Ug Practical Manual 2024-1

MICROBIOLOGY PRACTICAL MANUAL
GOVT. DHARMAPURI MEDICAL COLLEGE
DHARMAPURI
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INTRODUCTION TO THE USE OF MICROSCOPE
Microbiologists use microscopes to enable them to study microbial cells as they are extremely
small and cannot be studied in detail without technological assistance. In our lab, we will be
working with a monocular compound microscope, which is used to view slides. The compound
microscope we will use is said to be parfocal, which means that when you switch from the low
power to the next-power lens the object will be kept in focus.
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PARTS OF THE MICROSCOPE AND THEIR FUNCTIONS
1. Eyepiece- Contains a magnifying lens that focuses the image from the objective into your eye.
2. Coarse Adjustment -For focusing under low magnification
3. Fine Adjustment-For focusing under high magnification or low
4. Low Power Objective-For large specimens or overview or to select a field in case of direct
clinical specimen smear.
5. High Power Objective-For detailed viewing or small specimens
6. Oil immersion objective- For a magnified view of the objects
7. Specimen on glass slide-What you want to look at
8. Stage-Supports specimen in correct location to lens
9. Condenser-Focuses the light on specimen
10. Diaphragm (iris or disc)-Regulates amount of light and contrast
11. Light Source-Illuminates the specimen for viewing.
HOW TO COMPUTE MAGNIFICATION
 Multiply the power of the ocular lens (usually ocular lenses are 10x) times the
power of the particular objective lens you happen to be using at that time.
 The power of each lens is printed on its outer surface: a number such as 10 or 40
followed by an X, meaning the magnification of 10 x 10 = 100, or 40 x 10 = 400.
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HOW TO STORE A COMPOUND MICROSCOPE
When you store a microscope make certain that the low-power lens is in position above
the opening at the center of the stage, the electrical cord is securely wrapped around the
base of the microscope (not too tight – the cord will fray), the microscope arm is upright
and not inclined and the body tube is adjusted to its lowest position.
HOW TO CARRY A COMPOUND MICROSCOPE
When you carry a microscope, make certain that you keep one hand under the base and the
other on the arm of the microscope and hold the microscope close to your body.
ADJUSTMENTS OF THE MICROSCOPE
10X OBJECTIVES:
When the specimens are examined under low power objective, the amount of light entering
the microscopic field must be reduced.
Use concave mirror to direct the light from the light source.
Lower the condenser
If needed, diaphragm may be closed.
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40X OBJECTIVE:
When the specimens are examined under high power, the amount of light entering the
microscopic field must be increased.
Keep the concave mirror position as such.
Slightly raise the condenser.
Open the diaphragm little more.
100X OBJECTIVE
When the specimens are examined under oil immersion objective power, the amount of
light entering the microscopic field must be very high. (OPR).
Use a plain mirror to direct the light.
Raise the condenser to the maximum.
Open the diaphragm completely.
FOCUSING A MICROSCOPE
1. Place the slide under the stage clips on the stage.
2. Put on the lowest power objective lens. (10x)

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3. Do the adjustments for x10 objective.
4. First, focus with the coarse focus adjustment.
5. Secondly, focus with the fine focus adjustment.
6. Move to the high power objective lens. (40x)
7. Do the adjustments for x40 objective.
8. Focus with the fine focus adjustment alone
9. Get the object to be studied focused under high power.
10. Turn the high power lens up to the right away from the surface of the slide but do not allow
the next objective lens to go into position over the slide.
11. Use the space that was created between the lenses over the slide to add a drop of immersion
oil (cedar wood oil) to the surface of the slide, making certain that the oil is placed over the
area to be examined.
12. Now bring the oil immersion objective to its position over the object. Oil should be intact
between the object and objective lens.
13. Do the adjustments required for oil immersion objective.
14. Observe the image and record your findings.
15. When you are finished with your immersion oil examination remember to clean the
objective oil-immersion lens with alcohol and lens paper.
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CARE OF THE MICROSCOPE
a) Keep the microscope at a uniform temperature. Avoid exposure to sun-light or heat source.
Surface must be free of any vibrations caused by other instruments.
b) While moving, lift it by holding the arm and support the foot/ base but not by any movable
part and hold upright.
c) Keep the microscope clean. Before and after use clean the microscope with gauze piece/
lens paper.
d) After use keep the microscope in the neutral position i.e. Lowest objective in focus, and
condenser lowered
FREQUENTLY ASKED QUESTIONS IN MICROSCOPY
1. What is a microscope?
A microscope is an instrument used to see objects that are too small for the naked eye.
2. What is the type of microscope you are using in the lab?.
compound microscope
3. What are the various objective lenses seen in your microscope?

 10x -Low power objective
 45x -High power objective
 100x -Oil immersion objective.
4. What is the use of 10x objective?
To select the field.
5. What is the use of 45x objective?
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To grade pus cells, to observe wet mount preparations
6. What is the use of oil immersion objective?

To view stained preparations in a higher magnification
7. What are the microscopic adjustments for low power objective?

• Close the diaphragm.
• Concave mirror as light source.
• Lower the condenser.
8. What are the microscopic adjustments for high power objective?

• Partially open the diaphragm.
• Concave mirror as light source.
• Slightly raise the condenser.
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9. What are the microscopic adjustments for oil immersion?
(OPR)
• Open the diaphragm.
• Plane mirror .
• Raise the condenser.
10. How will you clean new glass slide?

• Wiping with the cotton ball.
• Showing the flame of Bunsen burner both sides.
11. What are the advantages of smear fixation?

• Fixing the bacterial cellsto prevent it from washing by heat
fixation/methanol fixation .
• Killing the pathogens for safety handling.
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12. Why the cedar wood oil is used in oil immersion?
The cedar wood oil has the same refractive index as that of the glass slide.
To focus the light rays within the Objective lens.
13. What do you understand by “Resolution power” of a microscope?
The ability or the power of microscope to clearly delineate two closely situated objects is
called Resolving power, which is inversely proportional to the wave length of the light
used.
GRAM STAINING
AIM:
To perform Gram staining and to differentiate whether the given smear contains Gram positive or
Gram negative bacteria.
PRINCIPLE:
Gram positive cells which have a thicker cell wall retain the dye-Iodine complex , In Gram
negative cells the dye-Iodine complex will come out due to thinner cell wall and take up the
counterstain.
• Cell wall theory: Cell wall of Gram positive bacteria (peptidoglycan layer) are 40 times thicker
than those of Gram negative cells, hence they retain the dye-iodine complex.
• Lipid Content Theory: The high lipid content of Gram negative bacteria is washed out by the
organic solvents like acetone after decolourisation and permits the dye iodine complex to come out
and makes them permeable to the secondary dye
• Cytoplasmic pH Theory: Gram positive cells have more acidic protoplasm than the Gram
negative and hence retain the basic primary dye more strongly than the Gram negative one.
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PROCEDURE:
1. A smear is made, air-dried/ heat-fixed in a grease free slide.
2.Primary staining: with pararosaniline dye such as crystal violet / methylviolet / gentian violet.
Leave it for 1minute.
3.Mordant - Gram's iodine which fixes the primary stain is used and Leave it for one minute.
4. Decolourisation- with organic solvent such as alcohol (1min.) / acetone (2sec.)
5.Counterstaining - with Dilute carbol fuchsin / Safranin / neutral red. Leave it for one min.
6. The slide is air dried and observed it under oil immersion.
7. The Gram stain differentiates bacteria into two groups. Gram positive bacteria are those that
resist decolourisation and retain the primary stain appearing violet. Gram negative bacteria are
decolourised by the organic solvents and take up the counterstain, appearing pink.
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GRAM STAINING - GPC
AIM:
To demonstrate Gram’s reaction of the organism in the given smear by Gram staining.
OBSERVATION:
GRAM’S REACTION: Positive
COLOUR : Violet
SHAPE : Spherical
ARRANGEMENT : Clusters
INFERENCE:
The given smear contains Gram positive cocci arranged in clusters.
EXAMPLES: Staphylococcus aureus, Staph epidermidis.
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GRAM STAINING - GNB
AIM:
OBSERVATION:
GRAM’S REACTION : Negative
COLOUR : Pink
SHAPE : Rod
ARRANGEMENT : Discrete
INFERENCE:
The given smear contains Gram negative bacill with discrete arrangement.
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EXAMPLES: Escherichia coli, Pseudomonas aeruginosa, Salmonella typhi, Klebsiella
pneumoniae, Proteus mirabilis
GRAM STAINING - MIXTURE
AIM:
OBSERVATION:
I II
GRAM’S REACTION : Positive GRAM’S REACTION : Negative
COLOUR : Violet COLOUR : Pink
SHAPE : Spherical SHAPE : Rod
ARRANGEMENT : Clusters ARRANGEMENT : Discrete
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INFERENCE:
The given smear contains both Gram positive cocci arranged in clusters and Gram negative
bacilli in discrete pattern.
Eg: GPC- Staphylococcus aureus, GNB- E.coli
FREQUENTLY ASKED QUESTIONS IN GRAM STAINING
1. Who invented Gram stain?

Hans Christian Gram in 1884
2. Give example for differential stain.
Gram stain, acid fast stain, metachromatic granule staining.
3. State why Gram stain is said to be a differential stain.
Gram’s stain is said to be a differential stain because it differentiates bacteria as Gram positive
bacteria (appears violet) and Gram negative bacteria (appears pink).
4. Why bacteria are classified as Gram positive or Gram Negative?
Based on the cell wall constituents and differences in the thickening of the peptidoglycan layer, those
bacteria appearing violet are called Gram positive (retaining the primary dye) and those bacteria
appearing pink are called Gram negative (take up the counter stain).
5. What are the various steps /procedure in Gram staining?
 Primary staining
 Addition of Mordant
 Decolorization,
 Counterstaining
6. What are the various primary stains that can be used in Gram staining?
Crystal violet, Methyl violet and Gentian violet

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7. What is the function of Gram’s iodine?
It acts as a mordant.
8. What do you mean by mordant?
Fixing agent
9. What is the role of Grams iodine in Gram staining?
It helps to fix the stain in the bacterial cell and forms the dye-iodine complex.
10. What are the various decolourisers that are used in Gram staining?
Acetone(2-3sec.), Ethyl Alcohol(20-30 sec.) Acetone -alcohol (10sec.)
11. What are the various counter stains that are used in Gram staining?
Dilute carbolfuchsin, Safranine, Neutral red
12. What is the important step in Gram staining?
Decolourisation.
13. What happens if you over decolourise?
Over-decolourization can result in Gram positive bacteria appearing Gram negative.
14. What happens if you under decolourise?
Under-decolourization can result in Gram negative bacteria appearing Gram positive.
15. Which part of the bacteria actually gets stained?

It is the cytoplasm (especially the nucleic acid) that gets stained and not the cell wall. Presence of
an intact cell wall is important for retaining Gram positivity. Cell wall deficient forms such as
Mycoplasma and L forms are Gram negative.
16. Explain the mechanism of Gram staining.
Gram positive – acidic protoplasm - retain basic primary dye. Peptidoglycan of Gram-positive
bacteria thick – retain the dye iodine complex.
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High lipid content of Gram-negative bacteria washed out during decolourisation and takes up the
counter stain appearing pink.
17. Which are the bacteria or bacterial component that can’t be stained by Gram stain?
• Extremely slender bacteria such as Treponema.

• Cells containing waxy substances impermeable to stain such as Mycobacterium.
• Minute intracellular bacteria such as Chlamydia and Rickettsia.
• Cell organelles such as capsule, spore, flagella etc.,
18. What are the differences between cell wall of Gram positive and Gram negative bacteria?
 Gram positives have a thick peptidoglycan layer in the cell wall when compared to
Gram negatives which possess a thinner peptidoglycan layer.
 Teichoic acid is present in Gram positive cellwall while it is absent in Gram
negative cell wall.
 Lipopolysaccharide is absent in Gram positive cell wall and is present in Gram
negative cell wall.
19. Give example for Gram positive cocci
Staphylococcus aureus, Streptococcus pyogenes, Streptococcus pneumoniae.
20. Name the Gram positive cocci arranged in clusters.
Staphylococcus aureus
21. Name the Gram positive cocci arranged in chains.
Streptococcus pyogenes
22. Name the Gram positive lanceolate shaped cocci arranged in pairs.
Streptococcus pneumonia
23. Give example for Gram positive cocci arranged in pairsat an angles to each other and
short chains.
Enterococcus specie
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24. Name few Gram positive bacilli.
Corynebacterium diphtheriae, Bacillus anthracis, Clostridium spp.
25. Name few Gram negative cocci.
Neisseria meningitidis, Neisseria gonorrhoeae.
26. Name few Gram negative bacilli.
Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Salmonella spp.,

Shigella spp., etc.,
27. Name a comma shaped Gram negative bacilli.
Vibrio cholera
28. Name few intracellular Gram negative cocci arranged in pairs.
Neisseria meningitidis
Neisseria gonorrhoeae
29. Name few anaerobic Gram positive cocci
Peptococci, Peptostreptococci.
30. Name anaerobic Gram negative cocci.
Veillonella.
31. Namean anaerobic Gram negative bacilli.
Bacteroides
32. How do spores appear in Gram staining?
The spores do not take any colour and appear unstained in Gram staining.
33. Name some Gram positive yeast cells.
Cryptococcus neoformans, Candida albicans.
34. Name the bacteria that are used as the positive and negative controls for Gram stain?
Positive control: Staphylococcus aureus

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Negative control: Escherichia coli
35. What are the conditions when Gram positive bacteria can appear Gram negative?
 When prolonged exposure to decolourizer.

 When cell wall gets damaged by exposure to lysozyme or cell wall acting antibiotics.
such as Penicillin and Cephalosporins
 Old cultures, where cell wall is weakened or action of autolytic enzymes
36. What are Gram variable bacteria?
Gram variable bacteria are those Gram positive bacteria that have lost their cell wall
integrity because of antibiotic treatment, old age or action of autolytic enzymes. These
changes allow methyl violet to come out of the cell wall during process of decolourising
resulting in some cells staining pink and others staining purple.
37. What are the applications of Gram staining?
1. To differentiate bacteria into Gram positive and Gram negative
2. To identify the morphology (cocci/bacilli) of bacteria
3. Selection of empirical antibiotics.

4. Selection of suitable culture media.
5. Rapid presumptive diagnosis of diseases such as bacterial meningitis.

6. Screening of quality of clinical specimens, such as sputum that should contain many pus
cellsand few epithelial cells.
7. For quantitative estimation of bacteria in urine specimens:
 Identifies urine specimens that contain bacteria greater than 10 5 cfu/ml of
urine.
 Presence of at least 1 organism/oil immersion field correlates with
significant bacteriuria (105 cfu/ml).
38. what are the modifications of Grams staining ?
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1. Jensen’s modification – Absolute alcohol is used as decolouriser and Neutral red as counter
stain.
2. Kopeloff and Beerman ‘s modification – Methyl violet is used as primary stainwhereas basic
fuchsin as counterstain
3. Preston and Morrell’s modification – Iodine –acetone is usedas decolouriser.
ACID FAST STAINING
AIM:
To demonstrate the acid fast bacilli in the given smear by Ziehl – Neelsen/Kinyoun’s method.
OBSERVATION:
 Acid - fast organisms:
COLOUR : Pink
SHAPE : long, thin, slender, slightly curved rod
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ARRANGEMENT : Scattered
INFERENCE:
 The given smear contains pink coloured, long, thin, slender and slightly curved acid fast
bacilli seen against the blue background.
 Eg:Mycobacterium tuberculosis.
PRINCIPLE:
Certain species of bacteria especially Mycobacteria and Nocardia, are not easily stained by
ordinary staining methods, but once stained have the ability to retain the primary dye (strong
carbolfuchsin) and resist decolorization by weak mineral acids such as H2SO4, HCl. These
organisms are called acid fast organisms and this property is termed acid fastness. Other bacteria
readily lose the stain upon decolorization and take up the color of the counterstain are called as
non-acid fast organisms.
MECHANISM OF ACID FAST STAINING:
Acid fast organisms are not easily stained because they are coated with lipids, fatty acids
and higher alcohols in their cell wall. Mycolic acid (waxy substance) present in the cell wall does
not allow the stain to penetrate easily inside these organisms. In Ziehl-Neelsen staining, Strong
carbolfuchsin (basic fuchsin+phenol) is used as the primary stain.Heat and phenol facilitate
penetration of the dye. Intermittent heating done, not allowing to boil and letting the primary stain
inside the cell and stain the bacilli, during cooling the waxy substance solidifies.
In Kinyoun’s cold method heating is not required; phenol concentration is increased in

carbolfuchsin and prolonging the duration of carbolfuchsin staining.
PROCEDURE:
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Smear should be prepaed from the thick purulent part of the sputum, dried, heat fixed and stained.
Ziehl-Neelsen technique
1. The given smear is flooded with filtered 1% strong carbol fuchsin and heated slowly for 5-
7min.until steam rises and should not be boiled.
2. The slide is then washed with water and decolourised with 20% sulphuric acid for 2 min. or
with 3% acid-alcohol
3. The washed slide is counter stained with0.1% Loeffler’s methylene blue, or 1% picric acidor
0.2% malachite green for 1 min.
4. Under oil immersion objective, acid fast bacilli appear as rods while the background is blue,
yellow or green depending on the counterstain used.
Kinyoun technique
1. The given smear is flooded with filtered 1% strong carbol fuchsin and wait for 8-10min.
2. The slide is then washed with water and decolourised with 20% sulphuric acid or with 3% acid-
alcohol for 2 min.
3. The washed slide is counter stained with 0.1% Loeffler’s methylene blue, or 1% picric acid or
0.2% malachite green for 1 min.
4. Under oil immersion objective, acid fast bacilli appear as pink rods while the background is
blue, yellow or green depending on the counterstain used.
Examination and Reporting (ZN Microscopy)
• Use the objective 100x – Oil immersion
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• A negative report should not be given till at least 300 fields have been examined.
• A positive report can be given only if two or more typical bacilli seen
• Count AFB in low positive smears for quantification. (Scanty &1+)
• Grade the smear according to RNTCP guidelines in India (Table-1).
Table-1
ZN staining grading (RNTCP)
ZN staining grading (RNTCP) Reporting Grading No. of

fields
>10 AFB/field Positive 3+ 20
1-10 AFB/field Positive 2+ 50
10-99 AFB/100 field Positive 1+ 100
1-9 AFB/100 field Positive Scanty 100
No AFB in 100 field Negative - 100
ACID FAST STAINING
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1. Define acid fast staining.
Acid fast staining is the type of differential staining used to differentiate acid fast bacilli
from non-acid fast organism. Eg.Mycobacterium spp.
2. Why it is called as acid fast staining?
Certain bacteria have the ability to retain the primary dye (strong carbolfuchsin) even after
decolourization by weak mineral acids such as H2SO4/ HCl.
3. Who discovered the acid fast staining?
The acid fast staining discovered by Ehrlich in 1882.
4. Who modified the acid fast staining?
It was later improved by Ziehl and Neelsen.
5. What is the original method of AFB staining by Ehrlich?
The original method involved staining with aniline-gentian violet and decolourization with
strong nitric acid.
6. Why mycobacteria are acid fast?

The cell walls of Mycobacteria are made up of waxy substance (long chain unsaturated fatty acids)
i.e., Mycolic acid which resists decolourisation by weak mineral acids. When heating the smear
while staining, mycolic acid melts and allows the stain to enter inside the cell.
7. What does the cell wall of mycobacterium contains?
The cell walls of Mycobacteria are made up of waxy substance mycolic acid, fatty acids,
waxes, phosphatides, proteins and polysaccharides.
9. What are the various methods of acid fast staining?
 Hot method (Ziehl-Neelsen)

 Cold methods such as Kinyoun’s and Gabbott’s method
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10. What are the components of Ziehl-Neelsen staining procedure?
Primary stain: Strong Carbol Fuchsin (contains Basic fuchsin and Phenol)
Decolourizer: 20% sulphuric acid
Counter stain: Loffler’s Methylene blue
11. Why should the slide be flooded with strong carbolfuchsin?

For uniform distribution of heat, or else the slide may break.
12. What is the purpose of intermittent heating?
The purpose of heating is to soften the waxy material (mycolic acid) of the cell wall and
allow the carbol fuchsin stain to enter into the cell.
13. What is the decolouriser used for Mycobacterium tuberculosis?
The decolouriser used for Mycobacterium tuberculosis is 20% sulphuric acid (or)
3% acid alcohol.
14. What is acid-alcohol decolourizer?
3% HCl in 95% alcohol
15. What is the use of acid-alcohol decolourizer?
 This is useful in differentiating saprophytic Mycobacteria from pathogenic

Mycobacteria.
 Pathogenic Mycobacteria are both acid and alcohol fast
 Saprophytic Mycobacteria are only acid-fast.
16. What are the various modifications of acid fast staining with respect toH2SO4 used?
Mycobacterium tuberculosis - 20% H2SO4

Mycobacterium leprae - 5% H2SO4
Nocardia – 1 % H2SO4
Oocysts of Cryptosporidium, Isospora, Cyclospora - 0.5% H2SO4
Bacterial spores - 0.25- 0.5% H2SO4
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17. Whatare the counter stains used for acid fast staining other than methylene blue?
 0.2% Malachite green

 1% Picric acid (for persons with colour-blindness).
18. What is the disadvantage of Malachite green?
It is a strong counter stain and which may mask the presence of AFB.
19. Give example for acid fast bacilli.
 Mycobacterium tuberculosis
 Mycobacterium leprae
 Non tuberculous mycobacteria
20. Name the other acid fast organisms other than Mycobacterium sp.
 Oocysts of Cryptosporidium, Isospora belli

 Actinomycetes, Nocardia
 Bacterial spores
21. What are the precautions to be taken while preparing or observing smears for AFB?
• A new slide must be used for every specimen, because scratch marks may give false positive
• A uniform smear from thick purulent portion of the sputum must be made.
• Fresh blotting paper must be used for each smear for drying the slide to prevent transfer
from one slide to another.
22. Name the other staining method for Mycobacteria?

Fluorochrome stain (Auramine and Rhodamine)
23. What is beaded appearance of Mycobacteria?
Beaded forms are:
 Those that doesn’t stain uniformly, showing stained and unstained regions of
pathogenic bacilli
 Beaded forms are common in Mycobacterium tuberculosis.
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 Most saprophytic Mycobacteria and M. Bovis stain uniformly.
24. How is the ZN smear graded?
No. of Bacilli / OIF RNTCP guidelines for ZN smear evaluation
Result Grading No of fields examined

>10/F Positive 3+ 20
1-10/F Positive 2+ 50
10-99/100 F Positive 1+ 100
1-9/100 F Positive Scanty 100
No AFB in 100 F Negative - 100
25. What are the factors which affect the acid-fastness of an organism?
 Age of colonies
 Medium on which growth occurs
 Ultra violet light
 Lipid content and integrity of cell wall
26. What are the various methods of acid fast staining?
The Hot method (Ziehl-Neelsen) involves heating the slide while the cold methods
such as Kinyoun’s and Gabbott’s do not involve heating the slide.
27. What are cold methods of acid fast staining?

1. The two methods namely Kinyoun’s and Gabbott’s don’t involve heating of slides, hence
called cold methods.
2. Heating is substituted by increased concentration of phenol and prolonging the duration of

staining.
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3. Kinyoun's method is favoured for detection of Cryptosporidium oocysts in fecal samples.
4. Gabbott’s method has decolourizer and counterstain in one solution.
28. What other methods are available for staining Mycobacteria?
Sputum smears for Mycobacteria can be stained by fluorochromes dyes such as Auramine
O and Rhodamine as they have affinity for mycolic acid in their cell walls.
29. Which staining method is most useful for screening procedure?
Fluorochrome staining method.
30. What are the advantages of Fluorescent staining over Ziehl- Neelsen staining?
 More sensitive
 Heating is not required
 Smear scanned under low power and hence scanning is quick
 Positive smear can be restained by ZiehlNeelsen or Kinyoun procedure, thereby saving the
time needed to make a fresh smear.
 Mycobacteria appear as bright luminous yellow rods against a dark background.
31. What are the drawbacks of Fluorochrome staining?
 Many rapid growers may not appear fluorescent with these reagents
 Fluorescent microscope and reagents are expensive
 Expert hands are required to distinguish between positive bacilli and artefacts.
32. What are the decolourisers used for Acid fast staining other than acid alcohol
and Sulphuric acid?
Nitric acid and hydrochloric acid
33. What is the role of phenol in carbolfuchsin?
It acts as a mordant. It also makes the cell surface easily penetrable for basic fuchsin by
dissolving fats.
34. Which method is more sensitive in acid fast staining? (Ziehl- Neelsen or Kinyoun
method)
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Ziehl- Neelsen is more sensitive. Weak acid fast strains of rapidly growing
Mycobacterium species may stain better with Ziehl- Neelsen than Kinyoun method.
35. How can you differentiate Mycobacterium smegmatis bacilli present in the urine sample
from M. tuberculosis?
M.tuberculosis is acid and alcohol fast,
M.smegmatis (commensal) bacilli are only acid fast
36. If you do Gram stain on M.tuberculosis, what will be the Grams reaction?
Gram positive.
37. How do acid fast bacilli appear in fluorescent staining?
By this method acid fast bacteria fluoresces bright yellow or orange against a greenish
background.
38. What is beaded appearance of Mycobacteria?
Beaded appearance is used to describe the appearance of Mycobacteria when the cell doesn’t
stain uniformly, showing stained and unstained regions. These forms are common in
Mycobacterium tuberculosis while Mycobacterium bovis stains uniformly.
39. Which is considered as the presumptive evidence of active tuberculosis?
A finding of acid fast bacilli in the sputum combined with a history of cough and weight loss
and a chest x ray showing a pulmonary infiltrate is considered as presumptive evidence of
active tuberculosis and is sufficient to initiate therapy.
40. What are the differences between Mycobacterium tuberculosis and Mycobacterium leprae?
1. M. tuberculosis is 20% acid fast M.leprae is 5% acid fast
2 M. tuberculosis is 95% alcohol fast M.lepraeis non-alcohol fast
3 Thin, slender bacilli, causative agent Thick bacilli, arranged in cigar bundle
of tuberculosis (other name is Koch’s appearance, causative agent of leprosy (other
bacillus) name is Hansen’s bacilli)
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41. What are the frequently examined specimens for the detection of Mycobacterium
tuberculosis?
Pulmonary tuberculosis - Sputum, Bronchial or laryngeal washings, Gastric lavage (when

sputum is swallowed by children)
Miliary tuberculosis - Bone marrow, Liver biopsy
Tuberculous meningitis - Cerebrospinal fluid (CSF)
Renal tuberculosis – Urine
43. Name the acid fast bacteria
Mycobacterium tuberculosis, Mycobacterium leprae, Mycobacterium smegmatis,

Mycobacterium fortuitum, Nocardia asteroids, Nocardia brasiliensis, Nocardia caviae.
44. List down the acid fast parasites:
Cryptosporidium parvum, Cyclospora cayatanensis, Isospora belli
45. Name the fungal structures which are acid fast.
Fungal spores
46. Name the other structures which exhibit acid fastness.
Spermatozoa head.
47. Name the culture media used to grow M.tuberculosis?
 Solid media: LJ medium / Dorset egg medium / Middlebrook 7H10&11, Petragnini.

 Liquid media: Dubos, Middlebrook 7H9.
48. Name the selective media used for cultivation of Mycobacterium tuberculosis?
Lowenstein’s Jenson medium is the selective media used for cultivation of
Mycobacterium tuberculosis.
49. Mention the constituents of LJ medium?
 Hen’s Eggs
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 Aspargine
 Mineral salts
 Malachite green
 Glycerol
50. Name the solidifying agent in the LJ medium?
Egg
51. What is the indicator used in LJ medium?

The aniline dyemalachite green is used as an indicator which gives bluish green colour to
the medium.
53. What is the function of malachite green in LJ medium?
Malachite green inhibits the growth of the organisms other than mycobacterium and also
provides colour to the medium
54. What is the function of glycerol?
Glycerol is the hygroscopic agent and it improves the growth of M.tuberculosis
55.How long the Mycobacterium tuberculosis will take to produce single colony?
In Lowenstein Jensen medium the colony appear only in about two weeks and has to be
incubated upto 6-8 weeks at 37oC.
56. How will you sterilise LJ medium?
LJ medium is sterilised by inspissation using an instrument called inspissator.
57. What is meant by inspissation?

Inspissation is a method of moist heat sterilisation. Here the medium is kept in water bath
at 80 -85 °C for 30 minutes at 3 consecutive days.
58. What is the generation time for Mycobacterium tuberculosis?

30
The generation time for Mycobacterium tuberculosisis 14-15 hours.
59. Describe the colony morphology of M. tuberculosis on LJ medium?
Colonies are rough, tough and buff colour and has a luxuriant (eugonic) growth.
60. Describe the colony morphology of M.bovis on LJ medium?
Colonies are flat, smooth, moist and white, breaking up easily when touched. It has
dysgonic growth (sparsely grown).
61. What is meant by Eugonic and Dysgonic growth?

M. tuberculosis produces confluent growth on the surface of LJ medium called eugenic
growth, whereas M. bovis produces dis-continuous, sparse growth called Dysgonic growth.
62. How will you demonstrate growth in liquid media?

Growth begins at the bottom, creeps up and form surface pellicle in liquid media.
63. Name the various concentration techniques used in lab. diagnosis of M.tuberculosis.
 Petroff‘s method – widely used technique

 N-acetyl-L-cysteine with sodium hydroxide
 Pancreatin
 Dilute acids (5% oxalic acid, 3% hydrochloric acid, 6% sulphuric acid).
64. Enumerate the differences between M.tuberculosis and M.bovis?
S.No M.tuberculosis M.bovis

1 Long, slender and slightly curved Short, straight and stout bacilli
bacilli
2 Staining may be uniform or granular Stains always uniformly
3 Beaded or barred forms are Beaded forms are not seen
frequently seen
4 0.5% glycerol improves the growth 0.5% glycerol has no effect or it may impair
the growth
31
5 Shows rough, tough and buff Shows moist, smooth and white colour
coloured colonies in solid media colonies in solid media
65. Name the automated methods for diagnosis of tuberculosis?
 BACTEC
 BACT Alert
 MGIT (Mycobacterial growth indicator tube)
66. Name the molecular methods for diagnosis of tuberculosis?
 PCR (Polymerase Chain Reaction)

 LCR (Ligase Chain Reaction)
 RFLP
 CBNAAT
67. What is Ghon focus?
Sub pleural focus of tuberculous pneumonia commonly located in the lower lobe or lower part
of upper lobe
68. Define Primary complex?
Ghon focus together with the enlarged hilar lymph node constitutes Primary complex
69. Mention the various forms tuberculous infection?
Miliary TB, Meningeal TB and other forms of disseminated TB
70. What are the types of tuberculous infections?
 Exudative type
 Productive type
32
71. What is tubercle?
It is an avascular granuloma composed of a central zone containing giant cells with or without
caseation and a peripheral zone of lymphocytes and fibroblasts.
72. What type of immunity involved in TB?
Cell mediated immunity (CMI)
73. What is the principle of tuberculin test?
It is delayed or type IV hyper sensitivity reaction.
74. What is the other name of tuberculin test?
Mantoux test.
75. Name the reagent used for tuberculin test?
Purified protein derivative (PPD)
76. How will you do the tuberculin test?
0.1ml of PPD is injected intradermally in the anterior aspect of forearm and the results should
read after 48-72 hours.
77. What is positive tuberculin test?
Induration of 10 mm or more in diameter surrounded by erythema at the site of inoculation.
78. What is the significance of positive tuberculin test?
Positive test only confirms the positive infection with the tubercle bacilli but it does not
indicate the presence of active stage of the disease. The test is helpful in children under five
years for indication of active infection. The test becomes positive 4-6 weeks after BCG
vaccination.
79. Name the conditions in which false negative reactions may occur in tuberculin test?
33
Early TB
Military TB
Measles
Sarcoidosis
Patients with Immuno suppression - Hodgkin’s disease, sarcoidosis, lepromatous leprosy,
malnutrition, Administration of immunosuppressive agents and corticosteroids.
80. Name the conditions in which false positive reactions may occur in tuberculin test?
Infections related with atypical mycobacteria
81. How does tuberculosis spread?
Spreads by droplet infection by airborne route.
82. What is the source of tuberculosis infection?
Untreated & unprotected cough / sneeze of positive pulmonary TB patient.

83. What is the common mode of transmission? Why?
Coughing.Because there are millions of tubercle bacilli in the lungs. Coughing projects
droplets into the air that contain tubercle bacilli.
86. What is the indicator drug for drug resistance?
Rifampicin.
87. What does MDR-TB refer to?

MDR-TB means Multidrug resistant Tuberculosis.
88. What is multi drug resistance (MDR) TB?
It refers to resistance to Rifampicin and INH with or without resistance to one or more
other drugs.
89. What does XDR-TB refer to?

XDR –TB means ExtensivelyDrugResistant Tuberculosis.
90. What is extensively drug resistant (XDR) tuberculosis?

34
Strains which are resistant to any fluoroquinolne and at least one of the three injectable
second line drugs (capreomycin, Kanamycin, Amikacin) in addition to INH & Rifampicin.
91. What is DOTS?
Directly Observed Treatment Short Course chemotherapy.
92. What is RNTCP?
Revised National Tuberculous Control Programme
93. Name the vaccine used for the prophylaxis of TB?
BCG
94. Give full form of BCG?
Bacilli Calmette Guerin
95. What is the dose & route of administration of BCG?
0.1 ml intradermal route.
96. What type of vaccine is BCG and when it is given?
BCG is a live attenuated vaccine and it should be given immediately after birth , if not
given at birth, it can be given within one year of age.
97. What is the strain used for preparation of BCG vaccine?
BCG strain derived from M. bovis
98. Name the scientist who prepared an attenuated strain of M.bovis?
Calmette &Guerin
99. Name the specimens to diagnose tuberculosis?
Depends on the site of involvement:
1. Pulmonary TB – sputum (2 samples), Laryngeal swab, BAL(Broncho alveolar

lavage),Gastric lavage – in children
2. Extra Pulmonary TB
35
i) Meningitis – CSF
ii) Renal TB – urine (3 consecutive morning samples)
iii) Bone and joint TB – Aspirated fluid
iv) Tissue/Lymph node – Biopsy
100. Mention the laboratory diagnosis of TB?
1. Direct microscopy – ZiehlNeelsen technique, Kinyoun’s method, fluorescent

staining
2. Culture – LJ medium
3. Biochemical reactions
4. Molecular methods
5. Drug Sensitivity testing
101. What is the other method of investigation to diagnose pulmonary TB?
X-ray chest.
102. What happens when CSF is taken from TB meningitis and allowed to stand?
Formation of cob web on standing.
103. What number of bacilli necessary for sputum culture to be positive?
10 - 100 bacilli/ml
104. What number of bacilli necessary for direct microscopic examination?

10,000 bacilli / ml of sputum.
106. Name the two biochemical test for differentiating M. tuberculosis from M. bovis.
Mycobacterium tuberculosis is differentiated from M.bovis by positive nitrate reduction
test, sensitive to pyrizinamide test & resistant to thiophene-2 carboxylic acid.
36
107. What does M. tuberculosis complex comprise of?
M. tuberculosis complex includes M. tuberculosis, M. bovis, M. microti and
M.africanum.
114. Define NTM.

Non-tuberculous mycobacteria are atypical mycobacteria causing opportunistic infections.
115. Classify NTM.

Photochromogens, Scotochromogens, Non- Photochromogen and Rapid growers.
116. DefineMOTT.
MOTT (Mycobacteria Other Than Tubercle Bacillus) or NTM (Nontuberculous
mycobacteria) mostly found in soil and water and causes chronic pulmonary disease
resembling TB, in mmunocompromised individuals.
117. Give example for Photochromogens

M.kansasii , M.simiae & M.marinum.
118. Give example for Scotochromogens

M.scrofulaceum, M. gordonae , M. szulgai
119. Give example for Non – Photo chromogens

M. avium, M. intracellulare, M. xenopi M. ulcerans
120. Give example for Rapid growers.
M.chelonei, M.fortuitum.
121. Name the Battey Bacilli.

M. intracellulare, is called as Battey Bacilli, as it was first identified as human
pathogen, at the Battey state hospital for tuberculosis, Georgia, USA.
122. Name the causative agent of Buruli ulcer.
37
Buruli ulcer is caused by M. ulcerans. Leg is the most common site of lesion.
123. Name the causative agent of swimming pool granuloma.

Swimming pool granuloma is caused by M. marinum. The lesion appear as papule
which breaks down to form an ulcer.The disease is also called fish tank granuloma.
IDENTIFICATION OF BACTERIA
Exercise 1.
38
IDENTIFICATION OF STAPHYLOCOCCUS AUREUS
Catalase test: positive
Gram Staining: Gram positive cocci in clusters.
Oxidase test : Negative
Motility : Non – motile
Nutrient agar: Golden yellow colonies
MacConkey agar: small pink lactose fermenting colonies
Blood agar: Beta hemolytic colonies
Mannitol salt agar: Yellow colour colonies indicating mannitol fermentation
Biochemical reactions:
Indole test: Negative
Urease test: Positive
MR/VP ----- Positive
Liquifies gelatin
Tellurite Reduction ---- Positive
39
Coagulase test:
Slide coagulase test-Positive ------- Tube coagulase test-Positive
Sugar fermentation: Mannitol ---- fermented, no gas
Since the isolate is mannitol positive and Coagulase positive, the isolate is Staphylococcus aureus.
INFERENCE:The given isolate is identified as Staphylococcus aureus
STAPHYLOCOCCUS AUREUS
1. Namea pyogenic bacteria.
Staphylococcus aureus
2. Whatare the nature of lesions produced by staphylococcus aureus?
They are localized.
3. What is the characteristic feature of pathogenic staphylococcal infections?
Formation of thick creamy pus.
4. What are the characteristic features of Staphylococcus aureus?
 Coagulase production
 Deoxyribonuclease production
 Phosphatase production
 Mannitol fermentation
 Beta hemolysis on blood agar
 Golden yellow pigment production
 Gelatin liquefaction
 Tellurite reduction
40
5. Name the important virulent factor of S.aureus that is anti-phagocytic.
Protein –A
6. What is the characteristic feature of Protein-A?
Co- agglutination
7. What is clumping factor?
 It is a surface protein called as bound coagulase.

 It is responsible for slide coagulase test.
8. Name the toxin responsible for staphylococcal skin infections.
Epidermolytic/Exfoliative toxin
9. What are the superficial and deep infections caused by Staph.aureus?
 Skin infections
 Respiratory infections
 Central nervous system infections
 Endovascular infections
 Urinary tract infections
 Musculoskeletal infections
10. Whatare the toxins mediated diseases produced by S.aureus?
 Food poisoning
 Staphylococcal scalded skin syndrome
 Toxic shock syndrome
11. WhyStaphylococcus aureus is an important nosocomial pathogen?
Nasal carriage of S.aureusby health care workers serves as an important source of hospital
acquired infections.
12. What is the classical diagnosis of Staphylococcus aureus?
41
Depending upon the type of infection, an appropriate specimen is obtained and sent to the
laboratory.
 Gram stain -Gram positive cocci in clusters.

 Mannitol salt agar- yellow coloured colonies due to mannitol fermentation.
 Blood agar- beta hemolysis.
13. How will you differentiate Staphylococcus at species level?
 Catalase test -Positive for all Staphylococcus species

 Coagulase test - Positive for Staphylococcus aureus.
 DNAse test- Positive for Staphylococcus aureus
 Lipase test&Phosphatase test - Positive for Staphylococcus aureus
14. What do you mean by CoNS?
Coagulase negative staphylococcus.
15. Name some coagulase negative staphylococci.
 Staphylococcus epidermidis
 Staphylococcus saprophyticus
 Staphylococcus lugdunensis.
16. What do you mean by MRSA?
Methicillin resistant staphylococcus aureus.
17. Name the biomarker responsible for Methicillin resistance.
mecA gene responsible for resistance to methicillin and other β-lactam antibiotics.
18. What are the two types of MRSA?
Community acquired MRSA (CA-MRSA) and Hospital acquired MRSA (HA-MRSA).
19. How will you screen MRSAcarriers ?
42
Nasal swab cultures
20. How will you treat MRSA carriers ?
Mupirocin &Chlorhexidine (Topical application )
21. Name the surrogate marker used to detect MRSA?
Cefoxitin
22. What are the antibiotics acting against MRSA?
For MRSA, Drug of Choice (DOC) - Vancomycin,

Alternative Drugs for MRSA - Teicoplanin, Linezolid, Daptomycin, Dalfopristin
23. What do you mean by VISA / VRSA ?
Vancomycin Intermediate - resistant Staphylococcus aureus. (VISA)

Vancomycin Resistant Staph.aureus (VRSA)
24. What are the antibiotics acting against VRSA?
Linezolid, Quinupristin / Dalfopristin, Daptomycin, Telavancin.
25. Compare the disease causing potential of S.aureus and S.epidermidis.
S.aureus is a main pathogen.

S.epidermidis is a normal skin commensal that acts as an opportunistic pathogen in
prosthetic devices and in immunosuppressed individuals.
26. Which tests can distinguish Staphylococcus from Micrococcus?
Hugh – Leifson’s Oxidation-fermentation (O/F) test, Micrococci show oxidative

and Staphlococci show fermentative breakdown of sugars.
27. The Staphylococcus which is novobiocin resistant?

43
S.saprophyticus
28. The CoNS which causes UTI in child bearing women ?
S.saprophyticus
Exercise: 2
44
IDENTIFICATION OF ESCHERICHIA COLI
Catalase test: Positive
Gram staining: Gram negative rods
Oxidase test: Negative
Motility: Motile
So it could be a member of Enterobacteriaceae.
Mac Conkey agar: Lactose fermenting colonies.
Blood agar: Greyish white colonies with variable hemolysis.
Biochemical reactions :
Indole test ----- Positive
Methyl Red test ----- Positive / Voges-Proskauer test ------ Negative
Citrate Utilisation test -------- Negative
Urease test ----- Negative
TSI agar ------- A/A with gas production.
45
Sugar fermentation:GLSM (Glu.Lac.Suc.Mannitol, Maltose.) ------- ( + + - + + ) with gas
Since the isolate is a Gram negative, motile, lactose fermenting rod which is indole positive, TSI-
A/A with gas, All sugars fermented with gas production except Sucrose, the given organism is
identified as Escherichia coli.
INFERENCE:The given isolate is identified as Escherichia coli
ESCHERICHIA COLI
1. What are the infections caused byEscherichia coli?

 Diarrhoea
 Septicemia
 Neonatal meningitis
2. Define UTI.
UTI is defined as significant bacteriuria with symptoms of cystitis or pyelonephritis.
3. Name the strain of Escherichia coli causing UTI.

Uropathogenic Escherichia coli called as UPEC.
The serotypes responsible for UTI are O1, O2, O4, O6, O7,O18 and O75.
4. What are uropathogens?

Uropathogens are bacteria with specific virulence factors that facilitate their invasion of the
urinary tract.
5. What is asymptomatic bacteriuria?
46
Asymptomatic bacteriuria refers to urine cultures growing more than 1,00,000 colony-
forming units (105 CFU)/mL of a bacterial species in a patient lacking symptoms of a UTI.
It is significant in pregnant women, patient undergoing prostatic surgery & urologic
procedures.
6. How does E.coli spread in the urinary tract?

 Ascending route
 Descending route
7. What are the types of UTI?

 Lower UTI
 Upper UTI
8. Define cystitis.
Cystitis represents bladder mucosal invasion, most often by enteric coliform bacteria
(eg, Escherichia coli) that ascend into the bladder via the urethra.
9. What are the pre disposing factors that promote UTI?

 Females
 Urinary obstruction
 Pregnancy
10. What is the risk factor for nosocomial UTI?
The most important risk factor for bacteriuria is the presence of a catheter.
11. What are the samples collected for diagnosis of UPEC?

 Clean catch mid-stream urine
 Supra pubic urine
 Urine from catheter tube
47
12. How will you diagnose Escherichia in a laboratory?
 Gram staining: Escherichia is a gram negative rod
 Mac Conkey agar: Lactose fermenting smooth colonies.
 IMViC test:++--
 Urease Negative
 TSI: A/A with gas production.
13. What is significant bacteriuria?
 Significant bacteriuria is a concept put forth by Kass, who stated that there should
be at least 1,00,000(105) bacteria of per ml of clean catch mid stream urine is
significant indicating infection. Any growth obtained from urine collected via
suprapubic aspiration is significant.
 Lower counts may be significant when S. aureus is the pathogen.
14. What is significant pyuria?
Presence of 10,000 (10 4 ) pus cells per ml of uncentrifuged urine is significant pyuria
15. What is sterile pyuria?

Presence of plenty of pus cells in urine but lack of growth on culture is sterile pyuria. The
reasons for this condition include recent administration of antibiotics, UTI by a fastidious/
anaerobic bacteria, urethritis due to gonococci, non-gonococcal urethritis (due to
Ureaplasma, Chlamydia, Trichomonas, or viruses) or renal tuberculosis
16. What is bacteriuria without pyuria?

It is the presence of large numbers of bacteria in the urine but lack of significant numbers
of pus cells. This condition is usually seen in pregnant women where there is retention of
urine.
17. Name the several multidrug resistant Escherichia coli

48
 Extended spectrum beta lactamase (ESBL) producing Escherichia coli
 Carbapenem-resistant Enterobacteriaceae (CRE),
 New Delhi metallo-beta-lactamase (NDM) producing Escherichia coli
18. What are the drugs generally used to treat E.coli infections?
 Third-generation cephalosporins (eg, cefotaxime, ceftriaxone)
 Carbapenems (eg, Imipenem/Meropenem)
 Aminoglycosides (eg, Gentamicin, Amikacin) and
 Quinolones.
19. How will you treat non ESBL producing isolates?
Combination of an aminoglycoside and a third-generation cephalosporin
20. Name the newer antibiotics used to treat multidrug resistant (MDR)Escherichia coli
infections.
Carbapenem(Meropenem), combination of Betalactam/ Betalactamase inhibitor

(Piperacillin -Tazobactam)
49
Exercise: 3.
50
IDENTIFICATION OF KLEBSIELLA SPECIES
Motility: Non-Motile
Mac Conkey agar: Mucoid Lactose fermenting colonies.
Blood agar: Greyish white mucoid colonies
Biochemical reactions :
Indole test:Variable(K.oxytoca + ve , K.pneumoniae – ve)
Methyl Red :Negative
VogesProskauer :Positive
Citrate Utilisation test: Positive
51
Urease production test: Positive
TSI agar: A/A with abundant gas production.
Sugar fermentation: GLSM (Glu.Lac.Suc.Mannitol, Malt.) ------- ( + + + + ) with gas
Since the isolate is a Gram negative non-motile lactose fermenting rod which is indole
positive/Negative TSI-A/A with gas, All sugars fermented with gas production and urease
positive, it could be Klebsiella oxytoca / pneumoniae
INFERENCE:The given isolate is identified as Klebsiella oxytoca / pneumoniae
KLEBSIELLA SPECIES
1. Name the three important species of Klebsiella species.
Klebsiella pneumoniae, Klebsiella oxytoca,Klebsiella granulomatis
2. Name the three sub species of K.pneumoniae.
 Klebsiella pneumoniae subsp. pneumoniae

 Klebsiella pneumoniae subsp. ozaenae
 Klebsiella pneumoniae subsp. Rhinoscleromatis
3. Which is the most pathogenic species?
Klebsiella pneumoniae subsp. pneumoniae
4. What are the infections caused by Klebsiella pneumoniae subsp pneumoniae?
 Lobar pneumonia
 Neonatal meningitis
52
 Septicemia
 Pyogenic wound infections.
5. Describe the nature of sputum produced by patients infected with Klebsiella pneumoniae
subsp. pneumoniae.
They produce thick, mucoid, brick red sputum. Sometimes the sputum has thin,and currant
jelly like appearance.
6. What is the significance of Klebsiella pneumoniae subsp. pneumoniae?
It is a common nosocomial pathogen colonizing the oropharynx of hospitalized patients.

They are multidrug resistant.
7. Name the bacteria causing atopic rhinitis.
Klebsiella pneumoniae subsp ozaenae causes atopic rhinitis characterized by necrosis of

nasal mucosa and mucopurulent nasal foul smelling discharge.
8. What is rhinoscleroma?
A chronic granulomatous hypertrophy of the nose caused by Klebsiella pneumoniae

subspp.rhinoscleromatis.
9. What does isolation of Klebsiella oxytoca from clinical specimens implicate?
K. oxytoca has been implicated in neonatal bacteremia, especially among premature infants
and in neonatal intensive care units. Increasingly, the organism is being isolated from
patients with neonatal septicemia.
10. What is the significance of Klebsiella granulomatis?
53
Klebsiella granulomatis (formerly Calymmatobacterium granulomatis) is a fastidious
member of the genus that causes chronic genital ulcerative disease also known as
granuloma inguinale or donovanosis.
11. How will you diagnose Klebsiella in a laboratory?
 Gram staining: Klebsiella is a short,plump,straight Gram negative rod

 Mac Conkey agar: Lactose fermenting large mucoid colonies.
 IMViC test: --++
 Urease test : Positive
 TSI: A/A with gas production.
12. How will you differentiate Klebsiella pneumoniae from Klebsiella oxytoca?
 Klebsiella oxytoca - Indole positive.
 Klebsiella pneumoniae - Indole negative.
13. What are the risk factors for acquisition of Klebsiella strains?
 Length of hospital stay
 Invasive procedures
14. What are the factors that increase the likelihood of Klebsiella infection?
The presence of invasive devices, contamination of respiratory support equipment, use of

urinary catheters, and use of antibiotics
15. Name the several multidrug resistant Klebsiella pneumoniae
 Extended spectrum beta lactamase (ESBL) producing Klebsiella pneumoniae

 Carbapenem-resistant Enterobacteriaceae (CRE), which are sometimes known as K
pneumoniae carbapenemase (KPC) and
 New Delhi metallo-beta-lactamase (NDM) producing Klebsiella pneumoniae
54
16. What is the major site of colonization of these multidrug resistant Klebsiella?
The bowel is the major site of colonization with infection of the urinary tract, respiratory
tract and wounds.
18. Why are these bacteria are life threatening?
These strains are highly virulent, show capsular type and have an extraordinary ability to
spread.
19. What are the drugs generally used to treat Klebsiella infections?
 Third-generation cephalosporins (eg, cefotaxime, ceftriaxone)

 Carbapenems (eg, imipenem/cilastatin)
 Aminoglycosides (eg, gentamicin, amikacin) and
 Quinolones.
Combination of an aminoglycoside and a third-generation cephalosporin.
21. Name the newer antibiotics used to treat multidrug resistant Klebsiella infections.
 Carbapenems, beta-lactam/beta-lactamase inhibitor combination antibiotics (Pip-

Taz) available.
 Carbapenem resistant isolates are treated with Polymyxin & Tigecycline.
55
Exercise: 4
56
IDENTIFICATION OF PROTEUS SPECIES

57
Motility: Motile
Mac Conkey agar: Non Lactose fermenting colonies.
Blood agar: Greyish white colonies with fishy/seminal odour exhibiting swarming.
Indole test:Variable(Proteus vulgaris+ ve , Proteus mirabilis – ve)
Methyl Red :Positive
Voges Proskauer :Negative
Urease production test: Positive
TSI agar: K/A with abundant H2S.
PPA (Phenyl Pyruvic Acid) test ------ Positive
Sugar fermentation: GLSM (Glu.Lac.Suc.Mannitol) ------- (+- + -) withgas.
Since the isolate is a Gram negativerod, motile,non-lactose fermenting, exhibit swarming which is
indole positive/Negative TSI-K/A with H2S, Glucose & Sucrose fermented with gas production
and urease positive, it could be Proteus vulgaris/mirabilis.
INFERENCE:
The given isolate is identified asProteus vulgaris/mirabilis
PROTEUS SPECIES
58
1. Name the three important species of Proteus.
Proteus vulgaris, Proteus mirabilis, Proteus penneri
2. What are the infections caused by Proteus species?
 UTI in those with structural abnormalities of the urinary tract and stone formation
(Struvite stones in bladder )
 UTI in those who have urethral instrumentation. / Pyelonephritis
 Pyogenic infections / Respiratory tract infections /ear infections / Septicemia
4. What are the risk factors that increase UTI rate caused by Proteus?
Females, duration of catheterization, underlying illness, improper catheter care. Infection

occurs by upward migration of bacteria from the catheter.
5. How will you diagnose Proteusin a laboratory?
 Gram staining: Proteus is a Gram negative coccobacilli occasionally appear

bacillary and filamentous forms (pleomorphic).
 Mac Conkey agar: Non Lactose fermenting colonies with characteristic fishy /
seminal odour.
 Swarming in Blood agar
 Variable Indole and citrate
 MR Positive and VP negative
 Urease Positive
 TSI: K/A with abundant H2S.
6. How will you differentiate Proteus vulgarisfrom Proteus mirabilis?
 Proteus vulgarisis Indole positive.

 Proteus mirabilisis Indole negative.
7. What are the risk factors for acquisition of Proteusstrains?
59
Length of hospital stay and performance of invasive procedures are the major risk factors
for acquisition of Proteus.
8. What are the reservoirs of Proteus infection?
The principal pathogenic reservoirs of infection are the gastrointestinal tract of patients and
the hands of hospital personnel. Organisms can spread rapidly, often leading to nosocomial
outbreaks.
9. Name the organisms associated with formation of Calculi - post UTI.
Calculi related to UTIs most commonly occur in women who experience recurrent UTIs
with Proteus.
10. Name the several multidrug resistant Proteus species.
ESBL - Extended spectrum beta lactamase (ESBL) producing Proteus.
CRE - Carbapenem-resistant Enterobacteriaceae (CRE)
12. What are the drugs generally used to treat Proteus infections?
 Third – generation cephalosporins (Cefotaxime, Ceftriaxone, Ceftazidime)

 Fourth -generation cephalosporins (Cefipime,Cefpirome)
 Carbapenems (eg, Imipenem/Meropenem)
 Aminoglycosides (eg, gentamicin, amikacin) and
 Quinolones. .
Combination of an aminoglycoside and a third-generation cephalosporin
14. Name the organisms other than Proteus which produce swarming.
 Serratia marcescens
60
 Vibrio parahemolyticus
 Clostridium tetani
15. Name the strains of Proteus which forms the basis of Weil-Felix reaction.
Three non-motile Proteus strains OX2, OX19 (from Proteus vulgaris) and OXK (from
Proteus mirabilis) are used in Weil Felix agglutination.
16. What is the use of Proteus antigens in Weil Felix test?
Proteus antigens can be used to detect heterophile antibodies in sera of patients suffering
from rickettsial infections.
17. WhyProteusantigens are used in Weil Felix test?
Somatic antigen of certain non-motile Proteus strains called X strains cross react with the
antigen of some Rickettsiaspecies.
18. Which of the following have positive phenyl pyruvic acid test?
Proteus, Providentia & Morganella
19. Which of the following methods can be used to inhibit swarming ?
 Increasing the concentration of agar in the medium (From 2 to 6%)

 Addition of bile salts in the medium
 Incorporation of chloralhydrate in the medium
 Incorporation of boric acid in the medium
20. What is Dienes phenomenon?
Dienes phenomenon is used to find out the identity and non-identity of various strains of
Proteus
61
Exercise 5
62
IDENTIFICATION OF PSEUDOMONAS AERUGINOSA

63
Oxidase test: Positive
Motility: Motile
So it could be a non Enterobacteriaceae member
Nutrient agar: Large translucent irregular colonies with greenish blue pigmentation
Mac Conkey agar: Non lactose fermenting colonies.
Blood agar: Beta hemolytic with fruitish / earthy odour.
Indole test:Negative
Urease production test: Negative
TSI agar: K/K with no gas.
OF (Oxidative / Fermentative) test: Oxidative GLSM (Glu.Lac.Suc.Mannitol) --(+ - - -), no

gas
Since the isolate is a Gram negativerod, motile, non-lactose fermenting with bluish green colonies
which is oxidase positive, TSI-K/K no gas, Citrate positive, Glucose is utilized oxidatively with no
gas production. So, it could be a Non fermenter - Pseudomonas aeruginosa.
INFERENCE:
The given isolate is identified asPseudomonas aeruginosa.
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PSEUDOMONAS AERUGINOSA
1. Name some important human pathogens among non-fermenting Gram negative

bacilli.
Pseudomonas, Burkholderia, Acinetobacter, Stenotrophomonas, etc.,
2. What do you mean by non -fermenters?
Organisms that do not ferment any sugars but utilize sugars oxidatively.
3. Why is isolation of Pseudomonas important from an inpatient?
It is a major pathogen among the hospitalized patients and inpatients with cystic fibrosis.
4. Why isolation of Pseudomonas from clinical specimens is a cause of concern?
It develops widespread resistance to multiple antibiotics and disinfectants.
5. What are the pigments produced by Pseudomonas?
 Pyocyanin-Bluish green
 Fluorescein-Pyoverdin- Greenish yellow.
 Pyorubin- Red
 Pyomelanin- Brown black.
6. Why Pseudomonas form biofilms?

Mucoid strains of Pseudomonas have an alginate layer which facilitates biofilm formation.
7. Why Pseudomonas exhibits multidrug resistance?
It possess genes coding for resistance to several antibiotics, thereby helping the bacilli to
survive under antibiotic pressure especially in the hospital environment.
8. Why Pseudomonas is an important nosocomial pathogen?
Pseudomonas is multi disinfectant resistant helping to spread the infection in the hospitals.
9. Name the patients who are at risk of acquiring Pseudomonas infections.
 Patients with burn wounds

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 Patients under immunosuppression
 Post surgeries
10. What are the antibiotics used to treat Pseudomonas infections?
 Penicillins: Piperacillin, Mezlocillin, Ticarcillin

 Cephalosporins: Ceftazidime,Cefaperazone,Cefepime
 Carbapenems: Imipenem,Meropenem
 Monobactum: Aztreonam
 Aminoglycosides: Tobramycin, Amikacin
 Quinolones: Ciprofloxacin,Levofloxacin
 Polymyxins: Poymyxin B, Colistin.
11. List down the infections caused by Pseudomonas.
 Ventilator associated Pneumonia

 Chronic respiratory tract infections in patients with cystic fibrosis.
 Bacteremia / Infective endocarditis
 Ear infections like Swimmer’s ear and malignant otitis externa
 Eye infections / Burns wound infections / Bone and joint infections.
 Shangai fever
 Ecthyma gangrenosum
 Dermatitis / Green nail syndrome
 Cellulitis characterized by blue green pus
 UTI in catheterized patients
 Meningitis in post operative and post traumatic patients
12. How will you diagnose Pseudomonas infections?
 Gram negative motile bacilli
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 Beta hemolytic bluish green pigmented colonies in blood agar ,
 Non lactose fermenter,
 Catalase and oxidase positive
 Indole negative,
 Citrate positive
 Urease negative
 TSI: K/K
 OF test showing oxidative pattern.
13. The bacteria which are non motile?

Acinetobacter, Burholderia mallei
14. The following are characteristic features of Pseudomonas aeruginosa?

Gram negative bacilli, motile, oxidase positive, Utilisation of glucose oxidatively, ,
pyocyanin production.
15. The most popular method employed for typing of pseudomonas aeruginosa?
Pyocin typing
16. The causative agent of Shanghai fever?

Pseudomonas seruginosa
17. The bacteria formerly known as Whitmore’s bacillus?

B.pseudomallei
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Exercise: 6
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IDENTIFICATION OF SALMONELLA SPECIES

Motility: Motile
Mac Conkey agar: Non Lactose fermenting colonies.
Blood agar: Non hemolytic, greyish white moist colonies
DCA : NLF colonies with black centre
XLD agar: red colonies with black centre
Indole test:Negative
Methyl Red : Positive
Citrate utilization test–Variable (Salmonella typhi&Salmonella paratyphi A is citrate negative

and Salmonella paratyphi B is citrate positive)
TSI agar: K/A. (S.paratyphi A– AbsentH2S, S.typhi - with a speck of H2S, S.para B- Abundant
H2S)
Sugar fermentation: GLSM M (Glu.Lac.Suc.Mannitol )------- (+ - - +) ,
Para A &B - with gas , S.Typhi – No gas.
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- Since the isolate is a Gram negative rod, motile, non-lactose fermenting which is MR
positive, Citrate negative, TSI-K/A no gas, with a speck of H2S ,it could be Salmonella
typhi.
positive, Citrate negative, TSI-K/A with gas, absentH2S ,it could be Salmonella paratyphi
A.
positive, Citrate positive, TSI-K/A with gas, abundant H2S ,it could be Salmonella
paratyphi B.
INFERENCE:
The given isolate is identified asSalmonella Typhi / Salmonella paratyphi A/ Salmonella

paratyphi B.
SALMONELLA SPECIES
1. Name the bacteria responsible for typhoid fever.
Salmonella Typhi
2. Expand Salmonella Typhi.
Salmonella enterica subsp. enterica serovar Typhi
3. How will you group Salmonellae?
Salmonellae are divided into groups namely
 Typhoidal salmonella (enteric fever group) which includes the human pathogen,
typhoid and paratyphoid bacilli.
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 Non Typhoidal salmonella (food poisoning group) which includes animal
pathogen ( S.typhimurium, enteritidis, cholera-suis)
4. Name the two species of Salmonella.
Salmonella enterica and Salmonella bongori.
5. Name the two important antigens of Salmonella used in the diagnosis.

 Somatic O antigen
 Flagellar H antigen
6. Name the organisms belonging to the typhoidal group.
 Salmonella Typhi
 Salmonella Paratyphi A
 Salmonella Paratyphi B
 Salmonella Paratyphi C
7. Name the diseases produced by Salmonella?
Enteric fever, Gastroenteritidis & Septicemia
8. Which Salmonella ferments glucose with acid but no gas?

S.Typhi
9. Which Salmonella does not produce H2S in TSI?

Salmonella para A & S.cholera-suis
10. How is Salmonellae transmitted?
By oral route through contaminated food /water.
11. What is the infective dose of Salmonella?
Minimum of 103 to 106 bacilli.
12. What is the typical presentation in the first week?

In the first week, the body temperature rises slowly, and fever fluctuations are seen
with relative bradycardia (Step ladder pyrexia), malaise, headache, cough and Abdominal
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pain . A decrease in the number of circulating white blood cells (leukopenia) occurs and
relative lymphocytosis.
13. What test is positive during the first week?
Blood cultures are positive for Salmonella Typhi or S. Paratyphi. (90% positivity )
14. What test done during the second week of typhoid fever?
The Widal test is strongly positive, with anti-O and anti-H antibodies.
15. What will be the result of Blood culture during the second week of typhoid fever?
Blood cultures are sometimes still positive at this stage. (75% positivity)
16. Name the specimens used for the detection of carriers of Salmonella.
Bile / faeces / Urine
17. How will you detect a recent carrier?

By detecting antibodies to Vi antigen
18. How will you trace carriers in cities?

Sewer swab technique.
19. What are the biochemical reactions of Salmonella Typhi ?

 Catalase positive and Oxidase negative
 Nitrate is reduced to nitrite.
 Indole negative, Citrate positive, Urease negative.
 TSI : K/A, gas(+) , with a speck of H2S.
20. What is the positivity rate of blood culture during the course of typhoid fever?
 First week -90 % of cases positive.
 Second week-75% of cases positive.
 Third week -60% of cases positive.
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 Fourth week-25% of cases positive.
21. What is clot culture?
Blood is centrifuged and then the serum is separated and used for WIDAL test and the clot
is used for Culture. Clot culture shows higher rate of isolation than blood culture.
22. What are the specimens used in the diagnosis of enteric fever during the first week?
Blood culture, Bone marrow aspirate culture and Duodenal aspirate culture.
23. What are the specimens used in the diagnosis of enteric fever during the second week
and the third week?
 Serum for antibody detection by WIDAL test.
 Serum for antigen detection.
 Stool and Urine culture.
24. What are the specimens used in the diagnosis of enteric fever during the fourth week?
Stool and urine culture.
25. Name the two types of media used in blood culture bottles.
 Monophasic medium-50-100 ml of brain heart infusion broth.

 Castaneda’s medium-Consists of BHI agar slant and BHI broth (50-100ml).
26. How will you inoculate blood culture bottle?
5 ml of fresh blood is added in 50ml of broth aspetically in a ratio of 1:10.
27. Name an enrichment media to recover Salmonella from Stool specimen.
 Selenite F broth
 Tetrathionate broth
 Gram negative broth
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28. Name some selective media used to isolate Salmonella from stool specimen.
Low selective media: Mac Conkey agar
Highly selective media such as:
Deoxycholate citrate agar (DCA agar)

Xylose lysine deoxycholate agar (XLD agar)
Wilson Blair’s Brilliant green Bismuth sulphite medium.
29. What are the currently recommended drugs used in the treatment of enteric fever?
 Fluoroquinolones e.g., Ciprofloxacin

 Third generation cephalosporins e.g., Ceftriaxone
 Azithromycin
30. What are the drugs that are used in the past?
 Chloramphenicol, Amoxicillin, Cotrimoxazole
31. Vi antigen is present in ?

S.typhi, S.paratyphi C, S.dublin
38. Salmonella which are nonmotile?
S.gallinarum & S. pullorum
39. Salmonella responsible for food poisioning?
S.typhimurium, S.enteritidis
40. The vaccines used for the prevention of enteric fever?
TAB vaccine, Live oral vaccine, Purified Vi polysaccharide vaccine
Exercise: 7
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IDENTIFICATION OF VIBRIO CHOLERAE
Gram staining: Gram negative short curved / comma shaped rods (fish in stream appearance)
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Oxidase test: Positive
Motility: Motile (Darting motility)
So it could be a non Enterobacteriaceae member
Enrichment broth: Alkaline peptone water / Monsur’s taurocholate tellurite peptone water
Mac Conkey agar: Non lactose fermenting colonies which becomes pink on prolonged
incubation (Late lactose fermentation).
Blood Agar: Greyish white colonies with hemodigestion.
TCBS agar: Yellow colour colonies
Biochemical reaction:
Indole test:Positive
Voges Proskauer :Negative(El Tor Biotype – Positive)
Citrate Utilisation test:± (Variable)
TSI agar: A/A, No gas.
Sugar Fermentation test: GLSM ( Glu.Lac.Suc.Mannitol ) ----- ( + - + + ) , no gas.
Cholera red reaction ----- positive
String test ----- Positive
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Since the isolate is gram negative, Catalase positive, oxidase positive curved rod,which is indole
positive, TSI - A/A, No gas, with fermentative metabolism, Cholera red reaction – positive &
String test positive , it produces yellow colonies in TCBS agar, So , it could be Vibrio cholerae.
INFERENCE:The given isolate is identified asVibrio cholerae.
VIBRIO CHOLERAE
1. What is cholera?
Cholera is an infection of the small intestine by some strains of the bacterium Vibrio
cholerae.
2. What is the hallmark of cholera infection?
The hallmark of the disease is profuse secretory diarrhea. The disease may beasymptomatic
or mild. Severe cholera can cause dehydration and death within hours of onset.
3. How is cholera transmitted?
Cholera is transmitted by the fecal-oral route.
4. How will you manage a patient suspected to have cholera?
Definitive diagnosis is not a prerequisite for the treatment of patients with cholera. The
priority in management of any watery diarrhea is replacing the lost fluid and electrolytes
and providing an antimicrobial agent when indicated.
5. How many cholera pandemics have occurred so far?
7 pandemics since 1961.
6. Name the causative agent involved in the first six pandemics of cholera.
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The first 6 occurred from 1817-1923 and were probably the result of V.cholerae O1 of the
classic biotype.
7. Describe the seventh pandemic of cholera.
The seventh pandemic of cholera started in 1961 caused by the El Tor biotype of V
cholerae O1.
8. What do you know about V.cholerae serogroup O139?

A new strain of cholera, V cholerae serogroup O139 (Bengal Strain) emerged in 1992 and
caused outbreaks in Bangladesh and India. Disease from this strain has become endemic in
at least 11 countries.
9. What are the serogroups associated with epidemic cholera?
Although more than 200 serogroups of V cholerae have been identified, V cholerae O1
and V cholerae O139 are the principal ones associated with epidemic cholera.
10. What is the Grams reaction produced by Vibrio cholerae?
It is a Gram negative bacilli.
11. What is the shape of Vibrio cholerae?
Comma shaped.
12. Is Vibrio an aerobe or anaerobe?
Obligate aerobe.
13. What is the temperature and pH requirement of Vibrio cholerae?
37°C and pH 8.6.
14. Describe the colony morphology of Vibrio cholerae in Nutrient agar?
Translucent colonies with a bluish tinge in transmitted light.
15. Describe the colony morphology of Vibrio cholerae in Blood agar?
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Greenish zone of discolouration around colony which later on becomes clear due to
haemodigestion.
16. Describe the colony morphology of Vibrio cholerae in Mac Conkey agar?
Late lactose fermenting colonies
17. Name the selective media used in the isolation of Vibrio cholerae?
 Thiosulphate citrate bile salt sucrose agar (TCBS)

 Alkaline bile salt agar (BSA)
 Monsur’s gelatin taurocholate trypticase tellurite agar (GTTTA)
18. Name the transport media used to transport stool specimen to a laboratory?
 Venkatraman Ramakrishnan Medium
 Cary-Blair Medium
 Autoclaved sea water
19. Name two enrichment media used to recover Vibrio cholerae?
 Alkaline peptone water
 Taurocholate tellurite peptone water
20. What is cholera red reaction?
When 24 hour old liquid culture is mixed with few drops of concentrated sulphuric acid,
red pink colour develops due to formation of nitroso indole. The test is positive for
V.cholerae.
21. What is string test?
When growth is mixed with 0.5% sodium deoxycholate in saline, suspension loses its
turbidity and becomes mucoid to form string when loop withdrawn from suspension. Test
is positive in V.cholerae.
22. What is hemolytic reaction?
Equal volumes of broth culture and 1% sheep erythrocytes are mixed and incubated for 2
hours at 37°C,then kept in refrigerator for overnight at 4°C and examined for hemolysis.
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 Classical Vibrio- non hemolytic
 El Tor Vibrio- haemolytic
23. Does Vibrio choleraeproduces oxidase?
Yes, they produce oxidase and are oxidase positive.
24. How many serogroups are there in Vibrio cholerae?
More than 200 serogroups are there.
25. Name the two biotypes of Vibrio cholerae?
Classical and El Tor biotypes
26. What are El Tor variants?
Isolates of Vibrio cholerae that do not fit into both the biotypes are called as El Tor
variants.
27. Name the variants of El Tor variants.

 Matlab variants
 Mozambique variant
28. Name the three serotypes of Vibrio cholerae?
Organisms in both the classical and the El Tor biotypes are subdivided into serotypes
according to the structure of the O antigen, as follows:
 Serotype Inaba - O antigens A and C
 Serotype Ogawa - O antigens A and B
 Serotype Hikojima - O antigens A, B, and C
29. How will you classify Vibrio cholerae based on O antigen?
Based on O antigen Vibrio can be grouped into serogroups, 01 - 0139.
30. How will you classify Vibrios based on salt requirements?

 Non halophilic vibrios (Vibrio cholerae, Vibrio mimicus)
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 Halophilic vibrios (Vibrio parahemolyticus, V.alginolyticus, V.vulnificus)
31. Name the four tests used in biotyping of Vibrio cholerae?

 Beta hemolysis on blood agar.
 Chick erythrocyte agglutination.
 VP test.
 Polymyxin B (50 IU) susceptibility test.
32. What are the antibiotics used to treat Cholera?
Doxycycline,cotrimoxazole, erythromycin, tetracycline, chloramphenicol,

and furazolidone.
33. Type of motility in Vibrio cholera?
Darting
34. What are the bacteria producing green colonies on TCBS?
Vibrio parahaemolyticus
35. The bacteria associated with food poisioning due to consumption of sea food?
Vibrio parahaemolyticus
36. Cholera toxin resembles which toxin?
Heat labile toxin of Escherichia coli
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BASICS IN MYCOLOGY
1. What is medical mycology?

It is the study of fungi causing infections in man.
2. What are fungi?

They are eukaryotes which may be either unicellular or multicellular.
3. How will you classify fungi based on morphology?

Based on cell morphology, fungi is divided into four groups namely
 yeasts
 yeast like fungi,
 moulds
 dimorphic fungi.
4. What are yeasts?
Yeasts are unicellular fungi. They occur as spherical or oval cells. They reproduce by simple
budding.
5. Name some pathogenic yeasts.

Cryptococcus neoformans
Malassezia species.
6. Name a yeast cell producing capsule.
7. What are yeast like fungi?
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Fungi which are basically yeasts but are able to produce elongated cells called as
pseudohyphae.
8. Give an example of yeast like fungi.

Candida albicans.
9. What is pseudohyphae?
In some yeast cells the bud remains attached to the mother cell. They undergo repeated
budding to form chains of cells which are elongated in nature. These are known as
pseudohyphae. The cells are called as Blastoconidia.
10. Name a fungi producing pseudohypha.
Candida albicans.
11. What are moulds?
Fungi which form mycelia are called as moulds. They are also called as filamentous fungi.
They reproduce by forming different types of spores.
12. Name a pathogenic mould.
Dermatophytes.
13. What is mycelium?
A tangled mass of hyphae is called mycelium.
14. What is aerial mycelium?

 The portion that projects above the surface of the culture medium.
 Moulds reproduce by means of spores.
 These spores are derived from aerial mycelium only.
 Hence it is called as reproductive mycelium.
15. What is vegetative mycelium?
 The portions of the mycelium extending into the substratum of the culture medium.
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 They absorb water and nutrients from the media.
16. What are hyphae?
It is a tubular thread like structure produced by moulds.
It is the microscopic unit of a mould.
17. What is a septate hyphae?
Some moulds have cross walls in their hyphae called as septa.
Hyphae containing septa are said to be septate hyphae.
18. Give example for fungi having septate hyphae.
Aspergillus and Penicillium.
19. What is Aseptate hyphae?
Hyphae without septa are called as aseptate hyphae.
20. Give example for fungi having aseptate hyphae.
Mucor and Rhizopus
21. What is hyaline hyphae?
Colorless hyphae is called as hyaline hyphae.eg.,Aspergillus.
22. What is pigmented hyphae?
Dark colored hypha are said to be pigmented hypha. Eg: Dematiaceous fungi
23. What are dimorphic fungi?
Fungi which can occur as moulds at 22°C (room temperature/in vitro) or as yeasts at 37°C
(body temperature/in vivo).
24. Give examples for dimorphic fungi.

Histoplasma capsulatum.
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Blastomyces dermatidis.
Coccidioides immitis.
Paracoccidioides brasiliensis.
Penicillum marneffei.
Sporothrix schenckii.
25. Classify human fungal infections.
Superficial mycoses
Subcutaneous mycoses
Systemic mycoses
Opportunistic mycoses
26. What are superficial mycoses?
 Fungal infections of the skin,hair , nail & mucosa.

 No living tissues is invaded and there is no cellular response from the host.
27. Name some superficial mycotic infections.
 Pityriasis (Tinea) versicolor.

 Tinea nigra and
 Piedra (black and white).
28. Name the infections caused by Malassezia spp.,
Pityriasis (Tinea) versicolor, Seborrhoeic dermatitis including dandruff, Atopic dermatitis

and folliculitis.
29. What is Pityriasis versicolor? Name the causative agent.

 It is a mild, chronic superficial fungal infection of the stratum corneum.
 It is seen as scaly areas of depigmentation or hyperpigmentation of the skin.
 Mainly affects the neck,chest and upper limbs.
 Caused by the yeast Malassezia furfur.
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30. What is pityriasis folliculitis?
 Superficial fungal infection characterized by follicular papules and pustules.
 It is found in back, chest and upper arms.
 They often appear after sun exposure and often itchy.
31. What is Tinea nigra? Name the causative agent.

 It is a localized infection of the skin particularly palm & sole.
 It produces brown or black lesions.
 It is caused by Hortaea werneckii.
32. What is piedra?

 It is a fungal infection of the hair.
 It presents as firm nodules along the hairshaft.
33. What are the two varieties of piedra? List down the causative agents.
1.White piedra is caused by Trichosporon species affecting scalp, moustache, beard area.
2.Black piedra is caused by Piedraia hortae affecting axillary, pubic, beard and moustache
area.
34. What are cutaneous mycoses?
Fungal infections of skin, hair, nail and mucous membranes.
35. Name the most important cutaneous mycotic infection.
Dermatophytosis
36. What is dermatophytosis?
The variety of clinical conditions caused by dermatophytes involving skin, hair and nail is
called dermatophytosis.
37. What is the common name for dermatophytosis?
Tinea or ring worm

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38. Define dermatomycosis.
The infection of skin caused by non dermatophytic fungi and the cutaneous manifestations
of systemic mycoses are known as dermatomycosis.
39. What are dermatophytes?
A group of closely related filamentous fungi. They infect only superficial tissues like
skin,hair and nail.
40. Give some examples for dermatophytes.
Trichophyton – Skin, hair and nail
Microsporum – Hair, skin
Epidermophyton – Skin, nail
41. What are the two types of spores produced by dermatophytes?

Microconidia and Macroconidia.
42. Which factor is considered to differentiate three genera of dermatophytes?
The differentiation of genera is mainly based on the nature of macroconidia.
43. Distribution of conidia of dermatophytes.
Dermatophytes Macroconidia Microconidia

Trichophyton Rare, very few Abundant
Microsporum Numerous Rare
Epidermophyton Numerous Absent
44. What are the three groups of dermatophytes?
Geophilic, Zoophilic and anthropophilic.
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45. Enumerate anthropophilic dermatophytes.
Trichophyton rubrum
Microsporum audounii
Epidermophyton floccosum
46. Name some clinical types of dermatophytosis.
Disease Sites affected

Tinea capitis Scalp
Tinea barbae Bearded area of face and neck
Tinea corporis Smooth, non- hairy skin
Tinea cruris Groin and Perineum
Tinea pedis Feet
Onychomycosis Toe nails and finger nails
47. What are the specimens of superficial mycoses?
Skin scales, skin scrapings, hair, nail scrapings.
48. What are the prelimnary tests done for the examination of superficial mycoses?
 Woods lamp examination.
 KOH, 10% preparation.
 Lactophenolcotton blue(LPCB) mounting.
 KOH, 40% preparation.
49. What are subcutaneous mycoses?
Mycotic infections of skin, subcutaneous tissue and bone which is localized & enter the
body by trauma.
50. Name some important subcutaneous mycotic infections caused by fungi.

Mycetoma
Chromoblastomycosis
Sporotrichosis
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Rhinosporidiosis
51. Name the subcutaneous fungal infection common in Tamil Nadu.
Mycetoma
52. What are Mycetomas? Name the causative agents.
They are chronic, slowly progressive granulomatous infections of the subcutaneous tissue,
usually of the foot.
53. What are the types of mycetoma based on the causative agents?
Eumycetoma , Actinomycetoma
54. What is eumycetoma?
Fungal agents causing mycetoma.Eg.,Madurella mycetomatis, Exophiala jeanselmii,

Pseudollescheria boydii
55. What is actinomycetoma?

Bacterial agents causing mycetoma.Eg.,Actinomadura madurae, Nocardia brasiliensis
56. What is Botryomycosis.?
The causative agents are Staphylococcus , Streptococcus, E.coli, Proteus etc.,
57. What is the hall mark of mycetoma?
Clinical triad consisting of
 Tumour like swelling

 Discharging sinuses
 Granules in the discharge.
58. Name the various kinds of granules produced during mycetoma?

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Black granules, white granules, white to yellow granules and pink to red granules.
59. What are the specimens generally received for the diagnosis of mycetoma?
Pus or exudates with granules
60. What is Chromoblastomycosis? Name the causative agents.
It is a slowly progressing localized fungal infection of the skin and the subcutaneous tissue
of the feet and lower legs. This disease consists of warty nodules and formation of sclerotic
bodies in lesion.
It is caused by a group of dematiaceous fungi namely,

Fonsecaea pedrosoi
Phialophora verrucosa
Cladophialophora carrionii
61. What is Sporotrichosis? Name the causative agents.
It is a chronic granulomatous fungal infection of skin and subcutaneous tissue caused by

Sporothrix schenckii.
It is a dimorphic fungus.
The disease is also called as Rose Gardner’s disease.
62. What is Rhinosporidiosis? Name the causative agents.
It is a subcutaneous fungal infection caused by the fungus Rhinosporidium seeberi.
It is characterized by the formation of polyps, usually in nose.
63. What are the three developmental forms of Rhinosporidium seeberi?
Trophocyte, Sporangia, Endospores.
64. What is phaeohyphomycosis?

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It is a subcutaneous fungal infection caused by a group of dematiaceous fungi characterized
by presence of hyphal tissue forms.The causative agents are Bipolaris, Curvularia,
Alternaria etc.,
65. Name the agents causing subcutaneous zygomycoses.
Subcutaneous zygomycoses is caused by two organisms namely
Basidiobolus ranarum and Conidiobolus coronatus.
Basidiobolus ranarum causes basidiobolomycosis.
Conidiobolus coronatus causes conidiobolomycosis.
66. What are systemic mycoses? Name the causative agents.
Fungal infections affecting internal organs and usually spread via the blood stream are
called as systemic mycoses.
67. What are the two groups of systemic mycoses?
Those caused by true pathogens and those caused by opportunistic pathogens.
68. Name the true pathogens causing systemic infections.
The true pathogens are fungi able to cause infections in healthy individuals also.
They include, Histoplasma, Blastomyces, Coccidioides, Paracoccidioides.
69. Name the opportunistic pathogens causing systemic infections.
The opportunistic pathogens include Candida, Aspergillus and the zygomycete fungi.
These occur in severely ill or immunocompromised individuals.
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70. What is Cryptococcosis? Name the causative agents.
Cryptococcosis is an infection caused by the yeast, Cryptococcus neoformans. It can cause

infections in normal as well as immunocompromised individuals.
71. What is Histoplasmosis? Name the causative agents.
Histoplasmosis is a systemic fungal disease caused by the dimorphic fungus, Histoplasma

capsulatum. It is characterised by involvement of reticuloendothelial tissues. It is also called
as Darling’s disease.
72. What are the three varieties of Histoplasmosis?
Histoplasma capsulatumvarcapsulatum
Histoplasma capsulatumvarduboisii
Histoplasma capsulatumvarfarciminosum
73. What is Blastomycosis? Name the causative agents.
Blastomycosis is a systemic fungal infection that primarily involves lungs caused by a

thermally dimorphic fungus, Blastomyces dermatidis. It is called as Chicago’s disease.
74. What is Coccidiomycosis? Name the causative agents.
Coccidiomycosis is a systemic fungal infection that primarily involves respiratory tract

caused by a thermally dimorphic fungus, Coccidioides immitis. It is called as Valley fever or
California disease.
75. What is Paracoccidiomycosis? Name the causative agents.
Paracoccidiomycosis is a systemic fungal infection that primarily involves respiratory tract

caused by a thermally dimorphic fungus, Paracoccidioides brasiliensis. It is called as south
American blastomycosis.
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76. What is Candidiasis? Name the causative agents.
Candidiasis is the commonest fungal infection in humans, causing superficial and deep
infections caused by the fungi Candida albicans.
77. What is Aspergillosis? Name the causative agents.
Aspergillosis is a superficial and systemic fungal infection found in immunocompromised

as well as immunocompetent individuals caused by Aspergillus spp.
78. Comment about infections with Penicillium marneffei.
Penicillium marneffei is an important opportunistic pathogen in HIV infected patients.
79. What is mycotic keratitis?
Invasive fungal infection of cornea is called as keratomycosis or mycotic keratitis.
80. What is otomycosis?
Superficial fungal infection of external auditory canal is called as otomycosis.
81. What is Zygomycosis? Name the causative agents.
Zygomycosis is a rare invasive disease caused by Zygomycetes like Mucor ,Rhizopusor

Absidia.
82. What are the common fungal laboratory contaminants?
Aspergillus, Penicillium, Mucor ,Rhizopus.
83. List down the various stains used in direct fungal examinations.
 KOH mount
 Calcoflour white stain
 India ink stain
 Nigrosin stain
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 Gram stain
 Giemsa’s stain
 Wright’s stain
 Direct immunofluorescence stain
 Periodic acid schiff’s stain
 Gomori’s methenamine silver nitrate stain.
84. What are the different types of media used in fungal culture?
Saboraud’s dextrose agar with PH 5.4
Emmons modification of saboraud’s dextrose agar with PH 7.2
Saboraud’s dextrose agar with chloramphenicol and cycloheximide
85. Name the bacterial culture media that can be used for the cultivation of fungi.
Brain heart infusion agar with cycloheximide and chloramphenicol.
86. Why are antibiotics added to the fungal medium?
To prevent bacterial growth.
87. What is the use of cycloheximide in fungal media?
To suppress the growth of laboratory fungal contaminants
88. Enumerate fungi inhibited by cycloheximide.

 Cryptococcus spp.,
 Pseudollescheria boydii
 Aspergillus spp.,
 Fusarium spp.,
 Candida spp .,
89. How will you sterilize the fungal media?
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By autoclaving 121°C for 15 mins at 15 lbs.
90. Why test tube agar slants are preferred for isolation of fungi as compared to agar
plates?
Fungi generally need a longer time for incubation
91. How will you do Wood’s lamp examination?

 The material to be examined is kept in a dish and examined under the woods lamp
in a dark room.
 If fluorescent hairs are present, they are picked for further examination.
92. What is Wood’s lamp?
It is a glass device made of barium silicate containing 9 percent nickel oxide which can
transmit long wave UV light of 365 nm that shows a characteristic fluorescence.
93. Enumerate some fungi fluorescing under Woods lamp.

 Microsporum produces bright green fluorescence
 Trichophyton schoenleinii produces dull green fluorescence
 Other dermatophytes produce no fluorescence at all.
 Malassezia furfur produces golden yellow fluorescence.
94. Name the chemical substance responsible for positive fluorescence.

 Pteridine.
KOH PREPARATION
95. How will you perform 10% KOH mount?

 Keep the specimen on a clean glass slide.
 Add a drop of 10% KOH.
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 Warming the slide slightly on a low flame may accelerate the digestion of
keratin.
 Put on a clean cover slip and leave for 10-15 mins.
 Examine for 15 to 20 minutes for the fungal filaments under 10X and 45X.
96. What are the materials commonly examined under KOH?
Skin scrapings, Nail scrapings, Hair, Corneal scrapings, materials from external ear,
Aspirates from nasal sinus, Pus from draining sinus.
97. What is the use of KOH?
KOH dissolves proteinaceous tissues including keratin, clears the debris and
renders them transparent. This enables fungi to be visualized more easily.
98. How will you interpret results of a KOH wet mount?

 Dermatophytes in skin or nail are seen as branching hyphae or arthrospores.
 Yeasts are present as budding cells, pseudohyphae, or mycelium.
 Brown color hyphae is seen in infections with dematiaceous fungi.
99. What are the other modifications of KOH preparation?
 KOH-DMSO mount.
 KOH-Calcoflour mount.
100. What is KOH-DMSO mount?
 Dimethyl sulfoxide (DMSO) is added to the KOH reagent to enhance the
clearing of specimens without heating or incubation.
 This is generally done for keratinous or fibrous tissue.
101. How will you perform KOH-DMSO mount?
 Place specimen on the slide.
 Add a drop of KOH-DMSO.
 Add coverslip.
 Squash preparation.
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 Examine immediately as specimens in DMSO, degrade quickly.
102. What is KOH –Calcoflour mount?
 Calcoflour white added to 10 % KOH. It binds to the chitin of the fungal
wall and flouresce under UV light.
103. What is the advantage of calcoflour white?
 It detects even small amount of fungi.
 It helps in rapid screening of specimens by inexperienced people.
104. How will you perform KOH- Calcoflour mount?
 Place specimen on slide.
 Add I drop of calcoflour reagent and 1 drop of 10 to 20% KOH.
 Place a coverslip.
 Squash preparation firmly.
105. What is the color of the fluorescence exhibited by fungi in calcoflour white?
 Fungi appears yellow green when emission filter is used.
 Fungi appears bright white when no filter is used.
106. What is ectothrix?

Spores present outside the hair shaft.
107. What is endothrix?
Spores inside the hair shaft are called as Endothrix.
108. What is faviform endothrix?

If the endothrix hair is broken up into pieces, then it is called as faviform endothrix.
109. How will you prepare 10 percent KOH mounting fluid?
10 gms of potassium hydroxide is dissolved in 80 ml of distilled water and then
10 ml glycerol is added.
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110. What is the other alkaline solution that can be used instead of KOH?
Sodium hydroxide.
111. How will you prepare KOH- calcoflour white mounting fluid?
100 mg of calcoflour white and 50 mg of Evan’s blue is mixed in 100 ml of distilled

water. One drop of it is added to 10 percent KOH.
112. Why Evan’s blue is added to calcoflour white mounting fluid?

To reduce non-specific background fluorescence and reveal the surrounding tissue.They
also produce a contrasting orange to ruby red background thereby enhancing detection of
fungi.
113. What are the advantages of KOH preparation?
It clears the tissue and debris from all types of clinical specimens without damaging the
fungal cells.
114. What are the disadvantages of KOH wet mount preparation?
It is difficulty to differentiate hyphae from collagen fibres and other artifacts by KOH
method.In KOH preparation, crystals can be formed on standing so that interpretation of
the smear becomes difficult.
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CANDIDA ALBICANS
AIM:
To identify the given fungal culture
OBSERVATION:
Pseudohyphae
Gram +ve budding yeast cells
IDENTIFYING FEATURES:
Gram stain: Gram positive budding oval/spherical yeast like cells
Presence of elongated yeast like cells named as pseudohyphae is suggestive of pathogenicity.
CULTURAL CHARACTERISTICS :
ON SDA AT 370C:Cream colored smooth surfaced pasty colonies
 OBVERSE: Cream coloured pasty colonies

 REVERSE: White coloured
ON BLOOD AGAR: Cream coloured colonies
ON CORNMEAL AGAR: Chlamydospore formation at 250C by Candida albicans.
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INFERENCE:Based on the above identifying features, the given yeast is identified as Candida
albicans.
CANDIDA ALBICANS
1. Name the common pathogen among the several species of Candida.?
Candida albicans.
2. How will you describe it morphologically?
Yeast like fungi.
4. What is yeast like fungi?

Fungi which grow partly as yeasts and partly as elongated cells (chain like) resembling a
hyphae.
5. What can one observe in a direct specimen Gram stained smear believed to contain
Candida. ?
Gram positive budding yeast cells with pseudohyphae
6. What is pseudohyphae?
 Candida albicans, in addition to its usual oval budding form, is also able to produce
pseudohyphae.
 The buds elongate forming a tube-like structure and the elongated buds remain
attached to one another and eventually produce a filament called a pseudohyphae.
 It is called so because it resembles the hyphae.
 The cells are called as Blastoconidia.
7. Describe the culture findings of Candida albicans.

White- cream colored, smooth, butyrous, bacteria-like colonies are observed.
8. Name the culture media used.

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Sabaraud’s Dextrose agar
9. What will be your observation from the gram stained smear of the colony?
Gram stained smear of the colonies confirm Gram positive yeast-like cells.
10. How will you identify Candida albicans?
Positive Germ tube test,
Chlamydospore formation on cornmeal agar.
11. What is Germ tube?
A filamentous, tube-like structure arising from the yeast cell is called a germ tube.
12. Name the other fungi producing germ tubes.
Candida dublinensis.
13. How will you do germ tube test?
When a small inoculum (≤105/ml) of Candida is made in 0.5ml rabbit, fetal calf or human
serum and incubated at 37oC for 2 hours, Candida albicans produce germ tube.
14. How many germ tubes should be observed before calling the isolate positive?
Minimum 5 germ tubes should be seen.
15. What is the phenomenon involved in germ tube test?
Reynaulds –Braude phenomenon.
16. What is chlamydospore?

They are the asexual resistant spores produced from the wall of the hyphae by Candida
albicans.
17. Name the media used for chlamydospore formation.
Corn meal agar.
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18. What is the significance of Candida albicans?
Candida can cause a variety of opportunistic infections in people who are debilitated,
immunosuppressed, or have received prolonged antibacterial therapy. Since C. albicans is a
normal flora, it must be clinically correlated.
19. How do you treat this condition?

Topical treatment include polyene antifungals (Nystatin), Imidazole antifungals (Clotrimazole)
or oral medications such as Itraconazole, Fluconazole, Ketoconazole can be given in resistant
or frequent relapse cases.
20. Which are the other infections/diseases caused by this fungus?

Candida can cause mucocutaneous infections (vaginalthrush, glossitis, cheilitis, oesophagitis,
balanitis, chronic mucocutaneous candidiasis, gastrointestinal candidiasis), cutaneous
infections (onychomycosis, paronychia, candidal granuloma, diaperrash, intertrigo) and
systemic infections (endocarditis, urinary tract infection, meningitis, endophthalmitis,
osteomyelitis, peritonitis/intra-abdominal abscess, arthritis, septicemia).
21. Which are the other species of Candida?

The various species of Candida include C. tropicalis, C. krusei, C. parapsilosis, C. stellatoidea,
C. pseudotropicalis, C. guilliermondii, C. dubliniensis, and C. glabrata.
22. Which are the other yeast-like fungi?

Trichosporon, Torulopsis, Geotrichum, Rhodotorula etc., are the other yeast like fungi.
23. What is the significance of pseudohyphae in C.albicans?
Presence of pseudohyphae indicates colonisation
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CRYPTOCOCCUS NEOFORMANS
AIM:To identify the given fungal culture.
OBSERVATION:
Gram stain: Gram positive spherical budding yeast cells surrounded by a clear halo. No
pseudohyphae.
India Ink preparation/Negative staining: Positive as capsules are present which appears as a
clear halo around the yeast cell.
CULTURAL CHARACTERISTICS:
SDA: Moist white coloured mucoid colonies on SDA at 370C.
NIGER SEED AGAR: Brown colonies
BLOOD AGAR: Moist white coloured mucoid colonies
KEY FEATURES:
 Urease positive.
 Non fermentative.
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 Brownish colonies on Niger seed agar due to melanin production.
INFERENCE:Based on the above identifying features, the given yeast is identified as

CRYPTOCOCCUS NEOFORMANS
1. Enumerate true yeasts?

Rhodotorula
Saccharomyces cerevisae
2. Name the infection caused by Cryptococcus?
Subacute meningitis.
3. What is the specimen collected for meningitis?
Approximately 3-5 ml of CSF is collected following lumbar puncture.
Blood may also be collected for culture.
4. Which are the necessary investigations to be performed?

The CSF sample is divided into three;
 First part for cell type and cell count,
 Second for protein and glucose analysis
 Third part for microbiological examinations.
If the CSF is not turbid, it should be centrifuged.
5. What are the microbiological investigations generally done for a CSF sample?
Microbiological examinations include a Gram stained smear, wet India ink mount, bacterial
and fungal culture and antigen detection.
6. What are the media used here? Elaborate as per the findings.
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CSF has been inoculated onto Sabouraud's dextrose agar or Niger seed agar and incubated
at 37oC for 1-2 days.
Large, cream-coloured, mucoid colonies were obtained on Sabouraud's dextrose agar
whereas brown colonies were seen on niger seed agar.
7. What will be your observation from the Gram stain findings?
Gram stained smear of CSF sediment will show polymorphonucelar leucocytes and Gram
positive budding spherical yeast cells will be seen.
8. Name the wet mount preparation used.

India ink wet mount preparation and would show spherical yeast cells with large capsules.
9. Name the biochemical test done.

Christensen’s urease test. The fungus is identified by positive urease test, as Cryptococcus
neoformans.
10. What are the main predisposing factors for Cryptococcosis?

In healthy individuals, cryptococcosis is often asymptomatic.
Resistance to cryptococcosis depends primarily on cell-mediated immunity.
Most cases of cryptococcal meningitis occur in patients with immunocompromised and
under immunosuppression.
11. How do you treat Cryptococcosis?

Cryptococcus meningitis is invariably fatal without appropriate therapy; death may occur
from 2 weeks to several years after symptom onset.
Antifungal drugs such as amphotericin B, flucytosine, fluconazole must be promptly given.
12. Which are the other infections/diseases caused by this fungus?

Following pulmonary infection, cryptococci disseminate widely and may infect any organ.
The organs most often involved include the CNS, bones, prostate, eyes, and skin. Other
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infections include myocarditis, chorioretinitis, hepatitis, peritonitis, renal abscess,
prostatitis, myositis and adrenal involvement.
13. Differentiate between pathogenic and non-pathogenic species?
Ability to :
Grow at 370C
Hydrolyse urea
Produce brown colonies on nigerseed agar
14. Two variants of Cryptococcus.

C.neoformans var.neoformans
C.neoformans var.gattii
15. Name the reservoir of Cryptococcus noeformans?

Pigeon droppings
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RHIZOPUS
AIM: To identify the given fungal culture
OBSERVATION:
1. It is a filamentous fungus possessing broad aseptate hyaline hyphae.

2. Sporangiophores are unbranched and arise in groups directly above the Rhizoids (Root like
structures arising from hyphae)
3. Sporangiophores arise in groups directly above the Rhizoids
4. At the tip of sporangiophore, it has an apical globular sporangium filled with colourless to
brown sporangiospores
5. Sporangia are supported and elevated by column shaped structure called Columella.
CULTURAL CHARACTERISTICS ON SDA:
OBVERSE SIDE: White cottony, wooly colonies with tube filling growth (hence called lid
lifters). Colonies become grey to brown black later due to sporulation giving rise to “Salt and
Pepper Appearance”
REVERSE: White in colour
INFERENCE: Based on the above features, the given fungal culture is identified as Rhizopus sp.,
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MUCOR
OBSERVATION:
1. It is a filamentous fungi possessing thin walled broad aseptate hyaline hyphae from which
sporangiophore arises.
2. Sporangiophores are branched at wide angles with short branches.
3. At the tip of sporangiophore, it has an apical globular sporangium filled with
sporangiospores
4. Sporangia are supported and elevated by column shaped structure called Columella.
5. Root like structures called Rhizoids are absent.
OBVERSE: White cottony, wolly colonies with tube filling growth (hence called lid lifters).
REVERSE: Light white coloured
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INFERENCE: Based on the above identifying features, the given fungal culture is identified as
Mucor spp
MUCOR AND RHIZOPUS
1. Name the fungi which invade the blood vessel.
Aspergillus
Mucor
Rhizopus
Absidia
2. What is mucormycosis?
Mucormycosis refers to several different diseases caused by infection with fungi in the
order Mucorales.
3. List down the causative agents of Mucormycosis.

Rhizopus species are the most common causative organisms followed by Mucor.
4. What are the risk factors for mucormycosis?

Immunocompromising conditions are the main risk factor for mucormycosis.
Patients with uncontrolled diabetes mellitus, especially with ketoacidosis,
Patients with cancer and as well as individuals receiving immunosuppressive agents.
5. Classify mucormycosis based on anatomic location.

 Rhinocerebral,
 Pulmonary
 Cutaneous
 Gastrointestinal
 Disseminated and
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 Uncommon presentations.
6. Which the necessary investigations to be performed for a suspected case of
Mucormycosis?
The specimen is subjected to a KOH mount.

The specimen is inoculated on Sabouraud's dextrose agar without cycloheximide and
incubated at room temperature for 1-2 days.
7. How do you treat this condition?
Aggressive treatment include surgical debridement of the necrotic tissue, restoration of acid-
base balance, correcting the glucose levels and treatment with antifungal agents such as
Amphotericin B.
8. Name two species of Rhizopus.

Rhizopus oryzae and Rhizopus microspores
9. Name the important species of Mucor.

Mucor racemosus.
10. What are the key features of Rhizopus?
The genus Rhizopus is characterised by

 presence of stolons and pigmented rhizoids.
 formation of sporangiophores, singly or in groups from nodes directly above
the rhizoids.
 globose sporangia.
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 Colonies are fast growing and cover an agar surface with a dense cottony
growth that is at first white becoming grey or yellowish brown with
sporulation.
11. What are the key features of Mucor?
The genus Mucor ischaracterised by
 Absence of stolons and pigmented rhizoids.

 Branched sporangiophores.
 Colonies are very fast growing, cottony to fluffy, white to yellow, becoming
dark-grey, with the development of sporangia.
12. How will you differentiate Mucor and Rhizopus morphologically?
Rhizopus possess rhizoids.
Mucor does not have rhizoids.
Absidia have internodal rhizoids
13. What is the best stain for the diagnosis of mucormycosis?
H & E stain
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ASPERGILLUS FUMIGATUS
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OBSERVATION:
OBVERSE: Granular to cottony,Smokygreen, rugouse colonies
REVERSE: White coloured colonies
MICROSCOPIC FEATURES:
1. It is a filamentous fungus with hyaline septate hyphae.

2. A club shaped vesicle is present at the tip of a conidiophore.
3. Upper third of the vesicle is covered by a single row of phialides /Sterigmata
(Uniserriate)
4. Spherical conidia arise from the phialides that bend inwards.
INFERENCE:
Based on the above identifying features, the given fungi is identified as Aspergillus
fumigatus
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ASPERGILLUS NIGER
OBSERVATION:
OBVERSE: The surface of the colony is covered by Jet black conidia
REVERSE: Buff / White coloured colonies
1. It is a filamentous fungus possessing septate hyaline hyphae.

2. A globose vesicle is present at the tip of a conidiophore.
3. The entire surface of the vesicle is covered by a double row of phialides
(Biserriate)/Sterigmata
4. The conidia are spherical & black that become roughened with maturity
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INFERENCE:
Based on the above identifying features, the given fungus is identified as Aspergillus niger.
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ASPERGILLUS FLAVUS
OBSERVATION:
OBVERSE: The colonies are granular to wooly with a shade of yellow/yellow green
REVERSE: White in colour
1. It is a filamentous fungi possessing hyaline septate hyphae.

2. A globular vesicle is present at the tip of a conidiophore.
3. Single or double row of phialides (uniseriate/biseriate) covering the entire surface of the
vesicle.
4. The conidia are spherical ,smooth/roughened on maturity
INFERENCE:
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Based on the above identifying features, the given fungi is identified as Aspergillus flavus
ASPERGILLUS SPECIES
1. Name the various species of Aspergillus causing human infections.

 Aspergillus fumigatus
 Aspergillus niger
 Aspergillus flavus
2. Which are the four most common manifestations of Aspergillus?
Aspergillus primarily affects the lungs,

 Allergic bronchopulmonary aspergillosis (ABPA)
 Chronic necrotizing Aspergillus pneumonia (also termed chronic necrotizing
pulmonary aspergillosis [CNPA])
 Aspergilloma
 Invasive aspergillosis
 Endocarditis
3. How are aspergilli transmitted?
The transmission of fungal spores to the human host is via inhalation.
4. Who are at higher risk of acquiring Aspergillus infections?

In patients who are severely immunocompromised, Aspergillus may hematogenously
disseminate beyond the lung, potentially causing endophthalmitis, endocarditis and
abscesses in the myocardium, kidney, liver, spleen, soft tissue, central nervous system
(CNS) and bone.
5. Name the causative agent of fungal endocarditis.

 Aspergillus and Candida
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 Aspergillus -related endocarditis and wound infections occur in the context of
cardiac surgery.
6. What is allergic bronchopulmonary aspergillosis (ABPA) ?
ABPA is a hypersensitivity reaction to A. fumigatus colonization of the tracheobronchial
tree and occurs in conjunction with asthma and cystic fibrosis (CF). Allergic fungal
sinusitis may also occur alone or with ABPA.
7. What is Aspergilloma?
Aspergilloma is a fungus ball (mycetoma) that develops in a pre-existing cavity in the lung
parenchyma.
8. What are the underlying causes of Aspergilloma?

 Underlying causes of the cavitary disease may include treated tuberculosis or other
necrotizing infection, sarcoidosis, CF and emphysematous bullae.
 The ball of fungus may move within the cavity but does not invade the cavity wall.
9. What is chronic necrotizing Aspergillus pneumonia?
 It is a subacute process usually found in patients with some degree of
immunosuppression.
 Because it is uncommon, it often remains unrecognized for weeks or months and
can cause a progressive cavitary pulmonary infiltrate.
10. What is invasive aspergillosis?

 Invasive aspergillosis is a rapidly progressive, often fatal infection that occurs in
patients who are severely immunosuppressed and immunocompromised.
 It is characterized by invasion of blood vessels, resulting in multifocal infiltrates,
which are often wedge-shaped, pleural-based and cavitary.
11. Why Aspergillus fumigatus is the most common cause of infection in human beings?
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Because of the ability of A. fumigatus, but not most other Aspergillus species, to grow at
normal human body temperature.
12. What are the key features of Aspergillus fumigatus?

The colonies are typically smoky-green with a vetlvetty to powdery surface and reverse is
white.
Philides arranged in a single row-Uniserriate
Columnar conidial heads with the phialides limited to the upper two thirds of the vesicle.
Conidiophores are short.
Conidia are hyaline
13. What are the key morphological features of Aspergillus niger?

 The colonies are black, cottony type and reverse is white.
 Vesicle is globular shaped.
 Philaides in two rows-Biserriate.
 Conidia arise from entire vesicle.
 Conidia are black.
14. Comment on Aspergillus niger.

 This species is very commonly found in aspergillomas
 It is the most frequently encountered agent of otomycosis.
 It is also a common laboratory contaminant.
15. What are the key morphological features of Aspergillus flavus?

 Colonies are yellowish green,velvetty and reverse is white.
 Phialides in one or two rows.
 Conidia arise from entire vesicle and hyaline
 Conidiophore rough-walled .
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16. Comment on Aspergillus flavus.
 They produce aflatoxins.
 Aflatoxin B1 is the most toxic of the many naturally occurring secondary
metabolites produced by fungi. They grow on almost any crop or food.
 It is a causative agent of otitis, keratitis, acute and chronic invasive sinusitis,
and pulmonary and systemic infections in immunocompromised patients.
 A. flavus is second only to A. fumigatus as the cause of human invasive
aspergillosis
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PENICILLIUM SPP.,
OBSERVATION:

OBVERSE: The colonies are flat velvety to powdery with greenish colour
REVERSE: Buff/White in colour
1. It is a filamentous fungi possessing hyaline thin septate hyphae.
2. There is no vesicle present at the tip of a conidiophore.
3. The conidiophore directly divides into elongated metulae from which phialides arise.
4. The phialides are flask shaped & give the brush border appearance
5. Chain of spherical conidia arises from the phialides.
INFERENCE:
Based on the above identifying features, the given fungi is identified as Penicillum spp.,
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PENICILLIUM
1. What is penicilliosis?
It denotes the group of infections caused by Penicillium species.
2. Name the important human pathogen of penicillium.
Penicillium marneffei
3. What is the characteristic feature of Penicillium marneffei?
It is a dimorphic pathogen causing opportunistic infections in HIV patients.
4. List down the other important species of Penicillium causing Mycotoxicoses.
Penicillium cyclopium , Penicillium verrucosum.
5. Name the invasive infections caused by Penicillium species.
Endophthalmitis, endocarditis
6. Name the superficial infections caused by Penicillium.
Otomycosis, keratitis & Onychomycosis
7. Name the allergic diseases caused by Penicillium.
Allergic pneumonitis & Asthma
8. What is the key feature of Penicillium marneffei?
The colony produces a typical brick red pigment.
9. What are the morphological features of Penicillium species?
 Colonies are usually fast growing, in shades of green,

 Microscopically, chains of single-celled conidia are produced from a flask shaped
phialides arranged in groups from branched metulae forming a penicillus.
 Conidiophores are hyaline, smooth or rough-walled.
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SEROLOGY
1.ASO (ANTI STREPTOLYSIN O ) TEST
INTRODUCTION:
Most of the strains of Streptococcus which are pathogenic to human beings belong to group A. All
such organisms produce an exotoxin known as streptolysin O, which induces the formation of anti-
streptolysin O (ASO) which is helpful in the diagnosis of streptococcal infections.
AIM:
To perform the anti-streptolysin O test in the given patient serum both by qualititative and
quantitative methods.
PRINCIPLE:
This is slide agglutination test. The reagent contains an aqueous suspension of polystyrene latex
particles which are sensitized with streptolysin O. These particles agglutinate in the presence of
ASO present in patient serum.
REQUIREMENTS:
 Test serum
 Positive control
 Negative control
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 Plastic dropper
 Special slide
 ASO latex reagent
 Applicator stick
 Discarding jar
PROCEDURE:
A.QUALITATIVE SLIDE TEST:
 The test sera, controls and reagents were brought to room temperature testing and mixed
gently prior to use.
 With the help of disposable plastic dropper, one drop of undiluted test serum was placed
within the circled area on the special slide provided kit. Also add positive and negative
controls in the respective circles.
 One drop of well mixed ASO latex reagent was added to the test serum, mixed well using
the applicator stick.
 The slide was slowly rocked and observed for macroscopic agglutination and observed
within two minutes.
OBSERVATION:
Agglutination was visible within two minutes. The results are reported in Todd units/ml or
international units (IU) /ml.
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RESULT:The given test sera is positive for ASO test and corresponds to a titre of more than 200
IU/ml. or Todd units/ml.
B. SEMI QUANTITATIVE SLIDE TEST
1. The dilutions of serum sample was prepared with normal saline as follows:
2. Take 6 test tubes, label them 1-6 and keep them in a rack.
3. Pipette 0.5ml normal saline in each tube.
4. Pipette 0.5 ml of test serum in tube 1 and mix well. (Serum dilution 1:2)
5. Take 0.5 ml of diluted serum from test tube 1 and add to tube 2.Mix well and transfer
0.5.ml to test tube 3, mix well and go on adding 0.5ml of diluted serum to next tube till
tube 6 is reached.
6. The dilution obtained in these 6 tubes are 1:2,1:4,1:8,1:16,1:32,1:64 respectively. With the
help of disposable plastic dropper, one drop of diluted serum was dispensed into one of the
circled area on the special slide provided.
7. One drop of well mixed ASO latex reagent was added to all the circles.
8. Test sera (diluted) and reagent were mixed well using the applicator sticks.
9. The slide was rocked slowly for agglutination and observed within two minutes
Serum dilution IU/ml
1:2 400
1:4 800
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. 1:8 1600
1:16 3200
1:32 6400
OBSERVATION:
Record the dilution upto which agglutination was observed.
RESULT:
The ASO titre of the given test serum is --------- IU/ml.
ASO (ANTI STREPTOLYSIN O)
1.What is ASO?
Anti Streptolysin O
2. What is Streptolysin O?
It is a hemolysin O, heat labile protein produces complete hemolysis, strongly antigenic
and induces brisk antibody response, produced by group A, C, & G.
3. Name the conditions in which ASO titre is elevated?
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1. Rheumatic fever
2. Glomerulonephritis
3. Scarlet fever
4. Why this hemolysin is known as Streptolysin O?
Because it is Oxygen – labile
5. What is the significant ASO titre?
More than 200 Todd units /ml (or) 200 IU/ml
6. What is the significant titre indicates?
It indicates recent /recurrent infection with streptococci
7. Why plasma should not be used for ASO test?
Fibrinogen in the plasma can lead to non specific agglutination of the latex particles
8. What are the supporting evidences for acute Rheumatic fever?
 Positive throat culture for group A streptococci
 Recent scarlet fever
 Increased titre of ASO, CRP and ESR
9. What are the disadvantages of ASO?
ASO titre also rises in C and G infections

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10. Why ASO titre is not raised in skin infections?
ASO response following skin infection is poor because inactivation of antigen by the
cholesterol present in the skin.
11. WhyASO increased in Rheumatic fever?
Antigenic cross reactions between Streptococci and heart tissues.
12. What are the infections caused by Streptococcus pyogenes?
a) Suppurative:
 Respiratory infections - scarlet fever, pharyngitis, tonsillitis, otitis media
and Streptococcal pneumonia
 Skin and soft tissue infections – Erysipelas, impetigo, cellulitis,
Necrotizing fasciitis
b) Non Suppurative:
 Acute glomerulo nephritis.
 Acute rheumatic fever.
13. What are the sequalae in post Streptococcal infections?
 Acute rheumatic carditis
 Nephrotic syndrome
14. What is agglutination?
When a particulate antigen reacts with its antibody in the presence of electrolytes at an
optimum temperature and pH, forms an insoluble clumps.

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15. What is precipitation?
When a soluble antigen reacts with its antibody in the presence of electrolytes at an
optimum temperature and pH, forms an insoluble precipitate.
16. Which factors inhibit the activity of Streptolysin O?
 Cholesterol in the serum
 Bacterial contamination of the serum
 Chemical treatment of the serum
17. Anti-deoxyriboniclease B increased titre is significant in which condition?
Skin infection in retrospective diagnosis
18. What is the significant titre of anti-deoxyribonuclease B?
Titres higher than 300 or 350 are significant
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2.C REACTIVE PROTEIN (CRP)
INTRODUCTION :
CRP is an abnormal protein (β globulin). It appears in acute phase of the disease. It is known as C-
reactive protein , because it precipitates with C antigen of pneumococci. It is raised in bacterial
infections, inflammation, malignancy and tissue destruction. Monitoring the levels of CRP in
patient’s sera indicates the effectiveness of treatment and the assessment of patient recovery.
AIM:
To perform C-reactive protein latex test for the detection and the titre of C-reactive protein in
human serum .
PRINCIPLE:
It is a passive agglutination test using latex particles coated with antibodies to human CRP. When
the latex suspension is mixed with serum containing elevated CRP levels on a slide, clear
agglutination is seen within two minutes if CRP concentrations are greater than 6mg/L.
REQUIREMENTS:
 Patient serum
 Test card
 CRP latex reagent
 Disposable stirrer
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PROCEDURE:
 Bring the test reagents and sera to room temperature.
 Transfer one drop of patient’s serum to the circle on the slideand add positive and negative
control sera in the respective circles .
 Add one drop of latex CRP reagent (antigen) to the circle .
 Mix the drops using a disposable stirrer gently and evenly.
 Observe for for agglutination within 2 minutes
INTERPRETATION:
• Agglutination visible within two minutes is to be interpreted as positive reaction.
• No agglutination is to be considered as a negative reaction.
• Sensitivity of the test is 6 mg/L.
OBSERVATION:
Agglutination was visible within two minutes. The results are reported as mg/dl.
RESULT:
The given test serum may show agglutination if the CRP levels are more than 6mg/dl.
If agglutination observed, it is further proceeded for a quantitative test.
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SEMI QUANTITATIVE TEST
ADDITIONAL REQUIREMENTS:
 Test tubes of 9 mm diameter
 Isotonic saline.
 CRP Latex reagent .
 Test sera
 Micropipettes
 Test card.
PROCEDURE:
tube 6 is reached.
5. The dilution obtained in these 6 tubes are 1:2,1:4,1:8,1:16,1:32,1:64 respectively and from
final dilution 0.1ml is discarded.
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6. Place one drop of diluted serum sample using separate plastic droppers in each circle of the
slide.
7. Add one drop of latex gamma globulin reagent in each of the circle mix well with
applicator stick.
8. Rock the slide gently back and forth for two minutes and observe for agglutination
9. The test procedure was repeated for each dilution as described above.
10. The serum CRP concentration can then be calculated approximately by multiplying the
dilution factor (i.e., 2,4,6,8, 16 or 64) by the detection limit. i.e., 6 to give the mg/dl
concentration.
OBSERVATION:
Agglutination was observed till the --------- tube.
RESULT:
The approximate serum CRP level is ------x6 = ------ mg/dl.
C - REACTIVE PROTEIN (CRP)
1. What is CRP?
CRP is an abnormal protein (β-globulin)
2. What is the nature of this protein?
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β-globulin
3. Where CRP is synthesised?
Hepatocytes in the liver
4. What is the principle of CRP test?
Passive agglutination test
5. Why it is known as C-reactive protein?
Because it precipitates with the C antigen of Pneumococci
6. What is the upper limit of CRP?
6 mg/dl
7. What is the diameter of the latex particle?
0.8-1µm
8. Which are the carrier particles used other than latex?
Bentonite, gelatine and RBC
9. What is the significant titre of CRP?
6mg/dl
10. What is the diagnostic importance of CRP?
Detects the activity of inflammation and also prognostic indicator
11. Name the test based on the principle of latex agglutination?
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ASO, CRP, RF and HCG detection
12. What are the conditions in which CRP level is elevated?
Bacterial infection, inflammation, malignancy and tissue destruction
13. Mention the other acute phase reactant proteins?
Procalcitonin, Haptoglobin, Mannose binding protein, Serum amyloid A, α 1 acid
glycoprotein.
3. RHEUMATOID (RA) FACTOR
INTRODUCTION:
Rheumatoid factors are anti-globulins of the IgG, IgA or IgM which forms immune complexes in
serum or joint fluids. This result in complex deposition in tissues where blood flow is restricted
resulting in dermatitis (skin), nephritis (kidney), synovitis and arthritis (joints), vasculitis (vessels
in many tissues). So testing for RF has a high diagnostic value.
AIM:
To detect the rheumatoid factor (auto-antibody) in the given patient’s serum and to quantitate them
if they are present.
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PRINCIPLE:
Latex particles (polystyrene particles) coated with human gamma globulin (IgG) are agglutinated
when mixed with samples containing rheumatoid factors. Distinct agglutination indicates the
presence of rheumatic factors (RF).Patients specimens that do not react with the latex particles
contain either no rheumatoid factors or concentration below 20 IU/ml. Elevated RF titres often
indicate a severe course and a generalized pathological process.
REQUIREMENTS:
 Latex reagent coated with human gamma globulin.
 Test sera
 Slide dropper
 Applicator stick.
PROCEDURE:
QUALITATIVE METHOD:
1. Bring the patient serum and reagents to room temperature.
2. Apply one drop of patient serum undiluted, one drop of positive and one drop of negative
control serum onto separate circles of the test plate.
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3. Place one drop of the aqueous suspension of the latex reagent to each patient serum and
controls. Mix well with a stirring rod.
4. After 2 minutes, examine for agglutination. Do not examine beyond two minutes.
INTERPRETATION:
 Agglutination visible within two minutes is to be interpreted as positive reaction.
 No agglutination is to be considered as a negative reaction.
 Sensitivity of the test is 20 IU/ ml.
RESULT:
The given test serum is positive/negative for RF.
Marked agglutination indicates the presence of rheumatoid factor (≥ 20IU/ml).
QUANTITATIVE SLIDE TEST
ADDITIONAL REQUIREMENTS:
 Test tubes of 9 mm diameter
 Isotonic saline
 Latex reagent coated with human gamma globulin.
 Test sera
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 Micropipettes
 Test card.
PROCEDURE:
tube 6 is reached.
5. The dilutions will be 1:4,1:8.1:16,……… and from final dilution 0.1ml is discarded.
6. Place one drop of diluted serum sample using separate plastic droppers in each circle of the
slide.
7. Add one drop of latex gamma globulin reagent in each of the circle mix well with
applicator stick.
8. Rock the slide gently back and forth for two minutes and observe for agglutination.
INTERPRETATION:
Agglutination in the highest serum dilution corresponds to provide approximate amount of
rheumatoid factor concentration present in the serum.
Concentration of RF can be calculated by
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RF in IU/ml = Sensitivity of the test x highest dilution of the serum showing agglutination.
OBSERVATION:
Agglutination was observed till the --------- tube.
Titre of patient serum = X, The RF concentration in the given patient serum is 20 IU/ml* X
RESULT:
The approximate serum RA level is ------ x 20 = ------ IU/ml.
CLINICAL SIGNIFICANCE:
The rheumatoid factor test is done in order to detect the presence of rheumatoid factors in the
serum of patients with rheumatoid arthritis. Higher titre of RF is usually related to the severity of
rheumatoid arthritis. Rheumatoid factor may be present in low concentrations in 3.5 percent of the
normal population. This percentage increases with the age of population. This factor is non
specific. Positive results may occur occasionally in various pathological disease states including
systemic lupus erythematosus, hepatitis, cirrhosis, lymphomas, hyper-gammaglobulinaemia,
scleroderma and sarcoidosis. Quantitative analysis helps to monitor the therapy of rheumatoid
arthritis
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RHEUMATOID (RA) FACTOR
1. What is Rheumatoid factor?
It is an anti gamma globulin autoantibody acts as an antibody to human IgG
2. Rheumatoid factor belongs to which class of immunoglobulin?
Mostly IgM, may also be IgG or IgA.
3. What are auto antibodies?
These are antibodies produced against self antigens.
4. What is the principle of this test?
Passive agglutination
5. Name the disease in which RF is used as the diagnostic test?
Rheumatoid arthritis
6. Name the other conditions where RF test is positive?
SLE, hepatitis, dermatomyositis, cirrhosis, syphilis
7. What is nature of the latex Rheumatoid factor antigen?
It is an aqueous suspension of polystyrene particles coated with human IgG
8. Which test was used earlier for detection of RF?
Rose waaler test
9. What is amboceptor?
Sub agglutinating dose of rabbit anti sheep erythrocyte antibody
10. What is Rheumatoid arthritis?
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Symmetrical polyarthritis with muscle wasting with subcutaneous nodules with
involvement of small joints with residual deformity and disseminated lesions like serositis,
myocarditis.
11. What is the significant titre for RF test?
≥20 IU/ml
12. Classify Autoimmune diseases?
a) Localised (organ specific) – Hashimoto’s disease, Graves disease, Addison’s disease,
pernicious anaemia , Myasthenia gravis
b) Systemic (non-organ specific) – SLE, Rheumatoid arthritis, polyarthritis nodosa,
Sjogren’s syndrome.
c) Hemocytolytic - Autoimmune haemolytic anaemia.
13. What are the differences between Rheumatic and Rheumatoid arthritis?
 Rheumatic arthritis – migrating polyarthritis involving major joints, without
deformity
 Rheumatoid arthritis – symmetric polyarthritis involving minor joints with muscle
wasting and deformity.
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4.RPR (RAPID PLASMA REAGIN) TEST
AIM:
To screen for primary syphilis by detecting the reagin antibodies in the given patient
serum.
PRINCIPLE:
It is a non-treponemal test used to detect syphilis. This test involves an RPR antigen
suspension which is a carbon containing cardiolipin-lecithin antigen. It detects the reagin antibody
present in the serum of individuals with syphilis. When a specimen contains antibody, flocculation
occurs due to co-agglutination of carbon particles of the RPR antigen which appears as black
clumps against the white background of the card. This co agglutination is read macroscopically.
REQUIREMENTS:
 Test card
 Dispensing needle
 Syringe
 Cardiolipin antigen
 Patient serum
 Mechanical rotator
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PROCEDURE:
A.SCREENING TEST:
1. Place one drop (50 µl) of serum onto a circle. Spread the drop to fill the circle..
2. Add one drop of positive and negative controls onto separate circles of the test plate.
3. Add one drop of RPR reagent
4. Rotate the test card smoothly on the palm of the hand for 8 minutes (or rotate on a
mechanical rotator).
5. Observe for flocculation under bright lightwhich is visible with naked eye.
INTERPRETATION:
After 8 minutes rotation, inspect the card in good light. Turn or tilt the card to see whether
there is clumping (reactive result).Test cards include negative and positive control circles
for comparison.
Reaction type Appearance Interpretation
Non-reactive No clumping Non-reactive for syphilis.
Reactive Highly visible clumping Reactive for syphilis
Weakly reactive Minimal clumping Reactive for syphilis.
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II. QUANTITATIVE SLIDE TEST:
Additional requirements:
 Micropipette (1000 µl) with tips.
 Normal saline (0.95%)
 6-8 test tubes per reactive serum
 Rubber teats.
PROCEDURE:
A. Preparing sera in dilutions:
a. Take 6 test tubes, label them 1-6 and keep them in a rack.
b. Pipette 0.5ml normal saline in each tube.
c. Pipette 0.5 ml of test serum in tube 1 and mix well. (Serum dilution 1:2)
d. Take 0.5 ml of diluted serum from test tube 1 and add to tube 2.Mix well and
transfer 0.5.ml to test tube 3, mix well and go on adding 0.5ml of diluted serum to
next tube till tube 6 is reached.
e. The dilution obtained in these 6 tubes are 1:2,1:4,1:8,1:16,1:32,1:64 respectively.
B. Performing quantitative testing of diluted sera:
a. Take a RPR card and add 0.5 ml of serum from the sixth tube on one circle.
b. Similarly add 0.5 ml of serum from the tube no: 5, 4, 3, 2 and 1 in the remaining
circles respectively.
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c. Positive and Negative controls for each qualitative test should be incorporated.
d. Add 1 drop of RPR antigen to each circle.
e. Rotate the card on a RPR/VDRL rotator for 8 min , rotating at a speed of 180 rpm.
f. Observe the card for flocculation.
g. Report the titre as the highest dilution of serum that shows a reactive result.
INTERPRETING TEST RESULTS:
RPR RPR TITRE
Active infection + >1:8
Latent syphilis + Often <1:4
Successful treatment + or - +/- 2 titres decrease
(e.g., from 1:16 to 1:4)
VDRL/RPR TEST
1. What is VDRL Test?
It is the non treponemal slide flocculation screening test for the detection of syphilis. It is
nonspecific test because it uses only cardiolipin antigen.
2. What is the basic principle of VDRL Test?
When the patient's serum containing regain antibodies are mixed with cardiolipin antigen,
flocculation occurs.
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3. What type of test is VDRL?
Slide flocculation test.
4. What is the microorganism causing syphilis?
Treponema pallidum.
5. What are the various stages of syphilis?
•Primary syphilis.
•Secondary syphilis.
•Tertiary syphilis.
6. What are the samples collected for VDRL?
• Blood-serum, plasma
• CSF
7. What is the antigen used in VDRL Test? From where it is got?
Purified mixture of cardiolipin, lecithin and cholesterol.Extracted from beef heart.
8. What are BFP (Biological false positive) reactions?
Since the reagin antibodies can also found in patient with unrelated diseases are called BFP
reactions.
Eg: SLE/other collagen diseases , leprosy/malaria/hepatitis /relapsing
fever/HIV/pregnancy/IV drug abusers.
9. What are the modifications in VDRL testing?
Rapid plasma reagin test(RPR).
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10. What is prozone phenomenon? How to nullify them?
It is important in clinical serology, as sera rich in antibody (high titre sera) gives false
negative agglutination. To nullify use several dilutions.
11. What is the quantitative test in VDRL?
For quantitative reporting, the end point is given as the antibody titre . For eg: reactive in
1:4 dilution is reported as “Reactive 4 dilution” or R4.
12. What is the sensitivity of VDRL testing in various stages of syphilis?
•Primary stage: 75- 85%
•Secondary stage: 100%
•Latent stage: 75%
•Late: 71%
13. What do you mean if the test is Non Reactive?
•Negative for syphilis.
•Early primary stage.
•Sero conversion after treatment.
•Impaired Immune system of a patient
14. What are the other tests to confirm syphilis?
•FTA-ABS-Flourescent Treponemal Antibody Absorption test.
•TPHA-Treponema Pallidum Hemagglutination Assay- confirmatory test.
•TPHA-MHA-TP-Micro Hemagglutination test
• EIA.. Enzyme Immuno Assay
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15. What are the confirmatory tests for Neurosyphilis?
•FTS-ABS test.
•PCR.
17. What are the criteria for CSF collection in suspicious neurosyphilis?
•Meningeal involvement.
•Tabes dorsalis.
•General paralysis of insane.
•Any other neurological manifestations after the latent stage.
18. What are the criteria for pregnancy testing in syphilis
Screening by VDRL/RPR:
•I trimester
•III trimester
•At delivery
• High risk of exposure
20. What is the best test of choice in late latent syphilis?
PCR is the test of choice in late latent stage as both VDRL/RPR, treponemal / non -
treponemal tests become false negative.
21.What are the differences/advantages in VDRL over RPR?
VDRL RPR
Serum & CSF both can be tested Only serum can be tested
Serum inactivation is necessary No need for inactivation
Interpretation on microscope Naked eye examination
Neurosyphilis can be screened Not possible
Antigen contain no carbon particle Antigen contain fine carbon particles
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22. What do you mean by heterophile antigen?
The same or closely related antigens may occur in different biological species, classes and
Kingdoms -which are utilised for diagnostic serology.
Eg:
•Forssman antigen
•Weil-Felix reaction
•Paul Bunnel test
23. Define flocculation reactions in serology.
When a soluble antigen combines with its specific antibody in the presence of
Electrolytes (Nacl) at suitable temperature and pH, the antigen and antibody complex forms
insoluble precipitate which instead of sedimenting, suspended as flocculus , the reaction is
called as flocculation.
24. How to interpret the endpoint in VDRL testing?
Reaction is read under low power microscope, visible clumps on combing with reagin
antibody. Reported quantitatively as “reactive” / “weak reactive” and “non reactive”. In
quantitative reporting serial dilutions to report as titre of sera as reactive 4 dilution, /R4.
25. Give examples for carrier particles in serology testing.
To make the visible endpoint during agglutination serological tests,we use carrier proteins
like carbon particles ,RBCs, Bentonite.
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26. What are the other treponema?
•Treponema endemicum
•Treponema pertenue
•Treponema carateum
28. What is dark ground examination?
Very slender organisms like spirochaetes are examined under increased resolution by
improving the contrast using reflected light only falling on the object. The dark ground
microscope contains the condenser with central circular stop, so the object appears self
luminous against a dark background. Wet films are prepared from the exudate of the base of the
leison after applying thin coverslips examined under dark ground microscope.
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5.WIDAL TEST
AIM:
To determine the amount of antibodies against the typhoid bacilli in the patients serum by
agglutination method.
PRINCIPLE:
It is an agglutination test where H and O antibodies against S.typhi and S. paratyphi A & B are
detected and measured in the patients sera by using O & H antigens.
SELECTION OF ANTIGENS IN WIDAL TEST:
Typhoid is caused by any one of the four organisms namely S.typhi, S.paratyphi A, S.paratyphi B
and S.paratyphi C. S.paratyphi C is not common in India, hence not used. Each organism possess
O and H antigens. So there must be six antigens used, namely TO, TH (S.typhi), AO, AH, BO and
BH. The O antigens are related to each other due to sharing of factor 12.
Hence use of one O antigen eg. TO is sufficient. So the antigens used in the WIDAL test are TO
antigen, TH antigen, AH antigen and BH antigen. The phase I H of A and B are used.
ANTIGENS :S.typhi O, S.typhi H, S.paratyphi AH and S.paratyphi BH.
SAMPLE MATERIAL: Serum
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PROCEDURE:
QUALITIATIVE METHOD (SCREENING SLIDE TEST):
 Place one drop of undiluted patient serum into each of the first four circles and add one drop of
positive control and negative control normal saline in each of the two circles respectively.
 Add one drop of O, H, AH, and BH antigens on the first circles and one drop of any antigen in
remaining two circles respectively.
 Mix the contents of each circle with separate applicator stick and spread well.
 Rock the slide for one minute and observe for agglutination.
INTERPRETATION:
 Agglutination visible within a minute is to be interpreted as positive reaction.
 No agglutination is to be considered as a negative reaction.
 If agglutination appears in any one of the first four circles, one has to do quantitative test to
determine the antibody titre value.
SEMI-QUANTITATIVE TEST:
1. Pipette one drop of isotonic saline into the first reaction circle and then place 5, 10, 20, 40, 80 ul
of the test sample on the remaining circles.
2. Add to each reaction circle, a drop of the antigen which showed agglutination with the test
sample in the screening method.

158
3. Using separate mixing sticks mix the contents of each circle uniformly over the reaction circles.
4. Rock the slide gently back and forth; observe for agglutination macroscopically within one
minute.
INTERPRETATION:
 Agglutination is a positive test result and if the positive reaction is observed with 20 ul of test
sample, it indicates presence of clinically significant levels of the corresponding antibody in the
patient serum.
 No agglutination is a negative test result and indicates absence of clinically significant levels of
the corresponding antibody in the patient serum.
REPORTING :
 The Widal test should be reported by giving the titre for both O and H antibodies.
 The titre of each serum was read as the highest serum dilution giving visible agglutination.
QUANTITATIVE TUBE METHOD:
METHOD:
Tube agglutination method.
Dreyer’s tube used for H agglutinins (conical bottom),
Felix tube for O agglutinins (round bottom).

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PREPARATION OF MASTER DILUTION:
 Take a test tube
 Place 2.3ml of saline
 Add 0.2 ml of patient’s serum. This gives 1:12.5 dilution.
ARRANGEMENT OF TUBES:
 Take a test tube rack with four rows each.
 Keep 7 test tubes in each row.
 Label the rows as - a) TO b) TH c) AH and d) BH.
 From the master dilution, add 0.2 ml into the first and second tube in all rows of tubes
TO,TH, AH and BH.
 Add 0.2 ml of physiological saline (0.85% NaCl) into all tubes from 2-7 in all rows
including the controls (7 thtube).
DILUTION OF SERUM:
 Carry out the dilution as follows.
 Mix and transfer 0.2ml from tube 2 to 3 then from 3-4 etc., through tube 6.Discard 0.2 ml
from tube 6.
 Follow the same dilution for the TH, AH and BH rows.
ADDITION OF ANTIGEN:
 For the row TO, add 0.2 ml well mixed TO antigen into tubes 1-7.
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 For the row TH, add 0.2 ml well mixed TH antigen into tubes 1-7.
 For the row AH, add 0.2 ml well mixed AH antigen into tubes 1-7.
 For the row BH, add 0.2 ml well mixed BH antigen into tubes 1-7.
 The final dilution obtained are 1:25, 1:50, 1:100, 1:200, 1:400 and 1:800.Tube 7 in each
row acts as a negative control.
 For positive control proceed with the same dilutions with TO, TH, AH and BH positive
serum.
Widal test showing titre of TO 1:160 and TH 1:320
INCUBATION:
Incubate the rack at 37°C for 18-24 hours and read the results.
READING THE RESULTS:
H agglutination leads to the formation of loose , cotton wooly clumps.
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O agglutination is seen as disc like granular chalky clumps spread at the bottom of the tube.
Titre: The highest dilution of the sera at which agglutination occurs is taken as the antibody titre.
INTERPRETATION:
 The sample which shows the titre of 1:100 or more for O agglutinations and 1:200 or more for H
agglutination should be considered as clinically significant (active infection)
 Demonstration of 4-fold rise between the two titre is diagnostic.
 Agglutinin starts appearing in serum by the end of 1st week with sharp rise in 2nd and 3rd week
and the titre remains steady till 4th week after which it declines.
WIDAL TEST
1. What is WIDAL test?
Widal test is a tube agglutination test which detects the presence of serum agglutinins (H and
O) in patients serum with typhoid and paratyphoid fever.
2. Who developed Widal test?

It was developed by Fernand Widal in 1896.
3. When shall WIDAL test will be positive?Why?
The Widal test is positive after the tenth day of the disease and may be false positive if an
individual previously received a TAB vaccine. Salmonella antibody starts appearing in
serum at the end of first week and rise sharply during the 2rd week of endemic fever.
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4. How many serum samples should be tested to demonstrate infection?
It is preferable to test two specimens of sera at an interval of 7 to 10 days to demonstrate a
rising antibody titre.
5. What is the principle of the Widal test?
Tube agglutination test for enteric fever in which antibodies against S.typhi, S.Para A and
Para B infections are detected in patient’s serum.
6. Name the antigens used in Widal test?
TO,TH, AH, BH
7. Why Paratyphi O antigen is not employed? (OA,OB)
Because it cross reacts with Salmonella Typhi O antigen due to sharing of factor 12.
8. Enumerate various antigens of Salmonella?
 flagellar antigen(H)
 somatic antigen(O)
 fimbrial antigen(F)
 surface antigen (Vi,M)
9. Which of the somatic or flagellar antigen is more antigenic?
Flagellar antigen is more antigenic
10. Which antigen is heat labile?
H (flagellar) antigen
11. When do agglutinins (antibodies) appear during the course of enteric fever?
Second week
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12. Which agglutinins indicate recent infection?
Appearance of O antibodies
13. What is anamnestic response? How it differentiates from enteric fever?
Transient rise of O & H agglutinins of Salmonella during unrelated fever (malaria,dengue).
Rise is transient in anamnestic response whereas in enteric fever there is sustained rise of
antibody level in the serum..
14. How do you diagnose enteric fever?
 first week – blood culture
 second week – widal test
 third week - stool culture
 fourth week – urine culture
15. Which test is ideal to detect enteric fever during first week of infection?
Blood culture
16. What is ‘Castaneda Method’ of blood culture? What is the advantage of using it?
It is a type of blood culture method in which biphasic medium containing bile broth and agar
slant in a single blood culture bottle is used. This method is adopted to eliminate the chances
of contamination during repeated subcultures and also for economy and safety.
17. What is clot culture? What is the advantage?
An alternative method to blood culture is the clot culture in which 5ml of blood is
drawn into a sterile tube and is allowed to clot; the serum is pipette off and the clot is broken
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by a sterile glass rod. It is added with bile broth and incubated. Clot culture yields higher rate
of isolation of bacteria as the bactericidal effects of the serum is obviated.
18. Enumerate the conditions in which Widal test gives false positive reactions?
 Previous exposure to infection
 Anamnestic response
 prior immunisation with TAB vaccine
19. Enumerate the conditions in which Widal test gives false negative reactions?
 Patient on prior antibiotic therapy
 Widal test done in first & fourth week of infection
 Prozone phenomenon
20. How much titre is diagnostic in Widal test?
 O agglutinin ≥ 1:100
 H agglutinin ≥ 1:200
21. Mention the name of the tubes used in tube Widal test?
 Dreyer’s tube with Conical bottom for H Agglutinins - (DCH)
 Felix tube with Round bottom for O Agglutinins - (FOR)
22. Mention the patterns of O and H agglutinins formed in Widal test?
 H – Large, loose, fluffy cotton woolly clumps
 O - Compact, chalky, Granular clumps
23. What is endemic titre for Typhoid fever?
It is the baseline titre value present in a population living in Typhoid endemic area which is due to
previous infection or immunisation with typhoid vaccines or unrelated fevers.

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24. Which agglutinin is used to indicate the carrier state?
Vi agglutinins ≥ 1:10
25. How do you diagnose carrier state of Salmonella?
 Demonstration of Vi agglutinins (≥1:10)
 faecal culture
 bile culture
 urine culture
 Sewer swab technique /culture through Millipore membrane
26. Where do Salmonella present in the carrier state?
Biliary tract, Urine and faeces
27. Why do we collect repeated samples in a carrier?
Because of intermittent shedding of the bacilli
28. What is the main advantage of detection of carriers?
Helps in screening food handlers and cooks. So, detection of carriers is important for
epidemiological and public health purposes.
29. What is the treatment for chronic carriers?
 Antibiotics
 Cholecystectomy
30. Who is a famous typhoid carrier?
Typhoid Mary, a New York cook who caused 7 outbreaks over a period of 15 years affecting
200 persons
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31. What are the antibiotics used in the treatment of enteric fever?
• Third generation cephalosporins – Ceftriaxone (DOC) for empirical treatment
• Azithromycin
• Fluoroquinolones – Ciprofloxacin.
32. How do you prevent the disease?
By maintaining good hygiene (proper hand washing), vaccination and elimination of
carriers
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ENZYME LINKED IMMUNOSORBENT ASSAY (ELISA)
Eg; DETECTION OF HEPATITUS B SURFACE ANTIGEN (HB S Ag)
INTRODUCTION
Hepatitis B surface antigen is associated with type B viral hepatitis. In order to reduce the
incidence of post transfusion hepatitis, testing for HBS Ag is mandatory for blood products
intended for human use.
PRINCIPLE
It is based on ‘Sandwich’ enzyme labelled immunosorbent assay. Each test sample and
control is incubated in a well coated with polyclonal antibody to HBS Ag. If HBS Ag is present in
the serum ,it will combine with the antibody in the well. The well is then washed by using a wash
solution to remove residual test sample.Afterwards antibody to HBS Ag labelled with horseradish
peroxidase (HRP) (conjugate) is added to the well.The conjugate will bind immunologically to the
HBS Ag forming a ‘sandwich’ consisting of antibody to HBS Ag- HBS Ag conjugate.The well is
washed again and then incubated with tetra methyl benzidine (TMB).The enzyme present in the
sandwich ,hydrolyses TMB and yellow color is formed which can be read in a spectrophotometer
at 450 nm.
REAGENTS
The ELISA Kit contains the following reagents:
 Antibody wells
 Conjugate
 Substrate
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 Wash solution
 Stop solution
SPECIMEN: Serum
TEST PROCEDURE:
1. Bring the reagents and test specimens to room temperature. Shake well before use.
2. Add positive and negative controls
3. Place 100 µl of sample, mix well.
4. Keep at 37°C for one hour .
5. Aspirate the contents of the well. Wash the well with wash solutions. Drain the well completely.
6. Place 100 µl of conjugate in each well.
7. Keep at 37°C for 30 minutes
8. Aspirate the contents of the well. Wash the well with wash solutions. Drain the well completely.
9. Place 100 µl of substrate in the well.
10. Keep at 37°C for 30 minutes
11. Add 100 µl of stop solution.
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12 Read absorbance at 450 nm.
INTERPRETATION OF RESULTS:
 Test O.D value should be compared with cut off O.D value.
 If the test OD value is equal or greater than the cutoff O.D value, then it should be
considered as HBS Ag positive.
 If the test OD value is lesser than the cutoff O.D value, then it should be considered as HB S
Ag negative
DENGUE IgM Capture ELISA
Purpose:
NIV Dengue IgM capture ELISA kit is intended for qualitative detection of
IgM antibodies in serum of patients presenting clinical signs and symptoms
consistent with dengue.
Principle:
IgM antibodies in the patients serum, are captured by anti human IgM
coated on to the solid surface (wells).In the next step, Dengue antigen is added
which binds to captured human igM in the sample. Unbound antigen is removed
during the washing step. In the subsequent step biotinylated flavivitus anti DEN
monoclonal antibodies are added followed by AVIDIN-HRP. Subsequently,
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chromogenic substrate (TMB/H2O2) is added the reaction is stopped by 1N
H2SO4. The intensity of color / optical density is measured at 450 nm.
Reagents:
No. Contents
1. Anti human 12 strips with 8 wells each
IgM coated
wells (96)
2. Sample One bottle
diluents for
DEN Gimp (60
ml)
3. Wash buffer One bottle
concentrate
20X (60 ml)
4. Dengue one vial
antigen (6ml)
5. Anti DEN one vial
Monoclonal
antibody
(biotin
labeled) (6ml)
6. Avidin –HRP One vial
(6ml)
7. Liquid TMB One vial
Substrate
(12ml)
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8. Stop solution One vial
9. DEN Gimp One vial
Positive
Control (0.8
ml)
10. DEN IgM One vial
Negative
Control
(0.8ml)
Equipment’s:
Elisa reader
Elisa washer
Micro pipette
Titer plate
INTERPRETATION OF RESULTS:
 Test O.D value should be compared with cut off O.D value.
 If the test OD value is equal or greater than the cutoff O.D value, then it should be
considered as Dengue positive.
 If the test OD value is lesser than the cutoff O.D value, then it should be considered as
Dengue negative
ELISA
1. Expand ELISA.
Enzyme Linked Immunosorbent Assay
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2. What can you detect by using ELISA?
Antigen or Antibody
3. Give some examples for enzymes and substrates used in ELISA.
 Enzymes – alkaline phosphatase, horse radish peroxidase, β-Galactosidase

 Substrates – Para-Nitrophenyl Phosphate, Tetra Methyl Benzidine (TMB) ,O-
Phenylene diamine dihydrochloride.
4. Which colour formation indicates positivity in ELISA?
Yellow colour
6. How is the interpretation done in ELISA?
By measuring OD value (Optical Density)
7. What are the different types of ELISA?
Indirect ELISA
Sandwich ELISA
Competitive ELISA
Capture ELISA
Cylinder or Casette ELISA
8. Name the types of Sandwich ELISA.
 Single antibody or Direct Sandwich ELISA
 Double antibody or Indirect Sandwich ELISA
9. What is the difference between EIA and Radioimmunoassay?
Both the tests are based on the same principle. The key difference is that in enzyme
immunoassays the antigen or antibody is conjugated to an enzyme rather than a radioactive
isotope, which is used in radioimmunoassay

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10. What is the purpose of using an enzyme?
Enzyme converts a colourless substrate into a coloured one thereby increasing the
detectability.
11. List out the different generations of ELISA.
First generation ELISA – Crude Ag
Second generation ELISA – Recombinant Ag
Third generation ELISA - synthetic
Fourth generation ELISA- Ag & Ab
12. Name few Immunosorbents.
 Cellulose, agarose – Particulate type
 Polysterene, Polycarbonate, Polyvinyl – Solid phase type
 Polyacrylamide, Paper, Plastic - Membrane, Disc type
13. Why a micropipette is used?
Because only microlitre quantities of test reagents and sample are used.
14. Advantages of ELISA test?
Highly sensitive
Safe and economical
Simple instrumentation is required
Many tests can be performed at the same time
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WESTERNBLOT TEST
Introduction:
 It is a serological test to detect the presence of antibodies to specific antigens.
 The test identifies specific proteins from a complex mixture of proteins extracted from cells.
 It is a confirmatory and gold standard test to detect anti HIV antibody in a human serum
sample.
 It works on the principle of immunoblot technique.
Principle:
HIV proteins are separated according to their electrophoretic mobility(& molecular weight)
by Polyacrylamide gel electrophoresis and blotted onto strips of nitrocellulose paper
These strips are reacted with the test sera and then with enzyme conjugated antihumanglobulin
Suitable substrate is then added
Produces a prominent colour band
Position of the band on the strip indicates the antigen with which the antibody has reacted
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Procedure:
Infectious agent is lysed in solution with sodium dodecyl sulphate to release proteins
Lysate is placed into trough of Polyacrylamide slab gel
Electrophoresis done
Results in separation of proteins on molecular size and charge
Transferred to a sheet of nitrocellulose
Nitrocellulose sheet is cut into strips
Patient serum is incubated with the nitrocellulose strip
Antibodies to the specific proteins if present bind to homogenous bands
Washing removes non-specific antibodies
Antihuman antibody enzyme conjugate is added
Conjugate binds to antigen antibody complex
Substrate for the enzyme is added
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Enzyme catalyzes production of colored product
Detection of specific antibody is based on enzyme substarte colored reaction product which occurs
in a band pattern based on the position of the proteins on the strip
Interpretation:
Positive blot : Any two or more of the following bands present: p 24, gp 41 &gp 120 / 160
Indeterminate blot : some bands present, not the definitive bands
Negative blot : No bands present
Uses of westernblot:
Used to confirm antibodies to HIV – 1 & HIV – 2
Advantages :
100 % sensitive and specific test
100 % reproducible and reliable
It is more precise than ELISA and is used to check the validity of ELISA
Helps to resolve false positive EIA results due to cross reacting antibodies to other viruses
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WESTERN BLOT
1. Which is the confirmatory test for the diagnosis of HIV infection?
Western blot test
2. What is the principle of westernblot?
Immunoblotting
3. What is immunoblotting?
In Immunoblotting, antibodies can detect antigens (proteins) in a mixture
4. What are the different blotting techniques?
Southern blot, Western blot and Northern blot
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5. Which antibodies are detected in western blot?
HIV 1&2 antibodies
6. Mention the gene specific antibodies in HIV?
Antibodies to:
gag gene : p 55, p 40, p 24, p 18
pol gene : p 65/66, p 55/51, p 31
env gene : gp 120/160, gp 41
6. Interpretation of westernblot?
The test is considered to be positive if it shows bands atleast against two of the following
gene products : p24, gp 120/160, gp41.
7. What is the meaning of Immunosorbent?
An absorbing material which is specific for the components of the reaction-

antigen/antibody
COMB AIDS HIV I & II RAPID ASSAY
Principle
Combaids HIV 1&2 rapid assay is a dot immunoassay for detection of HIV antibodies, where the
immobilized antigen antibody complex is visualized by means of a color producing (chromogenic)
reaction.
Reagents required
 Antigen coated combs

 Sample Diluents
 Positive and Negative controls .
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 Wash buffer concentrate
 Colloid Gold Signal Reagent
 Micro test well strips
 Disposable plastic droppers
 Rubber teats
Remove Comb aids kit from refrigerator and leave it at room temperature for 30 minutes before
starting the test to bring all the reagents to ambient temperature
TEST PROCEDURE:
 Take the required number of strips from the foil sachet and place it in the strip holder.
 Record the date as well as the number of the test run on the top of the comb.
 Add 2 drops of sample diluents to the wells.
 Add two drops of sample, positive and negative kit controls to the respective test wells.
 Immerse the teeth of the comb into the sample in the micro test wells.
 Incubate the comb for 10 minutes at room temperature.
 During this time add four drops of colloidal gold signal reagent to a fresh set of micro test
wells. Add the wash buffer to the buffer tray.
 Remove the comb immersed in specimen from the micro test wells at the end of 10 minute
incubation period and wash the strips in the wash buffer trough containing wash buffer.
 Dry the comb thoroughly by blotting on a filter paper.
 Place the comb in micro test wells containing colloidal gold signal reagent for 10 minutes.
 Remove the comb from the micro test wells and wash it with wash buffer and blot dry the
comb.
 Record the results in the laboratory work sheet within 10 minutes.
Results
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A test is considered negative if only the upper dot (i.e. the control dot) turns pink and the lower dot
does not.
A test is considered positive if both dots (upper i.e. the control dot and lower i.e., test) of the
comb turns pink.
If no dots turn pink the test is considered invalid. The test should be repeated with another test kit.
Interpretation
If the test result is negative the sample is considered Negative for HIV 1 & 2 antibodies.
If the test result is positive the sample is Positive for HIV 1 and/or 2 antibodies
CHROMATOGRAPHIC LATERAL FLOW IMMUNOASSAY.
Principle
HIV 1 and 2 Rapid Test works on Chromatographic lateral flow immunoassay.
Basic components of test strips includes :

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a) Colloidal Gold conjugated recombinant antigens corresponding to HIV I gp120, gp 41 and HIV
2 gp36 are dry immobilized at the end of nitrocellulose membrane strip on conjugate pad.
b) HIV 1&2 antigens are bond at the test line (1 and 2)
c) Monoclonal antibodies are bond at the Control zone.
The test sample that is added to the sample well (S), with adequate amount of buffer migrates from
the sample pad along the conjugate pad where HIV 1& 2 specific antibodies present in the sample
well bind to colloidal gold conjugate antigens to form a complex. These particles then continue to
migrate along the strip until the test zone ( 1&2) where they are captured by the HIV1& 2 antigens
generating a visible red line. If there are no HIV 1or 2 antibodies in sample no line is formed in the
test zone (1&2) The sample then migrates further along the strip until it reaches the control zone,
where it produce another visible line on the membrane in control zone (C) . This control line
indicates that the sample has migrated across the membrane as intended.
KIT Components:
*HIV 1-2 card test device.
* Assay buffer
* Controls
Remove HIV 1-2 Kits from refrigerator and bring it to the room temperature
Test procedure:
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 Label the device with the control and sample ID number, Date & Time using a permanent
marker and place the devices on a flat surface.
 Add 5 ul of serum into sample well .
 When the sample is fully absorbed , add 2 drops of assay buffer.
 Wait for 15 minutes and Interpret the results.
Results
Non-reactive: The presence of only one colour band at ‘C’ indicates negative result.
Reactive: The presence of colour bands at ‘C’ along with band at 1 (HIV-1) or 2 (HIV-2)
indicates a positive result.
Invalid Result: If the control of colour band is not visible at C, the test is considered invalid
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Polymerase Chain Reaction (PCR)
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PCR is a technology in molecular biology used to amplify a single or few copies of a piece of
DNA to generate millions of copies of DNA. It was developed by Kary B Mullis (1983) for which
he and Michael Smith were awarded the Nobel prize in Chemistry in 1993
Principle of PCR PCR involves three basic steps.
1. DNA extraction from the organism: This involves lysis of the organisms and release of the
DNA which may be done by various methods—boiling, adding enzymes (e.g. lysozyme,
proteinase K), etc. DNA extraction kits are also available commercially.
2. Amplification of extracted DNA: This is carried out in a special PCR machine called
thermocycler. The extracted DNA is subjected to repeated cycles (30–35 numbers) of
amplification which takes about 3–4 hours.
Each amplification cycle has three steps.
1. Denaturation at 95°C: This involves separation of the dsDNA into two separate single strands
2. Annealing (55°C): Primer is a short oligonucleotide complementary to a small sequence of the
target DNA. It anneals to the complementary site on the target ssDNA
3. Extension (72°C): This step is catalyzed by Taq Polymerase enzyme which keeps on adding the
free nucleotides to the growing end of the primer. Taq Polymerase is a special type of DNA
polymerase (isolated from the plant bacterium Thermus aquaticus), capable of withstanding the
high temperature of PCR reaction.
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Steps of PCR – Amplification cycle
NORMAL CONSTITUENTS OF STOOL
AIM:
To examine the faecal specimens for its normal constituents
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PROCEDURE:
1. With an applicator stick, pick up a bit of the sample.
2. Take enough material and emulsify in a drop of normal saline & diluted Lugol’s iodine
on a slide, avoiding the production of bubbles.
3. Place a coverslip gently over the preparation. The preparation must not overflow.
Density should be sufficiently light to permit reading of newsprint held under the slide.
EXAMINATION:
4. The slide is first examined under low power (X10) objective starting from one end of the
coverslip, the whole slide is examined. Any suspicious object is focused under high power
objective(X40) for detailed diagnosis.
5. Entire area under one 22X22 mm coverslip should be examined systematically.
OBSERVATION:
The bulk of stool consists of granular debris. Among the recognizable microscopic structures
encountered in normal conditions are:
I. Remnants of food:
(a) Vegetable fibres: Spiral structure with pits, dots or reticular marking
(b) Vegetable cells: Show double contours and sometimes chlorophyll bodies.
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(c) Vegetable hairs: Are homogenous, elongated tube like structures with highly
refractile wall.
(d) Starch granules: Best identified by adding drop of Lugol’s iodine which stains them
blue.
(e) Muscle fibre: Usually appear as short, transversely striated cylinders.
(f) Fats: May appear as:
1. Neutral fats (droplets)
2. Fatty acids (Needle like crystals)
3. Soap (Yellow masses or coarse crystals)
(g) Elastic fibres and Connective tissue: Appear as colorless yellowish threads,
II. Pus cells:
Present in ulcerative conditions of the intestine. In amoebic dysentery, their presence indicates
superadded infection
III. Macrophages:
Are sometimes present and are liable to be mistaken as E.histolytica. They are most common in
bacillary dysentery.
IV. Epithelial cells:
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A few cells are always present. Increased number of epithelial cells is present in catarrhal
condition of the colon.
V. Red Blood Cells:
In amoebic dysentery RBC’S are present in clumps and are reddish yellow, while in bacillary
dysentery, they are bright, discrete or in rouleaux.
VI. Charcoat Leyden Crystals:
Particularly common in ulcerative conditions of colon, especially amoebic dysentery.
Absent in bacillary dysentery.
NORMAL CONSTITUENTS IN STOOL
1. YEAST CELLS
Yeast in an iodine-stained concentrated wet mount of stool. Yeast in wet mounts may be
confused for Giardia.
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2. FUNGAL SPORES
Fungal spores may be confused for helminth eggs, especially hookworm.
3. POLLEN GRAINS IN STOOL
Pollen grain in a concentrated wet mount of stool. This grain looks very similar to the
fertile egg of Ascaris lumbricoides, although the spine-like structures on the outer layer should
separate the two.
4. PLANT MATERIAL AND PLANT HAIR IN STOOL
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Plant cell in a concentrated wet mount of stool. Such material can be common in stool and may be
confused for helminth eggs, although they are usually much larger than the eggs of helminths .
Plant material in an iodine-stained concentrated wet mount of stool. This material can be
confused for a hookworm egg.
PLANT HAIR IN STOOL
Plant hairs in a concentrated wet mount of stool. Plant hairs can be common in stool and
may be confused for the larvae of hookworm or Strongyloides stercoralis. However, they are
often broken at one end, have a refractile center and lack the strictures seen in helminth larvae.
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5. MISCELLANEOUS ARTIFACTS IN STOOL
Unknown object in a concentrated stool specimen. This object looks like the egg of
Hymenolepis but lacks refractile hooks and the polar filaments seen in H.nana.
CHARCOT LEYDEN CRYSTALS
192
Charcot-Leyden crystals. These crystals are the breakdown products of eosinophils and maybe
found in the feces and sputum of people with tissue-invading parasitic infections or various
allergic reactions.
DIATOMS IN STOOL
Diatoms in stool. Diatoms do not specifically resemble any parasites of humans, but their
size and shape and apparent structure may cause the microscopist to take notice.
MITE EGGS IN STOOL
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Mite egg in a formalin-concentrated stool specimen. Mite eggs are similar to hookworm
eggs but are usually larger (but not always). In this specimen, leg buds can be seen in the lower
right area of the egg.
FREQUENTLY ASKED QUESTIONS
1. List the normal constituents of stool
The normal constituents of stool are yeast cells, cotton fibre, starch cell, parenchyma cell,
plant epidermal hairs, fungal spores, muscle fibres, vascular structure of plants, pollen,
plant cells, oil drops, rice starch, potato starch,corn starch, leucocytes, moulds and
bacteria.
2. What are the different wet mount preparations of stool?
Different wet mount preparations of stool are saline wet mount, iodine wet mount, LPCB
wet mount and buffered methylene blue.
3. What are the uses of Saline wet mount?
194
It is a colorless preparation that highlights the staining property of the egg, whether bile or
non bile stained, detects motility of trophozoites and facilitates demonstration of
chromatoidal bodies in the cyst.
4. Which are the structures that are visualized best in the saline wet mount?
Larvae and trophozoites
5. List the advantages and disadvantages of iodine wet mount.
Advantages:
1. The number of nuclei present in the protozoan cyst can be clearly distinguished.
2. It also demonstrates the yellowish cytoplasm and brown glycogen mass present in the
cysts.
Disdvantages:
1. Trophozoites are killed by iodine, hence cannot be demonstrated in iodine wet mount.
2. The bile staining property of the helminthic eggs cannot be made out.
3. Chromatoidal bars in protozoan cysts are not clearly demonstrable.
8. List out the various acid fast parasites.
The intestinal coccidian parasites
 Cryptosporidium parvum,
 Cyclospora cayatanensis,
 Isospora belli
The non-intestinal coccidian parasites.
195
 Toxoplasma gondii
 Sarcocystis hominis
Other parasitic components
 Scolices of Echinococcus granulosus

 Spores of Microsporidia (now classified under fungi)
 Eggs of Taeniasaginata.
9. Give an example for permanent staining of stool.
Modified acid fast staining
10. What are the advantages of floatation method?
 Simple procedure
 Inexpensive method and easy to perform
 Good method for concentration of parasitic eggs
 Can be done at any level of the laboratory
 Least subjective to technical error.
11. What are the disadvantages of floatation method?
 Not suitable for demonstration of unfertilized eggs of Ascaris, operculated eggs of

trematodes, larvae of Strongyloides and eggs of Taenia.
 High specific gravity of the fluid may cause distortion of the morphology of the
parasitic eggs and cysts.
12. What are the advantages of sedimentation method?
 More sensitive method

 Good method for both cysts and eggs
 Morphology of eggs and cysts preserved.
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13. What are the disadvantages of sedimentation method?
 Technically cumbersome
 Ether is highly inflammable and not easily available.
14. Name the other floatation methods that can be employed as stool concentration
method.
 Zinc sulfate floatation method

 Sheather’s sucrose floatation method
15. Name the stool concentration method employed for recovery of coccidian parasites.
 Sheather’s sucrose floatation method.
16. What is the indication for concentrating the stool specimen?
When the number of parasites in the stool specimen is less and cannot be demonstrated in
the stool specimen, it demands a necessity to concentrate the stool specimen
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IDENTIFICATION OF ASCARIS LUMBRICOIDES
AIM: To identify ova or cyst in the given stool specimen using iodine wet mount method.
OBSERVATION
IDENTIFICATION FEATURES:
1. The egg is round or oval in shape measuring 60-75μm in length and 40-50 μm in breadth.
2. The egg is bile stained and brown in colour.
3. It is surrounded by a thick transparent shell with three layers- a non-permeable innermost
lipoidal vitelline membrane, a thick transparent middle layer and an outermost coarsely
mammilated albuminoid layer.
4. A large conspicuous unsegmented ovum is present inside the egg shell.
5. There are clear, crescentric spaces between the ovum and the egg shell at both the poles of
the egg.
INFERENCE: Based on the above identifying features, the given stool specimen contains
fertilized eggs of Ascaris lumbricoides.
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IDENTIFICATION OF ANKYLOSTOMA DUODENALE (HOOKWORM)
OBSERVATION:
1. Eggs are oval or elliptical in shape with bluntly rounded ends measuring 65μm in length
and 40 μm in breadth.
2. They are colourless and not bile stained.
3. They are surrounded by a thin transparent hyaline shell membrane.
4. They possess a segmented ovum with usually four blastomeres.
5. There is a clear space between the segmented ovum and the egg shell.
6. The eggs float in saturated solution of common salt.
7. The eggs are non-infective to humans.
INFERENCE:
Based on the above identifying features, the given stool specimen contains eggs of Ancylostoma
duodenale.
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IDENTIFICATION OF TRICHIURIS TRICHIURA
OBSERVATION:
1. The eggs are barrel shaped 50 μm in length and 25 μm in breadth.
2. They have projecting mucous plugs on both the poles.
3. They are bile stained and brown in colour
4. The eggs contain an unsegmented ovum.
5. They float in saturated solution of common salt.
6. When freshly passed they contain an unsegmented ova and are not infective to humans.
INFERENCE:
Based on the identifying features, the given stool specimen contains eggs of Trichiuris trichiura
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FREQUENTLY ASKED QUESTIONS IN STOOL EXAMINATION
1. Which type of container is used for collecting faecal samples?
Stool sample should be collected in a wide mouthed clean container without

contamination with urine, water and disinfectants.
2. How many faecal specimens are essential to make the diagnosis of intestinal parasitic
diseases?
Three faecal specimens (2 specimens after normal bowel movement and 3rd after
magnesium sulphate purge) are sufficient except in suspected case of intestinal amoebiasis
or giardiasis, a total of six specimens collected on successive days is required.
3. When should you examine the faecal samples after collection?

 Liquid stools-within 30 min of passage
 Semiformed and soft specimens-within 60 min of passage
4. Enumerate commonly used preservatives
 Formalin solution-most commonly used

 Polyvinyl alcohol
 Merthiolate-iodine-formalin solution(MIF)
 Sodium acetate acetic acid formalin fixative(SAF)
5. What is the advantage and disadvantage of preservation of faecal specimens?
Preservation of faecal specimen maintains the morphology of parasites whereas it kills the
parasite and inhibits the motility of trophozoites.
6. How do you assess best smear for faecal examination?
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Direct smear for faecal examination should be so thin that newsprint can be read through
the smear.
7. Name some parasites which can be seen in saline mount.
 Trophozoites of Entamoeba histolytica, Giardia lamblia.

 Eggs and larvae of helminths
 Protozoal cysts
8. Name some iodine solutions which can be used for faecal examination?
 Weak iodine solution should be used as too strong iodine can obscure the details of
the parasite.
 Lugol´s iodine (five times diluted solution). Gram´s iodine is not suitable.
9. Name some parasites whose eggs do not float in saturated salt solution.
 Unfertilised eggs of Ascaris lumbricoides

 Eggs of Diphyllobothrium latum
 Eggs of Taenia solium, T.saginata
 Eggs of all intestinal flukes(Fasciolopsis buski, Gastrodiscoides hominis)
10. Name some parasites whose eggs are non-bile stained. (NEHA).
Eggs of :
 Necator americanus
 Enterobius vermicularis
 Hymenolepis nana
 Ancylostoma duodenale
11. Name one parasite whose eggs are detected in sputum
Paragonimus westermani
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12. Name one parasite whose eggs are detected in urine.
Schistosoma haematobium
13. Which is the heaviest of all helminthic eggs?

Unfertilised eggs of Ascaris lumbricoides
14. Name the methods of detecting the number of helminthic eggs in faeces.
 Direct smear egg count
 Stolls method
15. Name one ovo-viviparous nematode
Strongyloides stercoralis
16. Classify oviparous nematodes.
Laying unsegmented eggs
 Ascaris lumbricoides
 Trichuris trichiura
Laying eggs with segmented ova
 Ancylostoma duodenale
 Necator americanus
 Trichostrongylus sp
Laying eggs containing larvae
 Enterobius vermicularis
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17. Name some bile stained eggs of parasites.
Eggs of :
 Ascaris lumbricoides- fertilised, unfertilized & decorticated

 Trichuris trichiura
 Taenia solium, Taenia saginata
 Echinococcus sp.
 Diphyllobothrium latum
 Schistosoma mansoni,Schistosoma japonicum,Schistosoma hematobium
 Fasciola hepatica, Fasciolopsis buski
 Paragonimus westermani and Clonorchis sinensis.
18. Name some parasites found in blood.
Plasmodium sp, Trypanasoma sp, Wuchereria bancrofti, Brugia malayi,

Loa loa, Mansonella sp.
19. What are Romanowsky stains? Name some of them.
Romanowsky stains are stains with a combination of methylene blue and eosin.
Some examples are:
 Leishman´s stain
 Giemsa stain
 Field´s stain and
 JSB (Jaswant Singh Bhattacharya)stain
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LIST OF OBSERVED STATION
S.NO. NAME OF THE EXERCISE
1. MICROSCOPIC ADJUSTMENTS
2. BLOOD COLLECTION TECHNIQUE
3. NASOPHARYNGEAL SWAB COLLECTION
4. BIOMEDICAL WASTE MANAGEMENT
5. HAND WASHING
6. DONNING AND DOFFING OF PPE
LIST OF UNOBSERVED STATION
NAME OF THE EXERCISE

1. ANTIBIOGRAM
2. BIOMEDICALWASTE MANAGEMENT
3. USES OF DISINFECTANTS
4. CAUTI
5. SEPTICAEMIA
6. MENINGITIS
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OSPE OBSERVED STATIONS
1) MICROSCOPIC ADJUSTMENTS
High power:
 Fix the correct objective

 Raise the condenser
 Open the diaphragm
 Plane mirror
Low power:
 Fix the correct objective

 Lower the condenser
 Close the diaphragm
 Concave mirror
Oil immersion : (OPR)
 Open the diaphragm

 Plane mirror
 Raise the condenser
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2) BLOOD COLLECTION TECHNIQUE
 Label the test tube
 Wash hands & Wear the gloves
 Apply tourniquet
 Clean the skin with 70% isopropyl alcohol first and then with povidone-iodine in a
circular motion(5 cm in diameter). Discard the cotton in yellow bin
 Collect the blood sample and transfer to the test tube
 Needle to be disposed in the white puncture proof container
 The plastic syringe has to be disposed in the red bin
 The gloves has to be disposed in the red bin
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3. NASOPHARYNGEAL (NP) SWAB COLLECTION
208
Nasopharyngeal (NP) swab collection
• Ensure that personal protective equipment (PPE) is worn when collecting the specimen.
• Gloves must be changed for each patient and discard into the BMW bins
STEP 1
1. Tilt patient’s head back 70 degrees. Gently and slowly insert a swab with flexible shaft through
the nostril parallel to the palate until resistance is encountered.
2. The distance is equivalent to that from the nostril to the ear of the patient, indicating contact
with the nasopharynx
STEP 2
1. Gently rub and roll the swab , leaving it in place for severalsecondstoabsorb secretions.
2. If a deviated septum or blockagecreatesdifficulty inobtainingthespecimen
fromonenostril,usethe same swab to obtain the specimenfromtheother nostril.
3. Slowlyremovethe swabwhile rotating it.
STEP 3
1. Place the swab tip into the transport tube provided
2. Once the tip is near the bottom, break the swab handleattheswabbreakpoint
bybendingbackandforthor cutitwithsterilescissors.
3. Theswabshouldfit inthetube comfortably so that the cap can be screwed on tightly to
prevent leakage and contamination.
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4. BIOMEDICAL WASTE MANAGEMENT
210
211
5. HAND WASHING - STEPS
212
INDICATIONS (FIVE MOMENTS FOR HAND HYGIENE)
213
6. PERSONAL PROTECTIVE EQUIPMENT (PPE):
A. Gloves; B. heavy duty gloves; C. Surgical mask; D. N95 respirator; E. Plastic apron; F. Linen
gown; G. Disposable gown; H. Coverall; I. Goggles; J. Face shield; K. Cap; L. Shoes; M. Gum
boot; N. Shoe cover
Donning (wearing): Gown first → Mask or
respirator → Goggles or face shield →
Gloves
Doffing (removing): Gloves first → Face
shield or goggles → Gown → Mask or
respirator
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UNOBSERVED
1. ANTIBIOGRAM
Cefoxitin
Lz
Van
n
1. What is the culture media used? Name the method used. ?
Media used: Mueller Hinton agar.
Method: Kirby – Bauer Disc diffusion method
Other Methods of Antimicrobial Susceptibility Testing
 Antimicrobial susceptibility testing methods are divided into types based on the principle
applied in each system. They include:
1. Diffusion method - Kirby-Bauer method / Stoke’s method
2. Dilution method - MIC (Broth & Agar)
3. Dilution & Diffusion – E test
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2. What are the common drug resistant organisms?
Meticillin Resistant Staphylococcus aureus (MRSA)
Extended spectrum beta lactamase producing Klebsiella pneumoniae.(ESBL)
Extended spectrum beta lactamase producing Proteus mirabilis.
Extended spectrum beta lactamase producing Escherichia coli.
Vancomycin Resistant Enterococci. (VRE)
Metallo beta lactamase producing Pseudomonas aeruginosa.(MBL)
Extensively drug resistant tubercle bacilli. (XDR -TB)
3. What is the resistance pattern in the given antibiogram?
ESBL & MRSA
4. How to measure the zone of inhibition?
Using antibiotic scale measure the diameter of the zone from margin to margin
5. What is the name of the antibiotic chart used for comparing the zone?
Zone size interpretative chart (Kirby-Bauer chart )
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2. Biomedical Waste Management
Write the appropriate colour coded bins for the Bio medical wastes given below.
1. Soiled cotton - yellow
2. Glass slides - blue
3. Gloves - red
4. Needles – white translucent container
5. Used Blood bag - yellow
6. Biopsy specimen - yellow
7. Urine bags- red
8. Metallic body implants- blue
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3.USES OF DISINFECTANTS
MATCH THE USES OF THE FOLLOWING DISINFECTANTS:
I. Iodine (a) Disinfection of cystoscopes and bronchoscopes
II. Sodium hypochlorite – (b) Disinfection of clinical thermometers, Hand washing .
III. Formaldehyde - (c) Virucidal (against HIV)
IV. Glutaraldehyde - (d) OT fumigation,Preservation of specimens.
V. Isopropyl alcohol - (e) Skin disinfectants, Surgical scrubbing.
Ans : I- e, II – c, III – d, IV – a, V – b
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4. CAUTI – CATHETER ASSOCIATED URINARY TRACT INFECTIONS
35 years female with urinary catheter develops fever with rigor and burning urination
1. What could be the diagnosis ?What is nosocomial infection?
CA – UTI, infection that is acquired from the hospital or other health care facility is called
nosocomial infection.
2. What are the etiological agents?
 Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa
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 Coagulase negative staphylococci, Staphylococcus aureus
 Enterococci, Candida spp.,
3. How to prevent CA -UTI?
 Insert urinary catheter under aseptic precautions (with proper hand washing, wearing
gloves and face mask )
 Before catheterization clean the external genitalia properly with betadine solution
 Shortening the duration of catheterization
4. How to collect urine for culture?
 Clean catch midstream urine collection in patients without catheter

 In Patientswith catheter - Catheter tubing to be clamped off above the port to allow
collection of freshly voided urine.
 Catheter port or wall of the tubing to be cleaned vigorously with 70 percent ethanol and
urine aspirated via a needle and syringe. Urine specimens to be obtained without opening
the catheter collection tube junction.
6. Name the othe Health care associated Infections (HAI )?
 SSI - Surgical Site infections

 CLABSI – Central Line Associated Blood Stream Infections
 VAP – Ventilator Associated Pneumonia
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5.SEPTICAEMIA
1. Define septicaemia and pyemia.
Septicaemia is the condition where bacteria circulate and multiply in the blood,form toxic
products and cause high swinging type of fever.
Pyemiais a condition where pyogenic bacteria produce septicaemia with multiple abscesses in the
internal organs such as spleen,liver and kidneys.
2. What are the tests used for the diagnosis of Septicaemia?
 CRP
 Procalcitonin
 Blood culture
3. What are the signs and symptoms of septicaemia:
 Fever/Hypothermia
 Chills
 Hyperventilation
 Respiratory alkalosis
4. What are the Causes of neonatal septicaemia?
Early onset neonatal septicaemia:
 Group B Streptococcus
 Escherichia coli
 Haemophilusinfluenzae
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 Listeria monocytogenes
Late onset neonatal septicaemia:
 CoNS
 Staphylococcus aureus
 Klebsiella
 Pseudomonas
 Acinetobacter
5. What is the treatment of neonatal septicaemia?

Gram negative septicemia with drugs - Cefoperazone ,Amikacin
Gram positive septicemia with Ampicillin, Vancomycin
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6) MENINGITIS
1.What are the biochemical /pathological test to diagnose meningitis?
Appearance of CSF, Total WBC count and lymphocyte count and estimation of protein and
glucose concentration.
2.How much CSF is needed and how to collect CSF?
1-2 ml of CSF is collected in a sterile container by lumbar puncture under strict aseptic
precautions.
3.What are the microbiological tests done in CSF?
CSF is divided into three portions:
1st portion - centrifuge deposit :Grams stain,Zeihl-Neelsen stain
In centrifuge supernatant: meningococcal/pneumococcal antigen detection by latex

agglutination
2ndportion : Culture for Bacteria in blood / chocolate/ modified Thayer martin media
3rdportion : enrichment with glucose broth and sub culture in chocolate agar
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4.Name the etiological agents of bacterial origin causing meningitis.
Adults: Staphylococcus aureus,Haemophilus influenza, Neisseria meningitis
Streptococcus pneumonia, Mycobacterium tuberculosis
Neonate:
Escherichia coli, Group B streptococcus, Listeria monocytogenes.
6.How to demonstrate capsulated fungi in CSF?
By India Ink / Nigrosin staining
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SPOTTERS
1.INSTRUMENTS
1. BACTERIOLOGICAL LOOP
1. It is made up of nichrome wire and is sterilized by red heat.
2. Used to streak specimens on solid media, calibrated loop is used for colony counting for
urine cultures.
2. STERILE SWAB
1. Sterilization: Hot air oven at 160°c for 1 hour.
2. Use: For collection of clinical specimens like pus swab, conjunctival swab, ear swab, rectal
swab, vaginal swab etc.,
3. TUBERCULIN SYRINGE
1. Used for Mantoux test.
2. 0.1ml of ppd is injected intradermally on the flexor aspect of forearm.
3. Reading after 48-72 hours shows indurations and erythema with diameter ≥ 10mm is
considered positive.
4. CANDLE FILTER
1. Used for filteration of water for drinking purpose

2. Used in Industries
5. VDRL ROTATOR
1. It is an electronic device used for mixing of treponemal antigen with suspected
patient’s serum for diagnosis of primary syphilis.
2. For quantitative VDRL test, the VDRL slide with specific antigen and antibody should
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be rotated in VDRL rotator for 180 rpm for 4 mins.
6. VDRL SLIDE
1. It is used to perform both for qualitative and quantitative VDRL test for diagnosis of
syphilis.
2. VDRL test is a non-specific slide flocculation test used as a screening test for primary syp
hilis.
7. AUTOCLAVE
1. Used for sterilization of Culture media, Instruments, linen, etc.,
2. Sterilisation principle – moist heat sterilization at 1210C/15lbs/15min.
8. MICROTITRE PLATE
1. It is a plastic plate with 96 wells where large number of small quantities of samples can be
tested at a time and sterilized by ionising radiation.
2. Used to perform ELISA, complement fixation, haemagglutination test etc.,
9. DURHAM’S TUBE
1. It is a one inch tube used upside down in the sugar containing media like glucose, sucrose,
lactose etc, for detecting gas production of bacteria.
2. Gas production is indicated by the presence of air bubbles in the Durham’s tube and the
absence of air bubbles indicates no gas production
.
10. STERILE CULTURE PLATE
1. It is a sterile petri dish available in glass (sterilized by hot air oven) or plastic (disposable,
sterilized by gamma radiation)
2. It contains sterile media sterilized by autoclaving at 1210C for 15 mins.
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11. UNIVERSAL CONTAINER
1. Widemouthed sterile container used to collect biological specimens
2. Most commonly used for collection of Urine and sputum samples for culture
2.MEDIA
12. NURIENT AGAR PLATE

1. It is a commonest basal medium (2% agar+ nutrient broth) sterilized by autoclaving at
121°C for 15 minutes at 15 lbs.
2. Use: To study colony morphology and pigment production
13. BLOOD AGAR PLATE:

1. It is an enriched and indicator medium, used to study the hemolytic property of bacteria.
2. Use: For cultivation of fastidious organism like streptococci, staphylococci which produce
ß- hemolysis.
3. Pneumococci and Streptococcus viridians produce α-hemolysis.
14. CHOCOLATE AGAR PLATE

1. It is an enriched medium, prepared by heating blood agar at 75°Cfor 10 mins.
2. Use: For the growth of fastidious organisms like Neisseria and Haemophilus.
15. MAC CONKEY AGAR PLATE

1. It is a selective medium and differential medium, used for cultivation of enteric bacteria.
2. Use: For differentiating lactose fermenting organisms (Pink colonies) from non-lactose
fermenting organisms (Colourless colonies).
16. ROBERTSON COOKED MEAT MEDIUM (RCM)
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1. It is an anaerobic selective medium consisting of minced meat in nutrient broth with a
layer of sterile paraffin on top to prevent oxygen penetration.
2. Use: Growth and preservation of anaerobic bacteria like Clostridium spp., Bifidobacterium,
Propionibacterium etc.,
17. THIOSULPHATE CITRATE BILE SALT SUCROSE AGAR (TCBS)

1. It is a selective medium and differential medium, used for isolation of Vibrio cholerae
from stool specimen with a PH of 8.2.
2. Use: For differentiating sucrose fermenting organisms (Yellow colonies) from non-sucrose
fermenting organisms (green colonies).
18. TRIPLE SUGAR IRON AGAR MEDIUM

1. The medium in a test tube has a slant and a butt with phenol red as the indicator.
2. It is used to determine ability of an organism to break down the specific carbohydrates
incorporated in growth medium with or without gas production and H 2S production.
3. Composition: Glucose, lactose and sucrose in the ratio of 1:10:10
19. TRIPLE SUGAR IRON AGAR MEDIUM (TSI): A/A WITH GAS +.
1. Yellow (acidic) slant/ Yellow (acidic) butt with gas production.
2. The reaction indicates fermentation of glucose, lactose and /or sucrose. Bubbles in the butt
indicate gas production. Phenol red is the indicator. (K-alkaline; A-acidic).
3. Organisms showing this reaction: E.coli and Klebsiella.
20. TRIPLE SUGAR IRON AGAR MEDIUM (TSI): K/A GAS + AND H2S +
1. Pink (alkaline) slant/Yellow (acidic) butt with gas and H 2S production.
2. The reaction indicates fermentation of glucose with gas and blackening indicates
production of H2S.Bubbles in the butt indicate gas production. Phenol red is the indicator.
(K-alkaline; A-acidic).
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3. Organism showing this reaction: Proteus mirabilis, Salmonella paratyphi B.
21. TRIPLE SUGAR IRON AGAR MEDIUM (TSI): K/A GAS – and H2S –
1. Pink (alkaline) slant/Yellow (acidic) butt without gas and no H2S production.
2. The reaction indicates fermentation of glucose only without gas and there is no H 2S
production. Phenol red is the indicator. (K-alkaline; A-acidic).
3. Organism showing this reaction: Shigella flexneri.
22. TRIPLE SUGAR IRON AGAR MEDIUM (TSI) - K/K REACTION

1. Pink (Alkaline) slant/ Pink (Alkaline) slant is seen.
1. The reaction indicates neither glucose nor lactose nor sucrose fermented. Phenol red is the
indicator.(K-alkaline; A-acidic)
2. Organism showing this reaction: Pseudomonas and non-fermenting Gram negative bacilli
(NFGNB).
23. PEPTONE WATERPEPTONE WATER

1. It is a basal liquid medium (1% peptone, 0.5% NaCl) for growing pathogenic bacteria and
for preparing sugar fermentation media.
2. Sterilization by autoclaving at 1210C for 15 minutes.
24.INDOLE TEST- POSITIVE

1. Media used: Tryptophan broth or Peptone water ; Reagent used: Kovac’s reagent
2. Formation of a red coloured ring near the surface of the medium indicates positive reaction.
3. Indole positive organisms are E.coli, Proteus spp., except Proteus mirabilis.
25.INDOLE TEST- NEGATIVE

1. Media used : Tryptophan broth or Peptone water.; Reagent used: Kovac’s reagent
2. Presence of yellow colour ring near surface of the medium indicates negative reaction.
3. Indole negative organisms are Klebsiella, Salmonella.
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26.CITRATE UTILISATION TEST- POSITIVE
1. Media used : Simmon’s citrate agar slant:Indicator used: Bromothymol blue.
2. Citrate positive organisms produces growth and blue colour on the slant.
3. Example: Klebsiella, Citrobacter.
27. CITRATE UTILISATION TEST -NEGATIVE

1. Media used: Simmon’s citrate agar slant:Indicator used: Bromothymol blue.
2. Citrate negative organisms produces green colour.
3. Example: E.coli, Proteus, Salmonella. typhi.
28.OXIDASE TEST- POSITIVE

1. This test detects the enzyme cytochrome oxidase in the bacteria.
2. Reagent used: Tetramethyl paraphenylene diamine dihydrochloride.
3. Example for oxidase positive organisms :Vibrio, Pseudomonas, Neisseria spp.,
29.ANTIBIOGRAM
1. Principle: Disc diffusion method (Kirby-Bauer method).
2. Medium used: Mueller Hinton agar.
3. It is a type of qualitative antibiotic sensitivity test done as per CLSI guidelines.
30. MAC CONKEY AGAR PLATE WITH LF COLONY

1. In the Mac Conkey agar, lactose fermenting bacteria produces pink colour colonies.
Indicator used: Neutral red.
2. Example: Escherichia coli, Klebsiella sp., and Shigella sonnei is a late lactose fermenter.
31. MAC CONKEY AGAR PLATE WITH NLF COLONY

1. In the Mac Conkey agar, colourless colonies are produced by non- lactose fermenting
organisms; Indicator used: Neutral red.
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2. Example: Proteus, Salmonella, Shigella sp. (except Shigella sonnei)
32. CHRISTENSEN’S UREASE TEST – POSITIVE

1. This test determines the ability of some bacteria to produce an enzyme urease which
decomposes urea into ammonia thereby making the slant alkaline.
2. Medium used: Christensen’s urease agar Indicator used: Phenol red.
3. Development of pink colour indicates positive test. Example: Klebsiella, Proteus,
Helicobacter pylori.
33. CHRISTENSEN’S UREASE TEST – NEGATIVE

1. This test determines the ability of some bacteria to produce an enzyme urease which
decomposes urea into ammonia thereby making the slant alkaline.
2. Medium used: Christensen’s urease agar Indicator used: Phenol red.
3. If urease is not produced there is no color change from pale yellow to pink. Then it is
negative. Example: Escherichia coli, Salmonella sp.
34. LOWENSTEIN-JENSEN MEDIUM (LJ)

1. It is a solid, enriched and selective medium used for growing Mycobacterium
tuberculosis and performing drug testing of M.tuberculosis.
2. It is prepared in McCartney bottles as slopes and sterilized by inspissation. Colonies of
Mycobacterium tuberculosis appear in 3-6weeks.
4. MODELS AND SPECIMENS
35.TAPE WORM
1. Important species: Taenia saginata and Taenia solium
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2. They are segmented into scolex (head), neck and strobila.
3. They are tape like parasites (cestode) and usually measure 2-6 m in length.
4. Habitat: Small intestine of man.
36. ROUND WORM

1. Scientific name: Ascaris lumbricoides
2. They are unsegmented and rounded, tapering at both ends. Male is smaller than female.
3. Infective form: Embryonated egg.
4. Mode of infection: Ingestion of food/water contaminated with eggs.
36. EMBRYONATED EGG

1. Embryonated hen’s eggs are used for cultivation of some viruses, chlamydiae and
rickettsiae.
2.Route of inoculation may be chorioallontic membrane, allantoic cavity, amniotic cavity, yolk
sac
37. INFLUENZA VIRUS MODEL

1. It is an enveloped RNA virus; spherical in shape, the nucleocapsid has helical symmetry
with 8 segmented RNA and RNA dependent RNA polymerase.
2. Two spikes project on the surface – Haemagglutinin (triangular) & Neuraminidase
(mushroom shaped).
38. HIV MODEL

1. HIV is a spherical & enveloped RNA virus and the nucloeocapsid comprises of an outer
icosahedral & inner cone shaped core.
2. HIV causes acquired immune deficiency syndrome.
39. ADENOVIRUS MODEL

1. It is a non-enveloped double stranded DNA virus.
233
2. The nucleocapsid is icosahedral with 20 facets & 12 vertices with rod like projection called
fibre and so appears like space vehicle. It causes respiratory infections.
40. CORONA VIRUS MODEL
1. It is an enveloped spherical RNA virus having helical symmetry.
2. It is responsible for the outbreak of pandemic COVID – 19.
41. DPT VACCINE

1. It is a triple vaccine (Toxoid) given for diphtheria, pertussis and tetanus.
2. Given in 3 doses at 4-6 weeks interval by intra muscular route from 6 weeks of age 1 st
booster at1 ½ years & 2 nd booster at 5-6 years.
42. ORAL POLIO VACCINE (OPV)

1. Live polio vaccine when administered orally is called as OPV given against polio virus
causing poliomyelitis.
2. OPV used in India contains type 1 virus, type 2 virus & 2 type 3 virus in the ratio-1:2:2.
3. Three doses are given at 4-8 week intervals.
43. TETANUS TOXOID

1. It induces active immunization against tetanus caused by Clostridium tetani.
2. It is a formal toxoid or toxoid absorbed on aluminium phosphate or hydroxide.
3. Also given as triple vaccine.
44. MMR VACCINE

1. Included in Universal immunisaton programme given at 9 – 15 months
2. Measles Mumps Rubella vaccine given by subcutaneous route.
45. BCG VACCINE

1. Live attenuated vaccine used for prevention of tuberculosis
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2. Bacillus Calmette Guerin , Given immeadetely after birth by intradermal route
5. PARASITOLOGY SLIDES
46. TAPE WORM PROGLOTTIDS

1. The body of the tape worm is called strobila, made up of segments called as the proglottids.
2. Mature proglottids are pushed away from the neck consists of fully developed
reproductive organ. The proglottids with eggs are called gravid segments.
47. HOOK WORM (ADULT)

1. Common name : Ancylostoma duodenale
2. Infective form: Egg containing filariform larva
3. Mode of infection: Penetration of filariform larva through the intact skin.
48. TAPE WORM (SCOLEX WITH HOOKLETS)

1. It is the scolex of Taenia solium.
2. Definitive host: Man.
3. Infective form: Cysticercus cellulosae in pork.
4. Mode of infection: Ingestion of pork containing Cysticercus cellulosae
49. PIN WORM

1. Enterobius vermicularis causes infection frequently in children
2.NIH swabs used to collect samples
50. LIVER FLUKE

1. Trematode which is a leaflike structure called as Fasciola hepatica
2. Causes hepatobiliary infections
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51. PLASMODIUM FALCIPARUM GAMETOCYTE
1. It is sickle or crescent shaped. RBC’S are not enlarged.
2. It is seen inmalignant tertian malaria.
3. Infective form: Sporozoite.
4. Mode of infection: Bite of female Anopheles mosquito.
52. PLASMODIUM VIVAX – RING FORM

1. In Plasmodium vivax, the young erythrocytes are infected and are enlarged.
2. In Leishman’s stain the early trophozoites are ‘C’ shaped the cytoplasm opposite to the
nucleus is thicker. It causes benign malaria.
6. MYCOLOGY SLIDES AND CULTURE
53. PENICILLIUM Sp.,

1. It is normally a contaminant and non-pathogenic.
2. Hyphae- Septate and the conidiophores give rise to multibranching phialids thereby giving
a brush border like appearance.
3. Penicillium marneffei is an emerging dimorphic pathogen causing nasal and ocular
infections
54. CANDIDA Sp.

1. Candida albicans is a representative pathogen which produces germ tube &
chlamydospores.
2. Pseudohyphae in tissue indicate invasive nature of Candida.
3. Disease produced: oral thrush, vaginal thrush, endocarditis etc.,
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55. ASPERGILLUS FLAVUS
1. Vesicleis globose shaped.. Both uniserriated or biserriated sterigmata produced on the
entire or in the 3/4 th of vesicle.
2. The conidia are yellowish green in colour.
3. Disease produced: Otomycosis, Aspergillus asthma, Aspergilloma etc.
56. ASPERGILLUS FUMIGATUS

1. Vesicle is flask shaped .Uniserriate sterigmata produced only on the upper half of the
vesicle.
2. The conidia are smoky green in colour.
57. ASPERGILLUS NIGER

1. Vesicle is globose shaped. Biserriate sterigmata seen produced on the entire vesicle.
2. The conidia are black in colour.
58. MUCOR
1. Hyphae: Aseptate with branched sporangiophore containing globose sporangium.
2. Rhizoids are not produced and hence absent.
3. Disease produced: This is an opportunistic fungi causing skin infections in diabetic
patients and produce rhinocerebral mucormycosis.
59. RHIZOPUS
1. Hyphae: Aseptate with unbranched sporangiophore containing globose sporangium.

2. Rhizoids are present with four radial branches and is an important identifying feature.
3. Disease produced: involved in causing invasive disease of upper respiratory tract, nose,
lungs.
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7. SYSTEMIC BACTERIOLOGY
60. GRAM POSITIVE COCCI IN CLUSTERS

1. It is a Gram stained slide containing purple coloured cocci arranged in clusters.(Gram
positive)
2. An example of differential staining.
3. Example: Staphylococcus aureus, Staphylococcus epidermidis etc.,
61. GRAM NEGATIVE BACILLI

1. It is a Gram stained slide containing pink coloured rod shaped bacteria arranged
singly (Gram negative).
3. Example: E.coli, Salmonella, Proteus, Klebsiella etc.
62. GRAM POSITIVE BACILLI

1. It is a Gram stained slide containing purple coloured rod shaped bacteria (Gram positive)
3. Example: Corynebacterim diphtheriae, Bacillus anthracis, Clostridium sp.
63. GRAM POSITIVE COCCI IN CHAIN (STREPTOCOCCI)

1. It is a Gram stained slide containing purple coloured cocci arranged in chains (Gram
positive)
3. Example: Streptococcus pyogenes (causative agent of tonsillitis, pharyngitis, & Scarlet
fever and acute glomerulonephritis and rheumatic fever)
64. ACID FAST BACILLI

1. It is a sputum smear containing pink coloured acid fast bacilli seen among the blue
coloured multilobed pus cells.
238
2. An example of differential staining (Ziehl Neelson method)
3. Probably the smear contains Mycobacterium tuberculosis which causes tuberculosis.Other
examples: Atypical mycobacteria, lepra bacilli, bacterial spore, Nocardia,
Cryptosporidium.
65. CLOSTRIDIUM TETANI (BACTERIAL SPORES)

1. It is a Gram positive bacilli causing tetanus.
2. The presence of Clostridium tetani in theatre swabs indicates the need for fumigation.
3. The spores of non-toxigenic strains of Clostridium tetani are used to test the efficacy of hot
air oven.
66. GPC AND GNB:

1. GPC (Gram positive cocci) appear purple and GNB (Gram negative bacilli) appear pink in
Gram staining.
2. Both GPC and GNB are seen in direct gram stained clinical specimens like pus and wound
swabs.
3. Beta streptococci (GPC) and Klebsiella pneumoniae (GNB) cause co infections in diabetics
with foot ulcers.
8. IMMUNO SEROLOGY
67. RPR KIT:

1. RPR stands for rapid plasma reagin test based on the principle of slide flocculation.
2. It is a non-treponemal test used as a first line procedure for both routine diagnosis and mass
screening programme of syphilis.
3. Cardiolipin-Lecithin is the antigen used and detects reagin, a non-specific IgM antibody in
patient’s serum or plasma. Results are read in naked eye and are interpreted as reactive and
non- reactive.
239
68. WIDAL KIT
1. It is a febrile agglutination test done for the diagnosis of typhoid fever caused by
Salmonella spp.,
2. The test is based on demonstrating the presence of agglutinin (antibody) in the serum of an
infected patient, against the H (flagellar) and O (somatic) antigens of Salmonella typhi,
Salmonella paratyphi A and Salmonella paratyphi B.
69. ELISA KIT

1. ELISA stands for enzyme linked immunosorbent assay which uses enzyme labeled
antibodies to induce a visible reaction. This test can be designed to detect either an
antibody in patient’s serum or an antigen (viral) in patient’s specimen.
2. ELISA has been used for detection of antibodies of HIV and Mycobacterium and also in
detection Hepatitis B virus markers in serum.
70. RA KIT
1. RA stands for Rheumatoid arthritis. It detects the rheumatoid factor in the patient’s serum.
Rheumatoid factors are autoantibodies (IgM type) produced against one’s own IgG in
patients with rheumatoid arthritis.
2. This kit works based on the principle of latex agglutination slide test .When serum
containing RF is mixed with IgG coated latex particles, the RF bind to IgG and cause
agglutination.
71. ASO KIT

1. ASO stands for Antistreptolysin O test. This test method is based on the immunological
reaction (neutralization) between streptococcal exotoxins coated with latex particles and
streptococcal antibodies in the patient’s serum.
2. Normal levels of ASO in the blood are 120 Todd units per ml for adults and pre-schoolers
and 170 Todd units per ml in school aged children. An ASO level of 250 todd units per ml
indicates prior streptococcal infection.
240
72. CRP KIT
1. CRP stands for C-reactive protein. CRP is an acute phase reactant which appears rapidly
in the serum of patients with inflammation of any origin. It is absent in normal individuals.
2. It works on the principle of rapid latex slide agglutination where latex particles are coated
with anti CRP that agglutinates in the presence of CRP.
3. It is a non-specific test but the findings can be used as a simple index of disease activity
and treatment status in conditions like viral meningitis and bacterial infections.
241

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MICROBIOLOGY PRACTICAL MANUAL

GOVT. DHARMAPURI MEDICAL COLLEGE

2. Coarse Adjustment -For focusing under low magnification

3. Fine Adjustment-For focusing under high magnification or low

clinical specimen smear.

5. High Power Objective-For detailed viewing or small specimens

6. Oil immersion objective- For a magnified view of the objects

7. Specimen on glass slide-What you want to look at

8. Stage-Supports specimen in correct location to lens

9. Condenser-Focuses the light on specimen

10. Diaphragm (iris or disc)-Regulates amount of light and contrast

11. Light Source-Illuminates the specimen for viewing.

HOW TO COMPUTE MAGNIFICATION

HOW TO CARRY A COMPOUND MICROSCOPE

ADJUSTMENTS OF THE MICROSCOPE

Lower the condenser

If needed, diaphragm may be closed.

Keep the concave mirror position as such.

Slightly raise the condenser.

Open the diaphragm little more.

Use a plain mirror to direct the light.

Raise the condenser to the maximum.

Open the diaphragm completely.

1. Place the slide under the stage clips on the stage.

2. Put on the lowest power objective lens. (10x)

4. First, focus with the coarse focus adjustment.

5. Secondly, focus with the fine focus adjustment.

6. Move to the high power objective lens. (40x)

7. Do the adjustments for x40 objective.

8. Focus with the fine focus adjustment alone

9. Get the object to be studied focused under high power.

the next objective lens to go into position over the slide.

between the object and objective lens.

13. Do the adjustments required for oil immersion objective.

14. Observe the image and record your findings.

objective oil-immersion lens with alcohol and lens paper.

FREQUENTLY ASKED QUESTIONS IN MICROSCOPY

2. What is the type of microscope you are using in the lab?.

3. What are the various objective lenses seen in your microscope?

6. What is the use of oil immersion objective?

7. What are the microscopic adjustments for low power objective?

8. What are the microscopic adjustments for high power objective?

10. How will you clean new glass slide?

11. What are the advantages of smear fixation?

To focus the light rays within the Objective lens.

13. What do you understand by “Resolution power” of a microscope?

1. A smear is made, air-dried/ heat-fixed in a grease free slide.

6. The slide is air dried and observed it under oil immersion.

GRAM’S REACTION: Positive

The given smear contains Gram positive cocci arranged in clusters.

EXAMPLES: Staphylococcus aureus, Staph epidermidis.

GRAM’S REACTION : Negative

GRAM STAINING - MIXTURE

GRAM’S REACTION : Positive GRAM’S REACTION : Negative

COLOUR : Violet COLOUR : Pink

SHAPE : Spherical SHAPE : Rod

ARRANGEMENT : Clusters ARRANGEMENT : Discrete

Eg: GPC- Staphylococcus aureus, GNB- E.coli

FREQUENTLY ASKED QUESTIONS IN GRAM STAINING

1. Who invented Gram stain?