogenic except:
(a) Neisseriaflavescens (b) N. sicca (c) N. subflava (d) M oraxella catarrhalis
2. Which of the following is a selective medium for isolation of Neisseria gonorrhoeae?
(a) Modified Thayer-Martin medium (b) Blood agar
(c) MacConkey's agar (c) Potassium tellurite blood agar
3. All of the following bacteria are oxidase positive except:
(a) Neisseria gonorrhoeae (b) Neisseria meningitidis (c) Vibrio cholerae (d) Enterobacter
4. How many serogroups of Neisseria meningitidis are known?
(a) Nine (b) Twelve (c) Thirteen (d) Fifteen
5. Which of the following rapid tests may be used to detect meningococcal antigen?
(a) Countercurrent immunoelectropheresis (b) Coagglutination test
(c) Latex agglutination test (d) All of the above
6. Waterhouse-Friderichsen syndrome is caused by:
(a) Neisseria meningitidis (b) Leptospira (c) Streptococcus pyogenes (d) Borrelia
7. The causative agent/s of non-gonococcal urethritis is/are:
(a) Chlamydia trachomatis (b) Ureaplasma urealytium
(c) Mycoplasma hominis (d) All of the above
Answers (MCQs):
1. (d) 2. (a) 3. (d) 4. (c) 5. (d)
6. (a) 7. (d)
---- • ++ - - - -
Chapter 27
CORYNEBACTERIUM
I. Corynebacterium diphtheriae
A. Morphology B. Cultural Characteristics
C. Biochemical Reactions D. Toxin
E. Resistance F. Antigenic Structure
G. Bacteriophage Typing H. Pathogenesis
I. Laboratory Diagnosis J. Prophylaxis
K. Schick Test L. Treatment
II. Other Pathogenic Corynebacteria
Ill. Diphtheroids
Corynebacteria are Gram positive, non-acid-fast, non- shaped due to the presence of metachromatic granules at
sporing, non-motile bacilli with average size 3 µm x 0.3 one or both ends. With Loeffier's methylene blue stain,
µm. They frequently show club-shaped swellings (coryne granules take up a bluish purple colour and hence they
means club shaped). The most important member of the are named metachromatic granules. These granules are
genus is C. diphtheriae which causes diphtheria in humans also called volutin or Babes-Ernst granules. These are
but other species are increasingly assuming a pathogenic composed of polymetaphosphates and represent energy
role particularly in immunocompromised individuals. storage depots. The bacilli are usually seen in angular
Bretonneau (1826) called the disease 'diphtherite' because fashion resembling the letters V or L. This has been
of leathery pseudo-membrane produced in the disease called Chinese letter or cuneiform arrangement. This
(diphtheros meaning leather). The diphtheria bacillus was typical arrangement is due to incomplete separation of
first described by Klebs (1883), but was first cultivated by the daughter cells after binary fission (Fig. 27 .1 ).
Loeffler (1884). It is also known as Klebs-Loeffier bacillus. Special stains like Albert (malachite green and
Roux and Yersin (1888) discovered diphtheria exotoxin. toludine blue), Neisser or polychrome methylene blue
The antitoxin was discovered by van-Behring (1890) who are used for staining the bacilli. The bacilli look green
was awarded the Nobel Prize for this work. and metachromatic granules appear bluish black when
Albert stain is used. They are non-capsulated, non-acid-
I. CORYNEBACTERIUM DIPHIBERIAE fast and non-motile.
A. Morphology
These are thin, slender, Gram positive bacilli (but tend to B. Cultural Characteristics
be decolourised easily) and measure approximately 3- 6 C. diphtheriae are grown best on media enriched with
µm x 0.6-0.8 µm. They are pleomorphic. They are club- blood, serum or egg. Growth is scanty on ordinary
210
Ch 27: Corynebacterium _ _ _ __ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __ _ _ __ _ _ __ _ 211
most other bacteria and thus acting as a selective agent.

The organisms grow slowly on this medium and form
grey or black colonies due to reduction of potassium
tellurite to tellurium (Fig. 27.2) . The colonies may take
two days to appear on this medium. Based on colony
morphology on tellurite medium and other properties,
three main biotypes of C. diphtheriae- gravis, intermedius
and mitis are distinguished (Table 27.1).
Fig. 27.1 Morphology of C. diphtheriae
media. They are aerobic and facultatively anaerobic. The

optimum temperature for growth is 37°C (range 15-40°C)
and optimum pH 7 .2. The following are the usual media
employed for cultivation of diphtheria bacillus.
1. Hiss's Serum Water

Fig. 27.2 C.diphtheriae on potassium tellurite blood agar
This is a liquid medium containing serum. Growth 1s
seen as a turbidity and pellicle formation.
Tinsdale agar, which contains sheep's blood, bovine
2. Loeffler's Serum Slope serum, cystine and potassium tellurite, is one of the
Diphtheria bacilli grow on this medium very rapidly. selective media used for growing C. diphtheriae. Black
Colonies appear after 6 to 8 hours of incubation, long or brown colonies are formed on this medium. A brown
before other bacteria grow. The colonies are small, halo surrounding the colony is a useful differentiating
circular, white or creamy and glistening. feature, because only C. diphtheriae, C. ulcerans and C.
pseudotuberculosis produce a brown halo. On this medium
3. Tellurite Blood Agar Medium staphylococci can also grow as black colonies but without
It contains potassium tellurite (0.04%) which inhibits haloes around the colonies.
Table 27.1 Type Differentiation of C. diphtheriae Strains

Character Gravis Intermedius Mitis
1. Morphology Short rods, few metachromatic Long, poor granulation, barred forms Long rods, curved, prominent
granules, uniform staining with clubbed ends, pleomorphic granules, pleomorphic
2. Colony on tellurite In 18 hours, 1- 2 mm, grey, raised In 18 hours, 1 mm in size. In 48 hours, Size variable, shiny black. In
blood agar centre. In 48-72 hours, flat colony no change in size, dull granular centre, 48-72 hours, flat colony with
with raised centre and crenated more glistening periphery and a lighter central elevation-poached
edge with radial striation-daisy ring near the edge- frog's egg colony egg colony
head colony
3. Consistency of Brittle, not easily picked out or Intermediate between gravis and rnitis Soft, buttery, easily
colonies emulsifiable, like cold margarine emulsifiable
4. Haemolysis Variable Non-haemolytic Usually haemolytic
5. Growth in broth Surface pellicle Turbidity in 24 hours, clearing in 48 Diffuse turbidity
hours, with fine granular sediment
6. Biochemical tests
Glucose Acid without gas Acid without gas Acid without gas
Glycogen Acid without gas Negative Negative
Starch Acid without gas Negative Negative
7. Virulence Severe Moderate Mild

212 _ _ _ _ _ _ _ _ __ _ _ _ _ _ _ __ _ __ _ _ _ _ _ _ Unit Ill: Systemic Bacteriology
C. Biochemical Reactions is thus protective. The toxin is heat labile. It is extremely

They ferment glucose and maltose with the production potent (0.0001 mg kills a guinea pig of 250 gm weight).
of acid but without gas. They do not ferment lactose, It is converted into toxoid by heat (at 37°C for 4 to 6
mannitol or sucrose. The gravis biotype ferments starch and weeks), treatment with 0.2 to 0.4% formalin or by acidic
glycogen while intermedius and mitis have no such action pH. The toxoid is a toxin that has lost its toxicity but
(Table 27.1) . It is necessary to employ Hiss's serum water has retained the antigenicity. It is capable of producing
for testing fermentation of sugars. Proteolytic activity is antitoxin.
absent. C. diphtheriae reduces nitrates to nitrites. They Toxin production is also influenced by the concentration
do not hydrolyse urea or form phosphatase. Gelatin is of iron. 0.1 mg of iron per litre of culture medium is
not liquefied. optimum level for toxin production, while a concentration
of 0.5 mg or more per litre inhibits the toxin production.
PYRAZINAMIDASE (PYZ)TEST Reason for this is not known. The repressor of the tox
Pyrazinamide is converted into pyrazinoic acid by the gene appears to be an iron containing protein. When
organisms which produce pyrazinamidase (PYZ). This test level of iron is more, suppressor is formed which inhibits
is helpful to distinguish 'C. diphtheriae' (PYZ-negative) toxin production.
from other corynebacterium species (mostly PYZ-
positive). However, C. ulcerans and C. pseudotuberculosis 2. Mode of Action
are also PYZ-negative but they are urease test positive It acts by inhibiting protein synthesis. It inhibits polypeptide
which differentiate them from C. diphtheriae (urease chain elongation in the presence of nicotinamide adenosine
negative). dinucleotide (NAD) by inactivating the elongation factor,
EF-2. Fragment A splits NAD to form nicotinamide and
D. Toxin adenosine diphosphoribose (ADPR). ADPR binds to
The pathogenicity is due to production of a very powerful and inactivates EF-2 which is an enzyme required for
exotoxin by virulent strains of diphtheria bacilli. About elongation of polypeptide chain.
90- 95% gravis and intermedius strains are toxigenic, NAD+ + EF-2 ADPR-EF2 + Nicotinamide + H+
while only 80-85% of mitis strains are so. Avirulent Active Inactive
strains are frequent among convalescents, carriers and
contacts. The strain most widely used for toxin production E. Resistance
is Park- Williams 8 strain which has been described as a It readily dies when exposed to a temperature of 58°C
mitis strain (Topley and Wilson) and as an intermedius for 10 minutes or 100°C for one minute. It is resistant to
(Cruickshank) strain. drying. The organism remains virulent in blankets and
The toxigenicity of the diphtheria bacillus depends on floor dust for five weeks. It is quite susceptible to penicillin,
the presence of a tox gene which can be transferred from erythromycin and broad spectrum antibiotics.
one bacterium to another by lysogenic bacteriophages, of
which beta phage is the most important. Non-toxigenic F. Antigenic Structure
strains may be rendered toxigenic by infecting them C. diphtheriae possesses heat labile type-specific protein
with beta phage. This process is known as lysogenic or antigens (K antigen) and heat stable polysaccharide
phage conversion. The toxigenicity remains as long as 'O' antigen. By agglutination, gravis strain has 13,
the bacteria is lysogenic. When the bacteria is freed of intermedius 4 and mitis 40 types. A group specific heat
its phage, as by growing it in the presence of antiphage stable polysaccharide O antigen is shared by all the
serum, it loses the toxigenicity and becomes non-toxigenic strains.
strain. In addition to beta phage, other phages (a , P, L,
y) are also known to carry tox genes. G. Bacteriophage Typing
About 15 bacteriophage types have been described,
1. Properties type I to III strains are mitis, IV to VI intermedius, VII
The diphtheria toxin is a protein and has a molecular avirulent gravis and the remainder virulent gravis strains.
weight of about 62,000. It consists of two fragments A and The only other corynebacteria which are found susceptible
B, of molecular weights 24,000 and 38,000 respectively. to the C. diphtheriae typing phages are C. ulcerans and
Both fragments are required for toxicity. Fragment A C. pseudo tuberculosis. Bacteriocin (diphthericin) typing
has all the enzymatic activity whereas fragment B is has also been described. Other typing methods include
responsible for binding the toxin to the cells. The antibody DNA restriction patterns, bacterial polypeptide analysis
to fragment B prevents the binding of toxin to cells and and hybridisation techniques.

Ch 27: Corynebacterium _ _ _ __ _ _ __ _ __ _ _ _ _ _ __ _ _ __ _ _ _ __ _ __ 213
H. Pathogenesis I. Laboratory Diagnosis

Diphtheria is most commonly seen in children of 2 Laboratory confirmation of diphtheria is necessary for
to 10 years. Infection is confined to humans only. The control measures and epidemiological studies, but not
incubation period is 3 to 4 days, but may on occasion be for the treatment of cases. Specific treatment should
as short as one day. Infection occurs by way of droplet be started immediately after clinical diagnosis without
spread. Diphtheria may be of the following clinical types waiting for laboratory reports. Any delay may be fatal.
depending upon the site of infection. Laboratory diagnosis consists of isolation of organism
1. Faucial and demonstration of its toxicity by virulence tests.
2. Laryngeal
3. Nasal 1. Isolation of Organism
4. Conjunctiva! (i) Collection of specimen
5. Otitic Two swabs from the lesions (throat, nose, larynx, ear,
6. Vulvovaginal conjunctiva, vagina, or skin) are collected. One swab is
7. Cutaneous mainly around mouth and nose. used for smear examination and other for culture.
Faucial diphtheria is the commonest type. The toxin Local lesion is usually in the throat. Swabs are
has both local as well as systemic effects. collected prior to start of antibiotics and application of
antiseptics in form of gargles. The swabs are rubbed over
1. Local effects the affected area and pseudomembrane, if formed, should
The bacilli remain confined to the site of entry (usually be scraped with swab. If there is no definite localised lesion,
upper respiratory tract), where they multiply and start the swabs should be rubbed over tonsils and the posterior
producing toxin. The toxin causes local necrotic changes pharyngeal wall without touching the mouth parts.
along with superficial inflammatory reaction. The necrosed
epithelium together with fibrinous exudate, leucocytes, (ii) Direct microscopy
erythrocytes and bacteria, constitute the pseudomembrane, Smears are stained with both Gram and Albert stain.
which is a characteristic feature of diptheritic infection. Diphtheria bacilli show beaded slender green rods
The mechanical complications of diphtheria are due to in typical Chinese letter pattern on Albert's staining.
the pseudomembrane, whereas the systemic effects are However, they cannot be confidently differentiated from
due to toxin. some commensal corynebacteria normally present in the
throat. Hence it is reported as "the organisms resembling
2. Systemic effects C. diphtheriae" seen in direct smear examination. Gram
The diphtheria toxin diffuses into the blood stream and staining is done to identify Vincent's spirochaetes and
causes toxaemia. The toxin has got affinity for cardiac fusiform bacilli (other causes of sore throat).
muscle, adrenals and nerve endings. It acts systemically
on the cells of these tissues. The bacilli themselves do (iii) Culture
not play any part in systemic effects because they neither The swabs are inoculated on the following culture
penetrate into the tissues nor pass into blood stream media:
producing bacteraemia.
(a) Loeffier's serum slope
Growth appears within 6-8 hours on this medium.
Complications Subculture from Loeffier's serum slope is made on
1. Local tellurite blood agar and plate is incubated at 37°C for
The pseudomembrane may extend to the larynx which 48 hours.
may lead to laryngeal obstruction, asphyxia and death.
(b) Tellurite blood agar
2. Systemic These plates have to be incubated at 37°C for at least 48
(a) Diphtheritic myocarditis which may terminate m hours before declaring these as negative, as growth may
heart failure and death. sometimes be delayed.
(b) Polyneuropathy and post-diphtheritic paralysis of
palatine and ciliary muscles. (c) Blood agar
(c) Degenerative changes in adrenals, kidney and liver It is useful for differentiating streptococcal or
may occur. staphylococcal pharyngitis, which may simulate diphtheria.

214 _ _ _ _ _ _ __ __ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Unit Ill: Systemic Bacteriology
It may also help to differentiate mitis biotype which shows • haemorrhagic oedema and, often, necrosis at the site
haemolysis. of inoculation,
• swollen and congested lymph nodes,
(iv) Colony morphology and staining • congested abdominal viscera,
On Loeffier's serum slope, the colonies are small, circular, • peritoneal and pleural exudate.
white or creamy. Diphtheria bacilli grow as black or grey
coloured colonies on tellurite blood agar. (b) Intracutaneous test
Smears are prepared from suspected growth from Two guinea pigs (or rabbits) are injected intracutaneously
various media. These smears are stained with Albert and with 0.1 ml emulsion from growth on Loeffier's serum
Gram stain to confirm the morphology of C. diphtheriae. slope, one of these animals is protected with 500 units
Albert staining shows green bacilli with bluish black antitoxin the previous day (control) and the other is given
metachromatic granules. Gram staining reveals Gram 50 units of antitoxin intraperitoneally four hours after
positive bacilli that tend to be decolourised easily. the skin test, in order to prevent death. If the strain is
Diphtheroids cannot be decolourised as easily as C. toxigenic (virulent), the inflammatory reaction at the site
diphtheriae. of injection, progresses to necrosis in 48 to 72 hours in the
test animal but there is no change in the control animal.
(v) Biochemical reactions An advantage in the intracutaneous test is that 8 to 10
Hiss's serum water is used for testing fermentation of strains can be tested at a time on a pair of animals and
carbohydrates. Biochemical reactions of C. diphtheriae the animals do not die.
are as follows:
IN VITRO TESTS
Glucose Lactose Mannitol Sucrose (a) Elek's gel precipitation test
A This is an immunodiffusion test described by Elek (1949).
Maltose N0 3 reduction Indole Urease A rectangular strip of filter paper soaked in diphtheria
+ + antitoxin (1000 units per ml) is placed on the surface of
phosphatase Catalase Oxidase Glycogen* a 20% horse serum agar plate while the medium is still
+ + fluid. When the agar solidifies, the test strain is streaked
Starch* at right angle to the filter paper strip. The positive and
+ negative controls are also put up. The plate is incubated
*Only Gravis biotype is positive at 37°C for 24 to 48 hours. The toxin produced by the
bacterial growth diffuses in the agar and produces a line
2. Virulence Tests of precipitation where it meets the antitoxin at optimum
These tests demonstrate the production of exotoxin by concentration (Fig. 27.3) . Non-toxigenic strains will not
bacteria isolated on culture. Virulence testing may be produce any precipitation line.
done by in vivo or in vitro methods.
(b) Tissue culture test
IN VIVO TESTS This is done by incorporating the test strains into the agar
Guinea pigs and rabbits are susceptible to toxin produced overlay of cell culture monolayers. The toxin produced
by C. diphtheriae. Two types of test are used viz. diffuses into the underlying cells and kills them.
subcutaneous and intracutaneous.
(c) Other tests
(a) Subcutaneous test Enzyme linked immunosorbent assays (ELISA) and
The growth from an overnight culture on Loeffier's serum immunochromatographic strip assays are also available
slope is emulsified in 2- 5 ml broth and 0.8 ml of this for the detection of toxin. These are rapid tests.
emulsion is injected subcutaneously into two guinea pigs,
one of which has received an intramuscular injection of (d) Polymerase chain reaction (PCR)
500 units of diphtheria antitoxin 18- 24 hours previously C. diphtheriae tox gene can be detected by PCR. This test
(this protected animal acts as a control). If the strain is can also be applied directly to clinical specimen.
virulent, the unprotected animal will die within 2 to 3
days with evidence of haemorrhage in the adrenal glands J. Prophylaxis
which is the pathognomonic feature. Other features which 1. Active Immunisation
can be observed at autopsy are as follows: One attack of diphtheria provides life long immunity.

Ch 27: Corynebacterium _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __ __ _ _ _ _ _ _ 215
Fig. 27.3 Elek's gel precipitation test
For active immunisation, alum precipitated toxoid (APT), 1. Method

purified alum precipitated toxoid (PAPT) and toxoid A test dose of 0.2 ml diphtheria toxin containing 1/50
antitoxin floccules (TAP) are used. Children are generally minimum lethal dose (MLD) is injected intradermally on
immunised with formol toxoid. one forearm and a similar amount of inactivated toxin
Active immunisation is started at 6 weeks of age by (inactivated at 70°C for 30 minutes) is injected on another
toxoid in combination with tetanus toxoid and pertussis forearm. MLD is defined as the least amount of toxin
vaccine (DPT, Triple vaccine). Three doses of 0.5 ml each required to kill a guinea pig weighing 250 gm within 96
are given by intramuscular route at an interval of 4-6 hours by subcutaneous inoculation.
weeks. Booster doses of DPT of are given at 18 months
and at 5 years. 2. Results
Results are read after 1, 4 and 7 days. There may be four
2. Passive Immunisation types of reaction.
This is an emergency measure when susceptible persons
are exposed to infection, as when a case of diptheria is (i) Positive reaction
admitted to paediatric wards. In such cases 500-1000 units An area of swelling and erythema appears at the site of
of antitoxin (antidiphtheric serum, ADS) is administered injection of toxin after 24-48 hours, reaching maximum
subcutaneously. As this is a horse serum, precautions in 4-7 days, when it measures 1- 5 cm and then fading.
should be observed against hypersensitivity reactions. The control forearm injected with heated toxin will show
no reaction. A positive test signifies that the person is
3. Combined Immunisation susceptible to diphtheria due to either absence or lack
All persons receiving ADS as prophylactic measure should of adequate amount of circulating antitoxin.
receive combined immunisation. An alum containing
preparation like APT or PAPT should be preferred for (ii) Negative reaction
combined immunisation as the plain formol toxoid (FT) There is absence of any reaction in either forearm (control
response is unsatisfactory when given with antitoxin and test). It indicates that the toxin has been neutralised
(ADS). by sufficient amount of antitoxin present in the blood
and that the person is immune to diphtheria.
K. Schick Test
This is an intradermal test to demonstrate circulating (iii) Pseudoreaction
diphtheria antitoxin. This antitoxin may be present There is erythema occurring in both forearms within 6-24
either due to previous infection (clinical or subclinical) hours and disappearing completely from both forearms
or immunisation. This test demonstrates immunity or within four days. This indicates that the individual is
susceptibility of a person against diphtheria and is one immune to diphtheria and he is also hypersensitive to
example of neutralisation test (toxin-antitoxin). the components of diphtheria bacilli.

216 _ _ __ _ _ __ _ __ __ _ _ __ _ _ _ _ _ _ _ _ _ _ _ _ Unit Ill: Systemic Bacteriology
(iv) Combined reaction doubtful agent for mastitis. Many of them cause infections
The initial reaction is similar to that of pseudoreaction. in immunocompromised patients (Table 27.2). C. jeikeium
Erythema disappears in the control forearm within four is an important pathogen in neutropenic patients and
days but it progresses in the test forearm to a typical those receiving antibiotics. It is usually multiresistant and
positive reaction. It indicates that the individual is responds only to vancomycin.
susceptible to diphtheria and hypersensitive to bacillus.
It is necessary to immunise such persons but the vaccine Table 27.2 Medically Important Non-Diphtheria
may likely induce reaction. Corynebacteria and Diseases Caused by these Organisms
Disease Organisms
L. Treatment
C. diphtheriae is sensitive to penicillin, erythromycin 1. Acute pharyngitis C. ulcerans
and other antibiotics. Diphtheria patients are given 2. Cutaneous infections C. jeikeium
a course of penicillin, to stop cases from becoming
3. Lymphadenitis C. pseudotuberculosis
carriers. Erythromycin is more active in the treatment
of carriers. 4. Infective endocarditis C. jeikeium
The antibiotics are of little value as these cannot C. xerosis
inactivate the toxin already present in patient's body. C. bovis
When a case is suspected as diphtheria, antidiphtheric 5. Shunt or cannula infections C. jeikeium
serum should be given immediately as the mortality C. xerosis
rate increases with delay in starting antitoxin treatment. C. bovis
In moderate cases, the dosage recommended is 20,000
units intramuscularly and in severely ill cases 50,000 C. minutissimum and C. tenuis are associated with
to 100,000 units are injected, half the dose being given superficial skin infection.
intravenously. Some bacteria which were initially in genus
Corynebacteria include Arachnobacterium haemolyticum
(formerly C. haemolyticum) and Rhodococcus equi
II. OTHER PATHOGENIC (formerly C. equi). A . haemolyticum may cause pharyngitis,
CORYNEBACTERIA peritonsillar abscess, cervical lymphadenitis, osteomyelitis
C. ulcerans is a bacillus related to C. diphtheriae, which and brain abscess. R. equi is found in soil. Human
can cause localised ulcerations in throat, clinically infection is rare,although an increased incidence in
resembling diphtheria. It resembles the gravis type but immunocompromised patients, especially AIDS patients,
it liquefies gelatin, ferments trehalose slowly and does has been reported.
not reduce nitrate. It is PYZ-negative and urease positive.
It produces two types of toxins, one is identical with
diphtheria toxin and the other resembling the toxin of III. DIPHTHEROIDS
C. pseudotuberculosis. It is pathogenic to guinea pigs, Commensal corynebacteria are normally present in the
the lesions produced are similar to those produced by throat, skin, conjunctiva and other areas. These may
C. diphtheriae . It causes infections in cows and human sometimes be mistaken for C. diphtheriae and are called
infections may occur through cow's milk. Some consider diphtheroids (Table 27.3). The common diptheroids
C. ulcerans as a variant of diphtheria bacillus. Diphtheria include C. x erosis (found in the conjunctiva! sac) and
antitoxin is protective. C. pseudodiphtheriticum ( C. hofmannii) which is found
C. pseudo tuberculosis (Preisz-Nocard bacillus) also in the throat. In general, diphtheroids possess few or no
referred to as C. ovis causes pseudotuberculosis in sheep metachromatic granules and are arranged in palisades
and suppurative lymphadenitis in horses. It is PYZ- (parallel rows) rather than in Chinese letter pattern.
negative and urease positive. It produces a toxin which Some diphtheroids are indistinguishable from diphtheria
differs from that of C. diphtheriae. The toxin kills guinea bacilli microscopically and require to be differentiated by
pig in 24 hours. C. bovis, commensal of cow's uddar, is a biochemical tests and more reliably by virulence tests.

Ch 27: Corynebacterium _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ 217
Table 27.3 Distinguishing Features of C diphtheriae and Diphtheroids

Feature C. diphtheriae Diphtheroids
1. Morphology (i) Weakly Gram positive and thin bacilli Strongly Gram positive, short and thick bacilli
(ii) Metachromatic granules present Few or absent
(iii) Chinese letter pattern Pallisade arrangement
(iv) Pleomorphism present Very little pleomorphism present
2. Culture Grow on special enriched media Can grow on ordinary media
3. Biochemical tests Ferments glucose only and does not ferment sucrose Ferments both glucose and sucrose
4. Toxin production Toxic Non-toxic
KEY POINTS
1. Corynebacterium diphtheriae are bacilli usually seen in angular fashion resembling the letters V or L. This has been
called Chinese letter. These bacilli contain metachromatic granules.
2. Special stains like Albert, Neisser or polychrome methy lene blue are used for staining the bacilli. The bacilli look green
and metachromatic granules appear bluish black when Albert stain is used.
3. C. diphtheriae are grown best on media enriched with blood, serum or egg. Loef.fier's serum slope (LSS) and potassium
tellurite blood agar medium are employed for cultivation of this bacillus.
4. Potassium tellurite blood agar is a selective medium. The organisms grow slowly on this medium and form grey or
black colonies due to reduction of tellurite to tellurium.
5. C. diphtheriae causes diphtheria. The pathogenicity is due to production of a very powerful exotoxin. The toxigenicity
of the diphtheria bacillus depends on the presence of a beta phage which can be transferred from one bacterium to
another by lysogenic conversion. Diphtheria toxin acts by inhibiting protein synthesis. It inhibits polypeptide chain
elongation by inactivating the elongation factor, EF-2.
6. Virulence tests demonstrate the production of exotoxin by bacteria isolated on culture. These may be done by in vivo
or in vitro methods. Guinea pigs and rabbits are susceptible to toxin produced by C. diphtheriae. Two types of tests
are used i.e. subcutaneous and intracutaneous. In vitro methods include Elek's gel precipitation test and tissue culture
test.
7. Triple vaccine (DPT) is used for active immunisation. This vaccine contains diphtheria toxoid, tetanus toxoid and
pertussis vaccine.
8. Commensal corynebacteria are normally present in the throat, skin, conjunctiva and other areas. These may sometimes
be mistaken for C. diphtheriae and are called diphtheroids. In general, diphtheroids possess few or no metachromatic
granules and are arranged in pallisades (parallel rows) rather than in Chinese letter pattern. C. xerosis and C.
pseudodiphtheriticum are two examples of diphtheroids.
YOU MUST KNOW

1. Morphology, culture characteristics and biochemical reactions of Corynebacterium diphtheriae.
2. Differences between three biotypes gravis, intermedius and mitis of C. diphtheriae.
3. Laboratory diagnosis of diphtheria.
4. Virulence tests for demonstration of diphtheria toxin.
5. DPT and antidiphtheric serum (ADS).
6. Schick test.
7. Differences between C. diphtheriae and diphtheroids.

218 _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __ _ _ Unit Ill: Systemic Bacteriology
STUDY QUESTIONS
1. Name the different species of genus Corynebacterium. Discuss in detail the laboratory diagnosis of diphtheria.
(a) Diphtheria toxin
(b) Schick test
(c) Corynebacteria other than C.diphtheriae
(d) Diphtheroids.
(e) Toxigenicity tests of C. diphtheriae.

1. Chinese letter arrangement of bacilli is a characteristic feature of:
(a) Mycobacterium tuberculosis (b) M. leprae
(c) Corynebacterium diphtheriae (d) Bacillus cereus
2. Babes-Ernst granules are present in:
(a) Mycobacterium tuberculosis (b) M. laprae
(c) Corynebacterium diphtheriae (d) Bacillus cereus
3. What is the colour of colonies of C. diphtheriae on tellurite blood agar medium?
(a) White (b) Golden yellow
(c) Grey to black (d) Cream
4. Which is the selective medium used for isolation of C. diphtheriae?
(a) Tellurite blood agar (b) Loeffier's serum slope
(c) Lowenstein-Jensen medium (d) Chocolate agar
5. Which biotype of Corynebacterium diphtheriae ferments starch and glycogen?
(a) Gravis (b) Intermedius
(c) Mitis (d) None of the above
6. Diphtheria toxin has affinity for the following tissues:
(a) Cardiac muscle (b) Adrenals
(c) Nerve endings (d) All of the above
7. Production of diptheria toxin by bacteria can be demonstrated by:
(a) Subcutaneous test (b) Intracutaneous test
(c) Elek's gel precipitation test (d) All of the above
8. The most common site affected by Corynebacterium diphtheriae is:
(a) Upper respiratory tract (b) Conjunctiva
(c) Skin (d) Vulvovaginal
9. Schick test is an example of:
(a) Precipitation (b) Agglutination
(c) Neutralisation (d) None of the above
Answers (MCQs):
1. (c) 2. (c) 3. (c) 4. (a) 5. (a)
6. (d) 7. (d) 8. (a) 9. (c)
---•••---

Chapter 28
BACILLUS
I. Bacill us anthracis
E. Antigens F. Toxin
G. Pathogenesis H. Laboratory Diagnosis
I. Treatment J . Prophylaxis
\
II. Anthracoid Bacilli
Ill. Bacillus Cereus
A. Types of Food Poisoning B. Pathogenesis
C. Diagnosis D. Treatment
E. Control
Sporogenous, rod shaped Gram pos1t1ve bacteria are 1. It was the first pathogenic bacterium to be seen under
divided into two groups, the aerobic Bacillus and the microscope (Po/lender, 1849).
the anaerobic Clostridium. The spores of Bacillus 2. The first communicable disease to be transmitted by
(unlike the genus Clostridium) are of the same width inoculation of infected blood was anthrax (Davaine,
as that of bacteria and do not produce a bulge in 1850).
the bacterial cell. Members of the genus Bacillus are 3. It was the first bacterium to be isolated in pure culture
ubiquitous, present in soil, dust, air and water and are and shown to possess spores (Robert Koch, 1876).
frequently isolated as contaminants in bacteriological 4. B. anthracis was the first bacterium used for the
culture media. B. anthracis, the causative agent of preparation of attenuated vaccine (Louis Pasteur,
anthrax, is the most important pathogen of the group. 1881 ).
B. cereus can cause food poisoning. They are generally
motile with peritrichous flagella except the anthrax A. Morphology
bacillus which is non-motile. Bacillus anthracis is a Gram positive, non-acid-fast,
non-motile, large (3- 10 µm x 1- 1.6 µm), rectangular,
I. BACILLUS ANTHRACIS spore forming bacillus. The spores are refractile, oval
It is the causative agent of anthrax, a disease primarily of and central in position and are of the same width as
animals, and man gets infected secondarily. Considerable the bacillary body so that they do not cause bulging of
historical interest is attached to the anthrax bacillus due vegetative cell (Fig. 28.1) . In infected tissues, the bacilli are
to the following reasons: found singly, in pairs or in short chains, the entire chain
219
220 _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Unit Ill: Systemic Bacteriology
being surrounded by capsule. The capsule is polypeptide

(polymer of d-glutamic acid) in nature.
Fig. 28.2 Medusa head appearance of colony on nutrient agar
Fig. 28.1 Anthrax bacilli

2. Blood Agar Media
The colonies are non-haemolytic, though occasional
Spores are never found in the animal body during strains produce a narrow zone of haemolysis.
life but are formed in culture or in the soil. Sporulation
occurs under unfavourable conditions for growth and 3. Gelatin Stab Culture
is encouraged by distilled water and 2% NaCl. It is Gelatin is liquefied mostly at the top due to aerobic
inhibited by calcium chloride. In contrast, the capsules are environment and gives a characteristic inverted fir tree
formed in the tissues but are usually lacking in ordinary appearance (Fig. 28.3).
conditions of culture. Capsules are formed only if the
media contain added bicarbonate or are incubated under
10- 25 per cent CO 2. Capsule formation may occur in the
absence of CO2, when this bacterium is grown in media
containing serum, albumin, charcoal or starch.
When blood films containing anthrax bacilli are
stained with polychrome methylene blue for 10---12 seconds
and examined under the microscope, an amorphous purple
material is noticed around the bacilli. This represents
the disintegrated capsular material and is characteristic
of the anthrax bacillus. This is known as McFadyean's
reaction and is employed for presumptive diagnosis of
anthrax. When stained with Giemsa stain, the bacillus
stains purple and capsule red. Fig. 28.3 Inverted fir tree appearance in gelatin stab culture
In cultures, the bacilli are arranged end-to-end in
long chains. The ends of the bacilli are truncated or 4. Selective Medium
often concave and somewhat swollen so that a chain of A selective medium (PLET medium) cons1stmg of
bacilli presents a bamboo-stick appearance. heart infusion agar with polymyxin, lysozyme, ethylene
diamine tetracetic acid (EDTA) and thallous acetate has
B. Culture been devised for isolation of B.anthracis from mixtures
B. anthracis is an aerobe and facultative anaerobe, with containing other spore-bearing bacilli.
a temperature range for growth being 12-45°C (optimum
35-37°C). 5. Solid Medium Containing Penicillin
When B. anthracis is grown on a solid medium containing
1. Nutrient Agar Media 0.05- 0.5 units penicillin per ml, in 3- 6 hours the cells
Colonies are round, 2- 3 mm in diameter, raised, opaque become large, spherical, and occurs in chains on the
and greyish white. Under low power of the microscope, surface of the agar, resembling a string of pearls. This
the edge of the colony is found to be composed of long, string of pearls reaction is useful in differentiation of B.
interlacing chains of bacilli, resembling locks of matted anthracis from B. cereus and other aerobic spore bearers.
hair, the so called the Medusa head appearance (Fig. B. anthracis is susceptible to gamma phage. This is another
28.2). characteristic which differentiates it from B. cereus.

Ch 28: Bacillus - - - - - - - -- - -- - - - - -- - - - - - - - - - - - - - - - 221
C. Biochemical Reactions The capsular polypeptide is controlled by a plasmid

Glucose, maltose and sucrose are fermented with (p x 02) and aids virulence by inhibiting phagocytosis.
production of acid only. Catalase is formed and nitrates
are reduced to nitrites. G. Pathogenesis
Anthrax is primarily a disease of animals like cattle
D. Resistance and sheep, and less often of horses and swine. Infection
1. Vegetative form : It gets killed at 60°C in 30 occurs in susceptible animals by ingestion of the spores
minutes. present in the soil. Direct spread of disease from animal
2. Spore form : Spores of many strains resist dry heat to animal is rare. Infected animals discharge large number
at 140°C for 1- 3 hours and boiling for 10 minutes. of bacilli from the mouth, nose and rectum. These bacilli
They remain viable for years in environment such as sporulate in soil and remains as the source of infection.
in soil. Spores are destroyed by: The disease is usually septicaemic but may sometimes
(i) Four per cent potassium permanganate solution be localised.
in 15 minutes.
(ii) Autoclaving (at 121 °C, 15 lbs/in 2 for 15 Human Anthrax
minutes) Humans are occasionally secondarily infected from
They survive in 5% phenol for weeks. Destruction of diseased animals. There are three clinical type of disease
the spores in animal product is achieved by duckering in based on route of infection--cutaneous, pulmonary and
which 2% formaldehyde is used at 30-40°C for 20 minutes intestinal anthrax. All types lead to septicaemia and
for disinfection of wool while 0.25% solution is used at meningitis.
60°C for six hours for animal hair and bristles. Cutaneous anthrax follows entry of the spores
The anthrax bacillus is susceptible to penicillin, through the abraded skin. The face, neck, hands and
streptomycin, erythromycin, tetracycline and back are commonly affected sites. This is commonly
chloramphenicol. found in farmers and persons handling carcasses. The
lesion starts as a papule which becomes vesicle containing
E. Antigens fluid (pustule). The acute inflammatory reaction leads to
These are of three types: congestion and oedema of the area with a central necrotic
l. Capsular antigen: It is present in virulent strain and lesion, which is covered by a black eschar. The name
consists of a polypeptide and acts as hapten. anthrax, meaning coal, comes from the eschar which is
2. Cell wall antigen: polysaccharide black coloured. The lesion is called 'malignant pustule'.
3. Somatic antigen: It is heat labile protein present Cutaneous anthrax may resolve spontaneously, but may
in bacterial body. It stimulates immune system to sometimes lead to fatal septicaemia and meningitis.
produce the antibody which is protective in nature. Pulmonary anthrax occurs due to inhalation of
the dust or filaments of wool from infected animals,
F. Toxin particularly in wool factories. This is also called wool
The virulence of anthrax bacilli depends on two factors- sorter's disease. The infection is characterised by
an extracellular toxin and a capsular polypeptide. The haemorrhagic bronchopneumonia often followed by
toxin consists of three components namely the oedema septicaemia. It has a high fatality rate.
factor (OF or Factor-I), the protective antigen (PA Intestinal anthrax is rare in man and is found in
or Factor-II) and the lethal factor (LF or Factor-III). those who consume improperly cooked infected meat.
These are not toxic individually, but a mixture of all Ingestion of spores causes violent enteritis with bloody
three produces the anthrax tox ic complex. The production diarrhoea with high case fatality.
of toxin is controlled by a · plasmid (p x 01). These Cutaneous, intestinal or pulmonary anthrax, if not
factors have been characterised and cloned. PA binds treated in time, lead to septicaemic anthrax and death
to the receptor on the target cell surface, and provides occurs.
attachment sites for OF or LF. OF is an adenyl cyclase
which is activated only inside the target cells and lead to H. Laboratory Diagnosis
intracellular accumulation of cyclic AMP. This is thought 1. Specimens
to be responsible for the oedema and other effects of Swabs, fluid or pus from pustules; sputum and blood
the toxin. LF causes cell death but the mechanism of from pulmonary and septicaemic anthrax are generally
action is not known. collected. Faecal specimen or peritoneal fluid can be

222 - - - -- - -- - - -- - - -- -- - - - - -- -- - - Unit Ill: Systemic Bacteriology
collected from intestinal anthrax. Laboratory personnel 1. For presumptive identification of anthrax
should take additional protective precautions against (i) Large gram positive bacillus with the general
infection while handling the material. morphology of anthrax bacillus and non-motile.
(ii) Non-haemolytic on blood agar and cultural features
of anthrax bacilus.
2. Microscopy (iii) Catalase positive.
Gram stained smear from the specimen shows often chain
of large Gram positive bacilli. Capsule appears as a clear
halo around the bacterium by India-ink staining. 2. For initial confirmation
Direct fluorescent antibody test (DFA) for capsule (i) Lysis by gamma phage.
specific staining and for polysaccharide (cell wall) antigen (ii) Direct fluorescent antibody test (DFA).
confirms the identification.
3. For further confim ,
McFadyean's Reaction (i) Polymerase chain reaction.
When blood films are stained with the polychrome
methylene blue for 10- 12 seconds and examined under I. Treatment
the microscope, an amorphous purple material is noticed Doxycycline and ciprofloxacin are used for treatment.
around the bacilli. This represents the disintegrated Antibiotics are ineffective once toxin is formed, although
capsular material and is a characteristic of B. anthracis. it may reduce the case fatality rate.
This reaction is employed for the presumptive diagnosis
in animals.
J. Prophylaxis
Prevention is mainly by general methods such as
3. Culture improvement of factory hygiene and proper sterilization
Specimen is inoculated on nutrient agar medium and of animal products like wool. Carcasses of animals
incubated at 37°C for overnight. Medusa head colonies suspected to have anthrax are buried deep in lime.
appear on the medium. Smears made from these colonies Animals are to be protected by active immunisation.
show typical Gram positive spore bearing bacilli. As spore is the common infective form in nature, Sterne
Gelatin stab culture shows inverted fir tree vaccine contains spores of a non-capsulated avirulent
appearance. mutant strain. This avirulent mutant strain is obtained
by loss of two plasmids (p x 01 and p.x.02) which encode
4. Animal Inoculation the anthrax toxin (p.x.0l) and capsular polypeptide
White mouse or guinea pigs are injected with exudate or (p.x.02) production. The animal is protected for a year
culture. Animal dies in 36-48 hours. Smears made from with a single injection of spore vaccine. It is extensively
heart blood and sputum show bacilli. used in animals, however, it is not safe for human use.
Anthrax vaccines have been used in persons
5. Serology (Ascoli's Thermoprecipitin Test) occupationally exposed to anthrax infection. There are
The tissues are ground up in saline and boiled for 5 two types of vaccines used in humans, namely cell free
minutes and filtered. When this extract is layered over the protective antigen (PA) vaccines and live attenuated
anti-anthrax serum in a narrow tube, a ring of precipitate vaccines. In PA vaccines, protective antigen (PA)
appears at the junction of two liquids within five minutes component of the anthrax toxic complex is either adsorbed
in a positive case. It is mainly used for rapid diagnosis (AVA) on to aluminium hydroxide or precipitated (AVP)
when the sample received is putrid and viable bacilli are on to aluminium potassium phosphate. Both AVA and
unlikely to be found. AVP vaccines are given by intramuscular route. Five
doses (0 and 4 weeks and at 6, 12, and 18 months) are
6. Polymerase Chain Reaction administered in case of AVA vaccine while four doses
It is used for confirmation of anthrax bacillus. are given over a period of 8 months for AVP vaccine.
In the wake of bioterrorism experience in the USA Annual booster is required in both the vaccines.
in 2001 , the Centers for Disease Control (CDC) have Live attenuated vaccines contain spores from
prepared guidelines for identification of B. anthracis as attenuated strains of B. anthracis. Two doses are
mentioned below. administered subcutaneously (Table 28. 1).

Ch 28: Bacill us - - - -- - - - - - - - - - - - - - - - - - - - - - - - - - - - - 223
Table 28.1 Anthrax vaccines for humans spores survive cooking and germinate into vegetative
Types Vaccines Doses bacilli which produce enterotoxin that cause food
Cell free PA • Anthrax vaccine Five doses, one po1sonmg.
vaccines adsorbed (AVA) booster.
• Anthrax vaccine Four doses, one A. Types of Food Poisoning
precipitated (AVP) booster 1. Short Incubation Period Type (1-5 Hours)
Live vaccines • Russian vaccine Two doses It is characterised by acute nausea and vomiting, 1- 5
• PR China vaccine hours after the meal. Diarrhoea is not common. It is
usually associated with consumption of cooked rice,
II. ANTHRACOID BACILLI usually fried rice from Chinese restaurants. B. cereus is
Aerobic spore bearing bacilli resembling B. anthracis present in large numbers in the cooked rice as well as in
are called anthracoid or pseudoanthrax bacilli. Some faecal samples of these patients.
of them are frequent laboratory contaminants and have
to be differentiated from B. anthracis. B. subtilis may act 2. Long Incubation Period Type (8-16 Hours)
as opportunistic pathogen, causing eye infections and It is characterised by acute abdominal pain and diarrhoea,
septicaemia. B. licheniformis has also been incriminated 8- 16 hours after ingestion of contaminated food. Vomiting
in patients of food poisoning. Spores of Bacillus is rare. The faecal samples contain small number of
stearothermophilus are used to test the efficiency of B. cereus.
sterilisation by autoclaves. Some species of bacillus are
important as producers of antibiotics such as bacitracin, B. Pathogenesis
tyrothricin and polymyxin. Most of anthracoid bacilli The disease is due to enterotoxin production. The
can be differentiated easily from the anthrax bacillus, but long incubation type (diarrhoeal disease) is caused by
some, like non-motile strains of B. cereus may resemble serotypes 2, 6, 8, 9, 10 or 12 while the short incubation
B. anthracis. The main differentiating features between type (emetic type) is caused by serotypes 1, 3 or 5 of
anthracoid bacilli and B. anthracis are shown in Table 28.2. B. cereus strains. Isolates from the diarrhoeal type of
disease produce an enterotoxin which resembles heat
III. BACILLUS CEREUS labile enterotoxin of Esch. coli. Strains that cause the
B. cereus has assumed importance as a cause of food emetic type of disease produce a toxin which resembles
poisoning. It is widely distributed in nature such as soil, staphylococcal enterotoxin. Two mechanisms of action,
vegetables, milk, cereals, spices, meat and poultry. Some one involving stimulation of CAMP system and the other
Table 28.2 Differentiating Features of B. anthracis and Anthracoid Bacilli

Feature B. anthracis Anthracoid Bacilli
1. Motility Non-motile Generally motile
2. Capsule Capsulated N on-capsulated
3. Chain formation Long chains Short chains
4. Colony on nutrient agar Medusa head colony Not present
5. Growth in broth No turbidity Uniform turbidity
6. Gelatin stab culture Inverted fir tree appearance and slow gelatin Rapid gelatin liquefaction
liquefaction
7. Haemolysis on blood agar Absent or weak Usually well marked
8. Growth in Penicillin agar (10 units/ml) No growth Grow usually
9. Growth at 45°C No growth Grow usually
10. Fermentation of salicin Negative Usually positive
11. Susceptibility to gamma phage Susceptible Not susceptible
12. Pathogenicity test in animals Pathogenic Not pathogenic
13. McFadyean's reaction Positive Negative
14. Ascoli's precipitin test Positive Negative
15. Fl uorescent antibody test with anthrax antiserum Positive Negative

independent of it, have been described for the enterotoxin antibody conjugate. It produces lecithinase and ferment
of B. cereus. glucose but not mannitol.
C. Diagnosis D. Treatment
Suspected food, faeces and vomitus are cultured on Disease is mild and self limiting, requiring no specific
ordinary media or a special mannitol-eggyolk-phenol red- treatment.
polymyxin agar (MYPA) medium. Spore bearing Gram
positive bacilli may be seen on smear from colonies. B. E. Control
cereus is a motile bacillus, non-capsulated, not susceptible Adequate food hygiene is to be maintained while storing
to gamma phage and does not react with fluorescent cooked food especially rice and reheating should be rapid.
KEY POINTS
1. Members of the genus Bacillus are ubiquitous, present in soil, dust, air and water and are frequently isolated as
contaminants in bacteriological culture media. They are sporogenous, rod shaped, aerobic, Gram positive bacteria.
2. B. anthracis, the causative agent of anthrax, is the most important pathogen of the bacillus group. B. cereus can
cause food poisoning.
3. B. anthracis is a capsulated organism and McFadyean's reaction is positive. It grows as 'Medusa head' appearance on
nutrient agar medium.
4. Ascoli's thermoprecipitin test has been used for rapid diagnosis of anthrax.
5. Alum precipitated toxoid has been used in persons occupationally exposed to anthrax infection. It is given in
three doses intramuscularly at intervals of 6 weeks and 6 months. A booster dose may be given after one year.
6. Aerobic spore bearing bacilli resembling B. anthracis are called anthracoid bacilli.
7. B. cereus causes food poisoning usually associated with fried rice from Chinese restaurants. It produces enterotoxin.
The diseases is mild and self limiting, requiring no specific treatment.
YOU MUST KNOW

1. Morphology and culture characteristics of Bacillus anthracis.
2. Laboratory diagnosis of anthrax.
3. Bacillus cereus.
4. Anthracoid bacilli.
STUDY QUESTIONS
1. Discuss the laboratory diagnosis of anthrax.
(a) Anthracoid bacilli. (b) Bacillus cereus food poisoning

1. What is the chemical nature of capsule of B. anthracis?
(a) Polysaccharide (b) Polypeptide (c) Fatty acid (d) one of the above
2. McFadyean's reaction is used for presumptive diagnosis of:
(a) Tetanus (b) Anthrax (c) Cholera (d) Gas gangrene

Ch 28: Bacillus - - - - -- - -- - - - ' - - - - - - - - - - - - - - - - - - - -- - 225
3. 'Medusa head' appearance of colonies is a characteristic feature of:

(a) Clostridium perfringens (b) Bacillus anthracis
(c) Mycoplasma hominis (d) Ureaplasma urealyticum
4. 'Malignant pustule' lesion is found in:
(a) Cutaneous anthrax (b) Pulmonary anthrax
(c) Intestinal anthrax (d) None of the above
5. Ascoli's thermoprecipitin test is helpful in the laboratory diagnosis of:
(a) Tetanus (b) Anthrax
(c) Cholera (d) Gas gangrene
6. Which of the following characteristics can differentiate Bacillus cereus from Bacillus anthracis?
(a) Non-capsulated (b) Motile
(c) Not susceptible to gamma phage (d) All of the above
Answers (MCQs):
1. (b) 2. (b) 3. (b) 4. (a) 5. (b) 6. (d)
---•••---

Chapter 29
CLOSTRIDIUM
I. Classification
II. General Features
E. Toxin
Ill. Clostridium perfringens
E. Classification F. Pathogenesis
G. Laboratory Diagnosis H. Prophylaxis
IV. Clostridium tetani
E. Classification F. Toxins
I. Prophylaxis J. Treatment
V. Clostridium botulinum
C. Resistance D. Classification
E. Toxin F. Pathogenesis
G. Laboratory Diagnosis H. Prophylaxis and Treatment
VI. Clostridium difficile
A. Morphology B. Toxins
C. Pathogenesis D. Laboratory Diagnosis
E. Treatment
The genus Clostridium consists of anaerobic, spore is derived from the word 'Kl oster' (meaning a spindle).
forming, Gram positive baciJli. The spores are wider Most species are saprophytes found in soil, water
than the bacterial bodies, giving the bacillus a swollen and decomposing plant and animal matter. Some of
appearance resembling a spindle. The name Clostridium the pathogens (e.g. Cl. tetani and Cl. perfringens) are
226
Ch 29: Clostridium _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ 227
commensals of the intestinal tract of human and animals. media is incubated at 37°C for 2-3 days. Most clostridia
The genus contains bacteria that cause three major produce colonies surrounded by haemolysis on blood
diseases of man- tetanus, gas gangrene and food poisoning. agar.
Clostridial pathogenicity is mainly due to production of a Liquid media like cooked meat broth (CMB)
powerful exotoxin. Cl. botulinum is non-invasive, Cl. tetani or thioglycollate media (containing reducing agent
is slightly invasive and clostridia causing gas gangrene thioglycollate and 0.1 % agar) are very useful for growing
are not only toxigenic but are also invasive. clostridia. CMB contains unsaturated fatty acids which
Most species of the genus are motile with peritrichous take up oxygen, the reaction is catalysed by haematin in
flagella except Cl. perfringens and Cl. tetani type VI which the meat and also sulphydryl compounds which lower
are non-motile. All clostridia are non-capsulated with the down the redox potential. Growth appears as turbidity
exception of Cl. perfringens and Cl. butyricum. in the medium. Proteolytic clostridia turn the meat black
and produce foul odour whereas saccharolytic species
I. CLASSIFICATION turn the meat pink.
Clostridia of medical importance may be classified on
the basis of diseases they produce: · C. Biochemical Reactions
A. Tetanus Cl. tetani Most clostridia ferment a variety of sugars and hydrolyse
B. Gas gangrene protein.
1. Established Cl. perfringens
pathogens (Cl. welchii) D. Resistance
Cl. novyi The vegetative cells of clostridia do not differ from other
( Cl. oedematiens) non-sporing bacilli in their resistance to various physical
Cl. septicum and chemical agents. The spores exhibit a variable resistance.
2. Less pathogenic Cl. histolyticum Spores of Cl. tetani persist for years in dried earth.
Cl. fa/lax Cl. perfringens spores are destroyed by boiling for less
3. Doubtful pathogens Cl. bifermentans than five minutes, but spores of some strains (type A) that
Cl. sporogenes cause food poisoning survive for several hours. Spores
C. Food poisoning of Cl. botulinum are not killed completely even at 105°C
1. Gastroenteritis Cl. perfringens for less than 100 minutes. However all spores are killed
(Type A) at 121 °C within 20 minutes (autoclaving). Halogens and
2. Necrotising Cl. perfringens glutaraldehyde (2%) are very effective in killing spores.
enteritis (Type C) Aqueous iodine solution (1 %) kills spores within three
3. Botulism Cl. botulinum hours. Spores are resistant to phenolic disinfectants.
D. Acute colitis Cl. difficile Formaldehyde is not very active.
Clostridia are susceptible to metronidazole, penicillin,
chloramphenicol and erythromycin but are resistant to
II. GENERAL FEATURES aminoglycosides and quinolones.
A. Morphology
Clostridia are Gram positive, spore forming rods, usually E. Toxin
3- 8 µm x 0.4-1.2 µmin size and are highly pleomorphic. Most clostridia produce one or more protein toxins which
The shape and position of spores varies in different species are responsible for pathogenic effects. Toxins of Cl. tetani
and is useful in identification of clostridia. Spores may and Cl. botulinum attack nervous system and are called
be (1) central or equatorial in Cl. bifermentans (2) sub- neurotoxin. The gas gangrene clostridia toxin attacks soft
terminal in Cl. perfringens (3) Oval and terminal in Cl. tissues and is known as histotoxic.
tertium (4) spherical and terminal, giving a drumstick
appearance, in Cl. tetani.
III. CLOSTRIDIUM PERFRINGENS
B. Culture (CLOSTRIDIUM WELCHII)
Clostridia grow well on ordinary media under anaerobic The organism was originally cultivated by Achalme (1891)
conditions. Inoculated culture (e.g. blood agar) is placed and was first described in detail by Welch and Nuttal
in anaerobic jar (McIntosh Filde's jar) from which air is (1892). Cl. perfringens is the most important and common
removed and replaced with a mixture of nitrogen and aetiological agent of gas gangrene (60%), followed by Cl. ~-
carbon dioxide. The anaerobic jar containing culture novyi (30--40%) and Cl. septicum (10-20%). Cl. perfringens

228 _ _ _ _ _ __ _ _ _ _ _ __ __ _ _ _ _ _ __ _ _ _ _ _ Unit Ill: Systemic Bacteriology
also produces food poisoning and necrotising enteritis In litmus milk, lactose fermentation leads to formation
mman. of acid, which changes the colour of litmus from blue to
Cl. perfringens is a commensal in the large intestines red. The acid coagulates the casein (acid clot) and the
of man and animals. The spores are commonly found clotted milk is disrupted due to vigorous gas production
in soil and dust. and this is known as stormy fermentation.
A. Morphology D . Resistance
It is a large, stout, Gram positive bacillus measuring Spores are usually destroyed within five minutes by
4--6 µmx lµm with subterminal spore (Fig. 29.1) . It boiling but those of the 'food poisoning' strains (type
usually occurs singly or in chains and is pleomorphic. A and certain type C strains) resist boiling for 1- 3
Involution forms are common. It is capsulated and non- hours. Autoclaving at 121 °C for 15 minutes is lethal.
motile. The spores are rarely seen in artificial culture or Spores are resistant to commonly used antiseptics and
in tissue and their absence is one of the characteristic disinfectants.
features of Cl. perfringens. Spore formation can be
induced only on special media. E. Classification
Cl. perfringens produce at least 12 distinct toxins but
on the basis of production of four major toxins (alpha,
beta, epsilon and iota), these are classified into five types
A to E.
1. Type A strains produce alpha toxin.
2. Type B strains produce alpha, beta and epsilon
toxins.
3. Type C strains produce alpha and beta toxins.
4. Type D strains produce alpha and epsilon toxins.
5. Type E strains produce alpha and iota toxins
Type A causes gas gangrene in man and some
strains of type A produce enterotoxin which causes food
po1sonmg.
Fig. 29.1 Clostridium perfringens
Alpha (a) Toxin
It is produced by all types of Cl. perfringens but
B. Culture most abundantly by type A strains. Chemically it is a
It grows on blood agar, cooked meat broth (CMB) and phospholipidase (lecithinase.C) and is responsible for
thioglycollate broth within 24--48 hours. It grows best in profound toxaemia in gas gangrene. It is relatively heat
carbohydrate containing media like glucose blood agar. stable, lethal, dermonecrotic and haemolytic. The toxin
It is anaerobic and grows over a pH range of 5.5- 8.0 is haemolytic for red blood cells of most animal species,
and wide temperature range of 20°C- 50°C. The optimum except those of horse and goat. The haemolysis is of the
temperature for growth is 37°C. hot-cold variety i.e. best seen after incubation at 37°C
On blood agar, colonies of most strains show a target followed by chilling at 4°C. The toxin splits lecithin, an
haemolysis, resulting from a narrow zone of complete important constituent of mammalian cell membrane. This
haemolysis caused by theta-toxin and a much wider specific effect of toxin is utilised for rapid detection of
zone of incomplete haemolysis due to alpha toxin. In Cl. perfringens in clinical specimens (Nagler reaction).
cooked meat broth, the meat pieces turn pink but are
not digested. Nagler reaction
Cl. perfringens is grown on a medium containing 6% agar,
C. Biochemical Reactions 5% Fildes' peptic digest of sheep blood and 20% human
Cl. perfringens is predominantly saccharolytic but also serum or 5% egg yolk in a plate. Neomycin sulphate
have mild proteolytic action (gelatin liquefaction). It may be added to the medium to make it more selective
ferments glucose, lactose, sucrose and maltose with the by inhibiting aerobic spore bearers and coliforms. To
production of acid and gas. It is indole negative, H 2S one half of the plate, antitoxin is spread on the surface.
is formed abundantly. MR is positive and VP negative. The inoculated culture plate is incubated at 37°C for 24
Most strains can reduce nitrates. hours. Colonies on the half plate without the antitoxin

Ch 29: Clostridium _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ 229
will be surrounded by opacity while colonies on the other actions. The delta (8) toxin is lethal and haemolytic. The
half with antitoxin shows no opacity, due to specific theta (0) toxin is oxygen labile and is antigenically related
neutralisation of the alpha toxin (Fig. 29.2). Alpha toxin to streptolysin 0. The kappa (K) toxin is a collagenase.
(lecithinase C) splits lecithin into phosphoryl choline The lambda (A) toxin is a proteinase and gelatinase. The
and a diglyceride (lipid). The lipid deposits around the mu (µ) toxin is a hyaluronidase. The nu (v) toxin is a
colonies resulting in opacity. deoxyribonuclease.
Enterotoxin
Some strains of type A produce enterotoxin which causes
diarrhoea and other symptoms of food poisoning.
No antitoxin
Antitoxin present Other Soluble Substances
Haemagglutinins, neuraminidase, fibrinolysin, haemolysin
and histamine are produced.
F. Pathogenesis
Fig. 29.2 Nagler reaction Cl. perfringens produces the following human infections:
Certain lecithinase forming bacteria ( Cl. novyi, some 1. Gas Gangrene

vibrios, some aerobic spore bearers) may produce opacity Cl. perfringens type A is the predominant bacterial agent
in egg yolk media but is not neutralised by Cl. perfringens causing gas gangrene. When a wound gets contaminated
antitoxin, except with Cl. bifermentans which produces a by faecal matter or soil, it may lead to 'simple wound
serologically related lecithinase. contamination', anaerobic cellulitis or myonecrosis (gas
gangrene proper). It is only when muscle tissues are
Reverse CAMP test invaded that gas gangrene results. The incubation period
This test is similar to the CAMP test for identifying varies from six hours to six weeks.
group B streptococci (Refer Chapter 24) except that
Clostridium sp. is inoculated in place of Staph. aureus 2. Food Poisoning
and a known group B streptococcus is used. Although Some strains of type A produce food poisoning.
group B streptococci may show some enhanced haemolysis Incubation period varies from 8 to 12 hours. A heat
with other clostridia but only Cl. perfringens exhibits labile enterotoxin is liberated in the small intestine after
accentuated zone of haemolysis as butterfly appearance ingestion of a contaminated food, usually cooked meat
(Fig. 29.3). and poultry. The toxin acts like enterotoxin of V cholerae
and leads to fluid accumulation in the rabbit ileal loop.
3. Necrotising Enteritis
Butterfly
appearance Group B A severe and fatal necrotising jejunitis (enteritis
streptococci necroticans) is caused by type C strains of Cl. perfringens.
Active immunistation with Cl. perfringens type C toxoid
Cl. perfringens
offers protection against this condition.
4. Other Diseases
These include gangrenous appendicitis, urogenital
Fig. 29.3 Reverse CAMP test infections, brain abscess, meningitis, panophthalmitis
and puerperal infection.
Other Major Toxins G. Laboratory Diagnosis

Beta (~), epsilon (£) and iota (t) toxins have lethal and The diagnosis of gas gangrene must be made primarily
necrotising properties. on clinical grounds and the laboratory only confirms
the clinical diagnosis. The specimens to be collected
Minor Toxins are exudates from wound, necrotic tissue and muscle
Gamma (y) and eta (Tl) toxins have only minor lethal fragments.

230 - - - - - - - - - - - -- - -- -- - - -- - - - - - - Unit Ill: Systemic Bacteriology
1. Direct Microscopy 2. Antibiotics: Gas gangrene organisms are susceptible to

Gram stained smears give presumptive diagnosis. Large metronidazole, penicillin, sulphonamide, tetracycline
number of Gram positive bacilli without spores is and amoxycillin.
strongly suggestive of Cl. perfringens. 'Citron bodies' and 3. Antitox in: Passive immunisation with anti-gas gangrene
boat or leaf shaped pleomorphic bacilli with irregular serum is used prophylactically in cases with extensively
staining suggest Cl. septicum and large bacilli with oval, soiled wounds. Precautions must be taken to guard
subterminal spores indicate Cl. novyi. In case of anaerobic against hypersensitivity to horse serum .
streptococcal myositis, which may be indistinguishable 4. Hyperbaric oxygen: Hyperbaric oxygen is introduced
from gas gangrene clinically in the early stages, Gram in the depth of wound to reduce anaerobiosis.
stained smears shows large number of streptococci and 5. A ctive immunisation: Toxoids have been found ,
pus cells, but no bacilli. experimentally, to induce antitoxic response, but it
has not come into practical use.
2. Culture
IV. CLOSTRIDIUM TETANI
The specimens are inoculated on fresh and heated blood
agar and cooked meat broth (CMB). Growth in CMB is Cl. tetani is the causative agent of tetanus, a disease,
subcultured on blood agar plates after 24-48 hours. The which has been known since very early times. Cl. tetani
blood agar is incubated anaerobically for 48- 72 hours. is widely distributed in soil and intestine of man and
Most strains produce beta haemolysis on blood agar and animals. It is ubiquitous.
few are non-haemolytic. A plate of serum or egg yolk
agar is used for Nagler reaction. The bacterial isolates A. Morphology
are identified by morphology, cultural characteristics, It is a Gram positive, slender bacillus (measuring 4-8
biochemical reactions and reverse CAMP test. Toxigenicity µm x 0.5 µm) with spherical, terminal spores giving the
of the strain can be done by animal pathogenicity. bacillus the characteristic drumstick appearance (Fig.
29.4). It is non-capsulated and motile (except Cl. tetani
3. Animal Pathogenicity type VI) with peritrichate flagella. The young cultures
0.1 ml of 24 hours growth in cooked meat broth is injected are stongly Gram positive but older cultures may show
into a healthy guinea pig by intramuscular route. The variable staining and may even be Gram negative.
animal dies within 24 hours. A control animal protected
with antiserum prior to test is also included. On autopsy,
bacteria can be recovered from heart and spleen of the
test animal.
Laboratory diagnosis of food poisoning is made
by isolating heat resistant Cl. perfringens Type A from
the faeces and food . Laboratory methods are same
but selective media are used for direct plating. CMB
broth is inoculated and heated at 100°C for 30 minutes.
After cooling, it is incubated at 37°C for 18 hours and
subcultured on selective medium which is then incubated
anaerobically at 37°C for 18 hours. The bacterial isolates
are identified by morphology, cultural characteristics, Fig. 29.4 Clostridiurn tetani
biochemical reactions and Nagler reaction. As Cl.
pe,fringens may be present in normal intestines, isolation
from faeces, except in large numbers is not meaningful. B. Culture
It is an obligate anaerobe which grows on ordinary
H. Prophylaxis media. Growth is improved by the addition of blood
l. Surgery: As a prophylatic measure, all damaged or serum. The optimum temperature for growth is 37°C
tissue should be removed promptly and the wound and pH 7.4. It can grow well in cooked meat broth
is irrigated with antiseptic solution to remove (CMB) , thioglycollate broth, nutrient agar and blood
blood clots, necrotic tissue and foreign materials. In agar. In CMB, growth occurs as turbidity and there
established gas gangrene, uncompromising excision is also some gas formation . The meat is not digested
of all affected tissue may save life. but becomes black on prolonged incubation. The bacilli

Ch 29: Clostridium - - - - - - -- - - - - - - - - - -- -- - - - - - - -- - -- 231
produce a swarming (thin spreading film) growth on Tetanospasmin is a good antigen and 1s specifically
blood agar. However, on horse blood agar, they produce neutralised by the antitoxin.
ex-haemolytic colonies which subsequently develop into
~-haemolytic, due to the production of a haemolysin G. Pathogenesis
(tetanolysin). Cl. tetani has little invasive power. Tetanus develops
following the contamination of wound with Cl. tetani
C. Biochemical Reactions spores. The source of infection may be soil, dust, faeces
Cl. tetani has slight proteolytic, but no saccharolytic etc. Infection strictly remains localized in the wound.
property. Gelatin liquefaction occurs very slowly. It does Germination of spores and toxin production occur only if
not ferment any sugar. It forms indole but is MR and VP favourable conditions exist, such as reduced 0-R potential,
negative. H2S is not formed. Nitrates are not reduced. A devatilised tissues and foreign bodies. Pathogenic effects
greenish fluorescence is produced on media containing are mainly due to tetanospasmin (neurotoxin) of Cl. tetani.
neutral red (e.g. MacConkey's medium). The tetanus toxin specifically blocks synaptic inhibition
in the spinal cord. The toxin acts presynaptically. The
D. Resistance abolition of spinal inhibition leads to uncontrolled spread
The resistance of Cl. tetani spores to heat vary in different of impulses initiated anywhere in the central nervous
strains. Most of the strains are killed by boiling for 10-15 system. This results in muscle rigidity and spasms due to
minutes but some resist boiling for three hours. However, the simultaneous contraction of agonists and antagonists.
autoclaving (at 121 °C for 20 minutes) kills the spores
of most strains. Spores are also killed with Iodine (1 % H. Laboratory Diagnosis
aqueous solution), hydrogen peroxide (10 volumes) and The diagnosis of tetanus should always be made
glutaraldehyde (2%) within a few hours. The spores may clinically and laboratory tests are done only to confirm
survive in soil for years. it. Laboratory diagnosis may be made by demonstration
of bacilli by microscopy, culture or by animal inoculation.
E. Classification Specimens generally collected are wound swab, exudate
Ten serological types (types I to X) of Cl. tetani have been or tissue from the wound.
recognised based on type specific flagellar (H) antigens by
agglutination test. Type VI strains are non-flagellated. All 1. Microscopy
the types produce the same toxin, which can be neutralised Gram staining may show Gram positive bacilli with
by antitoxin produced against any one type. drumstick appearance but these are indistinguishable from
morphologically similar non-pathogenic bacilli like Cl.
F. Toxins tetanomorphum and Cl. sphenoides. Hence microscopy
Cl. tetani produces two distinct toxins- tetanolysin alone is unreliable but diagnosis by culture is more
(haemolysin) and tetanospasmin (neurotoxin). dependable.
1. Tetanolysin 2. Culture
Tetanolysin is a heat labile, oxygen labile toxin which Specimen is inoculated on freshly prepared blood agar
causes lysis of erythrocytes of several animal species, and incubated at 37°C for 24-48 hours under anaerobic
especially the rabbit and the horse. It may act as a conditions. The incorporation of polymyxin B in culture
leucotoxin but its pathogenic role is not clear. medium makes it more selective as clostridia are resistant
to this antibiotic. Cl. tetani produces a swarming growth.
2. Tetanospasmin The specimen is also inoculated in three tubes of cooked
Tetanospasmin is a heat labile, oxygen stable, powerful meat broth. One of these tubes is heated at 80°C for
neurotoxin and rapidly gets destroyed by proteolytic 15 minutes, the second tube for five minutes and the
enzymes. It is protein in nature. It is plasmid coded. third left unheated. Heating for different periods is to
This is responsible for clinical manifestations of tetanus. kill vegetative bacteria, while leaving tetanus spores
On its release from the bacillus, it is autolysed to form undamaged. These cooked meat broths are incubated
a heterodimer consisting of a large polypeptide chain at 37°C and subcultured on blood agar plates daily for
(93 ,000 MW) and a smaller polypeptide chain (52,000 upto four days.
MW) joined by a disulphide bond. It is lethal to mice and Gram stained smear from culture shows typical Gram
guinea pigs in minute doses (0.0000001 mg for mouse). positive bacilli with drumstick appearance.

232 _ __ _ _ __ __ __ _ _ _ _ _ __ _ _ _ __ _ __ _ _ Unit Ill: Systemic Bacteriology
3. Toxigenicity Test Tetanus toxoid is given along with diphtheria toxoid

Pathogenicity of the isolated organism is established with and pertussis vaccine (DPT) in children. Pertussis vaccine
demonstration of toxin production. It is best tested in acts as adjuvant. Three doses are given intramuscularly
animals. 0.2 ml of 2 to 4 days old cooked meat culture at interval of 4--6 weeks, starting at age as early as 6
is injected into the root of the tail of one mouse (test weeks. Booster doses are given at age of 18 months and
animal), same amount is injected into another animal then at five years.
(control) that has received tetanus antitoxin (1000 units)
an hour earlier. (b) Passive immunisation
In positive case, the test animal develops symptoms Antitetanus serum (ATS), prepared by immunising horses
within 12- 24 hours, beginning with stiffness of the tail. with toxoid, has been used for preventing tetanus. The
Rigidity proceeds to the leg on the inoculated side, the dose is 1500 IU by intramuscular route immediately
another leg, trunk and forelimbs, in that particular order. after the person is wounded.The dose is same for
Death occurs within two days, but may be killed earlier as adults and children. Being a horse serum, it carries the
the ascending tetanus is very much diagnostic. The control risk of hypersensitivity reaction, therefore, a skin test
animal does not show any change due to neutralisation (subcutaneous injection of 0.5 ml ATS) is recommended
of toxin by antitoxin. before administersing ATS.
Homologous serum prepared from humans, human
I. Prophylaxis antitetanus immunoglobulins (HTIG), is now being used
The available methods are: without the risk of hypersensitivity. Dose of 250 units is
1. Surgical used in prophylaxis.
2. Antibiotics
3. Immunisation (c) Combined prophylaxis
In non-immune person, it is ideal to immunise with first
(1) Surgical dose of tetanus toxoid in one site along with administration
It aims at removal of foreign body, blood clots etc., in of ATS or HTIG in another arm, followed by the second
order to prevent anaerobic conditions favourable for and third doses of tetanus toxoid at monthly interval. In
the bacillus. Depending on the type of wound, surgical combined prophylaxis, adsorbed toxoid should be used as
prophylaxis may vary from simple cleansing to radical the immune response to plain toxoid may be interfered
exc1s1on. with antitetanus serum.
(2) Antibiotics J. Treatment

Antibiotics destroy or inhibit tetanus bacilli and other Tetanus patients are treated in special isolated units. The
pyogenic bacteria in wounds and thus the production reason for isolation is to protect them from noise and
of toxin is prevented. Long acting penicillin injection or light which may provoke convulsions. These patients are
erythromycin may be given. Antibiotic prophylaxis does not infectious and person to person transmission does
not replace immunisation but is a useful adjunct. not occur at all.
Treatment consists of controlling spasms, maintaining
(3) Immunisation airway by tracheostomy and attention to feeding. The
Tetanus is a preventable disease. antitoxin may be used to neutralise unbound toxin.
Antibiotic therapy with penicillin or metronidazole should
(a) Active immunisation be started and continued for a week or more. Patients
It is the most effective method of prophylaxis. Tetanus recovering from tetanus should be immunised with full
tox oid (formol toxoid), which is available either as 'plain course of toxoid, as an attack of the disease does not
toxoid', or adsorbed on aluminium hydroxide or phosphate confer immunity.
(APT), is commonly used for active immunisation. Three
doses of 0.5 ml tetanus toxoid (APT) each are given
intramuscularly, with an interval of 4 to 6 weeks between V. CLOSTRIDWM BOTULINUM
first two doses and 6- 12 months between the second and Clostridium botulinum causes a severe fo rm of food
third dose. A full course of three doses confers immunity poisoning named botulism. The term botulism means
for a period of at least 10 years. A 'booster dose' of sausage (botulus, Latin for sausage) as poorly cooked
toxoid is recommended after 10 years. sausages were formerly associated with this type of food

Ch 29: Clostridium - -- - -- - - - - - -- - - - -- - - - - - - - - - - - - - - - 233
poisoning. It is a widely distributed saprophyte and is hours to kill. Type A toxin is the most potent. Botulinum
found in soil, animal manure, vegetables and sea mud. toxin is the most toxic substance known so far.
The toxin is relatively stable, being inactivated only
A. Morphology after 30-40 minutes at 80°C and 10 minutes at 100°C.
It is a Gram positive, non-capsulated bacillus about Preformed toxin in food is destroyed by boiling for 20
5 µm x 1 µm , motile by peritrichate flagella and produces minutes. It is a good antigen and can be neutralised
subterminal, oval, bulging spores (Fig. 29.5). specifically by its antitoxin.
The toxin acts by blocking the production or release
of acetylcholine at synapses and neuromuscular junctions.
Death occurs due to respiratory failure.
The toxin production is probably determined by the
presence of bacteriophage.
F. Pathogenesis
Cl. botulinum is non-invasive and its pathogenicity is due
to the action of preformed toxin, the manifestations of
which are collectively called botulism. Botulism is of three
Fig. 29.5 Clostridiurn botulinurn
types- foodborne, infant and wound botulism.
B. Culture 1. Foodborne Botulism

It is a strict anaerobe and can grow on ordinary media. It is due to preformed toxin in food contaminated with
Optimum temperature is 35°C but some strains may Cl. botulinum. Human disease is usually caused by types
grow at l-5°C. Commonly used media are blood agar A , B, E and very rarely by types C, F and G. The source
and cooked meat broth. Colonies are large, irregular, of botulism is usually various preserved foods- meat, fish,
semitransparent, with fimbriate border. On blood agar, vegetables etc. The contaminated food usually exhibits
haemolysis around the colonies is observed. signs of spoilage, and cans may be inflated and show
bubbles on opening. Often the food may look normal
C. Resistance and there is no alteration in taste.
The spores are highly resistant, and can withstand heat for The symptoms appear 12- 36 hours after ingestion of
several hours at 100°C and for upto 10 minutes at 120°C, contaminated food. Vomiting, thirst, constipation, ocular
whereas spores of types B, E and F are less resistant to paresis, difficulty in swallowing, speaking and breathing
heat. They are also resistant to radiation. are the common symptoms. Diarrhoea is not a symptom.
Fatality rate is 25- 70% and death results from respiratory
D. Classification failure.
Eight types- A, B, Cl, C2, D, E, F and G have been
identified on the basis of their antigenically distinct 2. Infant Botulism
toxins. The toxins of different types are identical in their It affects infants, usually below 6 months. It is a disease
pharmacological action, but are neutralised only by the due to ingestion of food contaminated by spores
homologous antiserum. of Cl. botulinum. Honey has been incriminated as a
source of infection. It is a toxico-infection. The disease
E. Toxin is characterised by constipation, weakness, lethargy
Cl. botulinum forms a powerful exotoxin. The toxin differs and cranial palsies. Infants exhibit flaccid paralysis,
from other exotoxins in that it is not released during usually with a weak sucking response, generalised loss
the life of the bacterium. It is produced intracellularly of tone ("the floppy baby syndrome") and respiratory
and appears in the medium on autolysis of the cell. It complications. Spores and toxin are excreted in the faeces
is believed to be synthesised as a non-toxjc protoxin or of the patient. Management is by supportive care and
progenitor toxin. Progenitor toxin is converted to active assisted feeding. Severity of illness varies from mild illness
toxin by trypsin and other proteolytic enzymes. The toxin to fatal disease.
has been isolated as a pure crystalline protein with a M.W
70,000. It has a lethal dose for mice 0.000,000,033mg and 3. Wound Botulism
a lethal dose for humans is probably one microgram. It It is a very rare condition which results from wound
is a neurotoxin and acts slowly, therefore, takes several infection with Cl. botulinum. Toxin produced is absorbed

y
234 - -- -- - - - - - - - - - - - - - - - - - - - ---r- - Unit Ill: Systemic Bacteriolog
into the blood and the symptoms similar to foodborne exposed to the risk, two injections of toxoid may be
botulism occur except for the gastrointestinal components administered at an interval of ten weeks, followed by a
which are absent. Type A has been recovered from most booster dose a year later.
of the cases studied.
VI. CLOSTRI DIUM DIFFICILE
G. Laboratory Diagnosis
Cl. difficile was first isolated from the faeces of newborn
Diagnosis may be confirmed by demonstrati on of the
infants. It was so named due to unusual difficulties
bacillus or the toxin in suspected residual food or in
involved in its isolation.
faeces.
1. Demonstra tion of the Organism A. Morpholo gy

Smears made from suspected food or faeces are examined It is a long, slender, Gram positive bacillus containing
by Gram's staining which may show Gram positive sporing oval and terminal spores. There is a tendency to lose its
bacilli. Culture is done on blood agar or cooked meat Gram reaction.
broth. Growth on culture media may be confirmed by
Gram's staining. Presence of bacilli in food or faeces in B. Toxins
absence of toxin is of no significance. Hence, toxin in It produces two toxins, an enterotoxin (toxin A) and
culture fluid must be demonstrate d by toxigenicity test a cytotoxin (toxin B). The enterotoxin is primarily
in mice. responsible for diarrhoea whereas cytotoxin is capable
of producing cytopathogenic effects in several tissue
2. Demonstra tion of Toxin culture cell lines.
Specimens (stool, food and vomitus) are macerated in
sterile saline and the filtered extract is divided into three C. Pathogene sis
parts. One portion of extract is heated at 100°C for The pathogenic role of Cl. difficile was established in
10 minutes and other two kept unheated. Two mice or 1977, when it was found to be responsible for antibiotic
guinea pigs are injected with unheated filtrate; one of associated colitis. Acute colitis with or without membrane
them (control) is protected with polyvalent botulinum formation is an important complication in patients on oral
antitoxin prior to injection. The third animal is injected antibiotic therapy. Many antibiotics have been incriminated
with heated filtrate. The test animal (unprotected) but lincomycin and clindamycin are particularly prone to
develops dyspnoea, flaccid paralysis and dies within 24 cause pseudomembranous colitis. Active multiplication of
hours whereas control animal (protected) remains healthy. Cl. difficile and the production by it of an enterotoxin as
The animal injected with heated filtrate also remains free well as a cytotoxin is responsible for antibiotic associated
of any toxic symptoms. colitis. If the condition is not recognised early and
Typing of Cl. botulinum is done by using a series of properly treated, it terminates fatally.
animals protected with type specific antitoxins instead of
polyvalent antitoxin. D. Laboratory Diagnosis
1. Demonstra tion of Toxin
H. J?rophylaxis and Treatment Toxin can be demonstrated in the faeces by its characteristic
As botulism follows consumptio n of canned or preserved effect on human diploid cells and HEp-2. ELISA can
food, control may be achieved by proper canning and also be used for the demonstrati on of toxin. The toxin
preservation . By the time symptoms appear, the toxin is specifically neutralised by the Cl. sordelli antitoxin.
is fixed to nervous tissue and it can no longer be
inactivated by antitoxin. Intensive supportive therapy 2. Nucleic Acid Amplificat ion Tests
may bring down the mortality rate. However, if outbreak Nucleic acid amplification tests (NAATs) which can
occurs, prophylactic dose of antitoxin should be given determine the presence of toxins A and B genes in faeces
intramuscularly to all who have consumed the suspected are becoming the new gold standard for the detection of
food. Antitoxin may be tried for treatment. Polyvalent toxin producing C. difficile.
antiserum to types A, Band E may be given. Supportive
therapy with maintenanc e of respiration is also very 3. Isolation of Bacilli
important. Cl. difficile can be grown from the faeces of · patients
If immunisation is needed, as in laboratory workers with subsequent test for toxigenicity. Cycloserine-

Ch 29: Clostridium _ _ _ _ _ __ __ _ _ _ _ _ _ _ _ _ _ __ _ _ _ __ __ _ _ _ 235
cefoxitin-fructose agar (CCFA) is a selective medium E. Treatment

to primarily isolate Clostridium difficile from faeces of Cl. difficile are generally resistant to most antibiotics,
patients suspected of antibiotic associated colitis or but these are susceptible to vancomycin. Clindamycin
pseudomembranous colitis. C. difficile colonies appear and lincomycin should be avoided. Metronidazole is the
yellow due to fructose fermentation. drug of choice.
KEY POINTS
1. The genus Clostridium consists of anaerobic, spore forming, Gram positive bacilli.
2. This genus contains bacteria that cause three major diseases of man-tetanus, gas gangrene and food poisoning.
3. Clostridial pathogenicity is mainly due to production of a powerful exotoxin.
4. The shape and position of spores varies in different species and is useful in identification of clostridia, spores may
be subterminal in Cl. perfringens and drumstick appearance in Cl. tetani.
5. Clostridia grow well on blood agar medium under anaerobic conditions. Liquid media like cooked meat broth (CMB)
is very useful for growing clostridia.
6. Nagler reaction and reverse CAMP test are useful in identification Cl. perfringens, a causative agent of gas gangrene
and food poisoning.
7. Cl. tetani produces two distinct toxins-tetanolysin (haemolysin) and tetanospasmin (neurotoxin). It is the causative
agent of tetanus. '
8. Tetanus toxoid is used for prophylaxis of tetanus.
9. Cl. botulinum forms a powerful exotoxin which is responsible for the disease botulism.
10. Cl. difficile has been found to be responsible for antibiotic associated colitis.
11. As clostridia are toxin producing bacteria, antitoxins are used for passive immunisation in the diseases associated
with them. Anti-gas gangrene and human antitetanus immunoglobulins (HTIG) are two examples of antitoxins used
in gas gangrene and tetanus respectively.
YOU MUST KNOW

1. Diseases produced by different Clostridia species.
2. Morphology, culture and biochemical reactions of Clostridium perfringens.
3. Nagler reaction.
4. Laboratory diagnosis of gas gangrene.
5. Morphology, culture characteristics and biochemical reactions of Cl. tetani.
6. Toxins produced by Cl. tetani.
7. Laboratory diagnosis of tetanus.
8. Immunisation against tetanus.
9. Morphology and culture characteristics of Cl. botulinum.
10. Cl. botulinum toxin.
11. Laboratory diagnosis of botulism.
12. Clostridium difficile.
ST DY UESTIONS
1. Classify clostridia. Discuss the laboratory diagnosis of gas gangrene.
(a) Nagler reaction (b) Laboratory diagnosis of tetanus
(c) Tetanospasmin (d) Prophylaxis against tetanus

236 _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Unit Ill: Systemic Bacteriology
(e) Stormy clot reaction (f) Alpha toxin

(g) Clostridium botulinum exotoxins (h) Laboratory diagnosis of botulism
(i) Clostridium difficile.
"l.J[ Jl->J CHOICE QUESTIONS (MCQs)

1. Which of the following species of Clostridium is non-motile?
(a) Clostridium perfringens (b) Clostridium botulinum (c) Clostridium novyi (d) None of the above
2. Which of the following species of Clostridium is capsulated?
(a) Clostridium perfringens (b) Clostridium botulinum (c) Clostridium novy i (d) None of the above
3. All of the following species of Clostridium may cause gas gangrene except:
(a) Clostridium perfringens (b) Cl. novyi (c) Cl. septicum (d) Cl. difficile
4. Following tests may be helpful in identification of Clostridium perfringens?
(a) Nagler reaction (b) Reverse CAMP test (c) Both of the above (d) None of the above
5. Clostridium perfringens may produce the following infections:
(a) Gas gangrene (b) Food poisoning (c) Necrotising enteritis (d) All of the above
6. Drumstick appearance of spores is a characteristic feature of:
(a) Clostrdium perfringens (b) Cl. tetani (c) Cl. septicum (d) Cl. novyi
7. Which type of Clostridium perfringens strains is responsible for food poisoning?
(a) Type A (b) Type B (c) Type C (d) Type E
8. Stormy fermentation is useful in identification of:
(a) Clostridium perfringens (b) Cl. tetani (c) Cl. botulinum (d) Cl. difficile
9. Which of the following species of Clostridium shows swarming on blood agar?
(a) Clostridium perfringens (b) Cl. novyi (c) Cl. tetani (d) Cl. difficile
10. Which of the following toxins is the most toxic?
(a) Botulinum toxin (b) Tetanus toxin (c) Diphtheria toxin (d) Cholera toxin
11. Floppy baby syndrome is associated with:
(a) Clostridium botulinum infection (b) Cl. perfringens infection
(c) Cl. difficile infection (d) Cl. novyi infection
12. Which of the following bacteria is responsible for pseudomembranous enterocolitis?
(a) Clostridium perfringens (b) Cl. tetani (c) Cl. botulinum (d) Cl. difficile
13. Which of the following types of toxins is/are most commonly associated with foodborne botulism in man?
(a) Type A (b) Type B (c) Type E (d) All of the above
14. Botulism is due to
(a) Preformed toxin of Clostridium botulinum (b) Invasion of Cl. botulinum in the intestine
15. Selective medium used for isolation of Clostridium difficile from faeces is:
(a) Cycloserine-cefoxitin-fructose agar (b) Monsur's gelatin taurocholate trypticase tellurite agar
(c) Cefsulodin-irgasan-novobiocin agar (d) All of the above
Answers (MCQs):
1. (a) 2. (a) 3. (d) 4. (c) 5. (d)
6. (b) 7. (a) 8. (a) 9. (c) 10. (a)
11. (a) 12. (d) 13. (d) 14. (a) 15. (a)
- - - - ++• ----

Chapter 30
NONSPORING ANAEROBES
I. Introduction
II. Anaerobic Cocci
A. Peptococcus B. Peptostreptococcus and Others
C. Veillonella and Others
Ill. Anaerobic Gram Positive Bacilli
A. Eubacteri um B. Lactobacillus
C. Bifidobacterium D. Propionibacterium
E. Acti nomyces F. Mobiluncus
IV. Anaerobic Gram Negative Bacilli
A. Bacteroides B. Fusobacterium
C. Leptotrichia
V. Anaerobic Infections
VI. Laboratory Diagnosis
A. Specimens B. Direct Microscopy
C. Culture D. Other Anaerobic Techniques
VII. Treatment
I. INTRODUCTION anaerobes are particularly likely to set up infections

Non-sporing anaerobes have been recognised as an where there is damaged and necrotic tissue. Anaerobic
important cause of human and animal infections. Many of infections of the head, neck and respiratory tract are
these bacteria form comrnensal flora of man and animals often associated with comrnensals in the mouth, while
in mouth, oropharynx, gastro-intestinal tract and genital infection in the abdominal and pelvic regions are more
tracts. In the gut the anaerobic bacteria outnumber the commonly associated with those of gut.
aero bes (Escherichia coli) by a ratio of 1000:1. Even in such On the basis of morphology and staining characters,
seemingly aerobic situations as the mouth and the skin, they have been classified as given in Table 30. l .
anaerobic bacteria are more frequent than aerobes (ratio Spirochaetes are described separately in Chapter 45.
may be 10:1 to 30:1). The number of anaerobes have been
estimated to be 104 to 105 per ml in the small intestine, II. ANAEROBIC COCCI
108 per ml in saliva and 10 11 per gm in the colon. Some The anaerobic cocci occur as normal flora of skin, mouth,
of these anaerobic bacteria act as opportunistic pathogens intestine and vagina. Recently, changes have been made
when body resistance is lowered. These non-sporing in classifying anaerobic cocci but there is no change in
237
238 _ _ _ _ _ __ _ __ _ _ _ _ _ _ _ _ _ __ _ _ _ _ _ __ Unit Ill: Systemic Bacteriology
Table 30.1 Classification of Nonsporing Anaerobes infections such as pleuropulmonary disease, brain abscess
A. Cocci and puerperal infections. Peptostreptococcus anaerobius
is most often responsible for puerperal sepsis. Finegoldia
1. Gram positive cocci (i)Peptococcus
(ii)Peptostreptococcus magna (formerly Pst. magnus) is commonly isolated
(iii) Parvimonas anerobic cocci from wound infections. Peptoniphilus
(iv) Peptoniphilus asaccharolyticus (formerly Pst. asaccharolyticus),
(v) Anaerococcus Anaerococcus prevoti (formerly Pst. prevoti) and
(vi) Finegoldia Anaerococcus tetradius (formerly Pst. tetradius) are
( vii)Gallicola
some other anaerobic cocci commonly found in clinical
(viii) Atopobium
(ix) Blantia
specimens.
(x) Ruminococcus Anaerobic cocci are present in large number in pus
from suppurative lesions, so a Gram stained smear may
2. Gram negative cocci (i) Veillonella
(il) Acidaminococcus be helpful in diagnosis. Infections are usually mixed, the
(iii) Megasphaera anerobic cocci being present along with clostridia or other
(iv) Negativococcus anaerobic Gram negative bacilli.
(v) Anaeroglobus
B. Bacilli C. Veillonella and Others
1. Gram positive bacilli (i) Eubacterium
Veillonellae are small (0.3- 2.5 µm), Gram negative cocci,
(i1) Lactobacillus occurring in short chains, pairs or clumps. They are usually
(iii) Bifidobacterium non-pathogenic but occasionally invade blood stream.
(iv) Propionibacterium Other important genera of anaerobic Gram negative cocci
(v) Actinomyces include Acidaminococcus and Megasphaera.
(vi) Mobiluncus
2. Gram negative bacilli (1) Bacteroides III. ANAEROBIC GRAM
(ii) Prevotella POSITIVE BACILLI
(iii) Porphyromonas
A. Eubacterium
(iv) Fusobacterium
(v) Leptotrichia
Eubacterium species are strictly anaerobic and grow
very slowly. They are members of the normal mouth
C. Spirochaetes
and intestinal flora. Some species (E..brachy, E..nodatum)
(i) Treponema are commonly seen in periodontitis.
(ii) Borrelia
B. Lactobacillus
genus Peptococcus. Genus Peptostreptococcus contains Lactobacilli are Gram positive bacilli that frequently
only two species Pst. anaerobius and Pst. stomatis. Other show bipolar and barred staining. Most strains are non-
species of Peptostreptococcus are reclassified in different motile. They form considerable amount of lactic acid
genera such as Peptoniphilus, Anaerococcus, Parvimonas, from carbohydrates and grow best at pH of 5 or less.
Finegoldia, Gallicola and Slackia. Lactobacilli are normally present in the mouth, gut
and vagina. They are widely distributed as saprophytes
A. Peptococcus and ferment material such as milk and cheese. Several
Peptococci are Gram positive, non-sporing, anaerobic species of lactobacilli are present in the intestine, the
cocci that do not form chains. They are small spherical commonest being L. acidophilus, they synthesise vitamins
measuring 0.5- 1 µm in size, arranged singly or in pairs such as biotin, vitamin B 12 and vitamin K, which may
or in clumps. They are coagulase negative. They may be absorbed by the host. In the mouth lactobacilli
cause pyogenic infections of wounds, puerperal sepsis have been incriminated in the pathogenesis of dental
and urinary tract infection. This genus contains only one caries. It is believed that the mineral components of
species i.e., P niger. enamel and dentine are dissolved by acid formed by the
fermentation of sucrose and other dietary carbohydrates
B. Peptostreptococcus and Others by lactobacilli.
Peptostreptococci are small Gram positive cocci, 0.3 Lactobacilli of several species occur in the vagina and
to 1 µm in size, arranged in chains. Carbohydrates are these are collectively known as Doderlein's bacilli. They
fermented with the production of acid, gas or both. They ferment the glycogen deposited in the vaginal epithelial
are the commonest anaerobic cocci isolated from human cell and form lactic acid, which accounts for the highly

Ch 30: Nonsporing Anaerobes - - - - - ~ - - - - - - - - - - - - - - - -- - - -- - 239
acidic pH of the vagina. They protect adult vagina from They were grouped previously in the Family
infections. In prepubertal and postmenopausal vagina, Bacteroidaceae and classified into the following genera
lactobacilli are scanty.
They grow best in media enriched with glucose or A. Bacteroides (bacilli wit/, rounded ends)
blood in presence of 5% CO 2 and at pH 6. Lactobacilli are 1. Fragilis group
generally non-pathogenic, however, they can be involved B. fragilis, B. vulgatus, B. distasonis, B. ovatus, B.
in serious infections, especially in immunocomprornised thetaiotaomicron (Sacchrolytic)
individuals. Lactobacilli are particularly associated with 2. Melaninogenicus group
advanced dental caries. B. melaninogenicus, B. oralis (Moderately sacchrolytic)
3. Asacchrolytic group
C. Bifidobacterium B. asacchrolyticus, B. gingivalis
Bifidobacteria are Gram positive, non-sporing, The new genus names for melaninogenicus
pleomorphic, non-motile bacilli showing true and false group is Prevotella and for asacchrolytic group is
branching. The name is derived from frequent bifid Porphyromonas.
Y-shaped cells. They occur as normal flora in the mouth
and gastrointestinal tract and are occasionally pathogenic. B. Fusobacterium (bacilli wit/, pointed ends)
Dental caries is the most common clinical entity in which Species are F. necrophorum, F. nucleatum,
B. dentium may have a pathogenic role. F. necrogenes
D. Propionibacterium C Leptotricl,ia (large bacilli)

Propionibacteria are Gram positive, pleomorphic, L. buccalis is the only species.
non-motile bacilli. They are related to corynebacteria
and are usually labelled as anaerobic diphtheroids. The A. Bacteroides
commonest species isolated in the clinical laboratory is P. Bacteroides occur as commensals in the mouth,
acnes ( Corynebacterium acnes). This is commensal found gastrointestinal and female genital tracts. Normal stools
abundantly on the normal skin. It is regularly found contain 10 11 organisms per gram. The most common
in acne and was thought to be its causative agent. It are B. fragilis group isolated from the large intestine.
has also been isolated from abscesses, in some cases of P. melaninogenica is isolated particularly from the
infective endocarditis and in infections associated with oropharynx, gut and vagina. These are Gram negative,
implanted prosthesis. Pathogenic role of Propionibacteria strict anaerobes, non-sporing, non-motile bacilli and
is not definite. are very pleomorphic. They require enriched media
containing blood or haemin for growth. They grow readily
E. Actinomyces in media such as brain-heart infusion agar in atmosphere
Genus Actinomyces is described in Chapter 47. containing 10% carbon dioxide.
They cause peritonitis following bowel injury and
F. Mobiluncus pelvic inflammatory disease (PID). They are also found
Mobiluncus species are motile, curved, anaerobic bacilli. in abdominal and brain abscesses and in empyema.
They may appear as Gram variable. They have been Pus is often foul smelling. B. fragilis is probably the
isolated from the vagina in bacterial vaginosis, along most frequent among non-sporing anaerobes isolated
with Gardnerella vagina/is. Mobiluncus is described in from clinical specimens. P. melaninogenica is recognised
Chapter 35. easily because of black or brown coloured colonies.
The colour is due to a haemin derivative and not by a
melanin pigment, as was once thought. Cultures of P.
IV. ANAEROBIC GRAM melaninogenica give a characteristic red fluorescence when
NEGATIVE BACILLI exposed to ultraviolet light. It has been isolated from lung
Anaerobic Gram-negative, non-spore forming bacilli or liver abscess, mastoiditis, lesions of intestine, mouth
of clinical relevance belong to families Bacteroidaceae, and gums. Anaerobic infections are generally mixed
Porphyromonadaceae, Prevotellaceae, Rikenellaceae and infections containing other organisms that are often part
Fusobacteriaceae. Important genera include Bacteroides, of the normal flora.
Porphyromonas, Prevo tel/a, Fusobacterium and Leptotrichia. Porphyromonas gingivalis is responsible for periodontal
Besides these genera many new genera have also been disease while P. endodontalis causes dental root canal
included in these families. infections.

240 _ _ _ _ _ _ _ _ _ _ __ __ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Unit Ill: Systemic Bacteriology
B. Fusobacterium when the host's resistance is lowered. Trauma, tissue

Fusobacteria are Gram negative bacilli which are strict necrosis, diabetes, malnutrition, malignancy or prolonged
anaerobes. Some species of Fusobacterium produce long treatment with antibiotics may act as predisposing factors.
slender rods that are wide at the centre and taper towards Anaerobic infections are usually polymicrobial, more than
the ends (fusiform). They are usually non-motile. They one anaerobe is involved along with aerobic organisms.
can be grown on blood agar containing neomycin and Infections are usually localised but general dissemination
vancomycin. may occur by bacteraemia. Pus produced by anaerobes
Fusobacterium species are commensals in the mouth is usually putrid with a nauseating odour. Cellulitis
and also cause infections of mouth and related sites. F is a common feature of anaerobic wound infections.
nucleatum is the most common species. It may cause Toxaemia and fever are not marked. Bacteria responsible
infections of head and neck, dental and periodontal for common anaerobic infections are listed in Table 30.3.
infections and cerebral abscess. Some of Fusoba cterium
species are commensals in the gastrointestinal and VI. LABORATORY DIAGNOSIS
genitourinary tracts. These species along with F nucleatum As anaerobic bacteria form part of the normal flora
may cause intraabdominal infections, osteomyelitis and of the skin and mucous surfaces, their isolation from
various soft tissue infections. clinical specimens has to be interpreted cautiously. The
mere presence of an anaerobe does not prove it as a
C. Leptotrichia causative agent.
Leptotrichia is a long, straight or slightly curved Gram
negative, anaerobic, non-sporing, non-motile bacillus. It A. Specimens
measures 5- 15 µm x 1- 1.5 µmin size. The genus has only Specimens are to be collected in such a manner to avoid
one species Leptotrichia buccalis. It is normally present normal resident flora. For example, material collected by
in mouth and gastrointestinal tract. In association with aspiration in case of lung abscess is acceptable but not
Borrelia vincentii it is believed to cause acute ulcerative the sputum. As these anaerobic bacteria die on exposure
gingivitis or Vincent's angina. Diagnosis can be made to oxygen, care has to be taken to minimise contact
by direct microscopy. They can also be grown on blood of specimen with air during collection, transport and
agar at 37°C in presence of 5- 10% CO 2 under anaerobic handling of specimens. A satisfactory method of collection
conditions. Leptotrichia buccalis was previously named is to aspirate the specimen into an airtight syringe. After
as Fusobacterium fusiforme . collection, the needle is plunged into a sterile rubber cork
and is sent immediately to the laboratory. Pus and other
V. ANAEROBIC INFECTIONS fluids may be collected in small bottles with air-tight caps
Anaerobic infections are usually endogenous and are caused and transported immediately. Specimens should fill the
by normal flora of body surfaces. Anaerobic bacteria bottles completely without leaving any airspace. Swabs
are normally present on the skin, mouth, nasopharynx, are unsatisfactory specimens, but where they are to be
intestines and vagina (Table 30.2). They produce disease used, they should be sent in Stuart's transport medium.
Other clinical samples unsuitable for anaerobic culture
Table 30.2 Normal Anaerobic Flora include sputum, urine, faeces and secretions obtained
Bacteria Skin Mouth and Intestine Vagina by nasotracheal suction. Specimens should be inspected
nasopharynx for characteristics that strongly indicate the presence
Clostridium ++ of anaerobes, such as foul odour; sulphur granules
Gram positive ++ ++ ++ associated with Actinomyces sp., Propionibacterium sp.
cocci or Eubacterium. Other clues suggestive of anaerobic
Gram negative ++ + ++ infection include:
COCCI 1. Failure to isolate organisms from pus (sterile pus)
Bifido bacterium + ++ + 2. Deep abscesses.
Propionibacterium ++ 3. Pus which shows red fluorescence under UV light.
Actinomyces + 4. Failure to respond to conventional antimicrobial
++
treatment.
Bacteroides fragilis
P melaninogenica ++ + ++
B. Direct Microscopy
Fusobacterium ++ + Examination of a Gram stained smear is very useful.
Spirochaetes + Pus in anaerobic infection shows a variety of organisms

Ch 30: Nonsporing Anaerobes _ __ __ __ _ __ _ __ __ _ _ __ _ _ __ _ _ _ _ _ 241
Table 30.3 Bacteria Responsible for Common Anaerobic Infections

Site Type of infection Bacteria responsible
Central nervous system (C.N.S.) Brain abscess B. fragilis, Peptostreptococcus
Ear, Nose, Throat (E.N.T.) Chronic otitis media, mastoiditis, orbital cellulitis Fusobacteria
Mouth and Jaw Vincent's angina, dental abscess, cellulitis, abscess and sinus Fusobacteria, spirochaetes, Mouth
of jaw anaerobes, Actinomyces
Respiratory tract Lung abscess, empyema, bronchiectasis, aspiration Fusobacteria, P melaninogenica,
pneumonia Peptostreptococcus, Peptococcus
Abdominal Subphrenic abscess, hepatic abscess, appendicitis, peritonitis, B. fragilis
ischiorectal abscess, wound infection after colo-rectal
surgery.
Female genital tract Puerperal sepsis, tubo-ovarian abscess, Bartholin's abscess, P melaninogenica, B. fragilis,
septic abortion, wound infection following genital surgery Peptostreptococcus, Peptococcus
Skin and tissue Infected sebaceous cyst, axillary abscess, breast abscess, Peptococcus, Peptostreptococcus,
cellulitis, diabetic ulcer, gangrene P melaninogenica, B. fragilis
and numerous pus cells. Examination of specimen under takes time and is difficult, but it is possible to report on
ultraviolet light may show the bright red fluorescence in the following (1) whether the infection is solely aerobic,
cases of P melaninogenica. anaerobic or mixed (2) the identification of the commoner
anaerobes, particularly of B. fragilis (3) an indication of
C. Culture antimicrobial agents likely to be used. Antibiotic sensitivity
Freshly prepared blood agar with neomycin, yeast extract, tests can be done by disc or dilution methods.
haemin and vitamin K is a suitable culture medium.
Culture plates are incubated at 37°C in anaerobic jar with D. Other Anaerobic Techniques
10 per cent CO 2. Anaerobiosis can also be maintained by Gloved anaerobic chambers (refer Chapter 6) with
Gas-Pak system. Plates are examined after 24--48 hours. continuous gas flow may be used for culture of specimens.
Parallel aerobic cultures (such as Pseudomonas aeruginosa) Pre-reduced anaerobically sterilised media (PRAS) can
should always be set up to ensure the proper anaerobiosis also be employed but are not essential for rountine
inside the anaerobic jar. To verify the anaerobic conditions diagnostic procedures. Gas liquid chromatography (GLC)
in the jar, reduced methylene blue indicator is generally is also useful to identify anaerobes in specimens.
used for this purpose. This indicator remains colourless
anaerobically but turns blue on exposure to oxygen.
O ther anaerobic media, such as cooked meat broth VII. TREATMENT
(CMB) and thioglycollate broth, may also be used for Besides antibiotic therapy, most anaerobic infections
incoculating the specimens. For more details about the also require surgical intervention. Penicillin is effective
anaerobic jar, and the CMB, refer to Chapter 6. Specimens against most anaerobes except B. fragilis. Tetracycline and
should also be put up for growth of aerobic bacteria as chloramphenicol are also used. Clindamycin is effective,
in most anaerobic infections aerobic bacteria are also but is not commonly used because it is prone to cause
involved. pseudomembranous colitis. Metronidazole is the drug
D efinitive identification of the anaerobes depends of choice. It is also useful as a prophylactic measure in
on colony morphology and various biochemical tests. It large bowel surgery.
KEY POINTS
1. Non-sporing anaerobes form commensal flora of man and animals in mouth, oropharynx, gastrointestinal tract and
genital tracts. Some of these anaerobic bacteria act as opportunistic pathogens when body resistance is lowered. These
are particularly likely to set up infections where there is damaged and necrotic tissue.
2. Peptococcus, Peptostreptococcus and Veillonella are some examples of nonsporing anaerobic cocci.

242 _ _ _ _ _ _ _ _ _ _ _ _ _ __ __ _ _ _ _ _ _ _ _ _ _ _ Unit Ill: Systemic Bacteriology
3. Bacteroides, Prevotella, Porphyromonas and Fusobacterium are some examples of nonsporing anaerobic bacilli.
4. Anaerobic bacteria can be grown on blood agar under anaerobic conditions. Anaerobic jar is generally used for
producing anaerobiosis. Cooked meat broth (CMB) may also be used to grow these bacteria.
YOU MUST KNOW

1. Classification of nonsporing anaerobes.
2. Bacteria responsible for anaerobic infections.
3. Laboratory diagnosis of anaerobic infections.
STUDY QUESTIONS
1. Classify non-sporing anaerobes. Discuss the laboratory diagnosis of infections caused by non-sporing anaerobes.
(a) Anaerobic cocci (b) Bacteroides (c) Propionibacterium.

1. Which of the following bacteria are Gram negative cocci?
(a) Peptococcus (b) Peptostreptococcus
(c) Veillonella (d) All of the above
2. Which of the following bacteria protect adult vagina from infections?
(a) Lactobacilli (b) Peptococcus
(c) Veillonella (d) Bacteroides
3. Which is the commonest species of Bacterioides in fragilis group responsible for human infections?
(a) Bacterioides fragilis (b) B. distasonis
(c) B. thetaiotamicron (d) B. vulgatus
4. Which of the following bacterial cultures show red fluorescence when exposed to UV light?
(a) Bacteriodes fragilis (b) Prevotella melaninogenica
(c) Prophyromonas gingivalis (d) Fusobacterium nuc/eatum
Answers (MCQs):
1. (c) 2. (a) 3. (a) 4. (b)
____ ... ____

Chapter 31
ENTEROBACTERIACEAE
I. Classification
II. Escherichia coli
E. Antigenic Typing F. Resistance
G. Toxins H. Pathogenesis
I. Laboratory Diagnosis
Ill. Edwardsiella
IV. Citrobacter
V. Klebsiella
A. Classification B. Morphology
C. Culture D. Biochemical Reactions
E. Antigenic Structure F. Methods of Typing
I. Treatment
VI. Enterobacter
VII. Hafnia
VIII. Serratia
IX. Proteus, Morganella, Providencia
A. Classification B. Proteus
C. Morganella D. Providencia
E. Laboratory Diagnosis of Morganella
and Providencia
Members of the family Enterobacteriaceae are aerobic catalase positive except Sh. dyseneriae type 1 which is
and facultative anaerobic Gram negative enteric bacilli. catalase negative. They are non-capsulated and non-
They are motile by peritrichous flagella or are non-motile. sporing. Their natural habitat is usually the intestinal
They grow readily on ordinary media, ferment glucose
tract of man and animals. Some species are commensals
with production of acid or acid and gas, reduce nitrates
to nitrites, oxidase negative except. Pleiomonas sp. and and some are pathogenic for humans.
243
244 _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __ _ _ _ _ _ __ _ Unit Ill: Systemic Bacteriology
I. CLASSIFICATION tribe consists of one or more genera and each genus has
The classification of Enterobacteriaceae has been one or more subgenera and species.
controversial. There has been successive changes in the Tribe Ge11us
nomenclature. The oldest method was to classify these Escherichiae Escherichia, Shigella
bacteria on the basis of their action on lactose i.e. lactose Edwardsielleae Edwardsiella
fermenters, late lactose fermenters and non-lactose
Salmonelleae Salmonella
fermenters. The lactose fermenting property is observed
Citrobactereae Citrobacter
on a medium (MacConkey's agar) containing lactose
Klebsielleae Klebsiella, Entrobacter, Hafnia,
and neutral red. The organisms fermenting lactose form
Pantoea, Serratia
acid and in acidic pH, neutral red (indicator) becomes
Proteeae Proteus, Morganella, Providencia
red in colour, therefore, the bacterial colonies are red
Yersinieae Yersnia
or pink and those of non-lactose-fermenting bacteria
are pale. The major intestinal pathogens (Salmonella, The species are further classified into biotypes,
Shigella) are non-lactose fermenters. The ma_i ority of serotypes, bacteriophage types and colicin types.
the commensal intestinal bacilli are lactose fermenting The differentiating features of various genera of
and the most common member is the 'colon bacillus' en tero bactriaceae are shown in Table 31.1.
or Escherichia coli. All lactose fermenting enteric bacilli
(Escherichia, Klebsiella, Enterobacter and Citrobacter) II. ESCHERICHIA COLI
were called coliform bacilli. The heterogenous group The genus is named after Escherich who first isolated the
of late lactose fermenters was called paracolon bacilli. bacillus under the name Bacterium coli commune (1885).
Although classification based on lactose fermentation is Esch. coli is the type species of the genus Escherichia.
not acceptable yet this had a practical value in diagnostic Unlike other coliforms, Esch. coli is a parasite inhabiting
bacteriology to differentiate members of enteroba- only in the human or animal intestine.
cteriaceae.
Three widely used classification of Enterobacteriac eae A. Morphology
(Bergey's manual; Kauffmann; Edwards-Ewing) have Esch. coli is a Gram negative bacillus measuring 1- 3 µm
certain differences but the basic approach is the same. x 0.4-0.7 µm. Most strains are motile by peritrichate
The family is first classified into group or tribe. Each flagella. It is non-sporing and non-capsulated.
Table 31.1 Differentiating Features of Important Genera of Enterobacteriaceae
OS
:.e =
OS
a.,
.
a., OS
OS
OS
.
a.,
tlOS
OS
·oOS
tl
. =on
fl)
-e
·;;; =
i:.;
= =
.=
·;: ,e OS a.,
·;;; ,.Q Q,l a.,
Properties Q,l ,.Q 0
e OS ,e
-= :.e
-= on ·s:
,.Q
OS .§ a.,
i:.;
fl)
,e
u OS
00
00 ::;a
Q,l
0
e
Gas in glucose + + + + + + V + + V
Acid from lactose + + + +
Motility + + + + + + + + +
Indole + + V V V + +
Urease + V + + V
PPA test + + +
Citrate + + + + + V V V
H 2S + + + +
Lysine decarboxylase + + + V V +
V, Variable
Important exceptions:
I. S. typhi does not produce gas from sugars.
2. Sh. sonnei is late lactose fermenter.
3. S. typhi, S. paratyphi A and some other species of salmonella are citrate negative.

Ch 31 : Enterobacteriaceae _ _ _ __ _ _ _ _ __ _ _ _ _ __ __ _ _ __ _ _ _ _ _ _ _ 245
B. Culture K antigens were previously divided into three classes as L,

It is an aerobe and facultative anaerobe and grows A and B on the basis of (i) effect of heat on agglutinability
on ordinary culture medium at optimum temperature (ii) antigenicity (iii) antibody binding power of bacterial
of 37°C (temperature range 10--40°C) in 18- 24 hours. strains carrying them. Only one K antigen, L, A or B is
Colonies of some strains show beta haemolysis on blood present in any one strain.
agar. On MacConkey's medium, colonies are pink due to (i) L antigen: It is thermolabile and its capacity to
lactose fermentation (LF or lactose fermenter colonies). combine with specific antibody is lost.
In general, colonies are circular, moist, smooth with entire (ii) A antigen: It is thermostable and is usually associated
margin and non-mucoid unlike colonies of Klebsiella with well marked capsule.
which are mucoid. In liquid medium, growth occurs as (iii) B antigen: It is heat labile but antibody binding
uniform turbidity. power remains unaffected.
In the modern usage the term 'K ' antigen refers to
C. Biochemical Reactions the acidic polysaccharide capsular (surface) antigens. 103
They ferment most of the sugars (glucose, lactose, K antigens of Esch. coli are described. 'K' antigen is now
mannitol, maltose) with production of acid and gas. divided into two groups- I and II (Table 31.2). Group
Typical strains do not ferment sucrose. Indole and methyl I and Group II antigens correspond to the old A and
red (MR) reaction are positive but Voges- Proskauer (VP) L antigens.
and citrate utilisation tests are negative (IMViC ++ -
Table 31.2 Differentiating Properties of Group I and II
- ). Urea is not split, gelatin is not liquified, H 2S is not
antigens of Esch. coli
formed and growth does not occur in KCN medium.
The important biochemical reactions are summarised as Properties Group I Group II
follows: Molecular weight More than Less than 50,000
100,000
Glucose Lactose Mannitol Sucrose
Heat stability (100°C) Stable Labile
AG + +
Indole Urease Citrate MR VP 0 groups 08, 09 Many
+ +
Electrophoretic Low High
mobility
Acidic content Hexuronic Glucuronic acid,
Serotyping of Esch. coli is based on the presence of 0 acid, pyruvate phosphate, KDO
(somatic antigen), K (capsular antigen) and H (flagellar NeuNac
antigen) antigens detected by agglutination reactions.
Another antigen present is F (fimbrial) antigen. KDO Ketodeoxyoctonate
NeuNac N-acetylneuraminic acid
1. Somatic Antigen (0 Antigen)

These are heat stable, lipopolysaccharide antigens Role of K antigens
of cell wall and 173 0 antigens have been described (i) They cause 'O' inagglutinability by homologous
which are designated as 1, 2, 3, 4 and so on. For 0 antisera.
agglutination, the cultures should be boiled to overcome (ii) K antigens act as virulence factor by protecting
inagglutinability caused by K antigens. Numerous cross- bacilli from the killing action of antibody and
reactions occur between individual Esch. coli O antigens, complement and also by impending phagocytosis.
and between these and O antigens of other genera of Most strains of intestinal Esch. coli do not possess K
family Enterobacteriaceae ( Citrobacter, Salmonella , antigens. Of the strains possessing these antigens, most
Shigella and Yersinia). The normal colon strains of Esch. are of the L type.
coli belong to 'early' 0 groups (1 , 2, 3 etc.) and the
enteropathogenic strains belong to the 'latter' group (26, 3. Flagellar Antigen (H Antigen)
55, 86, 111 etc.) . These are thermolabile and 75 H antigens have been
described . All of them are monophasic. For H antigen
2. Capsular Antigen (K Antigen) determination, the organisms have to be grown in
This term was used collectively for the surface or capsular semisolid agar. There are very few cross-reactions between
antigens that cause O inagglutinability. These 'capsular' H antigens of Esch. coli and those of other members
structures are not demonstrable by light microscopy. Thus belonging to the family Enterobacteriacae.

246 - - - - - - - - - - - - - - - - - - - - - - - - - - - Unit Ill: Systemic Bacteriology
4. Fimbrial Antigen (F Antigen) (i) Heat labile toxin (LT)

These are thermolabile proteins and heating the organisms It is a heat labile protein and closely resembles
at 100°C leads to detachment of fimbriae. The F antigen enterotoxin produced by V. cholerae. LT is composed
has no role in antigenic classification of Esch. coli. of one enzymatically active polypeptide A (A for active)
Esch. coli possesses common fimbriae (pili) which subunit and five identical B (B for binding) subunits.
are chromosomally determined and sex pili, which The B subunits bind to the Gm 1 ganglioside receptor
are determined by conjugative plasmids and appear at the brush border of epithelial cells of small intestine
to be organ of conjugation. Type I fimbriae mediate and facilitates the entry of subunit A. The subunit A
adhesion of bacterium to cells that contain mannose is activated to yield two fragment- A 1 and A 2. The
residue. Such adhesion enhances bacterial pathogenicity A 1 fragment activates adenyl cyclase in the enterocyte.
e.g. type I fimbriae and its possible role in urinary tract The activation of adenyl cyclase converts adenosine
infection. triphosphate (ATP) to cyclic adenosine 5'- monophosphate
Several fibrin structures resembling fimbriae are (cAMP). The marked increase of cAMP results in intense
described in Esch. coli. These cause a mannose-resistant and prolonged hypersecretion of water and chlorides and
haemagglutination. These structures probably play a inhibits the reabsorption of sodium. The intestinal lumen
significant role in the pathogenesis of diarrhoeal disease is distended with fluid and hypermotility leads to profuse
and in urinary tract infection. These include K88, K99 watery diarrhoea.
and colonisation factor antigens (CFAs). They are Occasional strains of Esch. coli produce two types of
plasmid determined. K88 causes enteritis of piglets and LT i.e. LT-I and LT- II. LT- II has similar biological action
K99 produces diarrhoea in calves and lambs. CFAs are as LT-I but does not react with LT- I antiserum.
detected in some enterotoxigenic strains of Esch. coli that
cause human diarrhoea. Till now, four CFAs designated as (ii)Heat-stable toxin (ST)
CFA I, CFAII, CFA III and CFA IV have been described. In contrast to LT, ST is a low molecular weight polypeptide
They can be detected by serological tests (agglutination and poorly imrnunogenic. Two types of ST are known,
and immunodiffusion) with specific antisera. STI (or STA) and STII (or ST 8 ). STI stimulates fluid
secretion in the gut through the mediation of cyclic
E. Antigenic Typing guanosine monophosphate (cGMP). The mode of action
On the basis of O antigen, Esch. coli has been subdivided of STII is not known. ST acts more rapidly than LT.
into a number of O groups. Each O group is then divided STII cannot be detected by ligated rabbit ilea! loop test
into subgroups on the basis of K antigens. Each of these or infant mouse intragastric test, however these tests are
subgroups includes strains with different H antigens. useful in detection of STI.
Thus, the antigenic pattern of a strain is recorded as the Poorly immunogenic nature of ST initially prevented
number of the particular antigen it carries, as for example the development of immunological assays. This problem
0111 : K58 : H12. was overcome by preparing antiserum from toxin coupled
to a hapten. Subsequently ELISA with ST monoclonal
F. Resistance antibodies specific for ST have become available.
Esch. coli is excreted in faeces of humans and animals and
contaminate soil and water. It is killed by moist heat at 2. Haemolysin
60°C usually within 30 minutes. It can be killed by 0.5- 1 Some strains of Esch. coli produce a haemolysin which
part per million (ppm) chlorine in water. It can survive can lyse erythrocytes of some species. A larger proportion
for several days in soil, water, dust and air. of Esch. coli recovered from extra-intestinal lesions of
man are haemolytic than those isolated from faeces.
G. Toxins
Some strains of Esch. coli produce enterotoxins, haemolysin 3. Verocytotoxin (VT)
and Verocytotoxin. It is also called Shiga like toxin (SLT). Biological, physical
and antigenic properties·of VT are similar to Shiga toxin
1. Enterotoxins produced by Sh. dysentriae type 1. It is of two types,
Enterotoxigenic strains of Esch. coli (ETEC) produce one VTl which is neutralised by antiserum to Shiga toxin and
or both of two enterotoxins, a heat-labile toxin (LT) and VT2 that resists neutralisation by this antiserum. They
a heat-stable toxin (ST) . Production of both LT and ST are cytotoxic to Vero and He/a cells, enterotoxic in rabbit
is plasmid (ent plasmid) mediated. ilea! loop and show paralytic-lethality in mouse. VT is

Ch 31: Enterobacteriaceae _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ 247
also composed of A and B subunits. VT production is faecal flora spreading to the perineum and from there
found to be phage encoded in several Esch. coli strains. they ascend into the bladder.
Besides VTl and VT2, there is another toxin named The other commonly encountered bacteria in UTI
VT2v which is human and porcine variants. VT2v toxin are Klebsiella, Proteus, Citrobacter and those which
differs from VTl and VT2 in that it is not cytotoxic to rarely produce UTI are salmonellae, edwardsiellae and
Hela cells and is not phage encoded (Table 31.3). Enterobacter. The Gram positive organisms that can
cause UTI include Staph. aureus, coagulase negative
Table 31.3 Differentiating features of verocytotoxins staphylococci, Str. faecalis, Str. pyogenes, Str. milleri, Str.
VTl VT2 VT2v* agalactiae, other streptococci and anaerobic streptococci.
Synonym SLTI SLT2 SLT2v Rarely Gardnerella vagina/is may cause UTI. Among
Cytotoxicity
fungal causes, Candida albicans may cause UTI in
Vero cells + + +
Hela cells + + immunocompromised patients. The hospital-associated
Phage encoded + + infection following instrumentation, catheterization and
*Human and porcine variants. other procedures, is mostly caused by Pseudomonas and
Proteus.
H. Pathogenesis
Esch. coli forms a part of normal intestinal flora of 2. Diarrhoea
man and animal. There are four major types of clinical Esch. coli causing diarrhoeal diseases are of five groups.
syndromes which are caused by Esch. coli: (1) urinary They produce diarrhoea with different pathogenic
tract infection (2) diarrhoea (3) pyogenic infections, and mechanisms.
(4) septicaemia.
Pathogenic Esch. coli strains are grouped into two (i) Enteropatlwgenic Esch. coli (EPEC)
categories, namely extraintestinal pathogenic Esch. They cause enteritis in infants, especially in tropical
coli (ExPEC) and intestinal or diarrhoeagenic Esch. countries. In past, several serious institutional outbreaks
coli. ExPEC contains two groups 'urinary pathogenic of diarrhoea had occurred in babies less than 18 months
Esch. coli' (UPEC) and meningitis/sepsis associated old. EPEC adhere tightly to enterocytes, leading to
Esch. coli (MNEC). UPEC strains are a major cause inflammatory reactions and epithelial degenerative
of community acquired urinary tract infections. MNEC changes. Common serogroups of EPEC are, 026, 055, 086,
strains cause neonatal meningitis. There are five groups 0111 , 0114, 0119, 0125, 0126, 0127, 0128 and 0142.
under diarrhoeagenic Esch. coli, namely EPEC, ETEC,
EIEC, EHEC and EAEC. These have been described (ii) Enterotoxigenic Esch. coli (ETEC)
under heading 'Diarrhoea'. These are the strains that form a heat- labile enterotoxin
(LT) or a heat-stable enterotoxin (ST) or both. They are
1. Urinary Tract Infection now known to be a major cause of diarrhoea in children
Esch. coli is the commonest organism responsible for in developing countries and are the most important cause
urinary tract infection (UTI). Most frequent O serotypes of travellers diarrhoea or turista. The name 'travellers'
of Esch. coli causing UTI include 01, 02, 04, 06, 07, 018 diarrhoea refers to diarrhoea in persons from the
and 075. These are also named as nephritogenic strains. developed countries within a few days of their visit to one
Special nephropathogenic potential of these strains of the developing countries. ETEC sometimes causes a
appears to be due to following factors : diarrhoea similar to that produced by Vibrio cholerae.
(i) Polysaccharides of O and K-antigens protect the ETEC also possess colonisation factors which promote
organism from the bactericidal effects of complement their virulence by adhering the organisms to the epithelium
and phagocytes. Strains possessing Kl or K5 antigen of the small intestine. These colonisation factors may be
appear to be more virulent. pili or special type of protein K antigens. Most strains of
(ii) Fimbriae mediate the adherence of the organism to ETEC belong to the serogroups : 06, 08, 015, 025, 027,
uro-epithelial cells. The receptor to which it attaches, 063, 078, 0115, 0148, 0153, 0159 and 0167.
is believed to be a part of the P blood group antigen The identification of ETEC strains only depends on
and therefore it is termed as P fimbriae. the demonstration of toxins. The methods available for
Esch. coli that causes UTI often originates in the the detection of the toxins are listed in Table 31.4. There
intestine of the patient. Route of infection to reach is no biochemical markers available to differentiate ETEC
urinary tract is either the ascending route or the strains from other Esch. coli. In ELISA test, plates are
haematogenous route. The ascending route is through coated with ganglioside GM 1 which is used to capture
248 _ _ _ __ _ _ __ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Unit Ill: Systemic Bacteriology
Table 31.4 Methods for Detection of Enterotoxins of Esch. coli

Test Heat-labile toxin (LT) Heat-stable toxin (ST)
In vivo tests
Ligated rabbit ilea! loop
Reading at 6 hours ± +
Reading at 18 hours +
Infant rabbit bowel + +
Infact mouse intragastric test +
(fluid accumulation)
Adult rabbit skin for vascular permeability factor +
In vitro tests
Steroid production in YI mouse adrenal cell culture +
Changes in Chinese hamster ovary (CHO) cells +
Enzyme linked immunosorbent assay (ELISA) + +•
Solid phase radioimmunoassay (RIA) +

DNA probes + +
Biken test +
• ST ELISA with monoclonal antibodies
LT if present in the specimen. This bound LT is detected EIEC outbreaks have been mistaken for shigellosis.
by adding toxin (LT) specific rabbit antibodies.
(iv) Enterohaemorrhagic Esch. coli (EHEC) or
Biken test Verocytotoxin producing Esch. coli (VTEC)
It is a precipitin test to detect LT directly on bacterial These strains cause haemorrhagic colitis (HC) and
colonies. Bacteria are grown on a special agar medium haemolytic uraemic syndrome (HUS). It is most common
containing rabbit antibodies specific for LT. As bacteria in infants and young children but can occur in all ages.
grow and secrete LT, this toxin binds to the anti-LT It is characterised by marked haemorrhage but fever is
antibodies to form a precipitin line. not always present. Toxin responsible is called 'Verotoxin'
because of its effect on vero cells in culture. These strains
(iii) Enteroinvasiv e Esch. coli (EIEC) belong to serogroup 0157.
Some strains of Esch. coli invade the intestinal epithelial
cells as do dysentery bacilli and produce disease identical to (v) Enteroaggrega tive Esch. coli (EAEC)
shigella dysentery. These have been named enteroinvasive These strains are so named because they appear aggregated
Esch. coli (EIEC). They belong to serogroups: 028, 0112, in a 'stacked brick' formation on HEP- 2 cells or glass.
0124, 0136, 0143, 0144, 0152 and 0164. On instillation into Most of the strains are O-untypable, but many are
the eyes of guinea pigs, EIEC cause keratoconjunctivitis, H-typable. They form a heat stable enterotoxin called
this diagnostic test for EIEC is called Sereny test. Another enteroaggregative heat stable enterotoxin- 1 ( EAST 1).
diagnostic method is the invasion of HeLa or HEp-2
cells in tissue culture. This ability to penetrate cells is 3. Pyogenic Infections
determined by a large plasmid. The plasmid codes for Esch. coli may cause wound infection, peritomt1s,
outer membrane antigens called the virulence marker cholecystitis and neonatal meningitis. It is an important
antigens (VMA) which can be detected by ELISA test. cause of neonatal meningitis.
Recently DNA probes have become available to screen
faeces specimens for EIEC. 4. Septicaemia
EIEC strains are often atypical in biochemical Esch. coli is a very common cause of septicaemia in
reactions such as these may be late lactose fermenter many hospitals and leads to fever, hypotension and ·
or non-lactose fermenter and anaerogenic. These also disseminated intravascular coagulation (endotoxic shock).
resemble antigenically to shigellae. Because of their This condition usually occurs in debilitated patients and
biochemical and antigenic similarity to shigellae, many mortality is very high.
Ch 31: Enterobacteriaceae _ _ _ __ _ _ _ _ _ __ _ _ __ _ _ _ _ _ _ _ _ __ __ _ _ 249
I. Laboratory Diagnosis per ml is calculated. The blood agar gives a quantitative

URINARY TRACT INFECTION measurement of bacteriuria, while MacConkey's medium
Normal urine is sterile, but during voiding may become enables the presumptive diagnosis of the bacterium.
contaminated with commensals of genital tract.
Interpretation of results
1. Specimen Collection Kass (1956) gave a criterion for active bacterial infection
(i) Midstream urine specimen (MSU) of urinary tract as follows:
It is collected preferably prior to administration of Significant bacteriuria - When bacterial count is
antibiotics. Specimen is collected in a sterile container. more than 10 5/ml of a single species.
Before collecting a sample, genitalia should be cleaned Doubtful significance - between 104 to 105 bacteria
with soap and water and men are instructed to retract per ml. Specimen should be repeated for culture.
the foreskin of glans penis whereas women should keep No significant growth - < 103 bacteria per ml and
the labia apart. The first portion of urine is allowed are regarded as contaminant.
to pass, then without interrupting the urine flow, mid-
portion of the stream is collected. The first portion of (iii) Identification
urine adequately flushes out the normal urethral flora. The organisms are identified by colony characters,
Gram's staining, motility, biochemical reactions and slide
(ii) Catheter specimen agglutination test.
Urine should be collected directly from the catheter and
not from the collection bag. The catheter should not (iv) Antibiotic sensitivity test
touch the container. Although a catheter specimen yields Esch. coli and other common urinary pathogens develop
excellent results but catheterisation to obtain urine is not multiple drug resistance and of the transferable variety.
justified because of risk of introducing infection. Antibiotic sensitivity is necessary to administer proper
antibiotics.
(iii) Urine specimens from infants
A clean catch specimen after cleansing of genitalia is (v) Other methods of diagnosing urinary tract
preferred. Another procedure of collecting specimen in infection
infants is by supra-pubic aspiration. Several screening methods have been introduced and these
are described in Chapter 69.
2. Transport
As urine is a good culture medium, specimens after DIARRHOEA
collection should reach the laboratory with minimum Laboratory diagnosis of Esch. coli diarrhoea has been
delay, if it is not possible, the specimen is to be refrigerated complex as there is no single test which can identify all
at 4°C. strains of Esch. coli responsible for diarrhoea. Faeces
or rectal swab is plated directly on blood agar and
3. Laboratory Methods MacConkey's medium. After overnight incubation, growth
Part of the specimen is used for bacteriological culture and on culture media is identified by colony morphology, Gram
the rest is examined immediately under the microscope. staining, motility and biochemical reactions. Esch. coli
colonies are emulsified in saline on a slide and tested by
(i) Microscopy agglutination with polyvalent and monovalent OB antisera
Urine is centrifuged and deposit is examined under the against entero-pathogenic (EPEC) serotypes. It is essential
microscope for detecting pus cells, erythrocytes, epithelial to test at least ten isolated colonies by agglutination as
cells and bacteria. more than one serogroups may be present in faecal plates.
Special indicator media may be employed for
(ii) Culture specific serotypes with distinctive properties. A modified
Most laboratories use a semiquantitative method (standard MacConkey's medium in which sorbitol is incorporated
loop technique) for culture of urine specimens. instead of lactose, has been employed for the detection of
Standard loop technique serotypes O157:H7, 055 :BS and 0lll:B4 as these do not
A standard calibrated loop is used to culture a fixed volume ferment sorbitol unlike most other Esch. coli strains.
of uncentrifuged urine. Blood agar and MacConkey's agar The identification of ETEC depends on demonstration
are used and incubated at 37°C for 24 hours. Next day, of LT and ST. The tests for detection of toxins are shown
the number of colonies grown is counted and total count in Table 31.4.

250 - - - - -- - - - - -- - -- - - - -- - -- - - -- - Unit Ill: Systemic Bacteriology
VTEC can be detected by its cytotoxic effect on only species of the genus and the term tarda refers to
Vero and HeLa cells, enterotoxin effect in rabbit ileal slow or weak fermentation of sugars by this bacteria.
loop and its lethality in mouse. It can also be detected It is a motile, Gram negative bacillus, non-capsulated
by DNA probes for the genes encoding VTl and VT2. and ferments only glucose and maltose. It forms indole
PCR with VT specific primers has also been used to and H2S, utilises citrate and decarboxylates lysine and
detect VTEC. ornithine.
The demonstration of EIEC is more difficult. E. tarda is normal intestinal inhabitant of cold
It can be detected by sereny test. Simpler tests using blooded animals. It has been recovered occasionally from
HeLa cells or HEp2 cells have now been standardised. the faeces of healthy persons. It is an occasional cause of
Monolayers of these cells are exposed to suspension of wound infection in man. However, its role in causation
organisms. After an appropriate infection period, cells are of diarrhoea, meningitis has yet to be established.
examined microscopically for the presence of intracellular
orgamsms. IV. CITROBACTER
EAEC can be detected on HEp-2 cells which are These motile, Gram negative, citrate positive bacilli
exposed to Esch. coli strains and 'allowed' to adhere to are normal inhabitants of intestine. They grow well on
these cell monolayers in-vitro. The pattern of adhesion ordinary media producing smooth convex colonies, 2-4
is observed by microscopy of these exposed cells. mm in diameter. On MacConkey agar, they form pale
An aggregative adhesion gene probe has also been colonies. They ferment lactose late or not at all. They
used as a rapid means of screening these strains. are indole positive or negative, MR positive, VP negative,
For more details on diarrhoeal diseases, refer to urease weak positive and do not decarboxylate lysine but
Chapter 70. most strains decarboxylate ornithine.
Citrobacter has three species. C. freundii, C. koseri
PYOGENIC INFECTIONS ( C. diversus) and C. amalonaticus. C. freundii gives typical
The specimens are usually pus and wound swab. Cultures reaction with production of H2S whereas C. amalonaticus
are made on MacConkey's agar and the isolate is identified does not form H 2S.
by colony morphology, staining, motility and biochemical Some strains (Ballerup-Bethesda group) exhibits
reactions. extensive antigenic sharing with salmonellae and may
lead to confusion in diagnostic laboratory. Vi antigen of
SEPTICAEMIA Bhatnagar strain of citrobacter is serologically identical
Diagnosis depends on the isolation of the organism by with the Vi antigen of S. typhi, and S. paratyphi C.
blood culture and its identification by colony morphology, However, these citrobacter strains can be distinguished
staining, motility and biochemical reactions. from salmonellae by their negative lysine decarboxylase
and positive KCN reactions.
Alkalescens-Dispar Group Citrobacter may cause urinary tract, gallbladder and
A group of non-motile bacilli associated with dysentery middle ear infections. C. koseri may occasionally cause
were called the Alkalescens- Dispar (AD) group. They neonatal meningitis.
were believed to be related to Shigella. These are lactose
fermenting (similar to Sh. sonnei) but are indole positive. V. KLEBSIELLA
Antigens of AD group appear to be identical with those The genus Klebsiella consists of Gram negative, capsulated,
of Esch. coli. The AD group is now considered as non- non-sporing, non-motile bacilli that grow well on ordinary
motile anaerogenic Esch. coli EIEC. It is now named as media, produce pink mucoid colonies on MacConkey's
Esch. coli inactive. agar. They are widely distributed in nature, occurring as
commensals in human and animal intestines and also as
E., e ·c11fo lber~ll saprophytes in soil.
It is a new species and has been associated with diarrhoeal
diseases in Bangladeshi children. It ferments mannitol but A. Classification
is indole negative. It was previously classified as Shigella The classification of Klebsiella has undergone various
boydii serotype 13. modifications. They have been classified into K pneumoniae,
and K oxytoca (Table 31.5). K pneumoniae is further
III. EDWARDSIELLA subdivided into three subspecies, namely, pneumoniae,
Genus Edwardsiella is separated from Escherichia by ozaenae and rhinoscleromatis. Unlike other subspecies,
its ability to produce hydrogen sulphide. E. tarda is the K oxytoca is indole positive. A third species Klebsiella

Ch 31: Enterobacteriaceae _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __ _ _ _ _ _ _ _ _ _ _ _ _ 251
Table 31.5 Biochemical Reactions of Klebsiella Species

K. pneumoniae subspecies
Property K. oxytoca
pneumoniae ozaenae rhinoscleromatis
Gas from glucose + V +
Acid from lactose + V +
Urease + +
Indole +
Citrate + V +
Malonate + + +
MR + + V
VP + +
Lysine decarboxylase + V +
V, variable
granu/omatis has been included. This was previously 2. Somatic (0) Antigen
known as Calymmatobacterium granulomatis. It causes Klebsiellae contain five (01-05) different somatic or 0
donovanosis and has been described in Chapter 48. antigens in various combinations. Four of these (01 , 03 ,
04 and 05) are identical or closely related to O antigens
B. Morphology of Esch. coli.
These are short, plump, Gram negative, capsulated, non-
motile bacilli. They are about 1- 2 µm x 0.5-0.8 µmin size. F. Methods of Typing
Phage typing, biotyping, bacteriocin (klebocin or
C. Culture pneumocin) typing and resistotyping have been tried.
Klebsiellae grow well on ordinary media at optimum Many Klebsiella strains produce bacteriocins known as
temperature of 37°C in 18- 24 hours. On MacConkey's klebocins or pneumocins which show a narrow range of
agar, the colonies appear large, mucoid and pink to red activity on other Klebsiella strains. Klebocin typing can be
in colour. Mucoid nature of colonies is due to capsular done by the help of liquid preparations of bacteriocins.
material produced by the organism. Klebocin typing and capsular serotyping together may
be very useful for epidemiological studies.
D. Biochemical Reactions
They ferment sugars (glucose, lactose, sucrose, mannitol)
with production of acid and gas. They are urease positive, G. Pathogenesis
indole negative, MR negative, VP positive and citrate K pneumoniae subsp. pneumoniae is the second most
positive (IMViC - - ++ ). These reactions are typical of populous member next to Esch. coli of aerobic bacterial
K pneumoniae subsp. pneumoniae. flora of intestine of humans. It is responsible for severe
bronchopneumonia, urinary tract infections, nosocomial
Glucose Lactose Mannitol Sucrose infections, wound infections, septicaemia, meningitis and
AG + + + rarely diarrhoea. Klebsiella pneumonia is a serious disease
Indole Urease Citrate MR VP with high case fatality. Positive blood cultures can be
+ + +
obtained in about 25 per cent cases of pneumonia. It is
very important pathogen causing nosocornial infections of
E. Antigenic Structure the lower respiratory tract. Some strains of K pneumoniae
1. Capsular (K) Antigen isolated from cases of diarrhoea produce an enterotoxin
On the basis of capsular (K) antigens, the klebsiellae have similar to the heat-stable toxin of Esch. coli.
been classified into 80 (1 - 80) serotypes. Identification of K pneumoniae subsp. ozaenae has been associated
these capsular antigens is usually done by capsular swelling with atrophic rhinitis, a condition known as ozaena. K.
reaction with capsular antiserum. Serotypes 1- 6 are found pneumoniae subsp. rhinoscleromatis causes a granulomatous
most frequently in human respiratory tract infection. disease of the nose and pharynx called rhinoscleroma.

252 _ _ _ _ _ _ __ _ _ _ _ _ _ _ _ __ __ _ _ __ __ __ Unit Ill: Systemic Bacteriology
H. Laboratory Diagnosis meninges. Septicaemia, endotoxic shock and endocarditis

Laboratory diagnosis is done by culturing appropriate have also been reported. In hospital strains, multiple drug
specimens on blood agar and MacConkey agar. The resistance is common.
isolate is identified by colony morphology, Gram staining,
test for motility and biochemical reactions. IX. PROTEUS, MORGANELLA ,
PROVIDENCIA
I. Treatment The tribe Proteeae, in the family Enterobacteriaceae,
Clinical isolates of Klebsiella are resistant to a wide range contains three genera: Proteus, Morganella , Providencia.
of antibiotics like ampicillin, amoxycillin and carbenicillin. The single biochemical character, phenyl pyruvic acid
This resistance is due to R plasmid. Klebsiellae are usually test (PPA test), distinguishes this tribe from all other
sensitive to cephalosporins, trimethoprim, nitrofurantoin, members of Enterobacteriaceae. These bacteria are PPA
co-amoxiclav and gentamicin. However, plasmid mediated test positive i.e. they contain an enzyme phenyl alanine
resistance to gentamicin and various cephalosporins have deaminase which converts phenyl alanine to phenyl
been reported in hospital strains. pyruvic acid (PPA). Most strains of Proteus, Morganella
and Providencia are widely distributed in nature such
as sewage, soil, decomposing organic matter, faeces of
VI. ENTEROBACTE R humans and animals.
Enterobacter is a motile, capsulated, lactose fermenting
bacillus which is indole and MR negative and VP and A. Classification
citrate positive. These characteristics are similar to The genus Proteus was previously classified into five
those of klebsiella species except motility. Two species biotypes namely Pr. vulgaris, Pr. mirabilis, Pr. morganii,
are most commonly encountered in clinical specimens, Pr. rettgeri and Pr. inconstans. Because of high G+C
E. cloacae and E. aerogenes. content of DNA and low level of genetic homology, Pr.
They are widely distributed in water, sewage, soil and morganii, P,: rettgeri and Pr. inconstans an~ placed under
on vegetables. They are also found in human and animal new genera Morganella and Providencia. Pr. vulgaris and
faeces. They may cause urinary tract infections and Pr. mirabilis which are very similar in their properties are
hospital infections. They are occasionally associated with included in the genus Proteus. Another clinically relevant
meningitis and septicaemia. Arninoglycosides are often species is Proteus penneri.
effective in the treatment of Enterobacter infections.
B. Proteus
VII. HAFNIA These are motile, Gram negative bacilli, characterised
Hafnia is a motile, non-lactose fermenting bacillus which by swarming growth on agar. They were named Proteus
is indole and MR negative and VP and citrate positive. because of their pleomorphic character, after the Greek
Only one species of this genus is recognised, H. alvei. God Proteus who could assume any shape.
Biochemical reactions are evident best at 22°C instead of
37°C. It is found in human and animal faeces, sewage, soil 1. Morphology
and water. It is believed to be an opportunistic pathogen These are Gram negative bacilli measuring 1- 3 µm x
and has been recovered from wounds, sputum, abscesses, 0.5 µm in size. They are non-capsulated, non-sporing
urine and blood. and actively motile. They possess peritrichate flagella.
They may present as short coccobacillary forms, long
VIII. SERRATIA and filamentous type especially in young cultures. Many
Serratia is characterised by forming a pink, red or strains possess fimbriae.
magenta, non-diffusible pigment called prodigiosin.
Pigment is formed optimally at room temperature. It is 2. Culture
small motile, Gram negative bacillus and ferments lactose They are aerobic and facultatively anaerobic. They grow
very slowly. It is a saprophytic, found in water, soil and on ordinary media and culture emits a characteristic
food. Only one species is of medical importance- S. putrefactive (fishy or seminal) odour. When grown on
marcescens. Infections due to this species are on increase nutrient agar or blood agar, Pr. vulgaris and Pr. mirabilis
in recent years particularly among hospital patients. It exhibit 'swarming', but it does not occur with Morganella
can lead to serious nosocomial infections particularly in and Providencia species. Swarming of Proteus appears
the newborn and the debilitated persons. These include to be due to vigorous motility of the bacteria although
infections of urinary tract, respiratory tract, wound and the exact cause is yet not established.
Ch 31: Enterobacteriaceae _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __ _ _ __ _ __ _ _ _ _ _ _ 253
Swarming growth presents problems in the diagnostic 5. Antigenic Structure

laboratory when mixed growth is obtained in which Proteus The bacilli possess thermostable, 'O' (somatic) and
is also present. Several methods have been used to inhibit thermolabile 'H' (flagellar) antigens, based upon which,
swarming. These are (i) increasing the concentration of several serotypes have been recognised. Weil and Felix
agar in the medium, raising it to 6% instead of 1 to 2% (1916) observed that flagellated strains of Proteus bacilli
(ii) by incorporation of chloral hydrate (1 :500), sodium grew on agar as a thin surface film resembling the mist
azide (1: 500), boric acid (1: 1000) in the medium. produced by breathing on glass and named this variety
They form smooth, pale or colourless (NLF) colonies as 'Hauch' form (from Hauch, meaning 'film of breath').
on MacConkey's agar and do not swarm on this medium. Non-flagellated variant strains grew as isolated colonies
In liquid medium (peptone water), Proteus produces without the surface film and were named 'Ohne Hauch'
uniform turbidity with a slight powdery deposit and an form (meaning 'without film of breath'). These names
ammonical odour. are abbreviated as the 'H' and 'O' forms. Subsequently,
the terms H and O were used to refer the flagellar and
3. Dienes Phenomenon somatic antigens of other bacilli as well. On the basis of
When two different strains of Proteus species are 0 and H antigens, several serotypes have been recognised
inoculated at different places of the same culture plate, within the different species.
swarming of the two strains remain separated by a narrow, Weil and Felix also noted that certain non-motile strains
visible furrow. However, in case of two identical strains of Pr. vulgaris, called the 'X' strains were agglutinated by
of Proteus, swarming of two coalesce without signs of sera of typhus patients. The sharing of polysaccharide
demarcation. It is known as Dienes phenomenon. It has antigens of Proteus with some rickettsiae forms the
been used to determine the identity or non-identity of basis of the Weil and Felix reaction for the diagnosis
different strains of Proteus. of some rickettsial infections. Three non-motile strains
of Proteus are employed as the antigens for this test- Pr.
4. Biochemical Reactions vulgaris strains OX 2, OX 19 and Pr. mirabi/is OX K.
The distinctive characters of this genus are:
(i) Deamination of phenyl alanine to phenyl pyruvic 6. Pathogenesis
acid (PPA test)-lt is always positive. Proteus species are saprophytic and widely distributed in
(ii) Hydrolysis of urea by enzyme urease-lt is another nature. They also occur as commensals in the intestine.
characteristic of Proteus, but is negative in some They are opportunistic pathogens and may cause many
Providencia strains. types of infections such as urinary tract infection (UTI),
They ferment glucose by producing acid and gas. pyogenic lesions, infection of ear, respiratory tract
Lactose is not fermented. Indole is formed by Pr. vulgaris infection and nosocomial infections. Pyelonephritis due
but is negative in Pr. mirabilis. H 2S is produced by Pr. to Proteus is particularly toxic as the ammonia produced
vulgaris and Pr. mirabi/is. They are MR positive and by the organism interferes with complement and other
VP negative. defence mechanisms. Pr. mirabilis is the predominant and
Species of Morganella and Providencia can be accounts for the most infections in man.
differentiated from Proteus by different biochemical Multiple drug resistant strains carrying R plasmids
reactions (Table 31.6). have become very important in nosocomial infections.
Table 31.6 Biochemical Reactions of Tribe Proteeae

Property Pr. vulgaris Pr. mirabilis Morg. morganii Prov. alcalifacieos Prov. stuartii Prov. rettgeri
Gas from Glucose + + + +

Indole + + + + +
Phenyl pyruvic acid (PPA) test + + + + + +
Urease + + + ± +
H 2S production + +
Ornithine decarboxylase + +
Fermentation of adonitol + ± ±
Fermentation of trehalose ± + ± +

254 _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __ __ _ _ _ Unit Ill: Systemic Bacteriology
7. Laboratory Diagnosis as proteus bacilli are resistant to many of the

(i) Specimens: common antibiotics.
(a) Mid stream urine sample in UTI;
(b) Pus in pyogenic lesions C. Morganella
(ii) Collection: Specimen should be collected in The genus Morganella has one species, M morganii. It
sterile container under all aseptic conditions and does not swarm. It causes urinary tract infection and
transported immediately. nosocomial wound infections.
(iii) Culture: It is cultured on MacConkey agar or
blood agar with 6% agar to inhibit swarming. D. Providencia
Culture media are incubated at 37°C for 18- 24 This genus contains three species seen in clinical infections.
hours. NLF colony are seen on MacConkey agar. Prov. alcalifaciens (formerly P,: inconstans) is sometimes
Peptone water is also inoculated. found in diarrhoeal stools though its aetiological role is
(iv) Gram's staining: Gram negative bacilli which are uncertain. Prov. stuartii is a common cause of urinary tract
non-capsulated and non-sporing. infection and of infection in bums. Prov. rettgeri (formerly
(v) Hanging drop preparation: Actively motile bacilli Pr. rettgeri) sometimes causes nosocomial infection of the
are observed. urinary tract, wounds and burns, Providencia are very
( vi) Biochemical reactions: Most important are PPA resistant, particularly Prov. stuartii which is also resistant
and urease tests. PPA test is positive in all Proteus to disinfectants, making it a major pathogen in burn units.
species. Urease test is also positive in all Proteus
species with exception of Providencia which is E. Laboratory Diagnosis of Morganella and
negative. Other biochemical reactions can be used Providencia
to differentiate various species (Table 31.6). Laboratory diagnosis of species of these two genera is
(vii) Agglutination test: Strain may be agglutinated with similar to that of Proteus species. However, these can
polyvalent group specific sera to confirm it. be differentiated on the basis of different biochemical
( viii) Antibiotic susceptibility test: It is important reactions (Table 31.6).
KEY POINTS
1. Members of family Enterobacteriaceae are aerobic and facultative anaerobic Gram negative enteric bacilli. They
are motile by peritrichous flagella or are non-motile. They grow readily on ordinary media, ferment glucose with
production of acid or acid and gas, reduce nitrates to nitrites, oxidase negative and catalase positive except Sh.
dysenteriae type I which is catalase negative.
2. Some important genera of family Enterobacteriaceae include Escherichia, Salmonella, Shigella, Klebsiella, Proteus,
M organella and Providencia.
3. Escherichia coli forms pink colonies (lactose fermenter colonies) on MacConkey's medium. It ferments most of the
sugars (glucose, lactose, mannitol, maltose) with production of acid and gas. Typical strains do not ferment sucrose.
Indole and methyl red (MR) reactions are positive but Voges-Proskauer (VP) and citrate utilisation tests are negative
(IMVIC ++- ).
4. Toxins produced by Esch. coli include enterotoxins, haemolysin and verocytotoxin.
5. Enterotoxigenic strains of Esch. coli (ETEC) produce one or both of two enterotoxins, a heat-labile toxin (LT) and
a heat-stable toxin (ST).
6. Urinary tract infection, diarrhoea, pyogenic infections and septicaemia are the diseases caused by Esch. coli.
7. Midstream urine specimen (MSU) is collected for diagnosis of urinary tract infection.
8. Esch. coli causing diarrhoeal diseases are of five types namely Enteropathogenic Esch. coli (EP EC), Enterotoxigenic
Esch coli (ETEC), Enteroinvasive Esch. coli (EIEC), Enterohaemorrhagic Esch. coli (EHEC) or Verocytotoxin producing
Esch. coli (VTEC) and Enteroaggregative Esch. coli (EAEC).
9. Klebsiella is a short, plump, Gram negative, capsulated, non-motile bacillus. It forms large, mucoid, pink to red
colonies (lactose fermenter colonies) on MacConkey's agar. It ferments sugars (glucose, lactose, sucrose, mannitol)
with production of acid and gas. It is urease positive, indole negative, MR negative, VP positive and citrate positive
(/MVIC - - + +).

Ch 31 : Enterobacteriaceae _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ 255
10. K. pneumoniae and K. oxytoca are two species of genus Klebsie/la.

11. The tribe Proteeae in the family Enterobacteriaceae contains three genera : Proteus, Morganella, and Providencia.
Phenyl pyruvic acid test (PPA test) distinguishes this tribe from all other members of Enterobacteriaceae. Bacteria
of tribe Proteeae are positive for PPA test.
12. There are two species of proteus i.e. Pr. vulgaris and Pr. mirabilis. These species exhibit 'swarming' when grown on
blood agar. They form smooth, pale or colourless colonies (non-lactose fermenter colonies) on MacConkey's agar. They
are Gram negative, actively motile bacilli.
13. Hydrolysis of urea by enzyme urease is another characteristic of Proteus, but is negative in some providencia
strains.
14. Proteus may cause urinary tract infections ( UT[), pyogenic lesions, infection of ear, respiratory tract infection and
nosocomial infections.
YOU MUST KNOW

1. Classification of Enterobacteriaceae.
2. Morphology, culture characteristics and biochemical reactions of Escherichia coli.
3. Enterotoxins and verocytotoxin of Escherichia coli.
4. Diseases caused by Esch. coli.
5. Different groups of Esch. coli causing diarrhoeal diseases.
6. Differences between heat-labile toxin (LT) and heat stable toxin (ST) of Esch. coli.
7. Laboratory diagnosis of urinary tract infection and diarrhoea caused by Esch. coli.
8. Morphology, culture characteristics and biochemical reactions of Klebsiella sp.
9. Morphology, culture characteristics and biochemical reactions of Proteus sp.
10. Differences between Proteus sp., Morganella sp. and Providencia sp.
STUDY QUESTIONS
1. Discuss the laboratory diagnosis of urinary tract infections caused by Esch. coli.
2. Describe in detail the laboratory diagnosis of diarrhoea caused by Esch. coli.
(a) Antigenic structure of Esch. coli
(b) Enterotoxins of Esch. coli
(c) Verocytotoxin of Esch. coli.
4. Describe the various mechanisms by which Esch. coli produces diarrhoea.
(a) Citrobacter (b) Klebsiella pneumoniae
(c) Enterobacter (d) Serratia.
(a) Classification of tribe Proteeae (b) Dienes phenomenon
(c) Genus Morganella (d) Genus Providencia.

I. Which of the following bacteria is/are member/s of the family Enterobacteriaceae?
(a) Salmonella typhi (b) Citrobacter freundii (c) Yersinia enterocolitica (d) All of the above
2. Which of the following bacteria is/are called coliform bacilli?
(a) Escherichia (b) Enterobacter (c) Citrobacter (d) All of the above

Bacteriology
256 _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __ _ _ _ _ Unit Ill : Systemic
3. Heat labile toxin (LT) of Escherichia coli activates:

(a) Adenyl cyclase (b) Guanyl cyclase (c) Both of the above (d) None of the above
4. Verocytoto xin 1 (VT!) of Escherichia coli is similar to:
(a) Shiga toxin (b) Cholera toxin
(c) Enterotoxi n of Staphyloco ccus aureus (d) Toxin of Bacillus cereus
5. Travellers diarrhoea is caused by:
(a) Enteropath ogenic Escherichia coli (EPEC) (b) Enterotoxi genic Esch. coli (ETEC)
(c) Enteroinva sive Esch. coli (EIEC) (d) Enterohae morrhagic Esch. coli (EHEC)
6. Biken test is used for the detection of:
(a) Heat-labile toxin (LT) of Escherichia coli (b) Heat-stabl e toxin (ST) of Esch. coli
7. Sereny test is used for the identification of:
(a) Enteropath ogenic Escherichia coli (EPEC) (b) Enterotoxi genic Esch. coli (ETEC)
(c) Enteroinvasive Esch. coli (EIEC) (d) Enterohae morrhagic Esch. coli (EHEC)
8. The term 'significant bacteriuria ' is used in urinary tract infection when bacterial count is:
(a) more than 105 bacteria per ml (b) between 104 to 10 5 bacteria per ml
(c) less than 10 3 bacteria per ml (d) None of the above
9. MacConke y's medium with sorbitol instead of lactose is used for the detection of Escherichia coli strains:
(a) 055 : BS (b) 015 : Bl 1 (c) 08 : B70 (d) None of the above
10. Klebsiella may cause:

(a) Pneumoni a (b) Ozaena (c) Rhinoscle roma (d) M of the above
11. Which of the following bacteria produces indole?

(a) Klebsiella pneumonia e subspecies pneumonia e (b) K. pneumonia e subspecies ozaenae
(c) K. pneumonia e subspecies rhinosclero matis (d) K. oxytoca
12. Which of the following bacteria produces red pigment (progidiosin)?
(a) Enterobact er cloacae (b) Hafnia alvei (c) Serratia marcescens (d) Klebsiella pneumonia e
13. Which of the following genera are included in the tribe Proteeae of family Enterobacteriaceae?
(a) Proteus (b) Morganella (c) Providencia (d) All of the above
14. Which of the following has/have positive phenyl pyruvic acid test (PPA test)?
(a) Proteus (b) Morganella (c) Providencia (d) All of the above
culture media?
15. Which of the following methods can be used to inhibit swarming of Proteus strains on solid
(a) Increasing the concentrat ion of agar in the medium
(b) Incorporat ion of chloral hydrate in the medium
(c) Incorpora tion of boric acid in the medium
(d) All of the above
16. Dienes phenomen on is useful to find out the identity or non-identi ty of two strains of:
(a) Proteus (b) Escherichia coli (c) Citrobacter (d) Shigella
17. Which of the following Proteus strains is/are used in Weil-Felix reaction?
(a) Proteus vulgaris OX2 (b) Proteus vulgaris OX19
~M~ ~ili~
18. All of the following are urease test positive except:
(a) Proteus vulgaris (b) Proteus mirabilis (c) Helicobacter pylori (d) Escherichia coli
Answers (MCQs):
1. (d) 2. (d) 3. (a) 4. (a) 5. (b) 6. (a) 7. (c) 8. (a) 9. (a) 10. (d)
17. (d) 18. (d)
...
11. (d) 12. (c) 13. (d) 14. (d) 15. (d) 16. (a)
____ ____

Chapter 32
SHIGELLA
Shigella
C. Resistance D. Antigenic Structure
E. Classification F. Toxins
G. Bacteriophage and Colicin Typing H. Pathogenesis
I. Laboratory Diagnosis J. Treatment
K. Prevention and Control
SHIGELLA xylose lysine deoxycholate (XLD) agar is a better selective

The organisms of genus Shigella are exclusively parasites medium than DCA. It is less inhibitory to Sh. dysenteriae
of human intestine and other primates and cause bacillary and Sh. flex neri. On this medium colonies of shigella
dysentery in man. The name Shigella is named after Shiga appear red without black centres. The organisms which
who isolated the first member of this genus in 1896 from decarboxylate lysine, forming alkaline amines produce
epidemic dysentery in Japan. red colonies. The bacteria which also produce H 2 S have
black centres in the colonies.
A. Morphology Salmonella-Shigella (SS) agar is a highly selective
Shigellae are short, Gram negative bacilli measuring about medium for the isolation of Salmonella and Shigella.
1- 3 µm x 0.5 µm. They are non-motile, non-capsulated Colonies of Shigella on this medium are colourless
and non-sporing. (due to non-lactose fermentation) with no blackening,
while those of Salmonella are colourless with black
B. Culture centres. This medium contains bile salts, sodium citrate,
They are aerobes and facultative anaerobes and can grow sodium thiosulphate, ferric citrate, lactose and neutral
on ordinary media. Optimum temperature for growth is red (indicator). The high bile salt concentration and
37°C and pH 7.4. They can grow at temperature range of sodium citrate, in this medium, inhibit all Gram positive
10--40°C. After overnight incubation, colonies are about bacteria and coliforms. Sodium thiosulphate is a source
2 mm in diameter, circular, convex, smooth and translucent. of sulphur. Bacteria that produce H 2S are detected by
Colonies on MacConkey's agar and deoxycholate citrate the black precipitate formed with ferric citrate. Lactose
agar (DCA) are colourless (non-lactose fermenting- NLF) fermentation can be detected by the presence of neutral
except in case of Sh. sonnei which forms pink colonies red. Lactose fermenting colonies appear red.
due to late lactose fermentation . DCA is a useful selective Hektoen-enteric (HE) agar is a direct plating medium
medium to isolate these organisms from faeces. However, for the isolation of Salmonella and Shigella from the
257
Table 32.1 Differentiating features of Shigella species

Fermentation of
Lysine decar- Ornithine
Subgroup lndole Sero types
Lactose Mannitol Sucrose Dulcitol boxylase decar-boxylase
Sh. dysenteriae d 15
Sh. fiexneri A d 6+2 variants
Sh. boydii A d d 19
Sh. sonnei A* A A* + One
A- Acid; d- Variable; *- Late fermenter
faeces. Colonies of Shigel/a appear green with colour subgroup A (mannitol negative) from subgroups B, C,
fading to the periphery. Salmonella colonies are blue- and D (all of them are mannitol positive) (Table 32.1).
green typically with black centres. This medium contains
bile salts, lactose, sucrose, sodium thiosulphate, ferric 1. Subgroup A (Sh. dysenteriae)
ammonium citrate, acid fuchsin and thymol blue. The high It is divided into 15 serotypes. Serotype 1 is the bacillus
bile salt concentration inhibits Gram-positive bacteria and originally described by Shiga (Sh. shigae). It is the only
many coliforms. Sodium thiosulphate is a sulphur source member of the family that is always catalase negative.
and H 2 S gas is detected by ferric ammonium citrate. Serotype 2 is also known as Sh. schmitzi. It forms indole
in contrast to type 1 which is indole negative. Serotype
Enrichment medium 3- 7 used to be known as Large- Sachs group after the
Selenite F broth: Sodium selenite in this medium inhibits name of Large and Sachs who described these serotypes
coliform bacilli while permitting salmonellae and shigellae in India.
to grow. It is recommended for the isolation of these
organisms from faeces. 2. Subgroup B (Sh. flexneri)
Gram-Negative (GN) broth: Most strains of Shigella and This subgroup is named after Flexner, who first isolated
Salmonella species grow in this medium. It is very useful Sh. fiexneri in Philippines (1900). It is antigenically the
enrichment medium for the isolation of these organisms most complex among shigellae. They have been classified
from faeces. into six serotypes (1-6) based on specific antigens. Each of
these is further divided into subtypes (e.g. la and lb). In
C. Resistance addition to six serotypes, two antigenic 'variants' (X and Y)
Shigellae are killed at 56°C in one hour and by 1% are recognised, which do not possess type specific antigens.
phenol in 30 minutes. They remain viable in water and Serotype 6 occurs in three biotypes, some of which
ice for 1 to 6 months. Boiling or chlorination of water form gas from sugars (Table 32.2). Gas formation is
and pasteurisation of milk are effective and destroy the an important characteristic as all other Shigellae are
bacilli. anaerogemc.
Table 32.2 Biotypes of Sh. flexneri type 6

Shigellae possess a large number of somatic 'O' antigens Biotype lndole Fermentation of
and some strains produce K antigens which cover the Glucose Mannitol
O-antigen. K antigens are not relevant in typing but Boyd 88 A A
may interfere with agglutination by O antisera. Fimbrial
Manchester AG AG
antigens are also present. There is some antigenic sharing
within some members of the genus as well as between Newcastle A or AG
shigellae and Esch. coli. It is, therefore, important to A- Acid; AG- Acid and Gas
identify shigellae on the basis of antigenic and biochemical
properties and not by slide agglutination alone. 3. Subgroup C (Sh. boydii)
It is named after Boyd, who first described these strains
E. Classification from India ( 1931 ). It resembles Sh. fiexneri biochemically
Shigellae are divided into four subgroups (A, B, C & D) but not antigenically. Nineteen serotypes (numbered 1 to
based on a combination of biochemical and serological 20 except 13) of Sh. boydii have been identified. Sh. boydii
specificity. Mannitol fermentation reaction distinguishes serotype 13 has been reclassified as Esch. albertii.

Ch 32: Shigella _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ 259
4. Subgroup D (Sh. sonnei) host of shigellae. The minimum infective dose is as few as
Sonne described Sh. sonnei in Denmark (1915). It is 10-100 bacilli. Sh. dysenteriae type 1 causes most severe
indole negative but ferments lactose and sucrose late. It is form of dysentery usually associated with toxaemia.
antigenically homogeneous but may occur in two forms- The infection due to Sh. fiexneri and Sh. boydii are less
Phase I and Phase II. Sh. sonnei has been classified into severe and prevalent in tropical countries including India.
many colicin types. It causes the mildest form of bacillary Sh. sonnei causes the mildest form of the disease and is
dysentery. In many cases, the disease may occur as a common in Western countries like Britain. It is more
mild diarrhoea. Sh. sonnei infection is the most common frequently seen in children.
shigellosis in advanced countries. The incubation period of the disease is usually less
than 48 hours but varies between 1- 7 days. Mode of
F. Toxins infection is by ingestion of contaminated food .
1. Endotoxin The food or drink is contaminated by faeces of cases
All shigellae release endotoxin after autolysis. Endotoxin or of carriers. The organisms infect the epithelial cells of
has irritating effect on intestinal wall which causes the terminal ileum and colon and multiply inside them.
diarrhoea and subsequently intestinal ulcers. The distal parts of the colon are severely affected. Then
they spread laterally into adjacent cells as well as to lamina
2. Exotoxin properia. Inflammatory reaction develops and there is
Sh. dysenteriae type 1 produces a powerful exotoxin. It acts necrosis of surface epithelial cells. The necrotic epithelia
as enterotoxin and as well as neurotoxin. As enterotoxin, become soft and friable and are sloughed out leaving
it induces fluid accumulation, and as neurotoxin it behind transverse superficial ulcers. The organisms tend
damages the endothelial cells of small blood vessels of to remain localised in the gut wall and usually do not lead
the central nervous system which results in polyneuritis, to bacteraemia. Invasive property of Shigella is related to
coma and meningism. The exotoxin is not primarily a the presence in the bacillus of large plasmids coding for
neurotoxin. outer membrane proteins which are responsible for cell
penetration. These proteins are named virulence markers
3. Verocytotoxin (VT) antigens (VMA). VMA can be detected by ELISA. The
Sh. dysenteriae type 1 also produces a cytotoxin which invasive property can be demonstrated by its ability to
acts on Vero cells and is named verocytotoxin or VT. penetrate HeLa or Hep-2 cells.
Two verocytotoxins, VTl and VT2 are recognised. VTl is Sh. dysenteriae type 1 causes toxaemia due to
identical to exotoxin produced by Sh. dysenteriae type 1. production of exotoxin. The toxin causes accumulation
VTl comprises two subunits- A and B. Subunit B binds of fluid leading to severe diarrhoea (enterotoxic action)
the cytotoxin to cells while subunit A inhibits protein and may cause complications like polyneuritis, arthritis,
synthesis. Genes coding for VT production are located in conjunctivitis and parotitis. Haemolytic uraemic syndrome
the chromosome. VTl , VT2 or both are also produced by may occur as a rare complication in severe cases. The
certain strains of enterohaemorrhagic Esch. coli causing severity of the disease varies from acute fulminating
haemorrhagic colitis. dysentery to mild diarrhoea. The term shigellosis has
been employed to include the whole spectrum of the
G. Bacteriophage and Colicin Typing disease caused by shigellae. In India, Sh. fiexneri (50- 85%)
The determination of serotype within subgroup A, B is the most common isolate followed by Sh. dysenteriae
and C is sufficient for epidemiological study and further (8- 25%), Sh. sonnei (2- 24%) and Sh. boydii (0-8%).
subdivision is not necessary. However, each serotype of Several localised outbreaks of Sh. dysenteriae type 1
Sh. fiexneri is further subdivided into 123 phage types were observed in India from 1974, followed by extensive
by bacteriophage typing. epidemics in various states. These epidemic strains showed
Sh. sonnei consists of a single serotype which can multiple drug resistance.
be further subtyped either by phage typing or by colicin
typing. Phage typing being cumbersome, therefore, colicin I. Laboratory Diagnosis
typing has been adopted by most centres. Sh. sonnei Diagnosis depends upon isolating the bacillus from
strains are classified into 26 colicin types and each type faeces.
is characterised by the production of a specific colicin. 1. Specimens
Fresh stool is collected. Rectal swabs are not satisfactory.
H. Pathogenesis The ideal specimen is a direct swab of an ulcer taken
Shigellae cause bacillary dysentery. Man is the only natural under sigmoidoscopic examination.

260 _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __ __ _ _ _ __ _ Unit Ill: Systemic Bacteriology
2. Transport 7. Slide Agglutinat ion Test

Specimens should be transported immediately and Identification of shigella is confirmed by slide agglutination
inoculated without delay. If delay is inevitable, specimens with polyvalent antisera and monovalent sera. Then type
should be transported in a suitable medium such as Sach's specific antisera belonging to subgroups A, B or C is
buffered glycerol saline, pH 7.0-7.4. Alkaline transport used for agglutination test.
media used for vibrios are inhibitory for shigellae.
8. Colicin Typing
It is done for subgroup D (Sh. sonnei) strains.
3. Direct Microscop y
Saline and iodine preparation of faeces show large J. Treatment
number of pus cells, erythrocytes and macrophages. Uncomplicated shigellosis is a self limiting condition
Parasitic causes of dysentery may also be excluded by that usually recovers spontaneously. Dehydration has to
this examination. be corrected promptly particularly in infants and young
children.
4. Culture Routine antibacterial treatment is not indicated but
Specimen is inoculated on selective media like MacConkey's antibiotics should be reserved for the severe toxic cases. In
agar, DCA or XLD agar. Selenite F broth (0.4%) is used very serious infections, nalidixic acid has been life saving.
as enrichment medium which permits rapid growth of The wide prevalence in shigellae of R factors conferring
enteric pathogens while inhibiting the growth of normal resistance to multiple antibiotics makes antibiotic therapy
flora like Esch. coli for 6-8 hours. Subculture is done on futile. Many strains are still sensitive to nalidixic acid
solid media from selenite F broth. All the solid media or norfloxacin. There is no convincing evidence that
are incubated at 37°C for 24 hours. antibacterial therapy either hasten recovery or prevent
the carrier state.
5. Colony Morpholog y and Staining K. Prevention and Control

Colourless (NLF) colonies appear on MacConkey's agar As man is a major source of infection, control consists
which are further confirmed by Gram staining, hanging essentially in improvement of environmental sanitation.
drop preparation and biochemical reactions. Shigellae The role of vaccination in the prevention of dysentery
are Gram negative bacilli and are non-motile. is still uncertain. Killed vaccines proved to be toxic and
ineffective. Many live oral vaccines have been tested
6. Biochemical Reactions including avirulent mutants, streptomycin dependent
Any non-motile bacillus that is urease, citrate, H 2S and strain and Shigella-Esch. coli hybrids. The presence of
KCN negative should be further confirmed by various several sero-groups and types would appear to preclude
biochemical tests (Table 32.1) . effective immunisation.
KEY POINTS
1. The organisms of genus Shigella cause bacillary dysentery in man. They are short, Gram negative, non-motile
bacilli.
2. Shigella has four species i.e. Sh. dysenteriae, Sh. flexneri, Sh. boydii and Sh. sonnei.
3. They form colourless colonies (Non-lactose fermenting- NLF colonies) on MacConkey's agar. Exception is Shigella
sonnei which forms pink colonies due to late lactose fermentation.
4. Sh. dysenteriae type 1 produces a powerful exotoxin which acts as enterotoxin and as well as neurotoxin. It also
produces a cytotoxin which acts on Vero cells and is named Verocytotoxin or VT.
5. The term shigellosis has been employed to include the whole spectrum of the disease caused by shigellae.
6. Uncomplicated shigellosis is a self limiting condition that usually recovers spontaneously.

Ch 32 : Shigella _ __ __ __ __ _ __ _ _ _ _ _ _ __ _ _ _ _ __ _ _ __ _ _ 261
YOU MUST KNOW

1. Morphology and culture characteristics of Shigella sp.
2. Subgroups of shigellae.
3. Laboratory diagnosis of dysentery caused by shigella.
STUDY QUESTIONS
1. Discuss the laboratory diagnosis of dysentery caused by shigella.
(a) Classification of shigella (b) Colicin typing.

1. Which of the following is used as an enrichment medium for the isolation of Shigella from the faeces?
(a) Selenite F broth (b) Alkaline peptone water
(c) Taurocholate tellurite peptone water (d) None of the above
2. Which of the following Shigella species does not ferment mannitol?
(a) Shigella dysenteriae (b) S. boydii
(c) S. sonnei (d) None of the above
3. Which of the following biotypes of Shigella flexneri type 6 ferment mannitol with acid and gas?
(a) Boyd 88 (b) Manchester
(c) Newcastle (d) None of the above
4. Which of the following Shigella is also known as Shigella shigae?
(a) Shigella dysenteriae serotype 1 (b) Shigella dysenteriae serotype 2
(c) Shigella dysenteriae serotype 3 (d) Shigella dysenteriae serotype 4
5. Which of the following Shigella is also known as Shigella schmitzi?
(a) Shigella dysenteriae serotype 1 (b) Shigella dysenteriae serotype 2
(c) Shigella dysenteriae serotype 3 (d) Shigella dysenteriae serotype 4
6. Large-Sachs group of Shigella contains:
(a) Shigella dysenteriae serotype 1 and 2 (b) Shigella dysenteriae serotype 3 to 7
(c) Shigella flexneri (d) None of the above
7. Which of the following serotype of Shigella dysentriae produce verocytotoxin?
(a) Serotype 1 (b) Serotype 2
(c) Serotype 3 (d) Serotype 7
8. Which Shigella species is late fermenter of lactose and surcose?
(a) Shigella dysenteriae (b) Sh. flexneri
(c) Sh. boydii (d) Sh. sonnei
Answers (MCQs}:
1. (a) 2. (a) 3. (b) 4. (a) 5. (b)
6. (b) 7. (a) 8. (d)
____ ... ____

Chapter 33
SALMONELLA
I. Salmonella
E. Antigenic Structure F. Antigenic Variation
G. Classification H. Kauffmann-White Scheme
I. Toxin J. Pathogenesis
K. Epidemiology L. Laboratory Diagnosis
M. Bacteriophage Typing N. Biotyping
0. Plasmid Typing P. Treatment
Q. Drug Resistance R. Prophylaxis
II. Salmonella Gastroenteritis
A. Source of Infection B. Clinical Features
C. Laboratory Diagnosis D. Control
E. Treatment
Ill. Salmonella Septicaemia
The salmonellae are primarily intestinal parasites of I. SALMONELLA

vertebrates and which infect man, leading to enteric Salmonellae produce three main types of diseases m
fever, gastroenteritis and septicaemia. The most important man:
member is Salmonella typhi, the causative agent of typhoid 1. Enteric Fever
fever. Man is the only natural host for S. typhi and S. The causative agents of enteric fever are Salmonella
paratyphi A whereas most of the other salmonellae are typhi (causing typhoid fever) or S. paratyphi A, B, and
chiefly pathogenic in animals like poultry, pigs, cattle C (causing paratyphoid fever). The term enteric fever
etc. includes both typhoid and paratyphoid fever.
Salmon and Smith (1885) isolated the hog cholera 2. Gastroenteritis
bacillus (S. cholerae-suis) and the term Salmonella was Salmonellae under this group are essentially animal
coined from the name of Salmon. Interestingly, hog- parasites but can also infect man. These include
cholera disease was subsequently proved to be a viral S. typhimurium, S. enteritidis, S. newport, S. dub/in and
infection in which S. cholerae-suis was a common S. thompson.
secondary invader. 3. Septicaemia
262
Ch 33: Salmonella _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __ _ __ __ _ _ _ _ _ _ __ _ 263
The commonly associated Salmonella is S. choleraesuis D . Resistance

but other species may also cause septicaemia. The salmonellae are killed at 60°C in 15 minutes. Boiling,
chlorination of water and pasteurisation of milk destroy
A. Morphology the bacilli. They survive in water, ice and snow for weeks
Salmonellae are Gram negative bacilli measuring 1-3 and months. They are killed within five minutes by
µm x 0.5 µm. They are motile, non-sporing and non- mercuric chloride (0.2%) or phenol (5%).
capsulated. Motility is due to the presence of peritrichous
flagella except S. gallinarum and S. pullorum which are E. Antigenic Structure
non-motile. Salmonellae possess three types of antigens based on
which they are classified. These are (1) flagellar antigen
B. Culture 'H ', (2) somatic antigen 'O', and (3) a surface antigen
Salmonellae grow on ordinary culture media at optimum 'Vi', found in some species (Fig. 33.1). Several strains
temperature of 37°C (range 15--41 °C), pH 6-8 and are possess fimbriae. Fimbrial antigens are not important
aerobic and facultatively anaerobic. They produce colonies in identification, but confusion may be created due to
of 2- 3 mm in diameter, circular, translucent, low convex their non-specific nature and widespread sharing among
and smooth. On MacConkey's agar and deoxycholate enteric bacteria.
citrate agar (DCA), colonies are colourless due to non-
lactose fermentation (NLF). On Wilson and Blair bismuth
sulphite medium (selective medium for salmonellae), jet
black colonies with metallic sheen are formed due to
formation of hydrogen sulphide. S. paratyphi A and other
species which do not form H2 S produce green colonies.
Xylose lysine deoxycholate (XLD) agar is another
medium used for isolation of this organism. Most strains
of salmonellae produce red colonies with black centres,
when grown on this medium. H 2S negative serotypes of
Salmonella produce red colonies without black centres.
Selenite F broth and tetrathionate broth (TTB) are
commonly used enrichment media for inoculation of Fig. 33.l Cellular locations of H, 0 and Vi antigens
specimens especially faeces.
1. H-Antigen
This antigen is present on the flagella and is a heat labile
C. Biochemical Reactions protein. The H antigens of Salmonella are not shared
Salmonellae ferment glucose, mannitol and maltose by other enterobacteria. It is destroyed by boiling or by
forming acid and gas except S. typhi which produces only treatment with alcohol and acids, but not by formalin.
acid and no gas. They do not ferment lactose or sucrose. It is strongly immunogenic. When mixed with antisera,
Indole is not produced. Most salmonellae produce H 2S H agglutination occurs rapidly, producing large, loose,
in triple sugar iron (TSI) agar except S. paratyphi A and fluffy clumps. Flagellar antigens are present in two forms
S. cholerae-suis. They utilise citrate (except S. typhi and called phase 1 and phase 2.
S. paratyphi A) and are MR positive and VP negative.
Urea is not hydrolysed. Common biochemical reactions 2. O-antigen
of salmonellae are shown in Table 33.1 . The somatic O antigen 1s lipopolysaccharide complex
Table 33.1 Biochemical Reactions of S. typhi and S. paratyphi

Glucose Mannitol Lactose Sucrose Indole Citrate MR VP H 2S Xylose d-tartrate Mucate
S. typhi A A + + d A d
S. paratyphi A AG AG +
S. paratyphi B AG AG + + + AG AG
S. paratyphi C AG AG + + + AG AG
A- Acid, AG- Acid & gas, d-delayed

which forms an integral part of the cell wall. It is identical 1. H 0 Variation

with endotoxin. It is heat stable and is present both in Motile strains lose their flagella and become non-
motile and non-motile bacilli. It is resistant to boiling, motile. When salmonellae are grown on nutrient agar
alcohol and weak acids. When mixed with antisera, 0 media containing phenol (1 :800), flagella are inhibited.
antigen suspensions produce compact, chalky, granular This change is phenotypic and temporary since flagella
clumps. 0 antigen is less immunogenic than H antigen. reappear when the strain is subcultured on normal media
The titre of O antibody in serum after infection or without phenol. Salmonellae may rarely lose flagella by
immunisation is generally less than that of H antibody. mutation. A stable non-motile mutant is 901- 0 strain of
Anti O antibody is pre-dominantly IgM. 0 agglutination S. typhi which is widely used for the preparation of
occurs more slowly and at a higher optimum temperature O-agglutinable bacterial suspensions.
(50-55°C) than H agglutination (37°C). Sixty seven Generally, loss of flagella is not total but only
different O antigens have been recognised and they are a diminution in the number of flagella occurs. Such
designated as 1, 2, 3 etc. bacterial cultures contain small number of flagellated
cells. To obtain motile bacteria, rich in H antigen from
3. Vi-antigen such cultures, selection may be carried out by use of
Many strains of S. typhi possess surface antigen Craigie's tube. This consists of a wide tube containing
enveloping the O antigen, referred to as Vi antigen. 0.2% nutrient agar (soft agar) in which a short narrow
Hence, this antigen interferes with agglutination of freshly tube open at both ends is kept embedded in such a way
isolated strains with O antiserum. Felix and Pitt who that one end of it projects above the agar. The strain
first described the antigen believed that it was related is inoculated into the inner tube and after incubation,
to virulence (Vi for virulence). It is analogous to the K subcultures are taken from the top of agar outside the
antigens of coliform bacteria. Vi antigen is heat labile central tube. This yields motile bacteria with rich H antigen
and is destroyed by boiling or heating at 60°C for one (Fig. 33.2) .
hour. It is also destroyed by N HCI, 0.5N NaOH and Instead of the Craige's tube, a U-tube of soft agar
phenol. It is unaffected by 0.2% formol or alcohol. may also be used. Inoculation of strain is made into
Besides S. typhi, the Vi antigen with similar specificity one limb of U-tube and subculture is taken from the
is also present in S. paratyphi C, S. dub/in and certain other limb.
strains of Citrobacter (the Ballerup-Bethesda group). The
Vi antigen is lost on serial subculture. The Vi antigen 2. Phase Variation
acts as a virulence factor by inhibiting phagocytosis, The flagellar antigens of most salmonellae occur in one
resisting complement activation and lysis of bacteria by of two phases, phase 1 or phase 2. Phase 1 antigens are
the alternative pathway. It has been demonstrated that either specific or shared by a few species only and hence
strains with Vi antigen produce clinical disease more phase 1 is called specific phase. Phase 2 antigens are
consistently than those without Vi antigen. widely shared by several unrelated species of salmonellae.
The Vi antigen is poorly immunogenic and induces
production of low titres of antibody following infection.
Inoculation
Detection of Vi antibody is not helpful for diagnosis of
enteric fever and hence is not employed in the Wida! test.
The total absence of Vi antibody in a proven case of
typhoid fever indicates poor prognosis. The Vi antibody
disappears in early phase of convalescence. Persistence
of this antibody indicates the development of the carrier
Subculture
state. Specific Vi bacteriophages are used for epidemio-
logical typing of S. typhi.
In prophylactic vaccination with TABC, no Vi
antibody is induced by the phenolised vaccine, though
low titres of the antibody are produced by the alcoholised
vaccme.
F. Antigenic Variation
The antigens of salmonellae undergo several types of
Fig. 33.2 Craigie's tube
phenotypic and genotypic variations.

Ch 33: Salmonella _ _ _ __ _ __ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __ _ 265
Hence phase 2 is called the non-specific or group phase. 15. S. newington is further converted into S. minneapolis
Phase 1 antigens are designated as a, b, c, d etc. up to z (serotype, 3, 15, 34: e, h : 1,6) by phage 34. Such changes
and after z, as zl, z2, z3, up to zl4. Phase 2 antigens are are taking place in nature which might be responsible for
fewer and termed as 1, 2, 3, etc. In some species, antigens so many serotypes of salmonellae.
of phase 1 may occur as phase 2 antigens (e.g., e, n, x
and zl5). Strains that possess both phases are termed G. Classification
diphasic while some strains (S. typhi, S. enteritidis, S. The genus Salmonella is classified broadly into four
dub/in and S. paratyphi A) occur in phase 1 only and subgenera on the basis of biochemical reactions (Table
are called monophasic. 33.2) .
A culture will contain flagellar antigens of both
phases, but generally one of the two phases predominates Table 33.2 Classification of Salmonellae on the Basis of
so that the culture is agglutinated only by one of the phase Biochemical Properties
antisera. For identification of a serotype, it is necessary to Property Subgenera of Salmonella
identify the flagellar antigens of both phases. A culture
I II III IV
in one phase can be converted to the other phase by
passing the bacteria through a Craigie's tube containing Lactose +
the homologous phase antiserum incorporated in the agar Dulcitol + +
(Fig. 33.2) . Malonate + +
d- Tartrate +
3. V W Variation
Fresh isolates of S. typhi carry Vi antigen that completely Salicin +
masks the O antigen. These isolates are agglutinable with KCN +
Vi antiserum but not with the O antiserum. These are
called the V forms. Organisms lost their Vi antigen either Subgenus I is the largest and the most important
partially or completely after a number of subcultures. group containing all the species that are commonly
When there is a complete loss of Vi antigen, such responsible for human and animal infections. Other
cultures are agglutinable by O-antiserum but not with subgenera have little importance in human disease.
Vi antiserum. These are called W forms. With partial Subgenera II is common in reptiles and subgenera III in
loss of Vi antigen, the bacilli are agglutinable with both lizards. Subgenera IV strains are rarely encountered and
Vi and O antisera and are called VW forms. are considered as atypical members of subgenera II. The
Other Vi containing bacilli (S. paratyphi C and S. bacilli of subgenera III, formerly designated as Arizona
dub/in) seldom have the O antigen completely masked and later included in the genus salmonella because of
by Vi antigen. their antigenic similarity. About 60 per cent of them
ferment lactose promptly.
4. S R Variation Modern taxonomical techniques have shown that all
The smooth-to-rough variations occur due to mutation the members of the genus Salmonellae belong to two
and are associated with the change in the colony species, S.enterica and S. bongori. S. enterica is classified
morphology from smooth to rough, loss of the O antigen into six subspecies namely enterica, salamae, arizonae,
and of virulence of the strain. The rough (R) variants diarizonae, houtenae and indica. Subspecies enterica
have defective capacity to synthesise somatic (0) antigen corresponds to former subgenus I. S. enterica subsp.
and loss of O antigen may be partial or total. R forms enterica includes the typhoid, parathypoid bacilli and
may be common in laboratory strains maintained by serial most other serotypes responsible for human diseases.
subcultures. S • R variation may be prevented to some According to this, the taxonomically correct name for the
extent by maintaining laboratory cultures on Dorset's egg typhoid bacillus would be 'Salmonella enterica, subspecies
media in cold or by lyophilisation. enterica, serotype 'Typhi.' Serotype name should be given
in Roman and not in italics. This classification and
5. Variations in O Antigen nomenclature is taxonomically correct but would be too
Changes in the structural formulae of O antigen may complicated for use in clinical bacteriology. Therefore,
be induced by lysogenisation with some bacteriophages, the old species names are still being used in clinical
resulting in the alteration of the bacterial serotypes. bacteriology.
Thus, S. anatum (serotype, 3,10: e, h : 1,6) is converted Salmonellae are classified serologically by the
into S. newing ton (serotype, 3, 15: e, h : 1,6) by phage Kauffmann-White scheme.
266 - - -- - - - - - - - ~ ~- - - - -- - - - - - - - - - Unit Ill: Systemic Bacteriology
H. Kauffmann-White Scheme salmonellae contain lipopolysaccharide which acts as

It forms the basis of serotyping of salmonellae based endotoxin.
on identification of O and H antigens by agglutination.
Salmonellae are classified into groups based on distinctive J. Pathogenesis
0 antigen factors. The distinctive factor for group A is 2, S. typhi, S. paratyphi A and usually S. paratyphi B
for group B, 4 and for group D, 9. Any strain possessing are confined to human beings. The majority of other
factor 2 will be classified as group A and any strain salmonellae are primarily infective for animals and human
possessing factor 9 as group D. Salmonellae are divided beings are secondarily infected.
into 67 0-groups, each is designated by letters A to Z and Salmonellae cause three types of clinical syndrome
those discovered later by the numbers. Within each group, in human beings, enteric fever, septicaemia and
further identification and differentiation is by phase 1 gastroenteritis.
and phase 2 flagellar (H) antigens (Table 33.3) .
In Kauffmann-White scheme each serotype is 1. Enteric Fever
given a species status. There are about 2300 serotypes The term enteric fever includes typhoid fever (S. typh1) and
of salmonellae. Most serotypes of medical importance paratyphoid fever (S. paraptyphi A , B , C) . Infections due
belong to groups A to E. to S. typhi and S. paratyphi A are prevalent in India.
The Salmonella species were originally named after
the place of isolation (S. newport, S. panama, S. poona) , (i) Typhoid fever
after the disease caused by them (S. schottmulleri) and The infection is acquired by ingestion through contaminated
after the name of the person from whom the first strain food and water. The incubation period is usually 7- 14
was isolated (S. thompson). days but appears to be related to the dose of infection.
Sometimes, more than one species (S. gallinarum On reaching the small intestine the bacilli attach to the
and S. pullorum) may have the same antigenic epithelial cells of the intestinal villi and penetrate to the
formulae and cannot be distinguished serologically. lamina propria and submucosa. They are phagocytosed
Differentiation can be done by biochemical reactions (S. by neutrophils and macrophages. These bacteria resist
gallinarum is anaerogenic and ferments dulcitol unlike intracellular killing and multiply within these cells. They
S. pullorum). Important pathogens can be further typed enter the mesenteric lymph nodes, multiply there and, via
for epidemiological purposes by phage susceptibility, the thoracic duct, enter the blood stream. A transient
biochemical reactions, bacteriocin production and bacteraemia follows and internal organs like liver, gall
antibiogram. bladder, spleen, bone marrow, lungs, lymph nodes and
kidneys are infected. Towards the end of the incubation
I. Toxin period, a massive bacteraemia occurs from these sites of
Like all other Gram negative bacilli, the cell walls of multiplication and clinical disease sets in.
Table 33.3 Illustration of the Kauffmann-White Scheme.

0 group Serotype 0 antigens* H antigens
ew Old Phase 1 Phase 2
2 A S. paratyphi A I, 2, 12 a
4 B S. paratyphi B I, 4, 5, 12 b I, 2
S. typhimurium I, 4, 5, 12 1, 2
7 Cl S. paratyphi C 6, 7, (Vi) C 1, 5
S. cholerae- suis 6, 7 C I, 5
8 C2 S. muenchen 6, 8 d I, 2
S. newport 6, 8, 20 e, h 1, 2
9 D S. typhi 9, 12, (Vi) d

S. enteritidis I, 9, 12 g, m
S. ga//inarum 1, 9, 12
S. pullorum I, 9, 12
3, 10 El S. anatum 3, 10 e, h I, 6
Vi V antigen * umber in bold type a re the antigens characterising the O group

Ch 33: Salmonella _ _ _ _ _ _ _ _ _ __ __ _ __ _ _ _ _ _ _ __ _ __ _ _ __ _ 267
Salmonellae multiply abundantly in the gall bladder diarrhoea. Salmonella food poisoning is of infective type
as bile is a good culture medium for the bacillus. These in which the organisms not only grow in the food before
bacteria are discharged continuously into the intestine ingestion but also in the intestine. Generally, there is no
involving the Peyer's patches and lymphoid follicles of bacteraemia.
the ileum. These become inflamed, necrosed and slough
off, leaving behind the typhoid ulcers. These ulcers may K. Epidemiology
lead to two major complications-intestinal perforation Enteric fever is endemic in all parts of India. Typhoid
and haemorrhage. During the course of the disease (3-4 fever is commoner than paratyphoid fever. S. paratyphi. A
weeks), the intestinal lesions undergo healing. is prevalent in India. S. paratyphi B is rare and C very
The clinical course may vary from a mild pyrexia rare. Enteric fever occurs at all ages but is probably most
to a fatal fulminating disease. The illness is usually common in the 5- 20 years age group.
gradual, with headache, anorexia and congestion of The faeces of carriers are important source of
mucous membranes. The characteristic features are contamination of food and drink rather than the frank
hepatosplenomegaly, step-ladder pyrexia with relative clinical cases. Sewage contaminated by a carrier is
bradycardia and leucopaenia. Skin rashes known as responsible for polluting drinking water. Food material
rose-spots may appear during the second or third week. gets contaminated via polluted water or by the hands of
The infecting organisms appear in stool during second carriers. Mary Mallon ('Typhoid Mary'), a cook in USA,
to third week and in urine during third to fourth week. was a famous carrier who caused several outbreaks over
'Rose spots' appear on the skin during the second or a period of 15 years.
third week.
L. Laboratory Diagnosis
(ii) Paratyphoid fever Bacteriological diagnosis of enteric fever consists of
Paratyphoid fever resembles typhoid fever but is milder. 1. Isolation of bacilli
S. paratyphi A, B and C cause paratyphoid fever. S. 2. Demonstration of antibodies
paratyphi C more often leads to a frank septicaemia with 3. Demonstration of circulating antigen
suppurative complications. 4. Other laboratory tests
Some other salmonellae have also been reported to
cause enteric fever occasionally. These include S. dub/in, 1. Isolation of Bacilli
S. barielly, S. sendai, S. enteritidis, S. typhimurium, S. This may be done by culture of specimens like blood,
eastbourne, S. saintpaul, S. oranienburg and S. panama. faeces, urine, aspirated duodenal fluid etc. Selection of
relevant specimen depends upon duration of illness which
2. Septicaemia is very important for the laboratory diagnosis of enteric
Salmonella septicaemia is commonly caused by S. fever (Table 33.4, Fig. 33.3).
cholerae-suis or S. paratyphi C and occasionally by
other salmonellae. Infection occurs through oral route. Table 33.4 Relevance of Examination of Different
There is early invasion of blood stream and it produces Specimens at Different Phases of Enteric Fever
local suppuration in different organs. It may cause Duration of disease Specimen examination % Positivity
osteomyelitis, pneumonia, pulmonary abscess, meningitis 1st Week Blood culture 90
or endocarditis.
2nd Week Blood culture 75
Faeces culture 50
3. Gastroenteritis Wida! test low titre
Salmonella gastroenteritis or food poisoning is caused by
3rd Week Wida! test 80- 100
ingestion of food like meat, milk, egg contaminated by
Blood culture 60
certain salmonellae which are primarily animal pathogens. Faeces culture 80
Eggs and egg products are of great concern. Salmonellae
can enter through the shell if eggs are left on faeces
or contaminated feed of chicken. S. typhimurium is the BLOOD CULTURE
most frequently isolated in food poisoning. The other Blood cultures are positive in approximately 90 per cent
common species responsible are S. enteritidis, S. newport, of cases in first week of fever, 75 per cent in the second
S. senftenberg, S. dub/in, S. heidelberg and S. indiana. week and 60 per cent in the third week. Positivity rate
The incubation period is 12 to 24 hours. The illness declines thereafter and blood cultures remain positive in
is characterised by fever, vomiting, abdominal pain and 25 per cent of cases till the subsidence of pyrexia.

268 - - - - - - - - - - - - - - - - - - -- - - - - - - - - Unit Ill: Systemic Bacteriology
the culture as negative. Sometimes, Castaneda's method

100 of culture is practised instead of routine blood culture.
CLOT CULTURE
80
It is an alternative to blood culture. 5 ml of blood is
C withdrawn aseptically into a sterile container and allowed
(/)
Q) 60 to clot. The serum is separated and used for the Wida!
Cl
_ig test. The clot is broken up with a sterile glass rod and
C:
Q) added to bile broth containing streptokinase (100 units/
12
Q)
a.. 40 ml) which digests the clot causing its lysis and thereby
the bacteria are released from the clot. Serum used for
A
Widal test may be negative due to the early stage of the
20
disease but it may be useful as baseline titre against which
the results of tests performed later may be evaluated.
01234567
FAECES CULTURE
Weeks
Salmonellae are shed in the faeces throughout the disease
Fig. 33.3 Bacteriological diagnosis in typhoid fever. The and even in convalescence, due to lesions present in
approximate percentage of positivity during different weeks of
disease A- Blood culture, B- Faeces culture C- Widal test. the intestine. Hence, faecal cultures may be helpful in
patients as well as for the detection of carriers. These are
10 ml of blood is collected by venepuncture under particularly valuable during antibiotic therapy when blood
aseptic conditions and transferred into blood culture culture shows no growth of organisms. The antibiotic
bottles (glucose broth and taurocholate broth). Before does not eliminate the bacilli from the gut as rapidly
transferring blood into blood culture bottles, caps of as it does from the blood. Faeces cultures are generally
these bottles should be thoroughly cleaned with spirit or positive after the second week of illness.
alcohol. Blood should be transferred through a hole in As normal flora is present in faeces, successful culture
a cap by inserting the needle of the syringe rather than will depend on use of enrichment and selective media.
opening the bottle, thus it avoids contamination from Faecal samples are inoculated into one tube each of
the external environment. selenite and tetrathionate broth (both enrichment media)
Blood contains substances that inhibit the growth and are also plated directly on MacConkey's agar, DCA,
of the bacilli and hence it is essential to dilute out these XLD and Wilson-Blair media. Salmonellae appear as
substances (5 ml blood into the 50 ml culture media, pale yellow (NLF) colonies on MacConkey's agar and
1:10 dilution). Alternative is addition of liquoid (sodium DCA media. On Wilson-Blair medium, S. typhi forms
polyanethol sulphonate) which counteracts the bactericidal large black colonies with a metallic sheen whereas S.
action of these substances. paratyphi A produces green colonies due to the absence
Both blood culture bottles are incubated at 37°C for of H 2 S production.
overnight. The glucose broth is subcultured on blood Enrichment media (selenite F and tetrathionate
agar and the taurocholate (bile) broth on MacConkey's broths) are incubated for 6-8 hours before subculture
agar. Taurocholate broth is inhibitory to Gram positive on to selective media such as MacConkey's agar and
bacteria, therefore, Gram negative bacilli are selected out. DCA. These selective media are then incubated at 37°C
Pale non-lactose fermenting (NLF) colonies appear on for overnight.
MacConkey's agar and are picked out for biochemical
reactions and motility. Salmonellae will be Gram
negative, motile bacilli and fermenting glucose, mannitol URINE CULTURE
but not lactose or sucrose. S. typhi will be anaerogenic Urine culture is less frequently positive than the culture
but paratyphoid bacilli will form acid and gas from of blood or faeces. Cultures are generally positive only
carbohydrates (sugars). Final identification of the isolate in the second and third weeks. After third week, only
is by slide agglutination with O and H antisera. about 25 per cent cases may be positive. Repeated cultures
If salmonellae are not obtained from the first improve the chances of isolation.
subculture from taurocholate broth, subcultures should Urine samples are centrifuged and the deposit 1s
be done every other day up to ten days before declaring inoculated into enrichment and selective media.

Ch 33: Salmonella _ _ _ __ _ _ __ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ 269
OTHER SPECIMENS FOR CULTURE 2. Demonstration of Antibodies

Bone marrow culture is valuable as it is positive even WIDALTEST
when blood cultures are negative. Culture of bile is usually It is an agglutination test for detection of agglutinins
positive and may be useful in detection of chronic carriers. (H and 0) in patients with enteric fever. Salmonella
Other materials which may be used for culture are rose antibodies start appearing in the serum at the end of
spot's discharge, pus from suppurative lesions, CSF and first week (Fig. 33 .3) and rise sharply during the third
sputum. At autopsy, cultures may be done from the gall week of enteric fever. Two specimens of sera at an interval
bladder, liver, spleen and mesenteric lymph nodes. of 7 to 10 days are preferred to demonstrate a rising
antibody titre.
Colony Morphology and Staining Procedure
On MacConkey's agar or DCA, salmonellae grow as pale Two types of tubes were originally used for the test-
yellow, non-lactose fermenting (NLF) colonies. Gram Dreyer's tube (narrow tube with a conical bottom) for the
staining from these colonies show Gram negative bacilli H agglutination and Felix tube (short, round bottomed
and on hanging drop preparation, these are motile bacilli. tube), for the O agglutination. Equal volumes (0.4 ml) of
serial dilutions of the serum (1 :10 to 1:640) and the H
Biochemical Reactions and O antigens are mixed and incubated in a water bath
Salmonellae are catalase positive, oxidase negative, nitrate at 37°C for 4 hours and read after overnight refrigeration
reduction positive and ferment glucose, mannitol but not at 4°C. Control tubes containing the antigen and normal
lactose or sucrose. S. typhi ferments glucose and mannitol saline are included to check for autoagglutination. H
with production of acid only but paratyphoid bacilli (S. agglutination leads to the formation of loose, cottonwool
paratyphi A, B and C) form acid and gas. clumps, while O agglutination appears as a granular
deposit at the bottom of the tube. The highest dilution
Slide Agglutination Test of the serum showing agglutination (carpet formation)
A loopful of the growth from a nutrient agar slope indicates the antibody titre against that particular antigen
is emulsified in two drops of saline on a microscopic (Fig. 33.4). Control tubes show a compact deposit (button
slide. One emulsion acts as a control and other as a test. formation) .
Control is to show that the strain is not autoagglutinable.
Agglutination is first carried out with the polyvalent 0
and the polyvalent H antisera. Positive agglutination
indicates that the isolate belongs to genus Salmonella.
Further agglutination tests are done with single factor
sera for determining the O and H antigens. If S. typhi
is suspected (i.e. when no gas is formed from glucose
and mannitol), agglutination with O antiserum (factor
9) is done. Prompt agglutination indicates that the isolate
belongs to group-D salmonella. Its identity as S. typhi Negative Positive
(Button formation) (Carpet formation)
is established by agglutination with H antiserum (anti- d
serum). Sometimes fresh isolates of S. typhi are in V form Fig. 33.4 Wida! test (Negative and Positive tubes)
and do not agglutinate with O antiserum. Such strains
should be agglutinated with anti- Vi serum. Alternatively, The antigens used in the test are the H and O antigens
the bacterial suspension in saline is boiled for 20 minutes of S. typhi (TH and TO antigens) and H antigens of
which removes the Vi antigens from the organism. The S. paratyphi A (AH antigen) and S. paratyphi B (BH
boiled bacterial suspension is then tested with poly antigen). The paratyphoid O antigens are not employed
O- antiserum. as they cross react with S. typhi O antigen (TO) due to
When the isolate is a non-typhoid salmonella their sharing of factor 12.
producing gas from carbohydrates, it is tested for
agglutination with O and H antisera for groups A, B Preparation of widal antigen
and C. For identification of unusual serotypes, the help of The H agglutinable suspension of bacteria is prepared by
the National Salmonella Reference Centre at the Central adding 0.1 per cent formalin to a 24 hour broth culture or
Research Institute (CRI) Kasauli, may be sought. The saline suspension of an agar culture. For preparation of 0
reference centre for salmonellae of animal origin is located suspension of bacteria, the bacillus is cultured on phenol
at the Indian Veterinary Research Institute, Izatnagar. agar (1 :800). The growth is scraped off in a small volume

270 _ _ _ _ _ _ _ _ _ _ _ _ _ __ __ _ _ _ _ _ __ __ _ _ Unit Ill: Systemic Bacteriology
of saline and mixed with 20 times its volume of absolute • Fimbrial antigens: Bacterial suspensions used as
alcohol. It is then heated in a water bath at 40°C- 50°C antigens may contain fimbrial antigen which may
for 30 minutes, centrifuged and the deposit resuspended produce false positive results.
in saline to the appropriate density. Chloroform is then • Effect of treatment: Cases treated early with
added as a preservative. chloramphenicol may show a poor antibody
S. typhi 901 , 0 and H strains, are used for preparation response; if the antibody is already present, no
of antigens. Each batch of prepared antigen should be further rise in titre is expected.
compared with a standard. Wida! kits of stained antigens • Carriers: Test may be positive in many healthy
are available commercially. carriers.
Result
The highest dilution (titre) of patient's serum in which OTHER SEROLOGICAL TESTS
agglutination occurs is noted i.e. if the dilution is 1 in ELISA is a sensitive method of measuring antibody
160, the titre is 160. against the lipopolysaccharide of salmonellae. Indirect
haemagglutination test and CIEP are other serological
Interpretation of widal test methods of diagnosis.
(a) The agglutinin (antibody) titre depends on the stage Detection of porins, the outer membrane proteins of
of the disease. Agglutinins usually start appearing in S. typhi, by ELISA method is useful for early serodiagnosis
the serum by the end of the first week, so that blood of typhoid fever.
specimen taken earlier than first week may give a
negative result. The titre rises steadily till the third
or fourth week after which it declines. 3. Demonstration of Circulating Antigen
(b) Rising titre: Demonstration of a four-fold or greater Typhoid bacilli antigens are present in the blood in
rise in titre of both H and O antibodies at an interval the early phase of the disease, and also in the urine of
of 7- 10 days is more meaningful and diagnostic than patients. The antigen can be detected by coagglutination
a single test. test. Staph. aureus (Cowan I strain) containing protein A,
(c) Single test: 0 titre of 1:100 or more and H titre of is first stabilised with formaldehyde and then coated with
1:200 or more signifies presence of active infection, S. typhi antibody. These sensitised staphylococcal cells
but that has to be interpreted with caution considering (I % suspension) are mixed on slide with patient serum.
the following factors: The typhoid antigen present in the serum combines with
• Local titre: In endemic area, low titre of agglutinins the antibody coated on staphylococci producing visible
is present in the serum of normal persons. This agglutination within two minutes. The test is rapid,
normal titre differs from place to place. Local titre sensitive and specific, but is positive only during the first
of that place should be known before interpreting week of the disease.
the results of a single test. Counterimmunoelectrophore sis (CIEP) and ELISA
• Immunisation: In immunisation with TAB vaccine, have also been employed to detect typhoid antigens in
individuals may show high titres of antibody to blood and urine.
S. typhi, S. paratyphi A and B, while in case of
infection, antibodies will be seen only against the 4. Other Laboratory Tests
infecting organism. (i) Total leucocyte count (TLC)
• H- agglutination: In non-immunised individuals, Leucopaenia with a relative lymphocytosis is found.
presence of H agglutinin in serum indicates enteric
fever or a latent infection. H agglutinin is more (ii) Diazo test in urine
reliable than O agglutinin as O antigen is common This test becomes positive generally between 5th and
in all the salmonellae. 14th day of fever and remains positive till the fever
• Anamnestic reaction: Persons who have had past subsides.
enteric infection or immunisation may develop
anamnestic reaction during unrelated fever like Procedure
malaria etc. In such cases, a transient rise in H Equal volumes of patient's urine and the diazo reagent
antibody occurs, whereas the rise is sustained are mixed and a few drops of 30% ammonium hydroxide
in enteric fever. This may be differentiated by are added. On shaking the mixture, a red or pink froth
repeating the widal test after a week. develops, if the test is positive.

Ch 33: Salmonella _ _ _ _ __ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ 271
Diazo reagent particular strain of S. typhi by serial propagation in that

Solution A Sulphanilic acid particular strain. As phage typing of S. typhi depends
Cone. H 2SO4 on the presence of Vi antigens, some Vi negative strains
Distilled water will be untypable. A phage type is stable. At present,
Solution B - Sodium nitrite 97 Vi II phage types of S. typhi are known, which are
Distilled Water designated by letter or number. Phage types A and El
For use, 40 parts of solution A are added to one are the most common in India. However, prevalence in
part of solution B. different regions is subject to change from time to time.
Phage typing is carried out at National Phage Typing
DIAGNOSIS OF CARRIERS Centres. The National Salmonella Phage Typing Centre
Carriers are important sources of infection and their for India is situated at the Lady Hardinge Medical
detection is necessary for epidemiological and public College, New Delhi.
health purposes. Laboratory methods are also very useful Bacteriophage typing schemes for S. paratyphi A,
in screening food handlers for detecting carrier state. S.paratyphi. B, S. typhimurium, S. enteritidis, S. dub/in
Even after clinical recovery, about 2 to 3% patients have also been developed. The predominant phage types
in convalescence phase (convalescent carriers) and some of S. paratyphi A isolated from India belong to types 1
persons with subclinical infection (healthy carriers) and 2.
continue to excrete the bacilli in faeces for about two
months to a year. Chronic carriers harbour the bacilli in N. Biotyping
gall bladder or rarely in intestine or urinary tract. Most The serotypes can be subdivided into biotypes based on
of these carriers continue excreting organisms after one fermentation of xylose and arabinose.
year of convalescence and may continue to excrete the
bacilli for several years or throughout life. In most of 0. Plasmid Typing
these cases, there is intermittent shedding of bacilli in Plasmids present in salmonellae are separated
stool, therefore, repeated stool cultures may be helpful electrophoretically and analysed.
to detect the bacilli. Cholagogue purgatives increase the
chance of isolation from stool specimens. Other useful P. Treatment
specimens for culture are bile and duodenal drainage. For Chloramphenicol has been the antibiotic of choice
the detection of urinary carriers, repeated urine cultures for enteric fever. Amoxycillin, ampicillin, furazolidone
should be done. and cotrimoxazole are also effective. Resistance to
Wida! test is of no value in detection of carriers in chloramphenicol and other antibiotics has been reported.
endemic countries like India. Antibody to Vi antigen in Fluoroquinolones (ciprofloxacin, pefloxacin, ofloxacin)
serum is claimed to indicate the carrier state. This may and the third generation cephalosporins (ceftazidime,
be used for mass screening, however, confirmation should cefotaxime, ceftriaxone cefoperazone) are useful in such
always be made by culture. multiresistant cases.
'Sewer-swab' technique is used for the tracing of Antibacterial therapy has been very effective in
carriers for epidemiological purposes. Gauze pads left the treatment of patients but it has been disappointing
in sewers and drains are cultured and a positive swab may in the treatment of carriers. A prolonged course of
lead to a house harbouring a carrier. Organism isolated antibacterial agents along with the vaccine has been
from carrier is to be phage typed to establish the carrier tried in the eradication of carrier state. Cholecystectomy
as a cause of an epidemic. is indicated for chronic carriers who do not show any
Another method of isolation is filtration of sewage response to antibacterials.
through millipore membranes and culturing the
membranes on highly selective medium e.g. Wilson and Q. Drug Resistance
Blair medium. Resistance to chloramphenicol became a problem in
1972, when resistant strains emerged in Mexico and in
M. Bacteriophage Typing Kerala (India). In Mexico, the resistant strain caused an
For epidemiological purposes, strains may be differentiated explosive epidemic.
into a number of phage types. Craigie and Yen (1937) In India, chloramphenicol resistant typhoid fever
originally observed that a bacteriophage (Vi phage II) appeared as epidemic in Calicut (Kerala) in 1972. It
was acting on Vi antigen of S. typhi. The parent phage became endemic and remain confined to Kerala till 1978.
is known as phage A. It could be made specific for a Subsequently, resistant S. typhi strains were reported in

272 _ _ _ __ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Unit Ill: Systemic Bacteriology
many other parts of India. Resistance was originally been used as a live oral vaccine. These mutants initiate
confined to phage type Dl-N, but later to types C5, infection in the intestine but 'self- destructs' after four
A and 0. Resistance is due to a transmissible plasmid to five cell divisions and therefore cannot induce any
carrying r determinants to chloramphenicol, streptomycin, illness.
sulphonamide and tetracycline. Dose schedule: Three doses are given on alternate day.
Multiple drug resistant S. typhi is increasingly The oral vaccine (typhoral) is available in capsule form
being recognised in many countries and has become a containing 109 viable lyophilised mutant bacilli.
problem in India, too. R factors carrying multiple drug Protection: It gives 65- 96% protection for 3- 5 years
resistance have become widely disseminated among and is safe.
salmonellae. These may cause nosocomial salmonellosis
particularly in neonates as septicaemia, meningitis and (iii) Purified Vi polysaccharide vaccine (typhim-Vi)
suppurative lesions. In India, hospital outbreaks of It contains purified Vi antigen.
neonatal septicaemia caused by multiple drug resistant Dose schedule: It is injected intramuscularly in a single
salmonellae have occurred in recent years. Mortality in dose of 25 µg. The efficacy is about 75%.
neonates is very high unless early treatment is started
with antibiotics.
II. SALMONELLA GASTROENTERITIS
R. Prophylaxis Salmonella gastroenteritis or food poisoning is generally
1. General Measures a zoonotic disease, therefore the source of infection is
Typhoid fever can be effectively controlled by sanitary animal product. All salmonellae can cause the disease
measures for disposal of sewage, clean water supply and except S. typhi. A large number of salmonellae have
supervision of food processing and handling. Infected been identified from cases of gastroenteritis and food
meats and eggs should be thoroughly cooked. poisoning. S. typhimurium is the commonest species.
Other common species include S. enteritidis, S. heidelberg;
2. Carriers S. indiana, S. newport, S. haldar, S. agona, S. senftenberg,
Carriers should not be engaged in food preparation and S. virchow and S. anatum.
should observe strict personal hygiene.
A. Source of Infection
3. Vaccination The most important and frequent sources are poultry,
Vaccine is indicated for travellers or who live in endemic meat, milk, cream and eggs. It is due to ingestion of
areas. contaminated food. Food contamination may result
from droppings of small animals like rats or lizards.
(i) TAB vaccine Salmonellae can enter through the shell of egg and grow
It is heat-killed whole cell vaccine which contains inside. It happens when eggs are left on contaminated
S.typhi.1,000 millions, S. paratyphi A and B, 750 millions chicken feed or faeces. Human carriers do occur but their
each per ml and preserved in 0.5 per cent phenol. role is minimal. Cross infections in hospitals may lead
Dose schedule: The vaccine is given subcutaneously in to gastroenteritis without food poisoning.
two doses of 0.5 ml at an interval of 4--6 weeks followed
by booster every three years. B. Clinical Features
Protection: It varies from 3- 7 years with an efficacy It presents with diarrhoea, vomiting, abdominal pain and
of 60- 80%. fever. It has a short incubation period of 24 hours or
Side Effects: Local discomfort and fever may occur less. Disease usually subsides in 2-4 days, but in some,
for one or two days. a prolonged enteritis develops.
In India, a divalent vaccine containing S. typhi and
S. paratyphi A are now in use. In Europe and USA, C. Laboratory Diagnosis
monovalent vaccine containing S. typhi is employed It depends on isolation of salmonellae from faeces and
as paratyphoid A and B infections are rare in these from food.
countries.
D. Control
(ii) Live oral (Ty 21a) typhoid vaccine It requires the prevention of food contamination. It may
Avirulent mutant strain of S. typhi (Ty 21a) lacking the be achieved by proper cooking of food and control of
enzyme UDP- galactose-4---epimerase (Gal E mutant) has natural infection in animals.

Ch 33: Salmonella _ __ __ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __ __ 273
E. Treatment pneumoma, endocarditis and meningitis. Among

Symptomatic treatment should be given. Antibiotics all salmonellae, S. cholerae-suis is particularly very
should not be used in uncomplicated, non-invasive important causative agent of salmonella septicaemia.
salmonellosis. They may not hasten recovery but may It is a fulminating blood infection. Infection occurs by
actually increase the period of faecal shedding of the oral route.
bacilli. But for serious invasive cases, antibiotics are
needed. Salmonellae may be isolated from the blood or from
the pus from the suppurative lesion. Rarely, faeces culture
III. SALMONELLA SEPTICAEMIA may be positive for salmonellae. These septicaemic cases
Certain salmonellae may cause septicaemia with focal should be treated with chloramphenicol or appropriate
suppurative lesions, such as deep abscesses, osteomyelitis, antibiotics as determined by susceptibility tests.
KEY POINTS
1. Salmonellae are Gram negative, motile bacilli. They grow on ordinary culture media. On MacConkey's agar and
deoxycholate citrate agar (DCA), colonies are colourless due to non-lactose fermentation (NLF).
2. On Wilson and Blair bismuth sulphide medium (selective medium for salmonellae), jet black colonies with metallic
sheen are formed due to formation of hydrogen sulphide. S. paratyphi A and other species which do not form H 2S
produce green colonies.
3. Xylose lysine deoxycholate (XLD) agar is another medium for isolation of this organism. Most strains of salmonellae
produce red colonies with black centres, when grown on this medium. H 2S negative serotypes of Salmonella produce
red colonies without black centres.
4. Selenite F broth and tetrathionate broth (TTB) are commonly used enrichment media for inoculation of specimens
especially faeces.
5. Salmonellae ferment glucose and mannitol forming acid and gas except S. typhi which produces only acid and no
gas.
6. Most salmonellae produce H 2S in triple sugar iron (TSI) agar except S. paratyphi A and S. cholerae-suis. They utilise
citrate except S. typhi and S. paratyphi A.
7. Salmonellae possess three types of antigens namely flagellar antigen 'H', somatic antigen 'O', and surface antigen
'Vi' (found in some species).
8. The antigens of salmonellae undergo several types of phenotypic and genotypic variations.
9. Modern taxonomical techniques have shown that all the members of the genus Salmonellae belong to two species
S. enterica and S. bongori. S. enterica is classified into six subspecies namely enterica, salamae, arizonae, diarizonae,
houtenae and indica .
10. Kauffmann-white scheme forms the basis of serotyping of salmonellae based on identification of O and H antigens
by agglutination.
11. Salmonellae cause three types of clinical syndrome in human beings, enteric fever, septicaemia and gastroenteritis.
12. The term enteric fever includes typhoid fever (S. typhi) and paratyphoid fever (S. paratyphi A, B, C). Infections due
to S. typhi and S. paratypi A are prevalent in India.
13. Salmonella septicaemia is commonly caused by S. cholerae-suis or S. paratyphi C and occasionally by other
salmonellae.
14. S. typhimurium is the most frequently isolated in salmonella food poisoning. The other common species responsible
are S. enteritidis, S. newport, S. senftenberg, S. dub/in, S. heidelberg and S. indiana.
15. The faeces of carriers are important source of contamination of food and drink. Mary Mallon ('Typhoid Mary'), a
cook in USA, was a famous carrier who caused several outbreaks over a period of 15 years.
16. Laboratory diagnosis of enteric fever consists of isolation of bacilli, demonstration of antibodies, and demonstration
of circulating antigen.

274 _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __ _ __ _ _ _ _ _ Unit Ill: Systemic Bacteriology
17. Isolation of bacilli may be done by culture of specimens like blood,faeces, urine, aspirated duodena/fluid etc. Selection
of relevant specimen depends upon duration of illness which is very important for laboratory diagnosis of enteric
fever.
18. Blood culture is very important specimen in first week of enteric fever.
19. Faeces culture may be helpful in diagnosis of enteric fever as well as for the detection of carriers.
20. Wida! test is an agglutination test for detection of antibodies in patients with enteric fever. Salmonella antibodies
start appearing in the serum at the end of first week.
21. Multiple drug resistant S. typhi is increasingly being recognised in many countries and has become a problem in
India too.
22. TAB vaccine is available for prophylaxis. It is heat killed whole cell vaccine which contains S. typhi 1,000 millions, S.
paratyphi A and B, 750 millions each per ml.
23. Live oral (Ty 21a) typhoid vaccine is another vaccine available.
YOU MUST KNOW

1. Morphology, culture characteristics and biochemical reactions of Salmonella sp.
2. Antigenic structure and antigenic variations of Salmonella.
3. Kauffmann- White scheme.
4. Diseases caused by Salmonella sp.
5. Laboratory diagnosis of enteric fever.
6. Vaccination for prevention of enteric fever.
STUDY QUESTIONS
1. Describe in detail the laboratory diagnosis of enteric fever.
(a) Vi-antigen (b) Clot culture
(c) Kauffmann-White scheme (d) Antigenic variations in Salmonella
(e) Wida! test (f) Salmonella septicaemia
(g) Salmonella gastroenteritis (h) Vaccination against enteric fever
J CHO CE QUESTIONS (MCQs)

1. Which of the following disease/s is/are produced by Salmonella?
(a) Enteric fever (b) Gastroenteritis (c) Septicaemia (d) All of the above
2. Which of the following culture media is/are used as selective media for Salmonella?
(a) Wilson and Blair bismuth sulphite medium (b) Bile salt agar
(c) Potassium tellurite blood agar (d) None of the above
3. Which of the following Salmonella ferments glucose with acid but no gas?
(a) S. typhi (b) S. paratyphi A (c) S. paratyphi B (d) S. cholerae-suis
4. Which of the following Salmonella does/do not produce H 2S in triple sugar iron (TSI) agar?
(a) Salmonella paratyphi A (b) S. cholera-suis
5. Which of the following Salmonella does/do not utilise citrate?
(a) S. typhi (b) S. paratyphi A (c) Both of the above (d) None of the above
6. Which of the following Salmonella possess flagellar antigen in one phase (monophasic)?
(a) S. typhi (b) S. paratyphi A (c) S. enteritidis (d) All of the above
7. Vi antigen is present in the following bacteria:
(a) S. typhi (b) S. paratyphi C (c) S. dub/in (d) All of the above

Ch 33: Salmonella _ _ _ __ _ _ __ _ _;___ _ _ _ _ _ __ _ __ _ _ _ _ __ _ __ _ 275
8. Which of the following Salmonella is/are non-motile?

(a) Salmonella pullorum (b) S. paratyphi B (c) S. typhimurium (d) All of the above
9. Which of the following Salmonella is/are primarily infective to human beings?
(a) S. typhi (b) S. paratyphi A (c) S. paratyphi B (d) All of the above
10. Bacteriological diagnosis of enteric fever consists of:
(a) Isolation of bacilli (b) Demonstration of antibodies
(c) Demonstration of antigen (d) All of the above
11. Which of the following Salmonella is/are responsible for food poisoning?
(a) Salmonella typhimurium (b) S. enteritidis .
(c) S. senftenberg (d) All of the above
12. Enteric fever can be diagnosed by isolation of Salmonella typhi from:
(a) Blood (b) Faeces (c) Urine (d) All of the above
13. The most important specimen for isolation of Salmonella typhi in first week of enteric fever is:
(a) Blood (b) Faeces (c) Urine (d) Pus
14. What type of test is Wida) test?
(a) Slide agglutination test (b) Tube agglutination test
(c) Co-agglutination test (d) Latex agglutination test
15. Which of the following specimens is the most important for diagnosis of carriers in enteric fever?
(a) Blood (b) Faeces (c) Urine (d) Pus
16. Which of the following vaccines can be used for prevention of enteric fever?
(a) TAB vaccine (b) Live oral vaccine
(c) Purified Vi polysaccharide vaccine (d) All of the above
Answers (MCQs):
I. (d) 2. (a) 3. (a) 4. (c) 5. (c) 6. (d) 7. (d) 8. (a) 9. (d) 10. (d)
1I. (d) 12. (d) 13. (a) 14. (b) 15. (b) 16. (d)
----+++- ---

Chapter 34
VIBRIO
I. Classification
II. Vibrio cholerae
E. Antigenic Structure F. Toxins
G. Phage Typing H. Pathogenesis
I. Carrier State J. Epidemiology
M. Prophylaxis
Ill. Vibrio cholerae Non-01
IV. Halophilic Vibrios
A. Vibrio parahaemolyticus B. Vibrio alginolyticus
C. Vibrio vuln ificus
V. Aeromonas and Plesiomonas
A. Ae romonas B. Plesiomonas
Vibrios are Gram negative, oxidase positive, short, rigid, separate families, Aeromonadaceae and Enterobacteriaceae
curved rods that are actively motile by a polar flagellum. respectively. Vibrio species are susceptible to the compound
The name 'vibrio' is derived from the characteristic 0/129 (2, 4,-diarnino-6, 7 diisopropylpteridine phosphate)
vibratory motility of the bacilli ( Vibrare meaning to at strength of 150 µg , which differentiates them from
vibrate). The most important member of the genus is aeromonas species which is resistant.
Vibrio cholerae. It is the causative agent of cholera. It Genus Vibrio has about 33 species and the important
was first observed by Pacini (1854) and later on Koch pathogens of man include:
(1 883) isolated the bacillus from cholera patients.
1. Non-Halophilic Vibrios
I. CLASSIFICATION These vibrios may grow in media without salt (NaCl).
Vibrios are grouped under family Vibrionaceae which (i) V. cholerae: 01 classical and El Tor biotype
initially contained three genera, Vibrio, Aeromonas and (ii) Non 01 V. cholerae: Non-cholera vibrio (NCV) or
Plesiomonas; now a days family Vibrionaceae contains non-agglutinating vibrio (NAG) (For details refer
single genus Vibrio. Aeromonas and Plesiomonas belong to page 280)
276
Ch 34: Vibrio - -- - - - - -- - - - - -- - -- - - - - - - - - - - - -- - - - 277
V. mimicus is a non-halophilic vibrio which is similar named Vibrio comma. S shaped or spiral forms may be
to V. cholerae except that it is sucrose negative. seen due to end to end attachment of two or more cells.
In old cultures, they are frequently pleomorphic.
2. Halophilic Vibrios In stained mucous flakes of cholera cases, the vibrios
They cannot grow in media without addition of salt are arranged in parallel rows, described by Koch as the
(NaCl). fish in stream appearance.
(i) V. parahaemolyticus
(ii) V. alginolyticus B. Culture
(iii) V. vulnificus V. cholerae is strongly aerobic, growth being scanty and
Heiberg (1934) classified vibrios on the basis of slow anaerobically. It grows within a temperature range of
fermentation of mannose, sucrose and arabinose. There 16°-40°C but optimum temperature is 37°C. It grows best
are eight groups (I to VIII). Cholera vibrios belong to in alkaline media, the optimum pH being 8.2 (pH range
Group I, fermenting mannose and sucrose with acid 7.4-9 .6). The organism is extremely sensitive to an acidic
production but not the arabinose. pH which kills it. V. cholerae is a non-halophilic vibrio,
Gardner and Venkatraman (1935) classified vibrios therefore, cannot grow in media with a concentration of
based on antigenic structure. Details of this classification sodium chloride more than 7%. However it can grow in
have been described under heading 'Antigenic structure' media without sodium chloride.
of this chapter.
Modern taxonomical criteria, particularly DNA
studies, have recognised that all the cholera vibrios that 1. Ordinary Media
belong to Group A of Gardner and Venkatraman's (i) Nutrient agar
classification, constitute a single species Vibrio cholerae. After overnight incubation, the colonies are moist,
This can be further classified into serogroups (or serovars), translucent, round disks, 1- 2 mm in diameter, with a
biotypes and serotypes. The present nomenclature of bluish tinge in transmitted light.
V. cholerae strain may be written as V. cholerae serovar
01 , biotype El tor, serotype Ogawa. (ii) MacConkey's agar
Phage typing can further classify these strains. Typing The colonies are colourless or pale at first, but become
of strains can also be done by molecular methods like reddish or pink on prolonged incubation due to late
ribotyping. fermentation of lactose.
(iii) Blood agar

II. VIBRIO CHOLERAE V. cholerae, classical biotype, does not produce hemolysis
A. Morphology although some strains produce greenish discolouration
V. cholerae is a Gram negative, curved or comma-shaped around colonies which later becomes clear due to
rod, non-sporing, non-capsulated, about 1.5 µm x 0.2-0.4 haemodigestion. However, colonies of El Tor biotype
µmin size (Fig. 34.1). produce haemolysis on blood agar.
The organism is very actively motile with a single
polar flagellum and movement is named as darting motility. (iv) Peptone water
Because of its typical comma shaped appearance, it is also It grows as a surface pellicle because of its aerobic
nature.
(v) Gelatin stab culture

At first a white line of growth appears along the track
of inoculation. Gelatin liquefaction begins at the top
which spreads downward in a funnel shaped form
(infundibuliform or napiform) in 4 days at 22°C.
2. Special Media
The special media are classified as follows:
(i) Transport or holding media
(ii) Enrichment media
Fig. 34.1 Vibrio cholerae
(iii) Plating media

278 _ _ _ _ _ _ _ _ __ __ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Unit Ill: Systemic Bacteriology
TRANSPORT OR HOLDING MEDIA of V cholerae are large, yellow, convex and turn green
Venkatraman-Ramakrishnan (VR) medium on continued incubation.
It contains 20 gm common salt and 5 gm peptone in one
litre of distilled water and pH is adjusted to 8.6-8.8. It is C. Biochemical Reactions
dispensed in screw-capped bottles in 10-15 ml amount. Carbohydrate breakdown is fermentative, producing acid,
About 1- 3 ml faeces is added to each bottle. Vibrios do but no gas. It is catalase and oxidase positive. It ferments
not multiply, but remain viable for several weeks. glucose, mannitol, sucrose, maltose and mannose, but
not lactose, though lactose may be split very slowly. It is
Cary-Blair medium indole positive and reduces nitrates to nitrites. These two
This medium is prepared by adding disodium phosphate, properties contribute to the cholera red reaction which is
sodium thioglycollate, sodium chloride and calcium tested by adding a few drops of concentrated sulphuric
chloride and pH is adjusted at 8.4. It is a suitable transport acid to peptone water culture. In case of Vibrio cholerae,
medium for Salmonella, Shigella and Vibrio. a reddish pink colour is developed due to the formation
If a transport medium is not available, a strip of thick of nitrosoindole. Other indole producing organisms can
blotting paper is soaked in the faecal matter, then placed also reduce nitrates and thus give this reaction positive
in a sealed plastic bag, and sent to the laboratory. e.g. Esch. coli. It is methyl red (MR) and urease negative.
Gelatin is liquefied. It decarboxylates lysine and ornithine
ENRICHMENT MEDIA but do not utilise arginine. Voges-Proskauer (VP) reaction
Alkaline paptone water (APW) and haemolysis of sheep erythrocytes are positive in
It is a peptone water at pH 8.6. Besides enrichment ELTor biotype and both these tests are negative in
medium, it is also an excellent transport medium. classical biotype. Important diagnostic biochemical tests
of classical biotype are summarised as follows:
Monsur's taurocholate tellurite peptone water
It contains peptone, sodium chloride, sodium taurocholate
Catalase Oxidase Glucose Lactose Mannitol
in one litre of distilled water and pH is adjusted at 9.2. + + A +
To this medium, sterile potassium tellurite solution is Sucrose Maltose Mannose Indole NO 3 reduction
added to give a final concentration of 1: 200,000. Like + + + + +
Urease Gelatin MR VP*
APW, it is not only a good enrichment medium but is
+
transport medium as well.
Lysine Ornithine Arginine Sheep RBCs haemolysis*
+ +
PLATING MEDIA
*In case of El Tor biotypes, all biochemical reactions are similar but VP and
Alkaline bile salt agar (BSA); pH 8.2
sheep RBCs haemolysis are positive.
It is modified nutrient agar medium containing 0.5%
sodium taurocholate (bile salt). The colonies are similar
to those on nutrient agar. D. Resistance
Vibrio chloerae is susceptible to heat and drying. It is
Monsur's gelatin taurocholate trypticase tellurite killed at 55°C in 15 minutes. It is sensitive to common
agar (GITA) medium; pH 8.5 disinfectants and a pH less than 5. It survives for months
The colonies are small, translucent with a greyish black in sea water. In grossly contaminated water, such as the
centre and a turbid halo around the colonies due to Ganges in India, the vibrios do not survive for any length
hydrolysis of gelatin. The colonies become 3-4 mm in of time perhaps due to the presence of large number of
size after 48 hour incubation. vibriophages in this water. ELTor vibrios are hardier and
survive for longer duration than the classical biotypes.
Thiosulphate citrate bile sucrose (TCBS) agar; pH 8.6 On fruits, they survive for 1- 5 days at room temperature
It is the most widely used selective medium for isolation of and for a week in the refrigerator.
vibrios. It contains sodium thiosulphate, sodium citrate,
bile salts, sucrose, bromothymol blue (indicator), yeast E. Antigenic Structure
extract, peptone, sodium chloride, ferric citrate and water. V cholerae contains somatic 'O' and flagellar 'H' antigens.
Vibrio cholerae forms yellow colonies due to sucrose The 'H' antigen is shared by all the strains. Gardner and
fermentation, while non-sucrose fermenters such as V Venkatraman (1935) classified vibrios based on antigenic
parahaemolyticus produces green colonies. The colonies structure (Fig. 34.2).

Ch 34: Vibrio - - - -- -- - - -- - - -- -- - - - - - - - - - - - -- - - 279
I VI BRIO I
I
I I
Group A Group B
Cholera vibrios Biochemically similar Biochemically and
Common H antigen antigenically heterogenous
(Vibrio cholerae)
I
I I
1 serogroup 01 f Non-01 (02-0138)
Classical cholera vibrios EL Tor vibrios
Ogawa Inaba Hikojima Ogawa ~ ba Hikojima
Fig. 34.2 Antigenic classification of vibrios (Gardner and Venkatraman, updated)
Group A vibrios have a common H antigen but Enterotoxin

distinct 'O' component. On the basis of O antigen, it It is a heat labile, protein in nature. It has a molecular
is divided into subgroups (now named O serogroups or weight of 90,000. It has two fractions: one A (active)
serovars). Both classical and ElTor biotypes belong to subunit and five B (binding) subunits. The cholera toxin
serogroups O 1 and are antigenically indistinguishable. (CT) resembles heat-labile toxin (LT) of Esch. coli
These are referred as V cholerae OJ. On the basis of chemically, antigenically and in its mode of action. CT
minor O antigens (A, B, C), V cholerae 01 are subdivided is more potent than LT of Esch. coli. CT production
into three serotypes- Ogawa (AB), Inaba (AC) and is determined by phage integrated with bacterial
Hikojima (ABC) (Table 34.1). chromosome while LT production is plasmid mediated.
Table 34.1 Serotypes of Cholera Vibrios
Mechanism of action
Serotype 0 antigen The B subunit binds specifically to the Gm 1 ganglioside
Ogawa AB receptor on the intestinal epithelial cell. The active A
Inaba AC
subunit then enters the cell and is cleaved to its fragments,
Al and A2. The A2 fragment links the active Al to the
Hikojima ABC
B subunit and thus the Al fragment stimulates adenyl
cyclase activity of the epithelial cell. This in turn, converts
ATP into cAMP. The cAMP concentration within the
At least 139 (01 to 0139) 0 serogroups are recognised. cells causes (i) hypersecretion of water and electrolytes
Serogroups 02 to 0138 are called non-O1 V cholerae. (Na, K, HCO3) within the intestinal lumen and
Since these organisms were not agglutinated by 0 -1 (ii) inhibition of reabsorption of sodium and chloride
antiserum, they were called non-agglutinating (NAG) by cells, which results in purging diarrhoea of rice water
vi brios. Though NAG vibrios are not agglutinable by stool. The fluid secreted is isotonic with plasma but
0- 1 antiserum, they are agglutinated by their specific contains more of potassium and bicarbonate.
homologous antisera. The enterotoxins of V cholerae and NAG vibrios
appear to be identical in antigenic specificity.
F. Toxins
Besides heat stable endotoxin, V cnolerae also produces ELTOR VIBRIOS
exotoxin (enterotoxin). The enterotoxin is also named as Gotschlich isolated a vibrio from six Haj pilgrims at the
cholera toxin (CT), choleragen or cholera enterotoxin. Tor quarantine station in Arabia and the organism was
Both V cholerae 01 and 0139 produce cholera toxin. called ElTor Vibrio. Antigenically it is identical to classical

280 _ _ __ _ __ _ _ _ _ __ __ __ _ _ _ __ __ _ _ _ Unit Ill: Systemic Bacteriology
cholera vibrio and is now proved to be biotype of V. NON-AGGLUTINATING VIBRIOS

cholerae-01. The following tests are used for identification (NON-01 V. CHOLERAE)
of El Tor biotype (Table 34.2). The somatic antigen 01 is specific to the classical and
El Tor biotypes of V. cholerae, while vibrios containing
Table 34.2 Differentiating Features of Classical and El antigen other than 01 and 0139 are called non-
Tor Vibrios agglutinating (NAG) or non-cholera vibrios (NCV) or
Classical El Tor
Non-O1 V. cholerae. There are 139 serogroups containing
Test
0 antigen, 01 to 0139. Serogroup 01 and 0139 contain V.
Haemolysis of sheep erythrocytes + cholerae, while 02 to 0138 are NAG vibrios. It is extremely
Agglutination of chick + difficult to distinguish most of the NAG vibrios from V.
erythrocytes cholerae by means of culture and biochemical reactions.
Voges-Proskauer (VP) reaction + They are not agglutinable by 01 antiserum, therefore,
Polymyxin B sensitivity + named as non-agglutinable (NAG) vibrios. However, they
are readily agglutinable by their homologous antiserum.
Susceptibility to Mukherjee Phage + I
IV
V. cholerae 0139
Modified CAMP test +
V. cholerae serogroup O 139 originated in Madras, India,
in 1992 and has led to a widespread occurrence of cholera
cases throughout India and Bangladesh. Later on V.
1. Chick Red Cell Agglutination Test cholerae 0139 emerged as aetiological agent in a series
A loopful of the organisms from an agar cultures is of outbreaks of cholera in the entire Indian subcontinent.
emulsified in a drop of saline on a slide and a drop of V. cholerae strains of serogroup 01 and serogroup group
2.5% chick erythrocyte suspension is added. Clumping 0139 cause classical cholera. V. cholerae O 139 is very
of erythrocytes within a minute indicates a positive test. similar to El Tor biotype of Vibrio cholerae. It does not
The test is positive with all El Tor strains but classical produce the O 1 lipopolysaccharide. Like El Tor biotype,
cholera vibrios are negative. it is also positive for modified CAMP test. It makes a
polysaccharide capsule like other non-O1 V. cholerae
2. Sensitivity to Polymyxin B strains, while V. cholerae O 1 does not make a capsule.
The organism is tested by the disc diffusion method using Like V. cholerae O1, it ( V. cholerae O139) also produces
discs containing 50 units of polymyxin B. All strains of enterotoxin (cholera toxin).
classical cholera vibrio are sensitive and all strains of El
Tor vibrio are resistant. G. Phage Typing
The classical strains of cholera vibrios are classified into
3. Sensitivity to Cholera Phage IV five types (1 to 5) by using Mukherjee's phages I to IV.
All strains of classical cholera vibrio are lysed by Phage IV lyses all classical but not EL Tor strains (Table
Mukherjee's group IV phage routine test dilution (RTD), 34.3). El Tor strains can be divided into six types (1 to 6)
while all El Tor strains are not lysed. This is considered on the basis of lysis by five phages (I to V). All El Tor
to be the most dependable test for differentiating between strains are lysed by phage V (Table 34.4). National Institute
El Tor and classical strains. of Cholera and Enteric diseases (NICED), Kolkata is the
International Reference Centre for Vibrio phage typing.
4. Modified CAMP Test Table 34.3 Phage Types of Classical Biotype of V. cholerae
This test is similar to the CAMP test for identification of Sensitivity to phage group
group B streptococci. It is performed by inoculating a beta- Phage type
I II III IV
lysin producing Staph. aureus strain onto a blood agar plate
by making a single straight line streak and then inoculating + + + +
the vibrio species to be tested in a line prependicular to it. 2 + + +
The test is positive with El Tor strains but classical cholera 3 + + +
vibrios are negative. However, 0139 strains also demonstrate
4 + +
a strong positive CAMP reaction while non-O1 and non-
O139 vibrios give a weak positive reaction. 5 + + +

Ch 34: Vibrio-- -- - - -- - - - -- - - - -- -- - - - - - - - - - - - - 281
Table 34.4 Phage Types of El Tor Biotype of J. Epidemiology

V. cholerae Cholera is an epidemic disease and man is the only
Phage type Sensitivity to phage group natural host of V. cholerae. Cholera has been endemic
I II m IV V in the Ganges and Brahmaputra deltas in Bengal and
neighbouring parts of the Indian subcontinent. Before
+ + + + +
1817, cholera was confined to these endemic areas. Between
2 + + + + I 817 and 1923 cholera spread to all over the world in six
3 + + + + pandemics. All these pandemics were caused by classical
4 + + + biotype of V. cholerae. Between 1923 and 1961, the disease
remained confined almost to endemic areas except for
5 + + one isolated epidemic in Egypt in 1947.
6 + + The recent pandemic, the seventh, occurred in 1961
and this pandemic has been caused by the EL Tor
H. Pathogenesis biotype. The El Tor vibrio is hardier and more capable of
V. cholerae (both 01 and 0139) causes an acute diarrhoeal surviving in the environment. It leads to mild cases, higher
disease known as cholera and it occurs only in man. The incidence of carriers and greater chances of endemicity as
human infection occurs by ingestion of contaminated compared to classical vibrio. Whenever the El Tor vibrio
foods and drink. The ingested organisms pass through has caused infection, it displaces the classical vibrio in
the acid barrier of the stomach and multiply in the all those areas.
alkaline medium of the small intestine. The vibrio,s do not Epidemic cholera has a seasonal distribution, the
penetrate deep into the gut and there is no bacteraemia. epidemic seasons are different in different areas. In India
Vibrios become adherent to the epithelium by special (Calcutta) the epidemic season is in the hot dry months
fimbria such as the tox in co-regulated pilus (TCP). Once of March to May and ends with onset of monsoon in
epithelial cell attachment occurs, V. cholerae produces June, while in Bangladesh the cholera season (November
enterotoxin and the disease. Mechanism of action of to February) follows the monsoon rains. Epidemic cholera
enterotoxin has been described earlier. Enterotoxin and has been associated with fairs, festivals during which
TCP are regulated by the Tox R gene product, Tox R sanitary conditions tend to be unsatisfactory. The only
protein. natural reservoir is man in the form of convalescent
The massive loss of water and electrolytes (sodium and · chronic carriers. The transmission is maintained
and bicarbonates) by action of enterotoxin, leads to : by a cycle involving the excretor (carrier) and the
I. dehydration causing haemoconcentration, anuria and environment, particularly a water source. Since vibrios
hypovolaemic shock do not survive in water for a long period, so it should
2. base-deficit acidosis and be repeatedly contaminated to act as a prolonged source
3. muscle cramps due to hypokalaemia. of infection.
In untreated cases the mortality rate is 60- 70% due In 2012, the World Health Organisation reported an
to renal failure. Both the biotypes of V. cholerae O 1 outbreak of cholera in Sierra Leone, causing 280 deaths
produce equally severe disease but in El Tor strains, the among 20,700 cases.
incidence of mild and asymptomatic infections is more
frequent. The NAG vibrios may produce a clinical disease K. Laboratory Diagnosis
indistinguishable from cholera. 1. Specimens
(i) Watery stool
I. Carrier State (ii) Rectal swab
1. Convalescent Carrier
After treatment, few cases may continue to excrete vibrios 2. Collection and Transport
for 4 to 5 weeks due to survival of the bacilli in gall Specimens should be collected preferably prior to start of
bladder. These are known as convalescent carriers. antibiotics. These should not be collected from bedpans
due to risk of contamination. Specimens should be
2. Chronic Carrier immediately transported to the laboratory for processing.
It is found in endemic areas and the vibrios are excreted In case of delay, stool samples may be preserved in holding
in faeces intermittently from the gall bladder. Chronic media such as VR fluid or Cary-Blair medium for long
carriers have been found in ElTor cholera and serve to periods. If the specimen can reach the laboratory in a
perpetuate inter-epidemic infections. few hours, enrichment media such as alkaline peptone

water or Monsur's medium may be used as transport 7. Agglutination Test

media. When transport media are not available, strips of Colonies are picked up with a straight wire and tested
blotting paper may be soaked in watery stool and sent to with V cholerae 01 antiserum. If positive, the test is
the laboratory after proper packing in plastic envelopes. repeated with monospecific Ogawa and Inaba sera for
If possible, specimens should be plated at bedside and serotyping. The test should be repeated with at least five
the inoculated plates sent to the laboratory. colonies as V. cholerae 01 and non-O1 V cholerae may
co-exist in the same specimen.
3. Direct Microscopy The H antigen is shared by both V cholerae 01 and
It is not a reliable method. For rapid diagnosis, the non-O1 V. cholerae. Any vibrio which is agglutinated
characteristic darting motility of the Vibrio and its by H antiserum, but not by V cholerae O 1 antiserum is
inhibition by adding antiserum can be demonstrated considered to be NAG vibrio. These strains should be
under the dark field or phase contrast microscope, using tested with other O antiserum to establish their identity
cholera stool. as they may be belonging to one of 02- 0139 serogroups.
Specific antiserum against O 139 is available.
4. Culture
(i) Stool sample is directly cultured on following 8. Phage Typing
media. It is helpful in epidemiological study of cholera. It is of
(a) Selective media (BSA, TCBS or Monsur's little use in diagnosis.
GTTA) and non-selective media (blood agar
and MacConkey's agar) are inoculated. These 9. Serological Tests
plates are incubated at 37°C for overnight. These are of little use in diagnosis of cholera though it may
(b) Enrichment media such as alkaline peptone water help in assessing the incidence of cholera in an area. The
or Monsur's liquid media are inoculated. These tests used are agglutination, indirect haemagglutination,
media are incubated at 37°C for 6-8 hours before vibriocidal tests and antitoxin assay of serum.
subculturing on to selective media.
(ii) When the specimen has been collected in holding DETECTION OF CARRIERS
medium, it is first inoculated into enrichment medium The procedures are similar to those of an infective case
and incubated at 37°C for 6-8 hours before plating except:
onto a selective medium. 1. repeated stool examination may be needed as the
(iii) The specimen collected in enrichment medium should vibrio is excreted intermittently.
be incubated for 6-8 hours including transit time 2. it gives better result if stool is collected after
before subculturing onto a selective medium. administration of a purgative.
3. bile may be examined after duodenal intubation.
5. Colony Morphology and Staining 4. serological tests are useful in detecting chronic
After overnight incubation, culture media are examined earners.
for typical colonies of V. cholerae. On MacConkey's agar,
colonies are pale and on Monsur's medium the colony BACTERIOLOGICAL EXAMINATION OF WATER
has a black centre with a turbid halo around the colony. AND SEWAGE
TCBS shows yellow colonies and on BSA, translucent 1. Water sample: 900 ml water is added to 100 ml of
colonies are present. ten fold concentrated peptone water (pH 9.2) and
Gram staining from colony shows typical Gram incubated at 37°C for 6-8 hours. After incubation,
negative comma shaped bacilli. These show darting motility it is inoculated on selective media.
on hanging drop preparation. Further confirmation is 2. S ewage: It is diluted in saline and filtered through
done by biochemical reactions and agglutination test. gauze. The filtrate is treated as water.
6. Biochemical Reactions L. Treatment

V. cholerae ferments glucose, mannitol, sucrose maltose, 1. Oral Rehydration Therapy
mannose with acid production. Lactose is usually not The most important is prompt water and electrolyte
fermented. Catalase, oxidase and cholera red reactions replacement to correct the severe dehydration and salt
are positive. The El Tor biotype is usually haemolytic, VP depletion . This can be achieved by oral rehydration
positive, agglutinate chick erythrocytes and is resistant therapy, either alone or supplemented by intravenous
to polymyxin B and group IV cholera phage. fluids.

Ch 34: V i b r i o - - - - - - -- - - - -- - - - - -- - - - - - - - - - - - -- - - 283
2. Antibiotics CHEMOPROPHYLAXIS
Antibiotics are of secondary importance. Tetracycline In endemic areas, chemoprophylaxis with anti-microbial
is useful in reducing the number of stool and it also drugs is useful for attendants, nurses etc.
shortens the period of excretion of vibrios.
III. VIBRIO CHOLERAE NON-01

M. Prophylaxis These strains are identical to V cholerae- OJ biochemically
1. General Measures and in other characters. Their isolation and identification
(i) Purification of water supplies. procedures are same as those of V cholerae-OJ. There
(ii) Better provision for sewage disposal. are 137 (02- 0138) serotypes that occur under this
(iii) Infected patients should be isolated, their excreta group. Most infections are due to contact with saline
disinfected. environments and consumption of seafood.
(iv) Contacts and carriers are followed up. Non-OJ Vcholerae are less virulent than
V cholerae-OJ and cause mild diarrhoea. Occasionally the
symptoms may be as severe as cholera. Wound infection
2. Specific Measures with contaminated water, bacteraemia and meningitis are
ACTIVE IMMUNISATION other infections reported by these organisms.
Killed parenteral vaccine Non-OJ V cholerae strains may produce entero-toxins,
This vaccine contains 12,000 million V cholerae per ml, haemolysins and cytotoxins. A few strains produce cholera
composed of equal number of Inaba and Ogawa strains. toxin which can cause cholera like disease.
It is widely used for active immunisation. Two injections
are given intramuscularly at an interval of four weeks.
Degree of protection does not exceed 50- 60% and the IV. HALOPHILIC VIBRIOS
duration of protection is only 3- 6 months. Halophilic vibrios cannot grow in media lacking sodium
A single dose confers good protection in adults chloride. They grow best in the presence of sodium
due to its action as a booster on top of prior natural chloride. Their natural habitat is sea water and marine life.
immunisation. In children below five years of age, a Some halophilic vibrios which cause human diseases are
single dose is ineffective. There is a good cross protection V parahaemoly ticus, V alginolyticus and V vulnificus.
between classical and El Tor vibros.
The aluminium hydroxide and phosphate adjuvant A. Vibrio parahaemolyticus
vaccines have been tried. These vaccines have induced a It was first isolated in Japan in 1951 as causative agent
high degree of immunity, particularly in young children. of food poisoning due to consumption of seafood. It
Cholera toxoid has been tried as vaccine but without was named as V parahaemoly ticus by Sakazaki (1963)
any success. due to haemolysis produced by this organism on blood
agar. It has been isolated from shell fish, shrimps, crabs
Killed oral vaccine and molluscs in many countries.
B subunit whole cell ( BS- WC) vaccine has undergone a Morphologically, it resembles V cholerae and other
field trial in Bangladesh. The vaccine contains cholera vibrios. Unlike other vibrios, it produces peritrichous
toxin B subunit, heat killed classical vibrio (both Ogawa flagella when grown on solid media but polar flagella are
and Inaba) and formalin killed ElTor vibrio (both Ogawa formed in liquid media. It grows only in media containing
and Inaba). Degree of protection was 85% for one year sodium chloride (optimum concentration 2 to 4 per cent).
and 60% for two years. This vaccine also conferred It can tolerate sodium chloride concentration up to 8 per
significant protection against diarrhoea due to Esch. coli cent but not ten per cent. They do not ferment sucrose,
(LT- ETEC). therefore, produce green colonies on TCBS medium.
Biochemical reactions are similar to classical V cholerae
Live oral vaccine except it is sucrose negative.
Recombinant DNA vaccine with expression of It is killed by heat at 60°C in 15 minutes. It does not
V cholerae in attenuated strain Ty2la of Salmonella grow at 4°C but can survive freezing. Drying destroys it.
typhi has been developed. This vaccine colonises Peyer's All strains of V parahaemolyticus are not pathogenic
patches and induces IgA response by local immunity. for man. Strains isolated from environmental sources are
Live attenuated vaccine against O 139 is also available nearly always non-haemolytic when grown on a high
in some countries. salt blood agar (Wagatsuma agar) , while strains isolated

from human cases are almost always haemolytic. This medically important species include Aeromonas hydrophila
is called the Kanagawa phenomenon. It is due to heat and Plesiomonas shigelloides.
stable haemolysin. Kanagawa positive strains are being
considered pathogenic for man and negative strains non- A. Aeromonas
pathogenic. A. hydrophila is found in water. It is Gram negative bacilli
The organism causes food poisoning associated or coccobacilli. It is motile by single polar flagellum. It
with marine food . It can also cause acute diarrhoea is catalase and oxidase positive. It can reduce nitrate
unassociated with food poisoning. Abdominal pain, to nitrite. It grows well on nutrient agar, blood agar,
diarrhoea, vomiting and moderate degree of dehydration MacConkey agar, DCA and TCBS. It ferments glucose,
are the dominant features. Recovery occurs within three sucrose, maltose and starch with the production of acid
days. and gas. It produces yellow colonies on TCBS agar.
Aeromonas is resistant to the compound 0/129 at strength
B. Vibrio alginolyticus of 150 µg.
It is frequently found in sea fish and its role in human It may cause diarrhoea, wound infections, pyogenic
lesion is uncertain. It resembles V parahaemolyticus in infections and septicaemia in humans. It produces
many aspects. It has a higher salt tolerance (up to 10 per an enterotoxin which resembles cholera toxin and is
cent), is VP positive and, ferments sucrose. These features neutralised by V cholerae antitoxin.
differentiate it from V parahaemolyticus. (Table 34.5).
It has a property to swarm on the surface of non- B. Plesiomonas
selective media. It has been incriminated in marine wound The genus Plesiomonas has now been classified in family
infection. Enterobacteriaceae. It has only one species P shigelloides.
The strains of P shigelloides share antigens with Sh. sonnei
Table 34.5 Differentiating Features of and some are agglutinated by Shigella sonnei. antiserum.
V. parahaemolyticus and V. alginolyticus It has derived its name 'shigelloides' from this property.
V. para- V. algino- They are Gram negative bacilli. They are motile by polar
Feature
haemolyticus lyticus flagella usually lophotrichous. They are oxidase positive.
V.P. reaction + They grow well on blood agar, MacConkey agar but
do not grow on TCBS medium. Most strains ferment
Sucrose fermentation +
inositol with the production of acid only. They do not
Swarming + ferment sucrose.
Growth in 0% NaCl P shigelloides is commonly found in surface waters
7% NaCl + + and in soil. It has also been isolated from a variety of
+ mammals. It is rarely recovered from human faeces.
10% NaCl
Man is infected by ingesting contaminated water or
food. It causes gastroenteritis but in immunosuppressed
C. Vibrio vulnificus individuals a cholera-like illness may be seen. It may also
It was previously designated as L+ vibrio for its ability cause septicaemia, neonatal meningitis and cellulitis.
to ferment lactose. It resembles V parahaemolyticus Aeromonas and plesiomonas are differentiated from
but ferments lactose and has a salt tolerance of less Vibrio cholerae by biochemical reactions such as utilisation
than eight per cent. It may cause wound infection and of aminoacids, lysine, arginine and ornithine (Table 34.6).
cellulitis following exposure of wounds to seawater. Vibrio cholerae is susceptible to the compound 0 /129,
Following ingestion of under-cooked or raw seafood, it while aeromonas is resistant.
penetrates the gut mucosa without causing gastrointestinal
manifestations and enters the blood leading to septicaemia Table 34.6 Utilisation of Aminoacids in Vibrio,
with high mortality. Aeromonas and Plesiomonas
Organism Lysine Arginine Ornithine
V cholerae + +
V. AEROMONAS AND PLESIOMONAS
A eromonas +
These are Gram negative, motile bacilli which are oxidase
positive. They are aerobes and facultative anaerobes. The Plesiomonas + + +

Ch 34: Vibrio------ - - - - - -- - - - - -- - - -- - - - - - - - - - 285
KEY POINTS
1. Vibrios are Gram negative, oxidase positive, short, rigid, curved or comma-shaped rods that are actively motile by a
polar .flagellum.
2. Vibrio cholerae is the causative agent of cholera. Two serogroups 01 and 0139 are responsible for the disease. Both
serogroups produce cholera toxin (CT).
3. Two biotypes of Vibrio cholerae include classical and El Tor. Three serotypes are Ogawa (AB), Inaba (AC) and
Hikojima (ABC) on the basis of minor 'O' antigens.
4. Special media with alkaline pH are required for growth of vibrios.
5. Venkataraman-Ramakrishnan (VR) medium and Cary-Blair medium are transport media for V cholerae while alkaline
peptone water (APW) is an enrichment medium.
6. Alkaline bile salt agar (BSA) and Thiosulphate citrate bile sucrose (TCBS) are two important solid media for growing
V cholerae.
7. Classical and El Tor vibrios can be differentiated by haemolysis, agglutination of chick erythrocytes, VP reaction,
polymyxin B sensitivity, susceptibility to Mukherjee phase IV and modified CAMP test.
8. Killed parenteral vaccine is widely used for protection against cholera.
9. Non-01 V cholerae are also named as non-agglutinating vibrios (NAG).
10. Halophilic Vibrios cannot grow in media lacking sodium chloride. V parahaemolyticus, V alginolyticus and
V vulnificus are some examples of halophilic vibrios. V cholerae is a non-halophilic vibrio.
YOU MUST KNOW

1. Morphology, culture characteristics and biochemical reactions of Vibrio cholerae.
2. Serotypes of V cholerae.
3. Mechanism of action of cholera toxin (CT).
4. Differences between classical and El Tor vibrios.
5. Laboratory diagnosis of cholera.
6. Cholera vaccines.
7. Non-agglutinating vibrios.
8. Halophilic vibrios.
STUDY QUESTIONS
1. Discuss the laboratory diagnosis of cholera.
(a) El Tor vibrios (b) Non-agglutinating vibrios
(c) Prophylaxis against cholera (d) Halophilic vibrios
(e) Aeromonas (f) Plesiomonas.
ULT! LE C O CE QUESTIONS (MCQs)

1. Which of the following bacteria has darting motility?
(a) Escherichia coli (b) Vibrio cholerae
(c) Salmonella typhi (d) Pseudomonas aeruginosa
2. Which of the following media can be used as transport medium for Vibrio cholerae?
(a) Nutrient broth (b) Venkatraman-Ramakrishnan medium
(c) Selenite F broth (d) None of the above

286 _ _ __ _ _ __ _ _ __ _ __ _ __ _ __ _ _ __ _ _ _ Unit Ill: Systemic Bacteriology
3. Which of the following bacteria produce yellow colonies on thiosulphate citrate bile sucrose agar?
(a) Vibrio cholerae (b) Vibrio parahaemolyticus
(c) Klebsiella (d) All of the above
4. Which of the following bacteria produce green colonies on thiosulphate citrate bile sucrose agar?
(a) Vibrio cholerae (b) Vibrio parahaemolyticus
(c) Klebsiella (d) All of the above
5. Cholera toxin resembles:
(a) Heat-labile toxin of Escherichia coli (b) Heat stable toxin of Esch. coli
6. Which of the following tests can be used to differentiate Classical and ElTor vibrios?
(a) Haemolysis of sheep erythrocytes (b) Vogas-Proskauer reaction
(c) Modified CAMP test (d) All of the above
7. Stool specimens from cholera cases can be collected and transported to the laboratory using:
(a) Venkatraman-Ramakrishnan medium (b) Cary-Blair medium
(c) Strips of thick blotting paper (d) All of the above
8. Which of the following vaccines is/are available for prevention of cholera?
(a) Killed parenteral vaccine (b) Killed oral vaccine
(c) Live oral vaccine (d) All of the above
9. All of the following are halophilic vibrios except:
(a) Vibrio alginolyticus (b) V parahaemolyticus
(c) V vulnificus (d) V cholerae
10. Vibrio parahaemolyticus strains isolated from environmental sources can be differentiated from those isolated from
humans cases by:
(a) Kanagawa phenomenon (b) Modified CAMP test
(c) Reverse CAMP test (d) AJI of the above
11. Which of the following bacteria is associated with food poisoning due to consumption of sea food?
(a) Vibrio parahaemolyticus (b) V cholerae
(c) V vulnificus (d) All of the above
Answers (MCQs):
1. (b) 2. (b) 3. (a) 4. (b) 5. (a)
6. (d) 7. (d) 8. (d) 9. (d) 10. (a)
11. (a)
---•••---

Chapter 35
CAMPYLOBACTER, HELICOBACTER, MOBILUNCUS
I. Campylobacter
E. Laboratory Diagnosis F. Species Other Than C. jejuni
II. Helicobacter
A. Helicobacter pylori B. Helicobacter cinnaedi
C. Helicobacter fennelliae
Ill. Mobiluncus
E. Laboratory Diagnosis F. Treatment
I. CAMPYLOBACTER lari grow optimally at 42°C. However, C. fetus and most

Campylobacters are slender, spirally curved, Gram other campylobacters produce visible growth at 37°C.
negative bacilli. They resemble vibrios but differ in being Campylobacters growing at 42°C are referred to as the
microaerophilic, not fermenting sugars and having a lower thermophilic gro up.
G + C content of DNA. Colonies are circular and convex but those of
There are ten species in Genus Campylobacter but C. thermophilic group, particularly C. jejuni, are flat and
jejuni, C. coli, C. concisus, C. f etus, C. hyointestinalis, C. tend to swarm on moist agar. Well formed colonies are
/ari and C. sputorum are of medical importance. seen only after 48 hours.
A. Morphology C. Biochemical Reactions

Campylobacters are small, curved, Gram negative bacilli All produce oxidase and most species are catalase positive.
measuring 0.5- 5 µm x 0.2-0.5 µm. They are motile by single They are biochemically inactive and do not utilise sugars
polar flagellum (monotrichate) or amphitrichate flagella. or produce indole. Most species reduce nitrate to nitrite.
Of all the species of campylobacter, only C. jejuni can
B. Culture hydrolyse sodium hippurate.
Growth of campylobacters occurs under strict
microaerophilic conditions. They grow readily on simple D. Pathogenesis
media in an atmosphere of a mixture of oxygen (5%), C. jejuni has emerged as the most important human
CO 2 (10%) and nitrogen (85%). C. j ejuni, C. coli and C. pathogen in diarrhoeal diseases and accounts for 90%
287

Chapter 36
PSEUDOMONAS, STENOTROPHOMONAS, BURKHOLDERIA
I. Pseudomonas aeruginosa
C. Pigment Production D. Biochemical Reactions
E. Resistance F. Antigenic Structure
G. Toxins and Enzymes H. Typing Methods
I. Pathogenesis J. Laboratory Diagnosis
K. Treatment L. Control
M. Epidemiology
II. Other Pseudomonas
Ill. Stenotrophomonas maltophilia
IV. Burkholderia mallei (Pseudomonas mallei)
C. An imal Pathogenicity D. Human Pathogenicity
E. Laboratory Diagnosis
V. Burkholderia pseudomallei (Pseudomonas pseudomallei)
C. Biochemical Reactions D. Toxins
E. Pathogenesis F. Laboratory Diagnosis
VI. Burkholderia cepacia (Pseudomonas cepacia)
C. Pathogenicity
VII. Non-Fermenters Other than Pseudomonas and Burkholderia
The genus Pseudomonas comprises of aerobic, Gram or wherever decomposing organic matter is found.
negative, non-fermentative, non-spormg, oxidase The genus Pseudomonas belongs to the family
positive bacilli which are motile by polar flagella. Pseudomonadaceae which contains over 200 species.
Many species produce water soluble pigments which Human disease has been caused by Ps. aeruginosa, Ps.
diffuse through the culture medium. Majority of maltophilia, Ps. ma/lei, Ps. pseudomallei, Ps. cepacia, Ps.
them are saprophytic being found in soil, water, sewage stutzeri, Ps. fiuorescens, Ps. multivorans and Ps. putida.
292
Ch 36: Pseudomonas, Stenotrophomonas, Burkholderia _ _ _ _ _ _ _ _ __ __ __ _ _ _ _ _ 293
Table 36.1 Differentiating Features of Commonly Encountered Species

Oxidation of Lysine Arginine
Species Growth
Motility Oxidase Pyocyanin Fluorescin decar- dihyd-
at 42°C Glucose Lactose Mannitol Maltose boxylase rolase
Ps. aeruginosa + + + + + + +
Ps. stutzeri + + + + +
Ps. putida + + + + +
Sten. maltophilia + + +/- + + +
B. ma/lei + + +
B. pseudomallei + + + + + + + +
B. cepacia + + + + + + + +
The most important among these is Ps. aeruginosa. 3. MacConkey Agar

Recently, Ps. mallei, Ps. pseudomallei and Ps. cepacia Colonies are pale or colourless (non-lactose fermenters,
have been assigned a new genus Burkholderia which also NLF).
belongs to family Pseudomonadaceae. New names for
these species are Burkholderia mallei, B. pseudomal/ei and 4. Cetrimide Agar
B. cepacia. B. mallei is non-motile. B. cepacia has been It is a selective medium for Ps. aeruginosa.
included under B. cepacia complex which contains about
16 more species of Burkholderia. Pseudomonas maltophilia 5. Peptone Water
has been named as Stenotrophomonas ma/tophilia Differen- It forms a turbidity with a surface pellicle. Pseudomonas
tiating features of commonly encountered species are being a strict aerobe tends to collect at the surface for
shown in Table 36.1. more oxygen hence forming surface pellicle.
C. Pigment Production
I. PSEUDOMONAS AERUGINOSA Ps. aeruginosa produces a number of pigments which
A. Morphology diffuse into surrounding medium. These pigments are:
It is slender, Gram negative bacillus, 1.5-3 µm x 0.5 µm,
non-capsulated, non-sporing and is actively motile by 1. Pyocyanin
a polar flagellum. Most strains possess pili. It is non- It is a bluish-green phenazine pigment soluble in
capsulated though mucoid strains may sometimes occur. chloroform and water. It is not produced by other species
Occasionally strains have two or three polar flagella. of the genus, hence, it is diagnostic of Ps. aeruginosa.
B. Culture 2. Fluorescin (Pyoverdin)

It is a strict aerobe and grows well on ordinary media It is a greenish yellow pigment insoluble in chloroform
like nutrient broth and nutrient agar. The optimum but soluble in water. It may be produced by many other
temperature for growth is 37°C, but growth occurs at a species also.
wide range of temperature 5°C to 42°C.
1. Nutrient Agar
Colonies are smooth, large, translucent, low convex,
2-4 mm in diameter. The organism produces a sweetish
aromatic odour. This is due to the production of
2-aminoacetophenone. There is greenish blue pigment
which diffuses into the medium (Fig. 36.1).
2. Blood Agar
Colony characters are similar to those on nutrient agar.
Fig. 36.1 Pseudomonas aeruginosa on nutrient agar
Many strains are haemolytic on blood agar.

294 _ _ _ __ __ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __ _ Unit Ill: Systemic Bacteriology
3. Pyorubin G. Toxins and Enzymes

It is a reddish brown pigment that is insoluble m Several toxins and enzymes produced by Ps. aeruginosa
chloroform but soluble in water. contribute to enhance its virulence.
4. Pyomelanin 1. Extracellular Products

It is a brown to black pigment and is chemically unrelated Pyocyanin inhibits mitochondrial enzymes in mammalian
to melanin. Its production is uncommon. tissue and causes disruption and cessation of ciliary beat
on ciliated nasal epithelium. Thus, this favours colonisation
D. Biochemical Reactions of these organisms in the nasal mucosa.
Ps. aeruginosa derives energy from carbohydrates
by oxidative breakdown rather than a fermentative 2. Extracellular Enzymes and Haemolysins
metabolism. Special 0-F medium of Hugh and Leifson It produces proteases (general protease, alkaline protease
must be used to find out oxidative metabolism. It utilises and elastase), haemolysins (phospholipase.C and heat
only glucose oxidatively with acid production. Lactose stable rhamnolipid) and lipase. These play a key role in
and maltose are not utilised. All strains of Ps. aeruginosa producing local lesions.
are oxidase positive and utilise citrate as the sole source
of carbon. They are catalase positive and indole, MR, VP 3. Exotoxin
and H 2S tests negative. They reduce nitrates to nitrites It produces two exotoxins, A and S. Exotoxin A is a
and further to gaseous nitrogen. Arginine dihydrolase polypeptide of molecular weight 66,000- 72,000 and
test is positive. Important biochemical reactions are inhibits protein synthesis. Mechanism of action of
summarised below. exotoxin A is similar to that of diphtheria toxin.
N03 4. Endotoxins
0 /F test Catalase Oxidase reduction Glucose Lactose It is a lipopolysaccharide (LPS) exhibiting many biological
Oxidative + + + A properties of enterobacterial LPS including pyrogenic
action etc.
Mannitol Sucrose Citrate lndole Urease H2S
+ - /+
H. Typing Methods
E. Resistance As Ps. aeruginosa is an important cause of hospital-
It is killed by heating at 55°C for one hour. It is resistant acquired infections, it is essential to type the strain for
to the chemical disinfectants and can even grow in certain epidemiological study.
antiseptics like quaternary ammonium compounds,
chloroxylenol and hexachlorophane. Indeed, selective 1. Bacteriocin (Pyocin) Typing
media have been devised for isolation of Ps. aeruginosa Three types of bacteriocins (pyocins) are produced by
incorporating dettol or cetrimide. It is sensitive to 2% Ps. aeruginosa. These pyocins are known as R , F and
aqueous alkaline solution of glutaraldehyde and also to S. Pyocin-producing strains are resistant to their own
silver salts. Due to its sensitivity to silver salts, silver pyocins though they may be sensitive to pyocins produced
sulphonamide compounds has been applied as topical by other strains. Pyocin produced by the test strain is
cream in burns. employed to assess the growth inhibition of 13 (1- 8 and A
It is intrinsically resistant to commonly used to E) indicator strains of Ps. aeruginosa. Depending upon
antibiotics. Examples of clinically effective antibiotics are the growth inhibition of these 13 indicator strains, 105
polymyxin B, colistin, piperacillin, ticarcillin, cefotaxime, types are recognised. Pyocin typing is the most popular
gentamicin, azlocillin, tobramycin and ciprofloxacin. method employed for typing of Ps. aeruginosa.
F. Antigenic Structure 2. Phage Typing

0 Antigens Considerable difficulties have been encountered m
It possesses 19 distinct, group specific O antigens. These bacteriophage typing.
antigens are heat stable
3. Serotyping
H antigens Based on O and H antigens, 17 serotypes of Ps. aeruginosa
Two heat-labile H antigens have been recognised in Ps. are recognised. It is said to be reliable but facilities exist
aeruginosa. only in reference laboratories.

Ch 36: Pseudomonas, Stenotrophomonas, Burkholderia _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __ 295
olc 1 la · \II ,tlzod hours. On nutrient agar, there is bluish green pigment
Restriction endonuclease typing with pulsed-field gel diffused in the medium. On MacConkey's agar they
electrophoresis (PAGE) is the most reliable typing grow as pale colonies (NLF). In peptone water, surface
method. pellicle and green pigment can be observed. Selective
media such as cetrimide agar may be necessary to isolate
I. Pathogenesis Ps. aeruginosa from faeces or other samples with mixed
It causes infections more common in patients with flora . As Ps. aeruginosa is a frequent contaminant,
neutropenia, cystic fibrosis, burns and those on ventilators. isolation of the bacillus from a specimen should not
It is the most important agent causing nosocomial always be taken as aetiological agent. Repeated isolations
infections. It is due to its resistance to common antibiotics would help to confirm the diagnosis.
and antiseptics that it establishes itself widely in hospitals.
Equipments such as respirators and endoscopes, articles 3 Gmm Stai 1i1tg n , I 1' 'il,t,
such as bed pans, and antiseptic or disinfectant solutions They are Gram negative bacilli and are actively motile.
may be frequently contaminated. The other common
infections caused by it are: 4. Biochemical Reactio, "
1. Urinary tract infections following catheterisation. The oxidase test is positive within 30 seconds. They are
2. Acute purulent meningitis following lumbar non-fermenter. They break down glucose oxidatively with
puncture. acid production only. Other biochemical reactions also
3. Post-tracheostomy pulmonary infection. help to confirm the isolate ( I 111 ).
4. Septicaemia in patients who are debilitated due to
malignancy or immunosuppresive therapy. 5. Typmg Method
5. Wound and burn infections. Pyocin typing is the most commonly used method. It is
6. Chronic otitis media and otitis externa. mainly used for epidemiological studies.
7. Eye infections.
8. Acute necrotising vasculitis which leads to haemorr- 6. Antibiotic Sensitivity Test
hagic infection of skin and internal organs. It is useful to select out proper antibiotic as multiple
9. Infantile diarrhoea. resistance to antibiotics is quite common in Ps.
In some tropical areas, Ps. aeruginosa has been aeruginosa.
found to be responsible for a self-limited febrile
illness (Shanghai fever) resembling typhoid fever. K. Treatment
In spite of its lack of invasiveness, Ps. aeruginosa does It is intrinsically resistant to most of the commonly
cause severe disease. The mechanisms of pathogenesis used antimicrobial agents. Ciprofloxacin, piperacillin,
are not clearly understood but it has been claimed that ticarcillin, azlocillin, cefotaxime, ceftazidime, gentamicin
pathological processes seen in infection are caused by and tobramycin are used in treatment of Ps. aeruginosa
exotoxins, proteases, elastases, haemolysins, lipases and infections.
enterotoxins. Exotoxin A is a lethal toxin acting like
the diphtheria toxin. Elastases may be responsible for L. Control
haemorrhagic lesions in skin infections. The enterotoxin Cross infections in hospital are to be prevented by
causes diarrhoeal disease. The slime layer acts as a capsule constant vigilance and strict attention to asepsis.
and enhances the virulence. A polyvalent vaccine made from the cell surface of 17
The organisms produce blue pus and the term recognised serotypes of Ps. aeruginosa has been claimed
aeruginosa, meaning verdigris which is bluish green in to stimulate active immunity in man.
colour and pyocyanea, being a literal translation of 'blue
pus'. M. Epidemiology
1. Cross Infection: Ps. aeruginosa is a saprophyte and its
J. Laboratory Diagnosis ability to persist and multiply in moist environment of
1 Specimens hospital wards, kitchens, equipments and antiseptics
Pus, wound swab, urine, sputum, blood or CSF or disinfectant solutions is of particular importance
in cross infection.
2. Cultu,-e 2. Epidemics: Epidemics and outbreaks of Ps. aeruginosa
Specimens may be inoculated on nutrient agar, blood agar infection have occurred amongst newborn nurseries
or MacConkey's agar and incubated at 37°C for 18- 24 and young infants in paediatric wards.

296 _ _ _ __ __ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Unit Ill: Systemic Bacteriology
3. Risk group: These include burn patients, patients with inflammation of tunica vaginalis. This is known as Straus
immunosuppression and patients who have undergone reaction. This may be followed by death of the animal
cardiac and renal surgery. in 1-2 weeks. The straus reaction is not diagnostic of
glanders because similar reactions in guinea pig may also
be observed with Bruce/la sp., Priesz-Nocard bacillus,
II. OTHER PSEUDOMONAS Actinobacillus and B.pseudomallei.
Ps. putida, Ps. stutzeri and Ps. fluorescens are becoming
clinically relevant in increasing percentages. . Hu n Pathogenkity
Humans may become infected via skin abrasions or
wounds through contact with discharge of a sick animal.
III. STENOTROPHOMONAS Human disease may present as acute fulminant febrile
MALTOPHILIA illness or a chronic indolent infection producing abscesses
(PSEUDOMONAS MALTOPHILIA) in respiratory tract or skin. The fatality rate in glanders
This is an opportunistic pathogen, causing wound is high. The laboratory cultures are highly infectious to
infection, urinary tract infection and septicaemia. It work with.
is oxidase negative. It acidifies maltose in addition to
glucose. E. boratory Diagnosi
1. Specimens
IV. BURKHOLDERIA MALLEI Purulent discharge from the lesion.
(PSEUDOMONAS MALLEI)
The bacillus was first isolated by Loeffler and Schutz 2. Direct Microscopy
(1882) from horse dung of glanders (malleus, in latin). Gram negative beaded bacilli are found on Gram staining
It causes a natural disease called glanders in animals of the specimen.
(horses, mules and asses), and man occasionally acquires
the infection from animals. 3. Culture
Culture is done on blood agar.
orp o g
It is a small 2.5 µm x 0.5 µm , non-motile, Gram negative 4. Animal Inoculation Test
bacillus often giving a beaded appearance. It is done in guinea pigs. Straus reaction can be
demonstrated.
ltur
It is an aerobe and facultative anaerobe, grows on ordinary 5. Allergy Test
culture media. Colonies are small and translucent initially Mallein test
become yellowish and opaque on ageing. Mallein is a preparation from the organism B. ma/lei
and is analogous to tuberculoprotein. Intracutaneous
C. i I' thog nki or subcutaneous injection of mallein evokes a delayed
In susceptible animals (horses, mules and asses), hypersensitivity reaction in animals suffering from
B. ma/lei produces two types of lesions: glanders.
1. Glanders
In glanders, the respiratory system is affected and the V. BURKHOLDERIA PSEUDOMALLEI
animal develops profuse catarrhal discharge from the (PSEUDOMONAS PSEUDOMALLEI)
nose. Nodule formation starts in the nasal septum and It was formerly known as Whitmore's bacillus,
later the nodules break down to form ulcers. Actinobacillus whitmori, Malleomyces pseudomallei,
Loefflerella pseudomallei.
2. Farcy It is the causative agent of melioidosis which is a
Farcy follows infection through the skin with involvement glanders like disease and epizootic in rodents in South-
of superficial lymph vessels and lymph nodes. The lymph East Asia. The name melioidosis is derived from me/is,
vessels become thickened and stand out as hard cords a disease of horses, and eidos meaning resemblance.
under the skin which are called farcy pipes. The organism was first described by Whitmore and
Intraperitoneal inoculation of bacilli in male Krishnaswami (1912) from a glanders like disease of man
guinea pig causes testicular swelling in 2- 3 days due to in Rangoon.

Ch 36: Pseudomonas, Stenotrophomonas, Burkholderia _ _ _ _ __ _ __ _ _ _ _ __ _ _ __ 297
A. Morphology VI. BURKHOLDERIA CEPACJA

It resembles B. ma/lei but differs in being motile. (PSEUD 01'.lAS Er
Burkholderia cepacia is one member of B. cepacia complex
B. Culture which contains about 16 more species.
It is similar to B. ma/lei.
A. Morphology
C. Biochemical Reactions It is a slender, Gram negative bacillus which is motile
It resembles B. ma/lei but differs in being liquefying by multitrichous flagella. The bacillus accumulates poly-
gelatin and forming acid from several sugars. ~- hydoxy- buty rate as granules, hence stains irregularly.
D. Toxins B. Culture
Two thermolabile exotoxins- one lethal and other It is aerobic and grows well on ordinary nutrient agar
necrotising have been identified in culture filtrates. optimally at 25- 35°C. Colonies appear in 48 hours.
Most strains do not grow on DCA medium. Cultures
E. Pathogenesis on blood agar die in 3--4 days. On prolonged incubation,
B. pseudomallei is a saprophyte of soil and water with a colonies become reddish purple due to formation of a
large animal reservoir. Melioidosis occurs in rats, rabbits non-diffusible phenazine pigment.
and guinea pigs. Human infection may occur by inhalation
or through skin abrasions, or by arthropod vector. Disease C. Pathogenicity
is epidemic in South-East Asia. Subclinical infections are It is a low grade human pathogen and an important
common in man. There may be an acute septicaemia, cause of nosocomial infection. It is being recognised
a subacute typhoid like disease, or pneumonia and as an opportunist environmental pathogen, particularly
haemoptysis resembling tuberculosis. Acute disease has in those with cystic fibrosis, in whom it causes fatal
a high case fatality rate. In chronic form, the organism necrotising pneumonia.It is resistant to a large number of
may localize in any tissue producing caseous necrosis or antibiotics and contaminate antiseptic solutions including
suppurative lesion. chlorhexidine and cetrimide. It can also survive in distilled
In India, cases of melioidosis have been reported water up to a year.
from Maharashtra, Tamil Nadu, Orissa, Kerala, Tripura
and West Bengal.
VII. NON-FERMENTERS OTHER THAN
F. Laboratory Diagnosis SEUDO AN U
The organisms may be detected as small, typical bipolar Several Gram negative bacilli may be non-fermenters of
safety pin appearance with methylene blue stain. Small carbohydrates and may be confused with Pseudomonas or
irregularly stained Gram negative bacilli in exudates may Burkholderia species. Some of these non-fermenters are
be isolated from sputum, pus, urine or blood. Antibody to Acinetobacter, Alcaligenes, Achromobacter, Flavobacterium
B. pseudomallei may also be detected in patient's serum by and Eikinella species. Some characteristics of these
ELISA and indirect haemagglutination assay (IHA). organisms are shown in Table 36.2.
Table 36.2 Some Characteristics of Non- Fermenters

Organisms Oxidase test Habitat Pathogenicity
Acinetobacter spp. Saprophytes of soil, sewage; commensal Pneumonia, Respiratory tract infections
of skin of groin and axilla of man (10%)
Alcaligenes spp. + Human faeces UTI, wound infection
Achromobacter spp. + CSOM, Post-operative meningitis
Flavobacterium spp. + Saprophytes of soil Opportunistic nosocomial infections,

particularly in infants.
Eikenella spp. + Commensal of mucosa! surfaces Infections of wounds and soft tissues,
endocarditis, meningitis and pneumonia

298 _ _ _ _ __ _ _ _ _ _ _ _ __ _ _ __ __ _ _ _ _ _ _ _ Unit Ill: Systemic Bacteriology
KEY POINTS
1. The genus Pseudomonas comprises of aerobic, Gram negative, non-fermentative, non-sporing, oxidase positive bacilli
which are motile by polar flagella .
2. The most important species is Pseudomonas aeruginosa. It is a strict aerobe and grows well on ordinary medium like
nutrient agar. It produces greenish blue pigment which diffuses into the medium.
3. Different pigments produced by Ps. aeruginosa include are pyocyanin, fluorescin , pyorubin and pyomelanin.
4. Cetrimide agar is a selective medium fo r Ps. aeruginosa.
5. Ps. aeruginosa is the most important agent causing nosocomial infections. It is due to its resistance to common
antibiotics and antiseptics. The other common infections caused by it are urinary tract infection, acute purulent
meningitis, septicaemia, wound and burn infections.
6. Stenotrophomonas maltophila is ox idase negative.
7. Burkoholderia ma/lei is non-motile. It is the causative agent of glanders and farcy .
8. Burkholderia cepacia is motile by multitrichous flagella.
YOU MUST KNOW

1. M orphology, culture characteristics and biochemical reactions of Pseudomonas aeruginosa .
2. Pyocin typing.
3. Infections caused by Pseudomonas aeruginosa.
4. D iseases caused by Burkholderia ma/lei.
STUDY QUESTIONS
(a) Pathogenesis of Pseudomonas aeruginosa
(b) Pyocin typing
(c) Pigments produced by Pseudomonas aeruginosa.
(a) S tenotrophomonas maltophila
(b) Burkholderia mallei
(c) Burkholderia pseudomallei.

1. Which of following bacteria is non-motile?
(a) Pseudomonas stutzeri (b) Burkholderia mallei
(c) B. pseudomallei (d) Stenotrophomonas maltophila
2. Which of the following pigment is diagnostic of Pseudomonas aeruginosa?
(a) Pyocyanin (b) Fluorescin
(c) Pyorubin (d) Pyomelanin
3. Which of the following tests is/are characteristic feature/s of Pseudomonas aeruguiosa?
(a) U tilization of glucose oxidatively (b) Oxidase positive
(c) Pyocyanin production (d) All of the above
4. The most popular method employed for typing of Pseudomonas aeruginosa is:
(a) Pyocin typing (b) Phage typing
(c) Serotyping (d) Antibiogram

Ch 36: Pseudomonas, Stenotrophomonas, Burkholderia _ _ _ __ _ _ _ __ _ _ __ __ _ _ _ 299
5. Which of the following infections can be caused by Pseudomonas aeruginosa?

(a) Urinary tract infection (b) Wound and burn infections
(c) Pulmonary infection (d) ALI of the above
6. The causative agent of Shanghai fever is:
(a) Pseudomonas aeruginosa (b) Pseudomonas putida
(c) Burkholderia mellei (d) B. pseudomallei
7. Which of the following bacteria was formerly known as Whitmore's bacillus?
(a) Burkholderia mallei (b) B. pseudomallei
(c) Pseudomonas putida (d) Stenotrophomonas maltophila
Answers (MCQs):
1. (b) 2. (a) 3. (d) 4. (a) 5. (d)
6. (a) 7. (b)
----++ +----

Chapter 37
YERSINIA, PASTEURELLA, FRANCISELLA
I. Yersinia pestis
E. Antigenic Structure F. Pathogenesis
I. Diagnosis of Plague in Rats J . Treatment
K. Prophylaxis
II. Yersinia pseudotuberculosis and Yersinia enterocolitica
A. Yersinia pseudotuberculosis B. Yersinia enterocolitica
Ill. Pasteurella multocida
IV. Other Pasteurella Species
V. Francisella tularensis
These are Gram negative, short bacilli showing bipolar animals and occasionally producing human infections,
staining with methylene blue. They are non-motile except grouped under a common species called P multocida.
Yersinia pseudotuberculosis and Y. enterocolitica. These The genus Francisella, consisting of the single species,
bacteria are primary pathogens of rodents and men are F tularensis is named after Francis for his contributi on
accidentally infected. They were previously considered on tularaemia, caused by this bacterium .
together under the genus Pasteurella. Based on their
cultural, biochemical and antigenic characters, this Table 37.1 Diseases Caused by Species of Yersinia,
group has been divided into three genera-Y ersinia, Pasteurella , Francisella
Pasteurell a and Francisella (Table 37 .1). The genus
Genera Species Diseases
Yersinia contains three medically important species-Y .
pestis (the causative agent of plague), Y. pseudotuberculosis Yersinia Y pestis Plague in man and
(a primary pathogen of rodents) and Y. enterocolitica rodents
Y pseudotuberculosis Pseudotuberculosis of
(causes human diarrhoeal diseases). The name Yersinia is
animals
given after Alexander Yersin, who discovered the plague Y enterocolitica Enteritis in man and
bacillus. The genus Yersinia now belongs to the tribe animals
Yersinieae and the family Enterobacteriaceae. The genus Pasteurella P multocida Haemorrha gic
Pasteurella is now restricted to several related bacteria septicaemia in animals
causing haemorrhagic septicaemia in different species of Francisella F. tularensis Tularaemia
300
Ch 37: Yersinia, Pasteurella, Francisella _ _ _ __ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ 301
I. YERSINIA PESTIS 1. Nutrient Agar

Yersinia pestis, the causative agent of plague, formerly Colonies are small, delicate, transparent after
known as Pasteurella pestis, was isolated independently 24-48 hours of incubation.
and almost simultaneously by Yersin (1894) and Kitasato
(1894). 2. Blood Agar
Colonies are non-haemolytic and dark brown due to the
A. • orphology absorption of the haemin pigment. Other characters are
Y pestis is a short, ovoid, Gram negative bacillus, about same as that of the colonies on nutrient agar.
1.5 µm x 0.7 µm in size, with rounded ends and convex
sides, occur singly, in short chains or in small groups. 3. MacConkey Agar
When stained with methylene blue, it shows bipolar Colourless colonies are formed.
staining (safety pin appearance) with two ends darkly or
densely stained and the central area clear ( r-- &· 4. Broth
In nutrient broth, a flocculent growth occurs at the
bottom and along the sides of the tube, with little or
no turbidity. A pellicle may form later.
0 5. Ghee Broth
When the organisms are grown in a flask with oil or ghee
floated on the top (ghee broth), a characteristic growth
occurs which hangs down from the under surface of the
oil, resembling stalactites (stalactite growth).
C. i C
Y pestis ferments glucose, mannitol and maltose with the
Fig. 37 Smear of Y. pestis with bipolar staining production of acid but no gas. Lactose and sucrose are not
fermented. It is catalase positive, indole negative, urease
Pleomorphism is very common. It is characteristically negative, methyl red (MR) positive, Voges Proskauer
enhanced in media containing 3% NaCl. In old cultures, (VP) and citrate negative. Gelatin is not liquefied.
involution forms are very common-coccoid, club shaped, On the basis of fermentation of glycerol, reduction
filamentous and giant forms. of nitrate, and breakdown of arabinose; there are four
The bacterial body is surrounded by a slime layer biotypes of Ypestis. This typing is of epidemiological
(envelope or capsule). It is non-motile, non-sporing and significance because of the different geographical
non-acid-fast. distribution of the types (Table 37.2).
B. ul e isunce
The organism is aerobic and facultatively anaerobic. The The plague bacillus is destroyed by heat at 55°C in 15
optimum temperature for growth (unlike most pathogens) minutes. It is easily destroyed by sunlight, drying and
is 27°C but the envelope develops best at 37°C. It grows chemical disinfectants (0.5% phenol in 15 minutes). It
on ordinary media. can survive for several months in the soil of rodent
Table 37.2 Biotypes of Y. pestis

Variety Glycerol Nitrate Arabinose Distribution
fermentation reduction breakdown
Y pestis var. orientalis + + Primary foci in India, China and Myanmar.
Responsible for wild plague in Western USA,
South America, South Africa
Y. pestis var. antiqua + + + Transbaikalia, Manchuria, Mangolia, perhaps
responsible for the Justinian plague.
Y. pestis var. mediaevalis + + South-East Russia
Y. pestis var. microtus +

burrows. It remains viable for long periods in cold and 1. Bubonic Plague
moist environments. As the plague bacillus usually enters through the bite of
infected rat flea on the legs, the inguinal lymph nodes
E. Antigen Structure are involved, hence the name bubonic (bubo meaning
Plague bacilli are antigenically homogenous and serotypes groin). The inguinal lymph nodes become enlarged and
do not exist. The antigenic structure is complex and suppurate. Patient develops fever, chills and malaise. Pain
about 20 different antigens have been identified. These may accompany the bubo. The infection may stop up to
include: lymph nodes, but often the bacteria reaches the blood
1. A heat-labile protein envelope antigen known as stream and gets widely disseminated.
Fraction l or F-1. It is best formed in cultures If the bubonic plague is not treated properly, it may
incubated at 37°C. It inhibits phagocytosis. This progress to meningitis (meningeal plague) after 7- 10 days
antigen is generally present only in virulent strains. of the onset of bubonic plague.
It has therefore been considered to be a virulence
determinant. It has an important role in stimulating 2. Pneu nonic Plague
protective immunity in mice and man. It is highly infectious form of plague involving the
2. The V and W proteins have been considered to lungs producing haemorrhagic pneumonia. It can be
be virulence factors as they inhibit phagocytosis. transmitted from man to man by droplet infection
These are also formed by virulent strains of (airborne route) and is virtually always fatal. Primary
Y. pestis. pneumonic plague is rare but it may sometimes occur
3. Virulent strains produce a bacteriocin (Pesticin I), secondary to bubonic plague. Pneumonic plague may
fibrinolysin and coagulase. also occur in epidemic form. Patient develops fever and
4. Other antigens elaborated by plague bacillus include cough with expectoration.
murine and guinea pig toxins. The endotoxin
(lipopolysaccharide) is similar to that of other enteric 3. Sept 1 e•11ic Plague
bacteria. Murine and guinea pig toxins are active in The presence of bacteria in blood denotes septicaemic
rodents and guinea pigs, their role in natural disease plague. This may occur as primary infection but it
of man is not known. These toxins are termed as usually occurs as the terminal event of bubonic and
plague toxins. pneumonic plague. Massive involvement of blood vessels
5. Virulence has also been associated with the ability results in haemorrhages in the skin and mucosa. Due to
to synthesise purines. this manifestation, the disease is given the name black
6. Production of coloured colonies (on medium death.
containing haemin) also appears to be associated with Mortality rate in bubonic plague is 50- 75%, in
virulence. It appears that plague bacillus possesses pneumonic plague almost 100% and with treatment it
an unidentified surface component which absorbs is 5- 30%.
haemin and basic aromatic dyes in cultured media Pestis minor or benign plague is also seen in some
to form coloured colonies. cases during an epidemic. Only clinical presentation may
be slight temperature with one or two swollen glands.
F. Pathogenesis
Y. pestis is a natural pathogen of rodents and causes G. Epidemiology
zoonotic disease called plague. Infection is transmitted Plague is a zoonotic disease and may become epidemic in
from one animal to another by the bite of flea. Two the rat population. The infection may spread to domestic
natural cycles of plague exist, the urban and sylvatic rats and man by flea bites or contact. The commonest
plague. Urban plague involves man and rodents (rats) vector is Xenopsie/la cheopis but other fleas like X.astia,
living with him. In addition cat, goat, sheep, dog and Ceratophylus may also transmit the infection.
camel are also susceptible. Wild or sylvatic plague occurs Plague is the greatest killer known to mankind. In
in wild rodents most important being mice, squirrels and 14th century, plague pandemic known as "black death" is
chipmunk. believed to have killed about a quarter of all mankind.
Plague is a natural disease of rodents and is Several pandemics have occurred between 1500 and 1720.
transmitted to man via the bite of infected rat flea Last pandemic occurred in 1894, causing more than 10
(X cheopis). The incubation period is 2- 6 days. In man, million deaths by 1918. During 1958-77, plague occurred
plague occurs in three forms: bubonic, pneumonic and in 29 countries and India remained free of plague from
septicaemic. 1967 to 1993.

Ch 37: Yersinia, Pasteurella, Francisella _ _ _ _ _ __ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ 303
An outbreak of plague was reported from India in exudates from bubo or with 24 hours broth culture. The
1994. A total of 876 cases of presumptive plague were animals die within 2- 5 days. Postmortem reveals marked
diagnosed on the basis of serological tests. These cases were local inflammatory reaction with necrosis and oedema.
reported from Maharashtra, Gujarat, Delhi, Karnataka, Regional lymph nodes are enlarged and congested.
Uttar Pradesh and Madhya Pradesh. Outbreak started Organisms may be demonstrated in smears from local
in Beed district (Maharashtra) for bubonic plague and lesions, lymph nodes, spleen and heart blood, after
Surat (Gujrat) for pneumonic plague. Of the total 54 staining with Gram and methylene blue stains.
fatal cases, 52 were in Surat. In 2002, a short outbreak
of plague was reported from Shimla, claiming 4 lives. 5. 1l11tigrn Detectio11
Plague has several scattered natural foci in many Fl glycoprotein Ag may be detected in aspirated fluid
countries. It survives in wild rodents, occasionally causing from bubo or sputum by immunofluorescence and ELISA
human infections. At least four such foci of plague are test.
known in India. These include the region near Kolar,
Bead-Latur belt in Maharashtra (from where the Surat ' ,, ltd ft<;t
epidemic started), Rohru in Himachal Pradesh (from Fl glycoprotein can be detected by dipstick test using
where the 2002 Shimla outbreak took place) and a small monoclonal antibodies. It is a rapid diagnostic test, which
pocket in Uttaranchal. produces reliable results within 15 minutes.
H. Laboratory Diagnosis 6. Serological Tests

Laboratory diagnosis of plague includes diagnosis of Antibodies to F-1 antigen may be detected by passive
plague in man as well as rodents. Early detection of haemagglutination or complement fixation tests.
infection in animals will help in prevention of plague. Antibodies appear towards the end of first week of the
illness. Serological tests are useful for identifying plague
1. Spec· 1 ,11~ foci as the tests remain positive for several years after
(i) Pus or fluid aspirated from bubo in bubonic plague. recovery from plague. It has a limited diagnostic value as
(ii) Sputum and blood in pneumonic plague. the titres reach diagnostic levels (1 :16 or four fold rise)
(iii) Blood in septicaemic plague. only after 2- 3 weeks following onset of disease.
(iv) Splenic tissue on postmortem.
(v) CSF in meningeal plague. 7. ?olylllernse Chain Reaction (PCR)
It is a rapid and sensitive method for diagnosis of plague
2 .:Jin...i ·o c~r in clinical specimens and fleas.
Sputum and aspirate from lymph nodes are stained with
Gram's staining and methylene blue. Gram's staining
shows characteristic Gram negative coccobacilli. With I. Diagnosis of Pla 0 Je in Rats
methylene blue staining, bacilli show typical bipolar Before examining rats, immerse them in disinfectant to kill
staining. Wayson's stain is a methylene blue stain used any flea. Necropsy reveals enlargement of lymph nodes
for direct microscopy. with periglandular inflammation and oedema. Generally
Microscopic examination of buffy coat smear of cervical lymph nodes are involved due to the tendency of
blood may show plague bacilli in septicaemic cases. the rat flea to bite rat's neck region. Other features include
Other more specific direct staining method include pleural effusion, enlargement of spleen and haemorrhages
the use of fluorescently labelled antibody to the envelope under the skin and in internal organs.
F-1 antigen. Specimens (heart blood, lymph nodes and spleen)
collected from the animals are utilised for smear
,, Ci d examination, culture and animal inoculation test.
Culture is made on blood agar medium and incubated
at 27°C. Biochemical tests are performed to confirm the J. Trea ment
diagnosis. On ghee broth, there is characteristic stalactite Streptomycin is the drug of choice in both bubonic and
growth. pneumonic plague. Early treatment is essential because of
high fatality of the disease. Combination of streptomycin
,.!,Lt 1I and tetracycline is very effective. Chloramphenicol and
Guinea pigs or white rats are injected subcutaneously with kanamycin are also effective.

304 _ __ __ _ __ _ _ _ _ _ _ __ _ _ __ _ __ _ _ __ _ Unit Ill: Systemic Bacteriology
K. Prophylaxis should also be given chemoprophylaxis when exposed

1. General Measures to plague.
General measures such as control of fleas and rodents are
of great importance. Spray insecticides (DDT) inside the (ii) Live vaccine
rodent burrows and houses to kill the fleas. After the fleas Live vaccines are prepared from two avirulent strains of Y.
have been killed, kill the rats with rodenticides. Attempt pestis, Otten's Tjiwidej strain from Jawa and Girard's EV
should not be made to kill the rats unless spraying has 76 strain from Malagasey. Since live vaccines are difficult
first been done otherwise the fleas will jump from the to prepare and carry some risk of unacceptable reactions,
carcasses of rats to man. Stores especially near railway killed vaccines are recommended for general use. However,
stations, airports and seaports must be rat-free, otherwise mass vaccination is no more recommended.
infected rats with rat fleas may travel along the cargo to
far off places. 3. Chemoprophy laxis
Close contacts of patients with plague should be given a
2. Specific Measures course of tetracycline (500 mg 6 hourly for one week).
Two types of vaccines can be used-killed and live
attenuated. II. YERSINIA PSEUDOTUBERCULOSIS
AND YERSINIA ENTEROCOLITICA
(i) Killed vaccine These two organisms resemble Y. pestis in that they are
Killed vaccine is widely used for active immunisation. small, Gram negative bacilli with bipolar staining and
It is a whole bacterial culture antigen of plague reservoirs of infection are rodents, wild and domestic
bacillus. It is prepared at Haffkine Institute, Mumbai. animals. They differ from Y. pestis by their motility
The plague bacillus (virulent strain) is grown in casein at 22°C (and not at 37°C), non-capsulated, urease
hydrolysate broth for 2--4 weeks at 32°C and killed by positive, oxidase negative and insusceptible to Y. pestis
0.05% formaldehyde and preserved with phenyl mercuric bacteriophage (Table 37.3).
nitrate (Sokhey's modification of Haffkine's vaccine). The
vaccine contains 2,000 million organisms per ml. A. Yersinia pseudotuberc ulosis
It ferments rhamnose and melibiose. It is antigenically
(a) Dose schedule heterogenous. On the basis of somatic and flagellar
• 0.5 ml is injected subcutaneously followed by 1 ml antigens, it is divided into six serological groups and nine
after 7-14 days. serotypes. Y. pseudotuberculosis exhibits antigenic cross
• Booster dose is given every 6 months. reaction with Y. pestis as well as with salmonellae.
Pseudotuberculosis is a zoonosis. The infection occurs
(b) Immunity in animals by alimentary route resulting in epizootic and
Immunity appears 5- 7 days after vaccination and lasts enzootic form of disease. A wide range of animals such
for 6 months. Vaccination may reduce the morbidity and as guinea pigs, rabbits, birds etc. are susceptible. The
mortality in bubonic plague but not in pneumonic plague. liver, spleen and lungs of infected animals show multiple
Vaccine should be given to health workers and to those nodules resembling tuberculosis lesions and hence the
planning to visit plague infected areas. name pseudotuberculosis.
Man acquires infection through ingested materials
(c) Side effects contaminated with animal faeces. Patients develop acute
Fever, headache, lymphadenopathy and erythema at the mesenteric lymphadenitis. Occasionally, it may result in
site of inoculation are some of the side effects. Vaccination a severe generalised disease. The term yersiniosis denotes
is not very effective, therefore, even vaccinated individuals infection with yersiniae other than Y pestis.
Table 37.3 Distinguishing Features of Three Species of Yersinia

Species Motility at Fermentation of Urease VP Ornithine
22°c 31°c Sucrose Rhamnose Cellobiose test decarboxylase
Melibiose
Y pestis
Y pseudotuberculosis + + + +
Y enterocolitica + + + + + +

Ch 37: Yersinia, Pasteurella, Francisella _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ 305
Laboratory diagnosis includes isolation of the P multocida is a non-motile, Gram negative rod
organisms from an excised mesenteric lymph node, or resembling Yersinia but differs from it in being oxidase
blood and demonstration of antibodies in patient's serum positive, producing indole and failing to grow on
during the acute illness. MacConkey's agar.
The bacillus is usually normal inhabitant of
B. Yersinia enterocolitica respiratory tract of a variety of animals such as dogs,
It is a Gram negative coccobacillus and resembles Y rats, cats, cattle and sheep. It may sometimes occur as
pseudotuberculosis in being motile at 22°C, but differs normal commensal in throat in humans. Human infection
from it in fermenting sucrose and cellobiose and is rare but it may cause septic wound following animal
decarboxylating ornithine. It does not ferment melibiose bites, meningitis following head injury, respiratory tract
or rhamnose (Table 37.3). infections or appendicitis. The bacillus is susceptible to
Many strains are indole and VP positive. It is aerobic tetracycline, streptomycin and penicillin.
and facultative anaerobe. Optimum temperature for
growth is 22°C. On blood agar, smooth translucent, non- IV. OTHER PASTEURELLA SPECIES
haemolytic colonies measuring 2- 3 mm in diamenter appear P canis, P dagmatis and P stomatis are other three
after 48 hours incubation at 22°C. On MacConkey agar, it important species of Pasteurella. There species have been
forms pin-point, non-lactose fermenting (NLF) colonies. isolated from humans.
Y enterocolitica has a distinct antigenic structure and
so far 60 O-antigenic groups and about 20 heat-labile V. FRANCISELLA TULARENSIS
flagellar (H) antigens have been identified. Most of the (PASTEURELLA TULARENSIS)
human infections are due to serogroups 03, 08 and 09. F tularensis is a causative agent of tularaemia, a major
It can be divided into six biotypes- 1A, lB, 2, 3, 4 and zoonotic disease of rabbits and other rodents. The disease
5 by using biochemical tests. was originally described in Tulare County, California.
Y enterocolitica has been isolated from a wide The disease is transmitted by ticks and several other
range of domestic animals. Disease occurs via faeco- arthropod vectors among the rodents. Human infection
oral route by contaminated food and water. In man it may occur by handling or eating infected meat or
may cause gastroenteritis, mesentric lymphadenitis and drinking contaminated water. It may also occur through
septicaemia. It affects children and adults of both sexes. tick bites.
The incubation period varies from 5 to 10 days. The It is a capsulated, non-motile, Gram negative bacillus
early symptoms consist of fever, abdominal pain and measuring 0.3-0.7 µm x 0.2 µm. It has fastidious growth
diarrhoea. Diarrhoea is probably due to an enterotoxin requirements and special media such as Francis blood
or invasion of mucosa by the bacteria. dextrose cystine agar is used for its isolation. Minute
The laboratory diagnosis consists of isolation of the transparent colonies appear after incubation for 3- 5
organism and indirectly by demonstration of antibodies days.
in the patient serum. The organisms can be isolated from In humans, tularaemia may present as a local
faeces, blood or from mesenteric lymph nodes. Blood agar ulceration with lymphadenitis, a typhoid-like fever with
and MacConkey agar are used for growing this organism. glandular enlargement or an influenza like infection. The
Cefsulodin-irgasan-novobiocin (CIN) agar is a selective disease has been reported from North America, Europe
medium for recovery of Y enterocolitica from faeces. and Asia. The organism is highly infectious and laboratory
Colonies of Y enterocolitica have a bull's eye appearance infection has been quite common.
with a red center on CIN agar. Laboratory diagnosis may be made by smear
examination, culture or by inoculation into guinea pigs.
Pus from local lesions, blood during bacteraemia and
III. PASTEURELLA MULTOCIDA sputum (in pulmonary involvement) are used for diagnosis.
S, CA) Patient's serum may be tested for agglutinating antibodies,
A group of related bacteria isolated from haemorrhagic a titre of 1:80 or higher is regarded as diagnostic. Since
septicaemia in various animals and birds had, in the F tularensis shares somatic antigens with both Br. abortus
past, been named according to their species of origin- P and Br. melitensis, false positive reactions may occur with
boviseptica, P lepiseptica, P aviseptica etc, but now they brucella agglutinins.
are regarded as strains of a single species designated P Streptomycin is the drug of choice. An attenuated
multocida. Pasteur developed first attenuated vaccine from vaccine is available which can be administered to persons
P aviseptica, hence the name Pasteurella. who are subject to high risk of infection.

306 _ _ _ __ _ _ _ _ _ _ _ _ _ _ __ _ _ _ __ _ __ _ _ _ Unit Ill: Systemic Bacteriology
KEY POINTS
1. Yersiniae are Gram negative, short bacilli showing bipolar staining (safety pin appearance) with methylene blue. These
are non-motile except Yersinia pseudotuberculosis and Y enterocolitica. They are primary pathogens of rodents and
men are accidentally infected.
2. The genus yersinia contains three medically important species-Y pestis (the caustive agent of plague),
Y pseudotuberculosis (a primary pathogen of rodents) and Y entercolitica (causes human diarrhoeal diseases).
3. Y pestis is Gram negative bacillus, non-motile and capsulated. It grows at 27 °C but envelope develops best at
37 °C. It grows on ordinary media.
4. Envelope antigen known as Fraction 1 or F-1 is an important antigen of Y pestis.
5. In man, plague occurs in three forms: bubonic, pneumonic and septicaemic.
6. Plague is a zoonotic disease. It is a natural disease of rodents and is transmitted to man via the bite of infected rat
flea (Xenopsiella cheopis).
7. Laboratory diagnosis of plague depends upon direct microscopy, culture and antigen detection.
8. Y enterocolitica differs from Y pestis by its motility at 22 °C (and not at 37 °C), non-capsulated, urease positive, VP
positive, and ornithine decarboxylase positive.
9. In man Y enterocolitica may cause gastroenteritis, mesentric lymphadenitis and septicaemia.
10. Laboratory diagnosis consists of isolation of the organism .
YOU MUST KNOW

1. Morphology, culture characteristics and biochemical reactions of Yersinia pestis.
2. Laboratory diagnosis of plague.
3. Vaccines used for prevention of plague.
4. Yersinia enterocolitica.
STUDY QUESTIONS
I. Describe the laboratory diagnosis of plague.
(a) Prophylaxis against plague (b) Yersinia pseudo tuberculosis
(c) Yersinia enterocolitica (d) Francisella tularensis.

1. 'Safety pin appearance' on methylene blue staining is a characteristic feature of:
(a) Yersinia pestis (b) Pseudomonas aeruginosa
(c) Burkholderia ma/lei (d) B. pseudomallei
2. What is the causative agent of plague?
(a) Yersinia pestis (b) Y enterocolitica
(c) Y pseudotuberculosis (d) Francisella tularensis
3. Plague is transmitted to man by bite of infected:
(a) Sandfly (b) Rat flea
(c) Hard tick (d) Mite
4. Bubonic plague is transmitted by:
(a) Infected rat flea (b) Infected sandfly
(c) Inhalation (d) Ingestion

Ch 37: Yersinia, Pasteurella, Francisella _ _ _ _ _ __ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ 307
5. Pneumonic plague can be transmitted from human to human by:

(a) Droplet infection (b) Ingestion
(c) Infected sandfly (d) All of the above
6. The name 'black death' has been given to:
(a) Plague (b) Miliary tuberculosis
(c) Tularensis (d) Legionnaire's disease
7. Which vaccine is recommended for prophylaxis against plague?
(a) Killed vaccine (b) Live vaccine
(c) DNA vaccine (d) None of the above
8. Yersinia enterocolitica can be differentiated from Yersinia pestis by:
(a) Motility at 22°C (b) Non-capsulated
(c) Decarboxylation of ornithine (d) All of the above
9. Which is the selective medium for growing Yersinia enterocolitica from faeces?
(a) Cefsulodin-irgasan novobiocin agar (b) Butzler's selective medium
(c) Tinsdale agar (d) All of the above
10. The diseases caused by Yersinia enterocolitica in man include:
(a) Gastroenteritis (b) Mesenteric lymphadenitis
(c) Septicaemia (d) All of the above
Answers (MCQs):
1. (a) 2. (a) 3. (b) 4. (a) 5. (a)
6. (a) 7. (a) 8. (d) 9. (a) 10. (d)
---++• - - -

Chapter 38
LEGION ELLA
Legionella pneumophila
C. Biochemical Reactions D. Susceptibility to Physical and Chemical Agents
E. Serogrouping F. Pathogenesis
G. Laboratory Diagnosis H. Treatment
I. Prevention
In 1976, 221 cases of pneumonia occurred in members B. Culture

of the American Legion who attended a convention These are fastidious organisms and do not grow on
in Philadelphia. Hence, this form of pneumonia was ordinary media but require the presence of cysteine and
named as Legionnaire's disease and the causative agent iron for primary isolation. They are strict aerobe and grow
as Legionella pneumophila. The aerosols of water from air- best at pH 6.8 to 7.8 and at 35-37°C temperature. They
conditioning system led to the isolation of the organism. can be grown on buffered charcoal-yeast extract agar
The genus Legionella contains 52 species of bacteria. (BCYE) and Mueller-Hinton medium supplemented with
L.pneumophila is the most important species. In addition ferric salts plus L-cysteine as essential growth factors.
to this, some other species associated with human Colonies of L. pneumophila usually appear in 3- 5
disease include L.micdadei, L. longbeachae, L.bozemanii, days but some species may require incubation up to 10
L. dumoffii, L. gormanii, L. anisa, L. birminghamensis, days. Colonies are circular, 1- 2 mm in diameter, grey
L. feeleii, L. hackeliae, L. jordanis, L. wadsworthii and or grey blue and low convex with a slightly irregular
L. tucsonensis. edge. Colonies have a classical cut glass appearance when
examined under the plate microscope. With continued
LEGIONELLA PNEUMOPHILA incubation, the colonies become larger and more opaque.
A. Morphology
L. pneumophila is a Gram negative bacillus measuring C. Biochemical Reactions
2- 5 µm x 0.3- 0.9 µm . In infected tissues, it appears as L. pneumophila is catalase positive and oxidase variable.
a short rod or coccobacillus but in culture it becomes It hydrolyses hippurate, starch and gelatin.
longer. It is motile with polar or subpolar flagella. It is
non-capsulated, non-sporing and non-acid-fast. D. Susceptibility to Physical and Chemical Agents
It is Gram negative but stains poorly. It stains better L. pneumophila is readily killed by 1% formalin, 2%
with silver impregnation method but is best visualised glutaraldehyde and 70% ethyl alcohol. It can be destroyed
by direct fluroescent antibody (DFA) staining with by 5 parts per million (ppm) of available chlorine in one
monoclonal or polyclonal sera. minute.
308
Ch 38: Legionella _ _ _ _ _ _ _ _ _ _ __ _ _ _ _ __ __ _ _ _ __ _ _ _ _ _ _ 309
E. Serogrouping (sputum, bronchial aspirate, pleural fluid, lung biopsy

L. pneumophila is divided into more than 14 serogroups. or autopsy material) by direct immunofluorescence test
All these serogroups have been associated with human using labelled specific monoclonal or polyclonal antisera.
disease but most infections are caused by serogroup 1. Gram-stained smears are of little value as legionellae
stain poorly.
F. Pathogenesis
The natural habitat of L. pneumophila is water. The 2. Culture
organisms have been isolated from cooling towers, Specimen is inoculated on BCYE agar and incubated
airconditioning systems, humidifiers, whirlpool baths, at 35- 37°C up to 10 days. Bacteria from colonies are
showers and respiratory ventilators. These sources identified by direct immunofluorescence test with specific
facilitate transmission of the organism to the human antisera as described above.
respiratory tract by generating infectious aerosols. Person-
to-person transmission has not been demonstrated.
Legionella infection may occur in two main forms 3. Detection of Legionella Antigen
which are designated as Legionnaire's disease and Pontaic Legionella antigen in the urine persists for months.
fever, together known as legionellosis. This antigen can be detected in the urine by enzyme
linked immunosorbent assay (ELISA). This is a rapid
1. Legionnaire's Disease and specific test for the identification of L. pneumophila
It is a pneumonic illness which may also progress to in cases of pneumonia.
involve virtually every system of the body. The incubation Immunochromographic assay that detects antigen in
period ranges from 2 to 10 days. Patient develops malaise, urine is also available. It takes only 15 minutes.
fever, headache, myalgia, respiratory distress and non-
productive cough. Case fatality may be 15-20 per 4. Serological Test
cent. Smoking, alcohol, diabetes, immunosuppressive Antibodies in serum can be detected by ELISA or indirect
therapy and pre-existing lung disease are risk factors fluorescent antibody test (IFAT). A four fold or greater
for legionnaire's disease. rise in antibody titre or a high titre (>256) of antibody
may be considered diagnostic of legionellosis.
2. Pontaic Fever
It is a milder, non-fatal 'influenza-like' illness with fever, H. Treatment
chills, myalgia and headache. Erythromycin is the drug of choice in legionellosis.
Rifampicin and ciprofloxacin are also effective.
G. Laboratory Diagnosis
Laboratory diagnosis of Legionella infections include: I. Prevention
Legionellae from water may be eradicated either by heat
1. Microscopy above 60°C or disinfection with chlorine or other biocides.
Legionellae can be detected m the clinical specimens No vaccine is available against Legionella infections.
KEY POINTS
l. Legionella pneumophila is the most important species of the genus Legionella.
2. It is a Gram negative bacillus, motile with polar or subpolar flagella. It stains better with silver impregnation method
but is best visualised by direct fluorescent antibody (DFA) staining with monoclonal or polyclonal sera.
3. The natural habitat of L. pneumophila is water. The organisms have been isolated from cooling towers, airconditioning
systems, showers and respiratory ventilators. These sources facilitate transmission of the organism to the human
respiratory tract by generating infectious aerosols. Legionella infection may occur in two main forms- Legionnaire's
disease and Pontaic fever , together known as legionellosis.
4. Laboratory diagnosis depends on microscopy, culture, detection of antigen and by DNA probes.

310 _ _ _ _ _ _ __ _ _ _ _ _ __ _ _ _ _ __ _ __ _ __ _ Unit Ill: Systemic Bacteriology
YOU MUST KNOW

1. Morphology, culture characteristics and biochemical reactions of Legionella pneumophila.
2. Diseases caused by L. pneumophila.
S UDY Qt,;ESTIONS
1. Discuss the laboratory diagnosis of infections with Legionella pneumophila.
M TL 'IPLE CHOICE QUES1 IO N', (M )
1. The causative agent of Pontaic fever is:

(a) Legionella pneumophila (b) Yersinia pseudo tuberculosis
(c) Pseudomonas putida (d) Francisella tularensis
2. Mode of infection in Legionnaire's disease is/are:
(a) Infectious aerosols (b) Skin inoculation
(c) Ingestion (d) All of the above
3. The natural habitat of Legionella pneumophila is:
(a) Water (b) Faeces
(c) Sputum (d) Urine
4. Which is the antibiotic of choice in Legionella infections?
(a) Erythromycin (b) Cephalosporins
(c) Penicillins (d) All of the above
Answers (MCQs):
1. (a) 2. (a) 3. (a) 4. (a)
---•••---

Chapter 39
HAEMOPHILUS AND GARDNERELLA
I. Haemophilus influenzae
E. Variation F. Sensitivity to Physical and Chemical Agents
G. Virulence Factors H. Pathogenesis
I. Laboratory Diagnosis J . Treatment
K. Prophylaxis
II. Other Haemophilus
A. Haemophilus aegyptius B. Haemophilus ducreyi
C. Haemophilus parainfluenzae D. Haemophilus haemolyticus
Ill. Hacek Group Bacteria
IV. Gardnerella vaginalis
E. Clinical Features F. Diagnosis
G. Treatment
The genus Haemophilus contains non-motile, non-sporing, meningitis, pneumonia, epiglottitis, bronchitis, otltls
Gram negative bacilli and require one or both of two media, septic arthritis and H. ducreyi is the causative
accessory growth factors (X and V) present in blood organism of chancroid.
(Haemophilus meaning blood loving).
Pfeiffer (1892) isolated the organism from the sputa of I. HAEMOPHILUS INFLUENZAE
patients from the influenza pandemic and proposed this A. Morphology
as the causative agent of human influenza. This came to It is a small (1.5 µm x 0.3 µm) , Gram negative, non-
be known as 'influenza bacillus' (Pfeiffer's bacillus). It was motile bacillus showing considerable pleomorphism .
renamed as H. infiuenzae. The discovery of influenza virus It is non-sporing and non-acid-fast. In young cultures
(causative agent of human influenza) in 1933 established (18- 24 hours), the cells are usually coccobacillary, while
that H. infiuenzae was only a secondary invader. in older cultures, long filamentous forms may be seen. In
H. infiuenzae and H. ducreyi are the major pathogens sputum, they occur as small clumps of coccobacilli and
in the genus Haemophilus. H. infiuenzae may cause in CSF from meningitis cases, long and filamentous forms
311
312 _ _ _ __ _ _ _ __ _ _ _ _ _ _ _ _ _ _ _ __ __ _ _ _ Unit
Ill: Systemic Bacteriology
predominate. Virulent strains possess capsule while the Staphylococci. Growth on blood agar can be improved
avirulent strains and older cultures are non-capsulated. by providing a source of V factor (Staph. aureus) . The
differential requirement for X and V factors helps to
B. Culture distinguish various species of Haemophilus (Table
H. influenzae has fastidious growth requirements. It grows 39.1). Species with prefix para-(H. parainfluenz ae, H.
better in aerobic than in anaerobic conditions. It requires : parahaemoly ticus and H. paraphrohaemoly ticus) and H.
enriched media such as blood agar or chocolate agar pittmaniae require V factor only for their growth.
because the accessory growth factors known as X ('X ' Table 39.1 Requirement of X and V Factor of
for unknown) and V ('V' for vitamin) present in blood Haemophilus Species
are essential for growth. The optimum temperature for Species X factor V factor Haemolysis
growth is 35- 37°C, some strains require 5- 10% CO 2. It
H. infiuenzae + +
cannot grow on nutrient agar which lacks the accessory
growth factors. H. aegyptius + +
H. ducreyi + ±
1. X Factor H . parainfiuenzae +
It is a heat stable protoporphy rin IX, haemin or other
H. haemoly ticus + + +
iron-containing porphyrin. It is necessary for the synthesis
of catalase and other enzymes cytochrome C, cytochrome H. parahaemoly ticus + +
oxidase, involved in aerobic respiration. H. paraphrohaem oly ticus + +
H. pittmaniae + +
Porphyrin test
The porphyrin test is an alternative method for
differentiating the haemin-producing species of Blood agar with Staph. aureus streak and chocolate
Haemophilus. This test can be performed in agar, in agar are routinely used for the identification of
broth, or on a disc. The principle of the test is based H. influenzae. The colonies are small, translucent and non
on the ability of the organism to convert the substrate haemolytic on blood agar. Capsulated strains produce
deltaaminolevulinic acid (ALA) into porphyrins or distinctive iridescent colonies.
porphobilinogen. Porphyrins and porphobilinogens are Better growth of H. influenzae is also obtained on
intermediates in the synthesis of X-factor. Porphobilinogen Levinthal's medium (prepared by boiling and filtering a
can be detected by the addition of Kovac's reagent mixture of blood and nutrient broth) or Fildes agar (by
(p-dimethyl aminobenzaldehyde). After adding Kovac's adding a peptic digest of blood to nutrient agar).
reagent, a red colour develops in the lower acqueous Satellitism
phase if porphobilinogen is present. Porphyrins can Although blood agar contains X and V factors, colonies
be detected using an ultraviolet light (Wood's lamp). of H. infiuenzae are small due to non-availability of
Porphyrins :fluoresce reddish-orange under ultraviolet V factor. After inoculating suspected H. influenzae on
~hl. . a blood agar plate, Staph. auerus is streaked across the
Species that are porphyrin negative cannot synthesise· same blood agar plate and incubated at 37°C for 18- 24
haemin and therefore require haemin (X-factor) for their hours. The colonies of H. influenzae will be large and
growth. well developed alongside the streak of staphylococci while
2. V Factor those further away from staphylococcal streak are smaller.
It is a heat labile (destroyed at 120°C in 30 minutes) factor, This phenomeno n is called satellitism and demonstrates
present in red blood cells and in many other animal and that V factor is available in high concentration near the
plant cells. It is synthesised by some fungi and bacteria staphylococcal growth and only in smaller quantities away
(e.g. Staph. aureus). It is either nicotinamide adenine from it (Fig. 39.1).
dinucleotide (NAD, coenzyme I) or NAD phosphate
(NADP, coenzyme II). It appears to act as a hydrogen C. Biochemical Reactions
acceptor in the metabolism of the cell. Ordinary blood H. influenzae is catalase and oxidase positive, ferments
agar is not suitable for the growth of H. influenzae where glucose and galactose, reduces nitrate to nitrite. It does
growth is scanty, as the V factor is not freely available, not ferment sucrose, lactose and mannitol. On the basis
being imprisoned inside the red blood cells. V factor of production of indole, urease, ornithine decarboxylase,
is released from erythrocytes in chocolate agar (heated it is divided into eight (I- VIII) biotypes (Table 39.2).
blood agar at 80-90°C). V factor is also synthesised by Majority of clinical isolates belong to biotype I to III and

Ch 39: Haemophilus and Gardnerella_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __ _ _ _" 313
OMP antigens of Hib have been classified into 13

subtypes. LOS are antigenically complex. OMP and LOS
subtyping are of f,pidemiological value.
The complete genome of H. infiuenzae has been
sequenced.
E. Variation
Colonies of H. infiuenzae show a smooth to rough (S
R) variation accompanied by loss of capsule and
virulence. Genetic transformation has been demonstrated
in H. influenzae to transfer the characters of capsular
Fig. 39.1 Satellitism antigens and antibiotic resistance. Non-capsulated strains
become capsulated by genetic transformation.
type b organisms belong to biotype I. However, ,
biochemical reactions are not helpful in identification. F. Sensitivity to Physical and Chemical Agents
It is a delicate organism. It is readily killed by heat (55°C
Table 39.2 Biotypes of H. injluenzae for 30 minutes), refrigeration (at 4°C), disinfectants and
Biotype lndole Urease Ornithine decarboxylase drying.Cultures may be preserved op. chocolate agar. For
I
long-term preservation, the culture may be lyophilised.
+ + +
II + + G. Virulence Factors
III + 1. Capsular poly saccharide: It resists phagocytosis. Loss
IV + + of capsule leads to loss of virulence.
2. Pili (Fimbriae): They help in attachment of organisms
V + +
to epithelial cells.
VI + 3. Outer membrane proteins: They contribute in adhesion
VII + and invasion of host tissues.
VIII
- 4. IgAI protease: H. influenzae produces a protease that
specifically cleaves the heavy chain of IgAl.
D. Antigenic Structure H. Pathogenesis

Isolates occur in capsulated and non-capsulated forms. It is an obligate human parasite. The organism enters
Three major surface antigens are present-capsular by respiratory route. The non-capsulated strains of H.
polysaccharide, outer tttembrane protein (OMP) and influenzae are regular commensal in the nasopharynx
lipo-oligosaccharide (LOS). or oropharynx. Colonisation by capsulated strains is
The capsulated strains produce a capsule which found in 5- 10% cases, of which type b strains are seen
is polysaccharide in nature. On the basis of capsular ' in 1- 5%. Capsulated strains cause invasive infections, type
material, Pitman divided H. infiuenzae into 6 serotypes- a, b accounting for most cases.
b, c, d, e, and f. Capsular serotype b strains (Hib) are The following infections are caused by
associated with most invasive infections. Serotyping of H. H. infiuenzae.
infiuenzae was orginally done by agglutination reaction
using type specific antisera. Other methods include, 1. Meningitis
Quellung reaction, coagglutination, CIEP and ELISA. This is the most serious disease occurring in children of 2
Diagnostic kits for the identification of H. influenzae type months to 2 years of age. The fatality rate is about 90%
b (Hib) are available. in untreated patients. The bacilli reach the meninges from
The type b capsular polysaccharide has a unique the nasopharynx, apparently through the blood stream.
chemical structure containing polyribosyl ribitol phosphate Majority of the cases are due to type b strains.
(PRP) which induces IgG, IgM and IgA antibodies. These
antibodies are bactericidal, opsonic and protective. Hib 2. Acute Epiglottitis
PRP is therefore employed for immunisation. It is the second most common infection caused by H.
Non-capsulated strains cannot be typed and are called infiuenzae. This is an acute inflammation of the epiglottis
'non-typable strains'. with obstructive laryngeal oedema, seen in children over

314 _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __ _ _ _ _ _ _ _ _ Unit Ill: Systemic Bacteriology
two years old. This condition is always associated with (a) Latex agglutination
bacteraemia and the organisms can be isolated from Latex particles coated with antibody to type b antigen are
blood cultures. mixed with the specimen. In positive test, agglutination
occurs.
3. Pneumonia
It typically occurs in infants and is accompanied by (b) Coagglutination
empyema and sometimes meningitis as well. While these Instead of latex particles, Staph. aureus is coated with
primary infections are caused by capsulated strains, antibody to type b antigen and mixed with specimen. If
bronchopneumonia may occur as secondary infection positive, agglutination occurs.
with non-capsulated strains.
(c) Counter immunoelectrophoresis (CIE)
4. Bronchitis Specific antiserum is put in one well of agarose gel
H. infiuenzae is an important pathogen associated with and specimen is put in other well. Current is passed.
pneumococci in the acute exacerbations of chronic In positive test, precipitation line occurs in between the
bronchitis. two wells.
5. Suppurative Lesions 4. Culture

Some of the suppurative lesions include septic arthritis, (i) CSF culture
endocarditis, pericarditis and otitis media. CSF should be plated promptly on blood agar or chocolate
agar. A strain of Staph. aureus should be streaked across
I. Laboratory Diagnosis the blood agar plate on which the specimen has already
1. Specimens been inoculated. Plates are then incubated at 35- 37°C,
Depending upon the type of lesion, the following aerobically with 5- 10% CO2, overnight. The isolate is then
specimens may be collected: identified by its colony morphology, satellitism, Gram
(i) Cerebrospinal fluid (CSF) staining and serotyping.
(ii) Blood
(iii) Throat swab (ii) Blood culture
(iv) Sputum It is usually positive m cases of epiglottitis and
(v) Pus pneumoma.
(vi) Aspirates from joints, middle ears or sinuses etc.
(iii) Sputum culture
2. Collection and Transport Sputum should be homogenised by treatment with
Specimens should be collected in sterile containers and pancreatin or by shaking with sterile water and glass
under all aseptic conditions. As H. infiuenzae is very beads for 15- 30 minutes. Several samples of the sputum
sensitive to low temperature, therefore, clinical specimens are to be cultured to increase the rate of isolation.
should never be refrigerated. For optimal yield, specimens
should be transported to laboratory without delay and 5. Colony Morphology and Staining
inoculated on culture media immediately. After overnight incubation, small opaque colonies appear
that show satellitism. A smear is made from colony and
3. Direct Microscopy stained with Gram stain. It shows small Gram negative
(i) Gram staining bacilli or coccobacilli
In meningitis, Gram stained smear of CSF shows
pleomorphic Gram negative coccobacilli. 6. Serotyping
Typing may be done with type specific antisera.
(ii) Immunofluorescence and quellung reaction
These can be employed for direct demonstration of H. J. Treatment
infiuenzae after mixing with specific type b antiserum. H. infiuenzae is susceptible to sulphonamides,
chloramphenicol, trimethoprim, ampicillin, tetracycline,
(iii) Antigen detection ciprofloxacin, cefuroxime, cefotaxime and ceftazidime.
Type b capsular antigen can also be detected in patient's Cefotaxime or ceftazidime is the drug of choice for the
serum, CSF, urine or pus by: treatment of Haemophilus meningitis.

Ch 39: Haemophilus and Gardnerella _ _ _ _ _ _ _ _ _ _ _ _ _ _
__________ _ 315
Beta-lactamase bearing strains have acquired resistance containing vancomycin as a selective agent. It requires
to ampicillin. Amoxycillin-clavulnate or Clarithromycin 10% CO 2 and high humidity for primary isolation.
is more effective. Optimum temperature for growth is 35°C for 2-8 days.
It may also be grown on chorioallantoic membrane of
K. Prophylaxis the chick embryo.
1. A purified type b capsular polysaccharide vaccine After 24 hours of incubation, the colonies of H.
is used in children of 18- 24 months. Vaccine is ducreyi are small, grey, pin point to 0.5 mm in diameter,
administered in two doses at an interval of two translucent, non-mucoid. After 48-72 hours the colonies
months. are 1- 2 mm in diameter and become semiopaque.
2. Hib PRP vaccine in which type b capsular poly-
saccharide is covalently coupled to proteins such as 3. Biochem ical Reactio ns
diphtheria toxoid, tetanus toxoid and N. meningitidis It is biochemically inert except positive nitrate reduction
outer membrane protein. Such Hib PRP are available test.
for use in young children .
3. Rifampicin is given for four days to prevent infection 4. Pathoge nesis
in contacts and also to eradicate carrier state. H. ducreyi causes chancroid or soft sore, a highly contagious
sexually transmitted disease (STD) characterised by
tender, non-indurated, irregular ulcers on the genitalia.
II. OTHER HAEMO PHILU S
The infection remains localised, spreading only to the
A. Haemo philus influenz ae biotype aegypti us inguinal lymph nodes which are enlarged and painful.
(Koch-Weeks bacillus; H.aegtJptius) Sometimes it leads to inguinal abscess called bubo. There
It was first observed by Koch (1883) in cases of acute is no immunity following infection but a hypersensitivity
conjunctivitis in Egypt and was first cultured by Weeks develops. Chancroid is also known as soft chancre.
(1887) in New York. It causes a highly contagious
form of conjunctivitis (pink eye). It is worldwide in 5. Laborat ory Diagnos is
distribution. (i) Specimens
The organism, formerly known as H. aegyptius, is now Scrape edge of the ulcer or aspirated material from
thought to be a biotype of H. influenzae. It has features bubo.
similar to that of H. influenzae biotype III.
It also causes Brazilian purpuric fever (BPF) which (ii) Direct microscopy
is characterised by conjunctivitis, high fever, vomiting, Gram staining of the smear shows typical Gram negative
purpura, petechiae, septicaemia and shock. It responds cocco bacilli.
to combination of ampicillin and chloramphenicol.
(iii) Culture
B. Haemo philus ducreyi Specimens are inoculated on chocolate agar with 1%
H. ducreyi was first demonstrated in 1890 by Ducrey in Iso Vitalex. Vancomycin is added to make the medium
chancroid lesions, a venereal disease transmitted by direct selective. Incubate the medium at 35°C in a humid
contact. atmosphere and 10% CO 2 for 2- 8 days and look for
characteristic colonies.
1. Morpho logy
H. ducreyi is a short, Gram negative coccobacillus, 1- 1.5 (iv) Colony morphology and staining
µm x 0.6 µm in size. They have a tendency to occur in Colonies are grey, small, translucent and non-mucoid.
groups or in parallel chains. They frequently take bipolar Gram staining shows Gram negative coccobacilli.
staining. The microscopic findings has been described as
resembling a 'school of fish' appearance. (v) Agglutin ation
H. ducreyi is antigenically homogeneous and cultures
2. Culture are identified by agglutination with the antiserum.
It does not grow in ordinary laboratory media but has
complex growth requirements. It requires X factor but 6. Treatment
not V factor for its growth. Primary isolation is difficult. Co-trimoxazole or erythromycin is the drug of choice but
It can be grown on rabbit-blood agar, fresh clotted rabbit resistant strains occur. Newer drugs including cefotaxime
blood or chocolate agar enriched with 1% Iso Vitalex, and and ciprofloxacin have been shown to be effective.
316 _ __ _ __ __ _ _ _ _ _ __ _ _ _ _ _ _ _ _ _ __
_ _ Unit Ill: Systemic Bacteriology
C. Haemop hilus parainfluenzae A. Morphol ogy

It differs from H. influenzae in requiring the V factor These are small, 1- 2 µm x 0.3- 0.6 µm in size, Gram
only, and not the X factor. It is a commensal in the negative, non-sporing, non-capsulated, non-motile,
upper respiratory tract and may occasionally cause pleomorphic bacilli. They are sometimes Gram variable.
acute pharyngitis, subacute bacterial endocarditis and
urethritis. B. Culture
The organisms grow on enriched media such as blood
D. Haemop hilus haemoly ticus agar or chocolate agar. They° are facultative anaerobe
It occurs as a commensal in the upper respiratory tract. and grow well at optimum temperature of 37°C and
It requires both X and V factors. It produces a zone of optimum pH of 6.8. The growth is enhanced by 5% CO 2
beta haemolysis around its colonies on blood agar and and humidity. The inoculated medium is incubated for
may be mistaken for haemolytic streptococci. It is non- 48 hours. Small beta- haemolytic colonies develop on
pathogenic. Similar strains that require only the V factor human or rabbit (but not horse) blood agar. The medium
and not the X factor are termed H. parahaem olyticus. of choice for G vagina/is is human blood bilayer Tween
(HBT) agar.
III. HACEK GROUP BACTERIA
HACEK refers to a group of fastidious bacteria, C. Biochem ical Reactions
normally resident in the mouth, but can sometimes It is catalase, oxidase, indole and urease negative.
cause severe infections such as endocarditis. This group
contains Haemophilus sp. (parainfluenzae), Aggregatibacter D. Pathogen esis
actinomycetemcomitans, Aggregatibacter aphrophilus G vagina/is causes non-specific vaginitis and cerv1c1tls,
(Previously Haemophi lus aphrophilus and paraphrophilus) usually in association with anaerobic bacteria (anaerobic
Cardiobacterium hominis, Eikenella corrodens and Kingella vaginosis). In vaginosis, the vaginal discharge emits a fishy
kingae. HACEK is an acronym consisting of the first odour. Gram staining shows clue cells, which are vaginal
initial of each genus represented in this group. Members epithelial cells covered with many tiny Gram variable
of this group are Gram-negative bacilli have in common rods- the so called clue cells of Gardner and Duke.
the need for an increased CO 2 (capnophilic). Blood
cultures from patients with these bacteria take 7 to 30 E. Clinical Features
days to become positive. As drug resistance is common, There is foul smelling vaginal discharge with raised pH
antibiotic sensitivity tests are essential. (more than 5.0). The odour is ,due to amines and that
gets intensified by mixing with a drop of KOH and is
IV. GARDN ERELLA VAGINALIS named amine test.
(Coryne bacteriu m vaginalis ;
Haemop hilus vaginali s) F. Diagnosi s
As this bacillus does not require X and V factors, it Gram staining of vaginal discharge smear shows clue
has been placed in the new genus Gardnerella. It has cells. G vagina/is can be isolated on blood agar.
been isolated from the normal vagina and male urethra.
G vagina/is is primarily known for its association with G. Treatment
bacterial vaginosis (BV) in humans. Metronidazole is generally effective.
EY POINT S
1. The genus Haemophi/us contains non-motile, non-sporing, Gram negative bacilli and require
one or both of two
accessory growth factors (X and V) present in blood.
2. H. inftuenzae and H. ducreyi are the major pathogens in the genus Haemophi/us.
and
3. H. inftuenzae may cause meningitis , pneumonia, epiglottitis, bronchitis, otitis media, septic arthritis
H. ducreyi is the causative organism of chancroid.
4. H. inftuenzae requires both X and V factor while H. ducreyi requires only X factor.
5. Sate//itism is an important phenomenon in H. inftuenzae.
Ch 39: Haemophilus and Gardnerella _ _ _ __ _ __ _ _ _ __ _ _ _ _ __ _ _ _ _ _ _ 317
6. Latex agglutination, coagglutination and CIE are rapid tests for H. infiuenzae antigen detection in patient's serum,
CSF, urine or pus.
7. CSF culture is an important test in laboratory diagnosis of meningitis.
8. Haemophilus infiuenzae biotype aegyptius causes a highly contagious form of conjunctivitis (pink eye).
9. The microscopic findings of H. ducreyi has been described as resembling a 'school of fish' appearance.
10. H. ducreyi causes chancroid or soft chancre, a highly contagious sexually transmitted disease (STD), characterised by
tender, non-indurated, irregular ulcers on the genitalia.
11. Gardnerella vagina/is causes non-specific vaginitis and cervicitis, usually in association with anaerobic bacteria (anaerobic
vaginosis).
12. In vaginosis, Gram staining of vaginal discharge shows clue cells, which are vaginal epithelial cells covered with many
tiny Gram variable bacilli.
YOU MUST KNOW

1. Morphology and culture characteristics of Haemophilus influenzae.
2. X and V factors.
3. Satellitism.
4. Serotypes of H. infiuenzae.
5. Diseases caused by H. influenzae.
6. Laboratory diagnosis of meningitis caused by H. infiuenzae.
7. Koch-Weeks bacillus.
8. Morphology and culture charact,eristics of Haemophilus ducreyi.
9. Disease caused by H. ducreyi and its laboratory diagnosis.
10. Gardnerella vagina/is.
STUDY QUESTIONS
1. Discuss the laboratory diagnosis of infections caused by Haemophilus influenzae.
(a) X and V factors (b) Satellitism
(c) Haemophilus influenzae biotype aegyptius (d) Haemophilus ducreyi.
(e) Gardnerella vagina/is.

1. Accessory growth factor/s required by Haemophilus influenzae is/are:
(a) X factor (b) V factor
(c) Both X and V factors (d) Neither X nor V factor
2. Which of the following bacteria shows phenomenon of satellitism?
(a) Haemophilus influenzae (b) Listeria monocytogenes
(c) Streptococcus pyogenes (d) All of the above
3. Which serotype of Haemophilus infiuenzae is associated with most invasive infections?
(a) Serotype 'a' strains (b) Serotype 'b' strains
(c) Serotype 'c' strains (d) Serotype 'e' strains
4. Which of the following infections is/are caused by Haemophilus influenzae?
(a) Meningitis (b) Acute epiglottitis

318 - - -- - -- - -- - - - -- - - -- - -- -- Unit Ill: Systemic Bacteriology
5. Which of the following rapid methods can be used to diagnose meningitis caused by Haemophilus influenzae?
(a) Latex agglutination (b) Coagglutination
(c) Counter immunoelectrophoresis (d) All of the above
6. What is the causative agent of Brazilian purpuric fever?
(a) Haemophilus influenzae (b) H. influenzae biotype aegyptius
(c) H. ducreyi (d) H. aphrophilus
7. Haemophilus influenzae biotype aegyptius was formerly known as:
(a) Koch-Weeks bacillus (b) Klebs-loeffier bacillus
(c) Haemophilus vagina/is (d) Haemophilus haemolyticus
8. The causative agent of chancroid is:
(a) Haemophilus influenzae (b) H. ducreyi
(c) H. haemolyticus (d) H. aphrophilus
9. Which one of the following bacteria is included in HACEK group bacteria?
(a) Escherichia coli (b) Enterobacter
(c) Eikenella corrodens (d) Edwardsiella
10. 'Clue cells' in Gram staining can be useful in diagnosis of:
(a) Gardnerella vagina/is (b) Haemophilus parainfluenzae
(c) Kingella kingae (d) Cardiobacterium hominis
An swers (MCQs):
1. (c) 2. (a) 3. (b) 4. (c) 5. (d)
6. (b) 7. (a) 8. (b) 9. (c) 10. (a)
--- • ++ - - -

Chapter 40
BORDETELLA
I. Introduction
II. Bordetella pertussis
E. Antigenic Structure F. Variation
G. Pathogenesis H. Epidemiology
I. Laboratory Diagnosis J . Treatment
K. Prophylaxis
Ill. Bordetella parapertussis
IV. Bordetella bronchiseptica
V. Bordetella avium
I. INTRODUCTION 6. B. avium----causes respiratory disease m turkeys.

The genus Bordetella contains very small, Gram negative 7. B. trematum
coccobacilli which were formerly included in the genus 8. B. ansorpic
Haemophilus. As they do not require X and V factors for 9. B. petrii
growth and are antigenically different from Haemophilus,
they are separated into another genus Bordetella. Bordet
and Gengou (1906) isolated the organism, Bordetella II. BORDETELLA PERTUSSIS
pertussis, from children suffering from whooping A. Morphology
cough. B. pertussis is a small, ovoid, 1- 1.5 µm x 0.3 µm, Gram
negative coccobacillus. It is non-motile and non-sporing.
Species It is capsulated but tends to lose the capsule on repeated
1. B. pertussis----causes whooping cough. subcultures. Bipolar meta-chromatic granules may be
2. B. parapertussis- responsible for milder form of observed on staining with toludine blue.
whooping cough.
3. B. bronchiseptica- resposible for about 0.1 % cases B. Culture
of whooping cough of milder type. It is aerobic and cannot grow anaerobically. Optimum
4. B. holmesii----can rarely be isolated from patients with temperature for growth is 35--36°C. The organism does not
pertussis like symptoms. grow on nutrient agar. It requires a complex medium for its
5. B. hinzii----can rarely be isolated from patients with primary isolation. Bordet-Gengou (glycerol- potato-blood
pertussis like symptoms. agar) is a commonly used medium. After incubation for
319
320 _ _ __ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Unit Ill: Systemic Bacteriology
48-72 hours, colonies on this medium are small, smooth, 3. Heat Labile Toxin (HLT)
opaque, greyish white, refractile, resembling bisected pearls Heat labile (inactivated at 56°C x 15 minutes) toxin is
or mercury drops. A hazy zone of haemolysis surrounds present in all Bordetellae. Pathogenic role of this toxin
the colonies. Blood in the medium is apparently not is doubtful. It is a cytoplasmic protein.
to provide additional nutritive factors, but neutralises
inhibitory substances like toxic fatty acids. Charcoal or
starch or both may also be incorporated to neutralise 4. Tracheal Cytotoxin (TCT)
these toxic constituents. Charcoal blood agar medium is It is derived from the peptidoglycan of cell wall and is
an alternative medium used for primary isolation of this present in all Bordetellae. It causes damage to respiratory
organism. Commonly used charcoal blood agar medium epithelial cells and therefore makes a person more prone
is Regan- Lowe (RL) medium. It i; available both as to secondary infection.
semisolid transport medium and as a solid medium for
isolation of organism. 5. Pertussis Toxin (PT)
It is a heat labile exotoxin called the pertussis toxin. It
is present only in B. pertussis. It is a major virulence
C. Biochemical Reactions factor. It is also known as lymphocytosis producing factor,
B. pertussis is biochemically inactive. It does not ferment histamine sensitising factor and islet activating protein. It
sugars, form indole, reduce nitrates, split urea or utilize has a wide spectrum of biologic activity. It is apparently
citrate. It is oxidase positive and usually produces responsible for many of the signs and symptoms of
catalase. pertussis. It also causes profound lymphocytosis m
pertussis patients.
D. Resistance PT has a molecular weight of 117,000 and is
It is killed by heating at 55°C for 30 minutes, drying composed of six polypeptide chains (Sl to S5 with 2
and disinfectants. It survives outside the body in droplets copies of S4). PT has got two units A and B (the A
for few hours. It retains viability at low temperatures unit being the enzymatically active moiety and B the
(0-4oC). binding component). The A unit is made up of Sl while
B unit consists of the remaining five polypeptide chains.
E. Antigenic Structure Pertussis toxin can be toxoided. PT toxoid is the major
1. Agglutinogens component of acellular pertussis vaccines.
Freshly isolated strains of B. pertussis possess heat labile
antigens associated with the capsule (K antigens), designated 6. Adenylate Cyclase (AC)
1 to 14 factors or agglutinogens. All strains of B. pertussis Only one of adenylate cyclases has the ability to enter
carry factor 1 and one or more of the other factors. Factor target cells and act as a toxin. This is known as AC toxin
1 to 6 are present only in strains of B. pertussis but factor (ACT). It acts by catalysing the production of cAMP by
7 is found in all strains of the three species of Bordetella. various cells. It is produced by all the three mammalian
Factor 12 is specific for B. bronchiseptica and factor 14 for bordetellae.
B. parapertussis. These antigens are useful in serotyping
of the strains for epidemiology purposes.
Some of the agglutinogens have been recognised on 7. Filamentous Haemagglutinin (FHA)
the fimbriae. B. pertussis have eight agglutinogens; 1 to It is present on the bacillary surface. It mediates the
6, 7 and 13. These type specific antigens are under the attachment of the bacterium to the ciliated epithelial cells
control of vir gene. Agglutinogens promote virulence by of the respiratory tract. It also adheres to erythrocytes.
helping organisms to attach to respiratory epithelial cells. Antibodies directed against FHA are protective. FHA
As strains causing infection are of types 1, 2 and 3, is used in acellular pertussis vaccines along with PT
it is essential that pertussis vaccine strains should have toxoid.
factors 1, 2 and 3.
8. Pertactin
2. Lipopolysaccharide It is an outer membrane protein (OMP) present in virulent
It is a heat stable lipopolysaccharide endotoxin of the strains of B. pertussis. It is included in acellular pertussis
cell wall. It is not protective. It is common to all smooth vaccines. Antibody to pertactin can be detected in blood
strains of all the three species of Bordetella. after infection or immunisation.

321
Ch 40: Bordetella _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _
F. Variation I. Laboratory diagnosis

Freshly isolated strains are in the smooth form (Phase I). 1. Microscop y
On subculture, they undergo loss of surface antigens and Microscopic diagnosis is made by demonstration of the
become phase IV, which is the rough avirulent form. They bacilli in respiratory secretions by the fluorescent antibody
have to pass through phase II and III before becoming technique.
phase IV
A reversible change in the capsular antigen has been 2. Culture
described. The organism may occur in one of three In the early stage (catarrhal), bacilli are most abundant
'modes', X, I and C. Each mode has a characteristic surface in the upper respiratory tract. They are scanty in the
antigen. These modes refer to the colour of the colonies paroxysmal stage and are not demonstrable during
on the Bordet-Gengou medium- X for xanthic (yellow), convalescence. For culture, following methods have been
C for cyanic (blue) and I for intermediate. On the used for collection of specimens.
Bordet-Gengou medium, fresh isolates always occur in .
(i) The pernasal swab
X mode.
A swab is passed along the floor of the nasal cavity and
G. Pathogenesis material collected from the pharyngeal wall. The swab
B. pertussis infection is predominantly a childhood disease is inoculated on Bordet-Gengou medium or charcoal
and is known as whooping cough. 95% cases of whooping blood agar. Nasopharyngeal aspirate collected through
cough are due to B pertussis, 5% by B. parapertussis a catheter attached to a syringe is a better alternative.
and only in about 0.1 % cases, B. bronchiseptica is
responsible. (ii) The cough plate method
In human beings, the source of infection is the patient A Bordet-Gengou culture plate is held 10- 15 cm in front
in the early stage. The incubation period is about one of the patient's mouth during a bout of coughing so
to two weeks. Infection is transmitted by droplets. The that the cough droplets directly inoculate the culture
disease usually lasts for 6 to 8 weeks. It consists of three medium.
stages namely, the catarrhal, paroxysmal and convalescent,
each lasting approximately for two weeks. Although (iii) The postnasal (peroral) swab
cases are maximally infective during the catarrhal stage A West's postnasal swab is passed through mouth to
but clinical diagnosis in this stage is difficult. During collect posterior pharyngeal wall secretions. Precautions
the paroxysmal stage, the violent spasms of continuous should be taken to avoid salivary contamination.
coughing is followed by a long inrush of air into the Fatty acids present in cotton may inhibit the growth
almost empty lungs, with a characteristic 'whoop'. During of the bacilli and so it is better to use dacron or calcium
convalescent stage, the frequency and severity of coughing alginate swabs for collection of specimen. The swabs are
gradually decrease. to be plated without delay. In case of delay, modified
Stuart's medium or charcoal agar should be used for
Complications transport of the swab.
The disease is self-limiting. Complications may be: Medium employed is Bordet and Gengou or charcoal
(i) subconjuctival haemorrhage due to the pressure blood agar [Reagan-Lowe (RL) medium]. Incorporatio n
effects of violent coughing of diamidine, flouride and penicillin (Lacey's DFP
(iz) bronchopneumonia and lung collapse medium) into Bordet and Gengou medium makes it
(iiz) convulsions and coma. more selective. Plates are incubated in high humidity at
35- 36°C for 3- 5 days. Typical pearl like colonies of B.
H. Epidemiology pertussis appear in 48- 72 hours. These are confirmed by
Whooping cough is predominantly a paediatric disease. microscopy and slide agglutination with specific antisera.
The incidence and mortality are highest in the first Imrnunofluorescence is useful in identifying the bacillus
year of life. The source of infection is the patient, most in smears from culture. The differentiating features of
infective during early stage (catarrhal stage). The mode of different species of bordetella are listed in Table 40.1.
infection is via droplets. Whooping cough is one of the
most infectious of bacterial diseases. Healthy carriers have 3. Polymeras e Chain Reaction (PCR)
not been identified. Although natural infection confers PCR is the preferred method for the direct detection of
immunity but second attack may occur. Reinfection in B. pertussis and B. parapertussis from a nasopharyngeal
adults may be of severe form. swab or a nasopharyngeal aspirate.
322 _ __ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __ _ _ _ _ _ _ _ _ Unit Ill: Systemic Bacteriology
4. Serology age, who are contacts of the case, should receive a booster
Rise in titre of antibodies may be demonstrated in paired dose alongwith a chemoprophylaxis of erythromycin for
sera samples by agglutination, complement fixation test, five days.
immunofluorescent test and ELISA. Acellular pertussis vaccines containing PT, FHA,
Detection of anti-pertussis toxin (PT) antibody levels pertactin, agglutinogens 1, 2, 3 of B. pertussis have been
can be detected in serum by ELISA. Immunoglobulin developed. They have fewer complications.
G (IgG) anti-PT antibodies levels of > 100 to 125 Prevention by isolation of cases is seldom practicable,
international units/milliliter (IU/mL) can be used as as infectivity is the highest during early stage (catarrhal)
recent contact with PT-producing bacteria. Paired sample of the disease when clinical diagnosis is not easy.
serology method can also be used but single-sample
serology also provides good sensitivity and specificity.
III. BORDETELLA PARAPERTU SSIS
J. Treatment They are responsible for about five per cent of whooptng
The organism is sensitive to various antibiotics such as cough cases and generally produce a mild disease.
tetracycline, chloramphenicol, erythromycin and ampicillin. Morphologically they resemble B. pertussis but differ by
However, erythromycin is the drug of choice. their ability to grow on nutrient agar and pigment production
(Table 40.1 ). They are also antigenically distinct from B.
K. Prophylaxis pertussis. The pertussis vaccine does not protect against
Immunisation of infants and children with killed B. parapertussis infection.
B. pertussis vaccine is very effective. Smooth phase I
strain of B. pertussis is used for vaccine production. Three
intramuscular injections at intervals of 4-6 weeks are IV. BORDETELL A BRONCWSE PTICA
given before the age of six month, followed by a booster It is motile with peritrichate flagella and has been found
dose at the end of the first year of life. Vaccination is to cause 0.1 % cases of whooping cough in humans. It can
usually started at age of 6 weeks. Pertussis vaccine is grow on nutrient agar and is antigenically related to B.
generally administered in combination with diphtheria pertussis and Bruce/la abortus. Differentiating characters
toxoid and tetanus toxoid (DPT). B. pertussis acts as of bordetella species are listed in Table 40.1 .
an adjuvant for the toxoids, producing better antibody
response. Two types of vaccine DwPT (with whole cell
pertussis vaccine) and DaPT (with acellular pertussis V. BORDETELL A AVIUM
vaccine) are available. It is the causative agent of coryza in turkeys. It can grow
Rarely neurological complications like encephalopathy on nutrient agar. It produces HLT and TCT but does
and convulsions may occur. Children below four years of not produce ACT and PT.
Table 40.1 Differentiating Characters of Bordetella Species

Character B. pertussis B. parapertussis B. bronchiseptica B.avium
Motility + +
Growth on nutrient agar + + +
Pigment production +
Oxidase + + +
Urease production + +
Citrate utilisation + + +
Nitrate reduction +
Toxins:
HLT and TCT + + + +
ACT + + +
PT +

Ch 40: Bordetella _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ __ _ _ _ _ _ __ _ _ _ _ _ 323
KEY POINTS
1. The genus Bordetella contains very small, Gram negative coccobacilli.
2. Three important species of Bordetella include Bordetella pertussis, B. parapertussis and B. bronchiseptica.
3. B. pertussis is the main causative agent of whooping cough. It is a small, ovoid, non-motile, Gram negative
coccobacillus. It is aerobic and cannot grow anaerobically. Bordet-Gengou (glycerol-potato-blood agar) is a commonly
used medium. Regan-Lowe (RL) medium is charcoal blood agar used for growth of B. pertussis. It is oxidase positive.
4. Pertussis toxin is exotoxin produced by B. pertussis.
5. Laboratory diagnosis depends on microscopy and culture.
6. Pertussis vaccine is generally administered in combination with diphtheria toxoid and tetanus toxoid (DPT). B. pertussis
acts as an adjuvant for the toxoids producing better antibody response. Vaccine is very effective for prophylaxis.
7. Acellular pertussis vaccines containing antigens and toxins are also available.
YOU MUST KNOW

1. Morphology, culture characteristics of Bordetella pertussis.
2. Laboratory diagnosis of whooping cough.
3. Pertussis vaccines.
STUDY QUESTIONS
1. Describe the laboratory diagnosis of whooping cough.
(a) Antigenic structure of B. pertussis (b) Vaccination against pertussis (c) Bordet-Gengou medium.
MU CHOICE QUESTIONS (MCQs)

1. Which of the following media can be used for isolation of Bordetella pertussis?
(a) Glycerol-potato blood agar (b) Lacey's DFP medium
(c) Regan-Lowe medium (d) All of the above
2. Which one of the following media is most frequently used for isolation of Bordetella pertussis?
(a) Chocolate agar (b) MacConkey agar (c) Bordel-Gengou medium (d) Tinsdale agar
3. Whooping cough can be caused by:
(a) Bordetella pertussis (b) B. parapertussis (c) B. bronchoseptica (d) All of the above
4. Which of the following species of Bordetella is motile?
(a) Bordetella pertussis (b) B. parapertussis (c) B. bronchiseptica (d) None of the above
5. Bordetella pertussis infection is transmitted by:
(a) Droplets (b) Inoculation of skin (c) Contact (d) All of the above
6. Which is the most infective stage in whooping cough?
(a) Catarrhal stage (b) Paroxymal stage (c) Convalescent stage (d) None of the above
7. Which of the following vaccines is/are available for prophylaxis of whooping cough?
(a) Whole cell pertussis vaccine (b) Acellular pertussis vaccine
Answers (MCQs):
1. (d) 2. (c) 3. (d) 4. (c) 5. (a) 6. (a) 7. (c)
----+++----
Chapter 41
BRUCELLA
Brucella
E. Antigenic Structure F. Biotypes
G. Phage Typing H. Classification
I. Pathogenesis J. Epidemiology
M. Prophylaxis
BRUCELLA arranged singly or in short chains. They are non-sporing

The genus Brucella consists of small, non-motile, aerobic, and non-capsulated.
Gram negative coccobacilli which are strict parasite
of animals (goats, sheep, cattle, pigs, buffaloes). Man B. Culture
acquires infection by contact with infected animals or Brucellae are strict aerobes. Br. abortus is capnophilic,
through their products. The human disease had been requiring 5-10 per cent CO2 for its growth whereas Br. suis
recognised from very early times in the Mediterranean growth is unaffected by CO2. The optimum temperature
countries and had been known under various names such for growth is 37°C (range 20-40°C) and optimum pH
as Mediterranean fever, Malta fever and undulant fever. 6.6-7.4.
Bruce (1887) isolated a small microorganism from the They may grow on ordinary media, though growth
spleen of fatal cases in Malta. This has been named is slow and scanty. Growth is improved by addition of
Bruce/la melitensis (Bruce/la after Bruce; melitensis glucose, serum, blood or liver extract. The media employed
after Melita, the Roman name for Malta). Bang (1897) for culture are serum-dextrose agar, serum-potato-infusion
described Br. abortus in Copenhagen, the cause of agar, trypticase soy agar or tryptose agar. The addition
contagious abortion in cattle. Traum (1914) isolated Br. of bacitracin, cycloheximide, polyrnyxin B or vancomycin
suis from pigs in USA. These three are the major species makes these media selective. On solid media, colonies are
in the genus. Three additional species Br. neotomae, small, moist and translucent. In liquid media, growth is
Br. ovis and Br. canis have been recognised which are uniform.
animal pathogens and except for Br. canis, they do not
infect man. Cultivation in presence of dyes
The three important brucellae (Br. abortus, Br. melitensis
A. Morphology and Br. suis) are differentiated on the basis of their ability
Brucellae are Gram negative, non-motile, coccobacilli or to grow in media containing aniline dyes such as basic
short rods measuring 0.5-0.7 µm x 0.6-1.5 µm. They are fuchsin (1: 50,000) and thionin (1: 25,000). Br. melitensis
324
Ch 41 : Brucella - - -- - - - - - - - -- -- - - -- - - - - - - - - - - - - - 325
is not inhibited by any of the dyes, while Br. abortus is for 10 days in refrigerated milk, for months in butter, one
inhibited by thionin and not by basic fuchsin. Br. suis month in ice cream. They die out in butter and cheese
is inhibited by basic fuchsin and not by thionin (Table undergoing lactic acid fermentation. They are sensitive
41.1). to direct sunlight and acid. Br.melitensis may survive for
six weeks in dust and ten weeks in water.
C. Biochemical Reactions They are resistant to penicillin but are susceptible
No carbohydrates are ordinarily fermented but they to streptomycin, tetracycline, chloramphenicol and
possess oxidative capacity. They are catalase and oxidase ampicillin.
positive (except for Br. neotomae and Br. ovis which are
negative). They are urease positive, nitrate reduction E. Antigenic Structure
positive, citrate negative, indole is not produced and MR Two main somatic antigens of brucellae, A and M are
and VP tests are negative. Urease test is very rapidly present in different amounts in the three major species.
positive particularly in Br. suis which is positive within Antigen 'Xis dominant (about 20 times as much as 'M'
30 minutes and Br. abortus takes 1- 2 hour to become antigen) in Br. abortus and antigen 'M' is dominant in
urease positive. Br. melitensis (about 20 times of 'A:. antigen). Br. suis
has an intermediate antigenic pattern. Absorption of the
D. Resistance minor antigenic components of antiserum will leave most
Brucellae are killed by heat at 60°C in 10 minutes, hence of the major antibody components. Such absorbed A
in milk they are rapidly destroyed by pasteurisation. They and M monospecific sera are useful for identification by
are killed by 1% phenol in 15 minutes. They may survive in agglutinatation test. The antigenic structure of brucellae
soil and manure for several weeks. The organism survives is more complex. Some strains behave biochemically as
Table 41.1 Differential Features of Brucella Species and Biotypes

Growth in the presence of Agglutination by Lysis by phage
Bio- CO 2 H2S Mooospecific Common
Species Basic Anti-rough RTD host
types requirement production Thionin sera RTD
fuchsin serum X }04
A M
Br. melitensis + + + Sheep, goats

2 + + +
3 + + + +
Br. abortus ± + + + + + Cattle
2 ± + + + +
3 ± + + + + + +
4 ± + + + + +
5 + + + +
6 ± + + + +
9 ± + + + + +
Br. suis + + + + Pigs
2 ± + + ± Pigs, hare
3 + + + + Pigs
4 + + + + + Reindeer
5 + + + Rodents
Br. canis + + D ogs
Br. ovis + + + + Sheep
Br. neotomae + + + Wood rat
Dannish strains of Br. suis differ from American strains in not producing H2 S.

326 - - - -- -- - - - - - - - - - - - - - - - - - - - - - Unit Ill: Systemic Bacteriology
Br. abortus and serologically as Br. melitensis and vice- 2. Types of Infection
versa. The battery of other tests in addition to biotyping Human infection may be of three types:
and serotyping is required for differentiation of various (i) Subclinical or latent infection: There is no clinical
species (Table 41.1). evidence of disease but is detectable only by
Antigenic cross reactions exist between brucellae and serological tests.
Esch. coli 0:116, 0 :157; Salmonella serotype N (0:30 (ii) Acute brucellosis: It is also known as undulant fever
antigen), V cholerae, Ps. maltophila, F. tularensis and or Malta fever. It is associated with prolonged
Y enterocolitica. bacteraemia.
(iiz) Chronic brucellosis: It is usually non-bacteraemic.
The symptoms are generally related to a state of
F. Biotypes hypersensitivity.
(i) Br. abortus-7 biotypes (1 to 9, number 7 and 8 are
deleted) 3. Course of Disease
(ii) Br. melitensis-3 biotypes The bacilli after entering the body pass via the lymphatic
(iii) Br. suis-5 biotypes channels, regional lymph nodes, and thoracic duct to the
Br. suis strains which produce H 2S are called American blood stream causing bacteraemia. Brucellosis is primarily
strains and those not producing H 2S are called Danish a disease of reticuloendothelial (RE) system, hence, the
strains. organisms are localised in the RE system. Brucellae have
a special predilection for intracellular growth especially
G. Phage Typing inside phagocytic cells. Cell mediated immunity plays an
The Tblisi (Tb) reference phage is used for phage typing. important role in recovery from brucellosis.
It lyses Br. abortus at both RTD (Routine test dilution) The incubation period varies from 2 to 3 weeks and
and 10,000 RTD but Br. suis is lysed at 10,000 RTD only may extend to 6 months. The onset of symptoms may
while Br. melitensis is not lysed at all. be acute or insidious. Acute brucellosis is chracterised by
fever, chills, shivering, malaise, severe headache, bone and
H. Classification joint pains, mild lymph node enlargement and sometimes
Brucellae may be classified into six species based on CO 2 hepatosplenomegaly. The name undulant fever has been
requirement, production of H 2S; sensitivity to aniline dyes attributed to acute brucellosis because of the periodic
(basic fuchsin and thionin), agglutination by monospecific nocturnal fever that may occur over weeks, months or
sera and phage lysis (Table 41.1) . years especially in untreated cases. When the disease
persists for 6 months or more, it is termed as chronic
I. Pathogenesis brucellosis.
All the three major species of brucellae are pathogenic
to human beings. Br. melitensis is the most pathogenic J. Epidemiology
species followed by Br. suis of intermediate pathogenicity The disease is worldwide m distribution and endemic
and Br. abortus is the least pathogenic. Brucellosis is a in certain areas such as Mediterranean countries (B,:
zoonotic disease. melitensis), certain swine rearing areas of USA (Br. suis)
and Great Britain (Br. abortus). In India, Br. melitensis is
1. Mode of Infection the predominant pathogen of human brucellosis acquired
Human infection occurs by direct or indirect contact with from goats or sheep which is followed by Br. abortus of
infected animals. Brucellae are transmitted to humans cattle origin.
by:
(i) Drinking contaminated (unpasteurised) raw milk K. Laboratory Diagnosis
or by ingestion of milk products from infected Brucellosis is a febrile disease with varied clinical
animals. manifestations and clinical diagnosis is often difficult and
(ii) Direct contact with infected animal tissues: butchers, laboratory aid is, therefore, essential. Laboratory methods
dairy workers, farm workers and veterinarians are include culture of brucellae, serology and hypersensitivity
particularly at risk. test.
(iiz) Accidental ingestion, inhalation, injection and
mucosa! or skin contamination may occur m 1. Specimens
laboratory personnel working with brucellae. Blood culture 1s the most definitive method for the

Ch 41: Brucella - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -- - 327
diagnosis of acute brucellosis. Brucellae can also be 4. Serology

isolated from bone marrow, liver, lymph nodes and Serological methods are more useful as cultures are
occasionally from CSF, urine, sputum, breast milk, negative in high percentage of cases. Antibodies (IgG
vaginal discharge and seminal fluid. and IgM) appear in 7- 10 days after clinical infection.
As the disease progresses, IgM antibodies decline,
2. Blood Culture while the IgG antibodies persist or increase. In chronic
Blood is collected during the pyrexial phase because brucellosis, only IgG can be demonstrated and IgM
chances of positive blood culture are more during this may often be absent. The agglutination test identifies
period. 5- 10 ml of blood is inoculated into 50-100 ml mainly the IgM antibody, while both J.gM_and_IgG can
of serum dextrose broth or trypticase soy broth and fix c o m ~ h e IgG and IgA antibodies may act
incubated at 37°C aerobically in presence of 5- 10% CO 2 . as blocking (incomplete)--aruibodies and may prevent
Subcultures are made on solid media every 3- 5 days for agglutination ~ The agglutination test is usually ~itive
8 weeks, and incubated at 37°C aerobically in presence in acut~on, but may be negati~ or w~akly positive
of 5- 10% CO2 . in chronic cases.
The need for frequent subcultures can be avoided by
use of Castaneda 's method of blood culture. It contains
both liquid (trypticase soy broth) and solid (trypicase soy (i) Serum agglutination test (SAT)
agar) media in the same bottle (Fig. 41.1). The blood is Serum agglutination test (SAT) is the most widely used test
inoculated into the broth (liquid medium) and the bottle for diagnosis of brucellosis.- This is a tube agglutination
incubated in the upright position. For subculture, the test in which equal volumes of serial two fold dilutions
bottle is tilted so that the broth flows over the surface (1 :20 to 1:640) of patient's serum and the standardised
of solid agar slant. It is again incubated in the upright antigen (heat killed Brucella suspension) are mixed and
position. In case of positive blood culture, colonies appear incubated at 37°C for 24 hours. A single titre of more than
on the agar slant. This method reduces the chances 1: 160 is presumptive evidence of Brucella infection. The
of contamination and risk of infection to laboratory test can also be done in microplate.
workers. Prozone phenomenon is very frequent in brucellosis,
Blood cultures are positive in 30-50% cases, even therefore it is essential that several serum dilutions should
when repeated samples are tested. Br. melitensis and Br. be tested. Another source of error is presence of blocking
suis are readily cultured than Br. abortus. or non-agglutinating antibodies in serum. The blocking
BACTEC and BacT Alert are rapid methods for effect may be removed by heating the serum at 55°C for 30
blood culture and majority of samples become positive minutes or by using 4% saline as diluent for the test. The
within 7 days. most important method for detecting these incomplete
antibodies is the antiglobulin (Coombs) test.
3. Identification A positive agglutination test may also be produced by
It depends on biochemical tests, dye test, agglutination cholera, tularaemia or yersinia infection or vaccination.
with monospecific sera and lysis by phage. Cholera induced antibodies may be removed by absorption
with 2- mercaptoethanol.
Agglutinin titres are expressed in International units.
This is done by using a standard reference serum for
companson.
(ii) Castaneda strip test

A loopful of patient's serum is placed on a filter paper
strip containing a coloured brucella antigen. Antibody is
•- - -- ----t--Solid medium able to 'fix ' the antigen and prevent its movement along
the paper. It is simple and suitable test for screening of
brucellosis cases.
Liquid medium (iii) Complement fixation test (CIT)

Fig. 41.1 Castaneda's medium It is more useful in chronic cases as it detects both IgG
and IgM.

(iv) ELISA and RIA Method

These tests are very sensitive and helpful to distinguish 1. A sample of whole milk is mixed well with a drop
acute and chronic brucellosis. ELISA can detect IgM of the stained brucella antigen (a concentrated
and IgG antibody separately. suspension of Br. abortus or Br. melitensis stained
with haematoxylin) in a narrow test tube.
(v) Indirect immunoftuorescent test 2. This mixture of milk and bacterial suspension is
It is specific and sensitive method for detecting antibodies incubated in a water bath at 70°C for 40---50 minutes.
and may be positive even when the agglutination test is
negative. Result
In a positive test, the bacilli are agglutinated and rise
with the cream to form a blue ring at the top, leaving
5. Hypersensiti vity Test (Brucellin test) the milk unstained. When antibodies are absent (negative
It is delayed (type IV) hypersensitivity to Brucella antigen. test), no coloured ring is formed and the milk remains
It is an intradermal test using protein extract of the uniformly blue.
organisms, called brucellin. Positive reaction is indicated Whey agglutination test is another useful test for
by an erythema and induration of 6 mm diameter within detecting antibodies in milk.
24 hours. This test is not useful for diagnosis of acute
infections because test may remain positive for years L. Treatment
after acquiring infection. It may be helpful in chronic Treatment consists of administration of tetracycline along
brucellosis where a positive skin test may sometimes be with streptomycin for a period of not less than three
the only indicator of infection. Persistent negative skin weeks. WHO has recommended a regimen of rifampicin
test helps to exclude brucellosis. (600---900 mg) and doxycycline (200 mg), both taken as
a single daily dose for a minimum of 6 weeks.
6. Detection of Animal Infection M. Prophylaxis

The methods used for the laboratory diagnosis of human 1. Persons handling the animals should use protective
infections may also be employed for the diagnosis of clothing and gloves.
animal infections. Culture of milk and urine may give 2. Pasteurisation or boiling of milk should be done.
positive results. In addition, rapid methods such as 'rapid 3. Vaccination: Cattle should be vaccinated with live
plate agglutination test' and the 'Rose Bengal card test' attenuated Br. abortus strain 19 vaccine and goats,
have been employed for the diagnosis of brucellosis in sheep with Br. melitensis Rev. I vaccine.
herd of cattle. For detection of infected animals in dairies, 4. Unimmunised infected animals should be
besides culture, pooled milk samples may be tested for slaughtered.
antibodies by milk ring test. 5. Br. abortus strain 19-BA, a more attenuated
variant of strain 19, has been widely employed for
Milk ring test human immunisation in USSR for protection of
It detects brucella agglutinins m the milk of infected population exposed to infection. Vaccine is given
dairy cattle. in tradermally.
KEY POINTS
1. The genus Bruce/la consists of small, non-motile, aerobic, Gram negative coccobacilli which are strict parasite of
animals (goats, sheep, cattle, pigs, buffaloes).
2. Bruce/la melitensis, B. abortus and B. suis are the three major species of the genus Bruce/la.
3. Brucellosis is a febrile disease. Man acquires infection by contact with infected animals or through their products.
Human infections may be of three types: subclinical, acute brucellosis, chronic brucellosis.
4. Brucellae are strict aerobes. B. abortus is capnophilic, requiring 5-10 per cent CO2 for its growth.
5. The media employed for culture are serum dextrose agar, serum-potato-infusion agar, trypticase soy-agar or tryptose
agar. The three important species of brucella are differentiated on the basis of their ability to grow in media
containing aniline dyes such as basic fuchsin and thionin.
6. Brucellae are oxidase positive and urease positive. Urease test is very rapidly positive particularly in B. suis.

Ch 41: Brucella - - - - - - - - -- - - - - - - - - - - - - - - - - - - - - - - - 329
7. Laboratory diagnosis depends on culture of brucellae and serology. Castaneda 's method of blood culture is used.
Castaneda's medium contains both liquid (trypicase soy broth) and solid (trypticase soy agar) media in the same
bottle. Serum agglutination test is used for detection of antibodies in patient's serum. A single titre of more than
1 : 160 is presumptive evidence of Brucella infection.
8. Brucellin test may be helpful in chronic brucellosis where a positive skin test may sometimes be the only indicator
of infection.
YOU MUST KNOW

1. Morphology, culture characteristics and biochemical reactions of Brucella sp.
2. Different species of Brucella.
3. Diseases caused by Brucella sp.
4. Laboratory diagnosis of brucellosis.
5. Castaneda's medium.
STUDY QUESTIONS
1. Discuss the laboratory diagnosis of brucellosis.
(a) Castaneda's medium
(b) Serodiagnosis of brucellosis
(c) Diagnosis of brucellosis in animals.

1. The causative agent/s of undulant fever is/are:
(a) Bruce/la abortus (b) Br. melitensis (c) Bl: suis (d) All of the above
2. Which of the following bacteria is capnophilic?
(a) Bruce/la abortus (b) Br. melitensis (c) Br. suis (d) None of the above
3. Which of the following dyes is/are useful for differentiation of various species of Brucella?
(a) Basic fuchsin (b) Thionin (c) Both of the above (d) None of the above
4. Which of the following media can be employed for isolation of Brucella?
(a) Serum-dextrose agar (b) Serum-potato-infusion agar
(c) Trypticase soy agar (d) All of the above
5. Castaneda medium can be used for blood culture in:
(a) Acute brucellosis (b) Bordetella infections (c) Yersina infections (d) None of the above
6. Brucellae are transmitted to humans by:
(a) Ingestion of contaminated raw milk
(b) Direct contact with infected animal tissues
(c) Accidental mucosa! or skin contamination in laboratory personnels
(d) All of the above
7. Which of the following animals are involved in causing Brucella infection?
(a) Sheep (b) Goats (c) Cattle (d) All of the above
8. All of the following diseases are zoonotic except:
(a) Brucellosis (b) Plague (c) Tularensis (d) Floppy baby syndrome
9. 'Rose Bengal Card' test is employed for which of the following infections?
(a) Brucellosis (b) Salmonellosis (c) Tularensis (d) None of the above
10. Vaccination of cattle against brucellosis is done with:
(a) Bruce/la abortus vaccine(b) Br. melitensis vaccine (c) Br. suis vaccine (d) B,: canis vaccine
Answers (MCQs):
1. (d) 2. (a) 3. (c) 4. (d) 5. (a) 6. (d) 7. (d) 8. (d) 9. (a) 10. (a)
---- +++ - - - -
Chapter 42
MYCOBACTERIUM TUBERCULOSIS
I. Introduction
II. Classification
Ill. Mycobacterium tuberculosis
C. Resistance D. Biochemical Reactions
E. Antigenic Structure F. Mycobacteriophages
G. Pathogenesis H. Immunity and Hypersensitivity
I. Koch's Phenomenon J. Tuberculin Skin Test
K. Laboratory Diagnosis L. Sensitivity Testing
M. Treatment N. Prophylaxis
0 . Revised National Tuberculosiss
Control Programme
I. INTRODUCTION The saprophytic mycobacteria were isolated from a

Mycobacteria are slender bacilli that sometimes show number of sources e.g. butter (M. buty ricum), grass (M.
branching filamentous forms resembling fungal mycelium phlei) , dung (M. stercoris) , smegma (M. smegmatis). A
(Myces meaning fungus). They are difficult to stain, but group of mycobacteria, distinct from human or bovine
once stained, resist decolourisation with dilute mineral tubercle bacilli isolated occasionally from human sources,
acids and are therefore called acid-fast bacilli or AFB. are grouped together under the term atypical mycobacteria,
These organisms are aerobic, non-motile, non-capsulated anonymous or unclassified mycobacteria. Most of these
and non-sporing. Growth is generally slow. The genus bacteria occur in soil, water and other sources. They are
includes obligate parasites, opportunistic pathogens and mostly opportunistic.
saprophytic mycobacteria. Seven species are responsible for mammalian
Hansen (1868) discovered the first member of the tuberculosis M . tuberculosis (human tubercle bacillus), M.
genus i.e. lepra bacillus. Robert Koch (1882) isolated the bovis (bovine tubercle bacillus), M . microti (vole tubercle
mammalian tubercle bacillus and Johne (1895) described bacillus) M. africanum (intermediate form between M.
M. paratuberculosis (Johne's bacillus) causing chronic tuberculosis and M . bovis), M. caprae, M. canettii and
enteritis in cattle. Subsequently other pathogenic tubercle M . pinnipedii.
bacilli such as the murine type (M microti) from voles, the
avian type (M. avium) from birds and the cold blooded The Mycobacterium Tuberculosis Complex
type (M. piscium, M. marinum) were isolated. The M. tuberculosis complex includes M. tuberculosis, M.
330
Ch 42: Mycobacterium Tuberculosis _ _ _ _ _ __ _ _ __ _ _ __ __ _ __ __ _ _ _ _ 331
bovis, M . microti, M. africanum M. caprae, M . canettii M. tuberculosis and M. bovis are typical tubercle
and M. pinnipedii. These species are closely related to bacilli and cause human lesions such as pulmonary
each other by antigenic analysis and DNA hybridisation. tuberculosis.
However, they can be distinguished by certain characters
(Table 42.1).
Table 42.1 Distinguishing Features of M. tuberculosis Complex

TCH Pyrazinamide Oxygen Cycloserine Nitrate Niacin
10 µg/ml 50 µg/ml preference reduction production
M . tuberculosis
Classical R s Aerobic s + +
South Indian s s Aerobic s + +
M . bovis
Classical s R Microaerophilic s
BCG s R Aerobic R
M . africanum
African I s s Microaerophilic s + +/-
African II s s Microaerophilic s +
TCH = Thiophen- 2-carboxylic acid hydrazide
S = sensitive R = resistant
II. CLASSIFICA TION

Mycobacteria causing human diseases may be classified as:
Cultivable 1. Typical tubercle bacilli (mammalian)

(i) Human type- M tuberculosis (ii) Bovine type-M bovis
(iii) Vole type-M. microti (iv) African type- M. africanum
(v) Cattle type- M. caprae ( vi) M canettii
(vii) Pinniped type- M pinnipedii
2. Atypical mycobacteria
(z) Photochromog ens (ii) Scotochromog ens
(iii) Non-photochro mogens (iv) Rapid growers
3. Mycobacteria causing skin ulcers
(i) M . ulcerans (ii) M. balnei
4. Saprophytic mycobacteria.
(i) M. smegmatis (ii) M butyricum
(iii) M. stercoris (iv) M. phlei
Non-cultivable M. leprae
III. MYCOBACTERIUM TUBERCULOSIS (average 3 µm x 0.3 µm) in size. These bacilli are acid-
The species contains two major types, classical and South fast, non-sporing, non-capsulated and non-motile. Ziehl-
Indian type of M . tuberculosis. Classical type is virulent Neelsen staining is useful to study the morphology of
to guinea pig but South Indian type is attenuated in this these organisms. With this stain, tubercle bacilli are seen
animal. South Indian type is prevalent in South India bright red (acid-fast), while the tissue cells and other
and in persons of Asian-ethnic origin living in other organisms are stained blue (Fig. 42.1 ). Tubercle bacilli
countries. may also be stained with the fluorescent dyes (aurarnine
0 , rhodarnine) and appear yellow luminous bacilli under
A. Morphology the fluorescent microscope. Beaded or barred forms are
M. tuberculosis is a slender, straight or slightly curved frequently seen in M tuberculosis. They are Gram positive
bacillus with rounded ends, occurring singly, in pairs or but are difficult to stain with the Gram stain due to the
in small clumps. It measures, 1-4 µm x 0.2 - 0.8 µm failure of the dye to penetrate the cell wall.
332 _ _ _ _ _ _ _ _ _ _ _ __ _ _ __ _ __ _ _ _ _ _ _ _ _ Unit Ill : Systemic Bacteriology
mineral salts, malachite green and glycerol or sodium

pyruvate It is solidified by heating (inspissation). It is
one of the media which are solid without incorporation
of agar. In this medium egg acts as a solidifying agent.
Malachite green inhibits the growth of organisms other
than mycobacteria and provides a colour to the medium.
The addition of glycerol improves the growth of human
type of M tuberculosis, while it is without any effect or
even inhibitory to M . bovis. Sodium pyruvate improves
the growth of both M. tuberculosis and M. bovis. Colonies
of M. tuberculosis are dry, rough, buff coloured, raised,
Fig. 42.l Mycobacterium tuberculosis in Ziehl- with a wrinkled surface (Fig. 42.2) . They are tenacious
Neelsen stained smear and not easily emulsified. In contrast, the colonies of M.
bovis are flat, smooth, moist and white, breaking up easily
Another method of staining is Kinyoun's method of when touched . M . tuberculosis has a luxuriant growth
acid fast staining. This is similar to conventional acid- (eugonic growth) in culture as compared to sparsely grown
fast staining (ZN staining) except that heating is not (dysgonic growth) M. bovis.
required. It is named as cold staining method. Carbo] In liquid media, the bacilli grow as surface pellicle
fuchsin used in cold staining has higher concentration due to hydrophobic properties of their cell wall. Diffuse
of phenol which differentiation it from carbol fuchsin
used in ZN staining.
M. bovis appears straighter, stouter and shorter with
uniform staining (Table 42.2) .
B. Culture
M. tuberculosis is an obligate aerobe whereas
M. bovis is microaerophilic on primary isolation,
becoming aerobic on subculture. The bacilli grow slowly
(generation time 14-15 hours) and colonies appear only
in about two weeks and sometimes it may take up to 6---8
weeks. Optimum temperature for growth is 37°C (range
3~0°C). Optimum pH is 6.4 to 7.0. Tubercle bacilli
can grow on a wide range of enriched culture media
but Lowenstein- Jensen (LJ) medium is most commonly Fig. 42.2 LJ media without growth and with growth
used. This medium consists of beaten eggs, asparagine,
Table 42.2 Differentiating Features of M . tuberculosis and M. bovis
Test M. tuberculosis M. bovis
Morphology Long, slender and usually curved Short, stout and straight
Staining Barred or beaded appearance Uniform staining
Growth on LJ medium Eugonic growth Dysgonic growth
Presence of glycerol in medium Enhances the growth Inhibits the growth
Colony characters Dry, rough, tough, raised and wrinkled, creamy Moist, smooth, flat, white and friable
white or buff coloured, difficult to emulsify
Biochemical reactions
Niacin test +
Nitrate reduction +
Animal pathogenicity test
In guinea pig + (progressive and fatal disease) + (Disease similar to that in M. tuberculosis)
In rabbit - or mild lesion + (generalised lesion)

Ch 42: Mycobacterium Tuberculosis _ _ _ _ _ _ __ __ _ _ _ _ _ __ __ _ _ _ _ __ _ 333
uniform growth can be obtained by addition of a detergent

Tween 80 (polyoxyethylene sorbitan mono-oleate) in
Dubo's medium. Tween 80 wets the surface and permits
them to grow diffusely. Virulent strains tend to grow as
serpentine cords in the liquid media, while avirulent strains
grow in a more dispersed fashion. The cord factor by
itself is not responsible for virulence. It is also present
in some non-pathogenic species of mycobacteria. The
cord factor consists of two mycolic acids linked to a
molecule of trehalose.
Liquid media are generally used for sensitivity testing,
preparation of antigens and vaccines. In automated culture
methods, liquid media are used for growing mycobacteria.
Various culture media used for mycobacterial isolation
include Lowenstein-Jensen (LJ) medium, Petragnani,
Dorset egg medium (all the three are egg based) and
Middle brook 7H10, Middle brook 7Hll (agar based
media). Dubas medium and Middle brook 7H9 are two
commonly used liquid media. 2. Arylsulphatase Test
Arylsulphatase is an enzyme formed by certain atypical
C. Resistance mycobacteria. The organisms are grown in a medium
Mycobacteria are killed at 60°C in 15- 20 minutes. They containing 0.001 Mtripotassium phenolphthalein disul-
are sensitive to ultraviolet rays and sunlight. Bacilli in phate. If arylsulphatase enzyme is produced it liberates
cultures may be killed by direct sunlight exposure for two free phenolphthalein from tripotassium phenolphthalein
hours, but bacilli present in sputum may remain alive for disulphate. This can be detected by adding 2N NaOH
20- 30 hours. They are relatively resistant to chemical dropwise to the culture. A pink colour develops in a
disinfectants and can survive exposure to 5% phenol, positive reaction.
15% sulphuric acid, 5% oxalic acid and 4% sodium
hydroxide. The bacilli are destroyed by tincture of iodine 3. Neutral Red Test
in five minutes and by 80% ethanol in 2- 10 minutes. 80% Virulent strains of tubercle bacilli can bind neutral red
ethanol has been recommended as disinfectant for skin in alkaline buffer solution, whereas avirulent strains
and rubber gloves. are unable to do so. Positive tests are obtained with
Bacilli may remain viable in droplet nuclei for 8- 10 M. tuberculosis, M. bovis, M. avium and M. ulcerans.
days. Cultures remain viable for 6-8 months at room
temperature. 4. Catalase-Peroxidase Tests
Most atypical mycobacteria are strongly catalase positive
D. Biochemical Reactions and peroxidase negative. In contrast, the tubercle bacilli
Mycobacterial species can be identified by several (M. tuberculosis and M . bovis) are peroxidase positive
biochemical tests. Some of the important biochemical and weakly catalase positive. Tubercle bacilli lose catalase
tests are discussed below: and peroxidase activity when they develop resistance to
INH. Catalase negative tubercle bacilli are avirulent for
1. Niacin Test guinea pigs.
Niacin is produced as a metabolic by-product by all A mixture of equal volumes of 30% H 20 2 and 0.2%
mycobacteria, but most species possess an enzyme catechol in distilled water is added to a 5 ml test culture
that converts free niacin to niacin ribonucleotide. and left for few minutes. Catalase production is indicated
M. tuberculosis, lacks this enzyme and accumulates niacin by effervescence while browning indicates peroxidase
in the culture medium. When 10% cyanogen bromide and activity.
4% aniline in 96% ethanol are added to a suspension of
bacterial culture, a canary yellow colour shows a positive 5. Amidase Tests
reaction (Fig. 42.3). The human tubercle bacilli give a Atypical mycobacteria can be differentiated by their
positive test, while the bovine type is negative. Positive ability to split amides. The commonly used amides
niacin test also occurs in M simiae and M. chelonei. include acetamide, benzamide, carbarnide, nicotinamide

334 _ __ __ _ __ _ _ _ _ _ _ _ _ __ _ _ _ _ __ _ _ __ Unit Ill: Systemic Bacteriology
and pyrazinamide. A 0.00165 M solution of amide is which modifies the optical characteristics of the test
incubated with the bacillary suspension at 37°C and to solution from a yellow to pink. A pink colour indicates
this is added 0.1 ml of MnSO 4 . 4H 2O, 1.0 ml of phenol hydrolysis of Tween 80. M . kansasii and M. gordonae are
solution and 0.5 ml of hypochlorite solution. The tubes positive, while M. bovis, M. africanum, M. avium complex
are placed in boiling waterbath for 20 minutes. A blue and M. scrofulaceum are negative. M . tuberculosis shows
colour indicates a positive test. M tuberculosis produces variable results.
nicotinamidase and pyrazinamidase, therefore, splits
nicotinamide and pyrazinamide. E. Antigenic Structure
Mycobacterial antigens are mainly of two types, cell wall
6. Nitrate Reduction Test (insoluble) and cytoplasmic (soluble) antigens.
This test depends on reduction of nitrate to mtnte
by an enzyme nitroreductase. This test is positive 1. Cell Wall Antigens
with M. tuberculosis and negative with M. bovis. The cell wall consists of lipids, proteins and polysaccharides.
Other mycobacteria, which may give positive test, are The lipid content accounts for 60% of the cell wall weight.
M. kansasii, M. fortuitum and M. chelonei. Lipids of the cell wall particularly the mycolic acid fraction
The test organism is suspended in a buffer solution are responsible for the acid-fastness of bacteria and the
containing nitrate and incubated at 37°C for 2 hours. cellular tissue reactions of the body. The cell wall is made
Then sulphanilamide and n-naphthyl-ethylene diamine up of four distinct layers (Fig. 42.5) .
dihydrochloride solutions are added. Development of pink (i) Peptidoglycan (murein) layer, the innermost layer
or red colour indicates positive reaction (Fig. 42.4) . which maintains the shape and rigidity of the cell.
(ii) Arabinogalactan layer lies external to the peptidoglycan
7. Susceptibility to Pyrazinamide layer.
M. tuberculosis is sensitive to 50 µg/ml pyrazinamide but (iii) Mycolic acid layer is the principal constituent of cell
other mycobacteria including M . bovis are resistant. wall. It is a dense band on long chain a - alkyl and
~-hydroxy fatty acids attached by ester bonds to
8. Susceptibility to Thiophen-2-Carboxylic Acid the terminal arabinose units of arabinogalactan.
Hydrazide (TCH) (iv) Mycosides (peptidoglycolipids or phenolic glycolipids)
M. bovis is usually susceptible to 10 µg/ml of TCH. form the outermost layer of the cell wall. The
M tuberculosis is usually not inhibited by this chemical, agglutinogen antigens have been identified as the
however, South Indian strains are susceptible. sugar moieties on mycosides.
The cell wall antigens include arabinomanan,
9. Tween 80 Hydrolysis arabinogalactan and lipoarabinomanan.
Certain mycobacteria possess an enzyme lipase that splits
Tween 80 into oleic acid and polyoxyethylated sorbitol 2. Cytoplasmic Antigens
Cytoplasmic antigens are protein antigens employed to
type the mycobacteria. These include antigen 5, antigen
6, antigen 14, antigen 19, antigen 32, antigen 38 and
antigen 60. All are protein in nature except antigen 60
which is a lipopolysaccharide protein complex.
Fig. 42.4 Nitrate reduction test Fig. 42.5 Cell wall of Mycobacterum

Ch 42: Mycobacterium Tuberculosis _ _ _ _ _ __ _ _ _ _ _ _ __ _ _ _ _ _ _ _ _ _ _ _ 335
F. Mycobacteriophages 2. Secondary (Post-primary) Tuberculosis

Some tubercle bacilli are infected with temperate phages. It is caused by reactivation of the primary lesion
Many mycobacteria infected with bacteriophage are not (endogenous) or by exogenous reinfection. Reactivation
truly lysogenic, the phage genome appears as independent tuberculosis is likely to occur in immunocompromised
plasmid. There is no integration of phage genome into individuals. Granulomas of secondary tuberculosis most
the host genome. This is called pseudolysogeny. Tubercle often occur in the apex of lungs. The necrotic element of
bacilli have been classified into four phage types- A, B, the reaction causes tissue destruction and the formation
C and I. Type I is intermediate between A and B. Type of large area of caseation termed tuberculomas. Presence
A is worldwide in distribution and is the commonest type. of caseous necrosis and cavities are two special features
Infection due to type I is more frequent in India and of secondary tuberculosis. Cavities may rupture into
neighbouring countries. Type B mainly occurs in Europe blood vessels, spreading mycobacteria throughout the
and North America. Type C is rarely seen. Phage 33 D body, and break into airways, releasing the organisms
(isolated from an environmental mycobacterium) can lyse in aerosols and sputum (open tuberculosis) .
all variants of M. tuberculosis, but not BCG.
H. Immunity and Hypersensitivity
G. Pathogenesis Two immunological responses develop simultaneously in
The infection is commonly acquired by inhalation naturally infected host, one is antitubercular immunity
of infected droplets coughed or sneezed into the air and other being tuberculin hypersensitivity. Both responses
by a patient with pulmonary tuberculosis. In bovine are mediated by T-lymphocytes sensitised to the bacterial
tuberculosis, infected cows develop lesions in the udder antigen. Hurnoral immunity appears to be of no relevance
and bacilli are excreted in the milk which can then infect in tuberculosis.
people who drink it raw. In developed countries, pasteu- Immunity (resistant to infection) and hypersensitivity
risation of milk has virtually eradicated this organism. (allergy) are two different manifestation of the same
Tubercle bacilli are engulfed by macrophages but mechanism in tuberculosis.
they survive and multiply in macrophages. These lyse 1. In non-immune host, the bacilli are readily
the host cell, infect other macrophages and sometimes phagocytosed and multiply inside the mononuclear
disseminate to other parts of lung and elsewhere in cells. This intracellular parasitism is associated with
the body. The cell mediated immunity (CMI) plays a the development of delayed hypersensitivity and
major role to interact with these macrophages whereas of activated macrophages which have increased
humoral immunity appears to be irrelevant, CD4+ helper ability to destroy the ingested bacilli.
T cells secrete interferon gamma, interleukin 2, tumour 2. After first infection the host acquires some
necrosis factor a and others exerting different biological resistance against reinfection. In the immune
effects. It may result in protective immunity or delayed host the sensitised T-lymphocytes (developed
type hypersensitivity (DTH) reaction. Th-1 dependent during primary infection) proliferate and release
cytokines activate macrophages to kill intracellular lymphokines which make the macrophages
mycobacteria and thus result in protective immunity. Th-2 bactericidal.
cytokines induce delayed type hypersensitivity (DTH), Delayed hypersensitivity can be induced by
tissue destruction and progressive disease. infection with virulent or avirulent tubercle bacilli. For
Human tuberculosis is divisible into primary and demonstrating delayed hypersensitivity, tuberculoprotein
secondary forms. (tuberculin) injection is commonly employed.
1. Primary Tuberculosis I. Koch's Phenomenon

Inhaled tubercle bacilli are engulfed by alveolar The response of a tuberculous animal to reinfection was
macrophages in which they replicate to form a lesion best explained by Robert Koch. When a healthy guinea pig
called Ghan focus. It is frequently found in the lower is inoculated subcutaneously with virulent tubercle bacilli,
lobe or lower part of the upper lobe. Some bacilli are the puncture site heals quickly and there is no immediate
transported to the hilar lymph nodes. The Ghon focus visible reaction. After 10--14 days, a nodule appears at the
together with the enlarged hilar lymph nodes is called site of injection which ulcerates and the ulcer persists till
the primary complex. In case of M bovis which enters the animal dies of progressive tuberculosis. The regional
through mouth, the primary complexes involve the tonsil lymph nodes are enlarged and caseous. If on the other
and cervical lymph nodes or the intestine, often the hand, virulent tubercle bacilli are injected in a guinea pig,
ileocaecal region, and the mesenteric lymph nodes. which had received a prior injection of tubercle bacilli

4-6 weeks earlier, an indurated lesion appears at the site 4. Result

of injection in a day or two which undergoes necrosis In the Mantoux test the site of injection is examined
in another day or so to form a shallow ulcer. This ulcer after 48- 72 hours and interpreted as follows:
heals rapidly without involvement of the regional lymph
nodes or tissues. This is called Koch 's phenomenon.
Koch's phenomenon is a combination of (i) Positive test
hypersensitivity and immunity and has got three In a positive reaction, there is induration (local oedema)
components: of 10 mm diameter or more surrounded by erythema
1. A local reaction of induration and necrosis at the site of inoculation. Positive test only confirms
2. A focal response in which there occurs acute past infection with tubercle bacilli but does not indicate
congestion and even haemorrhage around the presence of active stage of the disease. The test is helpful in
tuberculous foci in tissues. children under five years for indication of active infection.
3. A systemic response of fever that may sometimes The test becomes positive 4-6 weeks after infection or
be fatal. BCG vaccination.
J. Tuberculin Skin Test (ii) False negative

1. Principle The test may become negative in following conditions:
Tuberculin skin test (TST) 1s delayed or type IV (a) miliary tuberculosis
hypersensitivity reaction. (b) when anergy develops following overwhelming
infection of measles, Hodgkin's disease, sarcoidosis,
2. Reagents lepromatous leprosy, malnutrition, administration of
(i) Old tuberculin (OT) immunosuppressive agents and corticosteroids.
It was originally described by Robert Koch. It is a crude
preparation of 6-8 week culture filtrate of tubercle bacilli, (iii) False positive
concentrated by evaporation on a heated water bath. This This is observed in presence of related mycobacteria such
crude product may lead to serious complications in some as atypical mycobacteria.
patients, it is now rarely used.
5. Uses
(ii) Purified protein derivative (PPD) (i) To measure prevalence of infection in a community.
A purified preparation of the active tuberculoprotein was (ii) To diagnose active infection in young children.
prepared by Seibert (1941). It was prepared by growing (iii) It is used as an indicator of successful BCG
M tuberculosis in a semisynthetic medium. It is called vaccination.
purified protein derivative (PPD). The dosage of PPD is In recent years in vitro interferon-y release assays
expressed in tuberculin unit (TU). One TU is equal to (IGRA) have been introduced as a sensitive and more
0.01 ml of OT or 0.00002 mg of PPD-S. Another PPD specific alternative to tuberculin skin test (TST). This test
is RT-23 with tween 80. In India PPD RT-23 of strength is used in blood specimen which contains T-lymphocytes.
1 TU and 2 TU are available. 1 TU of PPD RT-23 is It uses ELISA to measure interferon-y (IFN-y) production
equivalent of 5 TU of PPD-S. by sensitised T-lymphocytes which are stimulated by
M . tuberculosis antigens. If an individual was previously
3. Method infected with M tuberculosis, exposure of sensitised
(i) Mantoux test T-lymphocytes to M. tuberculosis specific antigens results
0.1 ml of PPD containing 5 IU of PPD-S is injected in IFN-y production.
intradermally into flexor aspect of forearm. A PPD-S
dose of 1 TU is used when extreme hypersensitivity is K. Laboratory Diagnosis
suspected. In India 1 TU of PPD RT-23 is recommended Bacteriological diagnosis can be established by microscopy,
and not PPD-S. culture examination or by animal inoculation test.
(ii) Heaf test

This is done with a multiple puncture apparatus that 1. Specimen
pricks the skin. A drop of undiluted PPD is spread on the Specimen collection depends on the site of involvement.
area of skin. The multiple puncture apparatus is pressed Tuberculosis may involve lungs (pulmonary) or sites other
against this area of skin. than lungs (extrapulmonary).

Ch 42: Mycobacterium Tubercu losis _ _ _ _ _ _ __ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ 337
(i) Pulmonary tuberculosis urine specimen, care must be taken to exclude commensal
Sputum is the most common specimen. It is collected in a M. smegmatis bacillus which is only acid-fast and not
clean wide-mouthed container. A morning specimen may alcohol-fast, whereas M. tuberculosis is both acid and
be collected on three consecutive days. If sputum is scanty, alcohol fast.
a 24 hour specimen may be collected. When sputum is If a large number of smears are to be examined,
not available, laryngeal swab or bronchial washings are flu orescent microscopy is more convenient. Smears are
collected. In children, gastric washings may be examined stained with fluorescent dyes such as auramine 'O' or
as they tend to swallow sputum. auramine rhodamine and examined under ultraviolet
light. The bacilli appear as bright bacilli against dark
(ii) Meningitis background. WHO has recommended the use of LED
Cerebrospinal fluid (CSF) from tuberculous meningitis (light emmiting diode) fluorescence microscopy over
(TBM) often forms a spider web clot on standing, conventional fluorescent microscopy. Compared to
examination of which may be more useful than of conventional mercury vapour fluorescent microscopes,
fluid. LED microscopes are less expensive and are able to run
on batteries. LED microscopes can perform equally well
(iii) Renal tuberculosis without a dark room. It has more sensitivity than ZN
Three consecutive days morning samples of urine are staining.
examined. Demonstration of AFB by microscopy provides
only presumptive evidence of tuberculous infection,
(iv) Bone and joints tuberculosis as saprophytic mycobacteria may present a similar
Aspirated fluid appearance.
(v) Tissue 3. Concentration of Specimens

Biopsy of tissue. Concentration of a specimen is done to achieve:
(a) homogenisation of the specimen
2. Direct Microscopy (b) decontamination i.e. to kill other bacteria present in
Smear is made from the specimen on a new glass slide and the specimen.
stained by the Ziehl-Neelsen technique (refer to chapter (c) concentration i.e. to concentrate the bacilli in a small
2 fo r method of Ziehl-Neelsen staining). It is examined volume without inactivation.
under oil immersion lens. The acid-fast bacilli (AFB) Such concentrate is used for culture and animal
appear as bright red bacilli against a blue background. To inoculation tests besides smear preparation.
detect the bacilli microscopically, there should be at least Several concentration methods are in use:
10,000 bacilli per ml of sputum. A negative report should
not be given till at least 300 fields have been examined. (i) Petroff's method
Grading of smears is done according to number of bacilli It is a simple and widely used technique. Sputum is
seen: (Table 42.3) . mixed with equal volume of 4% sodium hydroxide and
is incubated at 37°C with frequent shaking for about 30
Table 42.3 Grading of Ziehl-Neelsen (ZN) Smear minutes. It is then centrifuged at 3,000 rpm for 30 minutes.
o. of AFB seen in oil Report The supernatant fluid is poured off and the deposit is
immersion field neutralised by adding 8% hydrochloric acid in presence
0 I 300 fields AFB not seen of a drop of phenol red indicator. The deposit is used
1-2 /300 fields Doubtful, repeat the smear for smear, culture and animal inoculation.
1-9 / l 00 fields l+
(ii) Other methods
1-9 /10 fields 2+ Dilute acids (5% oxalic acid, 3% hydrochloric acid or
1-9/field 3+ 6% sulphuric acid), mucolytic agents such as N-acetyl-
10 or more /field 4+ L-cysteine with sodium hydroxide and pancreatin are
used for concentration of specimens. In urine and CSF
Purulent part of the sputum is used for making a specimens centrifugation is done to concentrate the
smear. Microscopy is also performed on the smear which specimen. Centrifuged deposit is used for smear and
is prepared after concentration of specimen. In case of culture examination.

4. Culture (Fig. 42.6) M. bovis (i.e. growth index decreases) but not that of
Culture is a very sensitive method for detection of tubercle M. tuberculosis. This significant method has now been
bacilli. It may detect as few as 10 to 100 bacilli per ml. discontinued due to radioactivity.
The concentrated material is inoculated on two bottles of Mycobacterial growth indicator tube (MGIT) is
Lowenstein-Jensen medium. In case of gastric washings, another rapid method for detection of mycobacterial
alkali is added to neutralise the acid present in the growth. It is a non-radiometric, automated method. It
specimen and then inoculated on culture medium. Urine consists of tubes containing liquid culture media, and a
is centrifuged and then from deposit, culture medium fluorescent compound is embedded on the bottom of the
is inoculated. In case of CSF, it is centrifuged and the tube. The fluorescent compound is sensitive to dissolved
deposit is used for culture and smear examination. oxygen in the liquid medium. Thus the dissolved oxygen in
The culture media are incubated at 37°C in the dark the uninoculated medium quenches any fluorescence from
and in the light. Cultures are examined first after 4 days the compound. When mycobacteria grow they deplete the
(for rapid growing mycobacteria, fungi and contaminant dissolved oxygen in the liquid medium and allows the
bacteria) and thereafter weekly till 8 weeks. The tubercle compound to fluoresce brightly which can be detected by
bacilli usually grow in 2 to 8 weeks. Longer incubation observing the tube under ultraviolet light (wood's lamp) .
is necessary for strains originating from patients treated The result are obtained in 8 to 14 days.
with antitubercular drugs. BacT/Alert 3D system is a non-radiometric, rapid
In a positive culture, characteristic colonies appear on and fully automated. It uses liquid medium vials in which
culture medium. Smear is prepared from isolated colony specimen is inoculated. If mycobacteria are present in the
and stained with Ziehl-Neelsen technique. When acid-fast specimen, carbon dioxide (COi) is released by actively
bacillus (AFB) is slow growing, non-pigmented and niacin proliferating mycobacteria. The elevated level of CO 2
positive, it is regarded as M . tuberculosis. Confirmation lowers the pH in the medium which produces a colour
is done by biochemical reactions. change in sensor present in the liquid medium vial. This
In radiometric method such as BACTEC, the growth colour change is detected by the instrument.
may be detected in about a week by using 14C labelled
substrates. Culture media contains 14C-labelled palmitic 5. Animal Inoculation
acid. Mycobacteria metabolise the 14C-labelled substrates 0.5 ml of the concentrated specimen is inoculated
and release radioactively labelled 14CO 2 • The instrument intamuscularly into the thigh of two tuberculin negative
measures 14CO 2 and reports in terms of 'growth index'. healthy guinea pigs. Inoculation by subcutaneous route is
A growth index of 10 is considered as positive. This avoided as it causes local ulcer which may be infectious.
method can also differentiate between M. tuberculosis and The animals are weighed prior to inoculation and
M bovis. As M . bovis is susceptible to TCH, incorpora- thereafter at weekly interval. They are tuberculin tested
tion of TCH in the medium inhibits the growth of after 3-4 weeks. There is progressive loss of weight and
Growth on LJ medium
Slow growing Rapid growing (within 7 days)

I
Niacin test
Growth on MacConkey;
Aryl sulphatase test
+ +
Type of growth M. tuberculosis M. fortuitum complex Tellurite reduction
Scanty, smooth, flat Pigment production +

colonies M. smegmatis M.phlei
I
Rabbit In light In dark No pigment
pathogenicity I
Group I
I
Group II
I
Group Ill
+ Photo- Scoto- Nonphoto-
B.C.G. M. bovis chromogen chromogen chromogen
Fig. 42.6 Identification of Mycobacteria


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1.Find books by subject names.

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Unit I: General Microbiology 1-84

Unit II: Immunology 85-170

Unit III: Systemic Bacteriology 171-408

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Unit IV: Virology 409-540

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Unit VI: Clinical Microbiology _ _ _ _ _ _ _ _ _ _ _ _ _ _ __ 567-646

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1. Historical Introduction ................................................................................... 3

2. Microscopy and Morphology of Bacteria ...................................................... 9

3. Growth, Nutrition and Metabolism of Bacteria .......................................... 22

4. Sterilisation and Disinfection ..................................................................... 28

5. Culture Media .. .......................................................................................... 40

6. Culture Methods ........................................................................................ 46

7. Identification of Bacteria ............................................................................. 51

8. Bacterial Taxonomy .................................................................................... 59

9. Bacterial Genetics ...................................................................................... 61

10. Microbial Pathogenicity ............................................................................... 77

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I. INTRODUCTION of bacteria and communicated his findings to the Royal

Important Contributions of Louis Pasteur B. Joseph Lister (1827-1912)

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2. Introduced methods of obtaining bacteria in pu"(e Important Discoveries by other Scientists

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YOU MUST KNOW

MU TIPLE CHOICE QUESTIONS (MCQs)

MICROSCOPY AND MORPHOLOGY OF BACTERIA

I. INTRODUCTION Table 2.1 Differences between Prokaryotic and

Nuclear membrane Absent Present

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1 micron (µ) or micrometre (µm) = 1/1000 B. Phase Contrast Microscope

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focussed by circular electromagnets (magnetic condenser), Common Staining Techniques

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Gram positive 3. The stained smear is decolourised with 20% sulphuric

Fig. 2.1 Shapes and different arra ngements of bacteria

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bacteriophages. It carries bacterial antigens that are

Gram positive Gram negative

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(ii) Serological methods different. Specific flagellar antibodies are produced in

Parts and Composition

Fig. 2.6 Structure of bacterial flagellum Fig. 2.7 Arrangement of flagella

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Sporangium with completely enclosed forespore

Fig. 2.8 Sporulation

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20% sulphuric acid as used in conventional method)

YOU MUST KNOW

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2. Draw a labelled diagram of bacterial cell.

...

...

...