Unit-I Microbiology: Amit Kumar Rai

AMIT KUMAR RAI PHARMACEUTICAL MICROBIOLOGY
UNIT- I
MICROBIOLOGY
INTRODUCTION
➢ Microbes: Microbe or microorganism, is a microscopic organism that comprises either a single cell
(unicellular), cell clusters or multicellular.
➢ The study of microorganisms is called microbiology, a subject that began with Anton van Leeuwenhoek’s
(Father of Microbiology) discovery of microorganisms in 1675, using a microscope of his own design.
➢ Microorganisms include bacteria, fungi, algae, protozoa, microscopic plants (green algae) and animals such as
rotifers and planarians. Some microbiologists also include viruses, but others consider these as nonliving.
➢ Most microorganisms are unicellular or multicellular organisms.
HISTORY OF MICROBIOLOGY
➢ First observation of microbes: Anton van Leeuwenhoek (1632–1723) discovered the ‘invisible’ world of
microorganisms ‘animalcules’. He was the first microbiologist and the first person to observe bacteria using a
single-lens microscope of his own design.
➢ Cell Theory: Robert Hook (1665) reported that life’s smallest structural units were ‘little boxes’ or ‘cells’.
This marked the beginning of cell theory – that all living things are composed of cells
➢ Spontaneous generation: Until second half of nineteenth century many believed that some forms of life
could arise spontaneously from non-living matter. The hypothesis that living organisms arise from nonliving
matter is called spontaneous generation. According to spontaneous generation, a “vital force’ forms life.
• Francesco Redi (1668): Strong opponent of spontaneous generation. He demonstrated that maggots appear
on decaying meat only when flies are able to lay eggs on the meat.
• John Needham (1745) claimed that microorganisms could arise spontaneously from heated nutrient broth.
• Lazzaro Spallanzani (1765) repeated Needhams experiments and suggested that Needham’s results were due
to microorganisms in the air entering the broth.
➢ Biogenesis: Rudolf Virchow (1858) gave concept of biogenesis which states that the living organisms arise
from preexisting life.
• Louis Pasteur (1861) demonstrated that microorganisms are present in the air. His experiments on swan
shaped necks resolved the controversy of spontaneous generation. His discoveries led to the development of
aseptic techniques used in the laboratory and medical procedure to prevent contamination by microorganisms
that are in the air.
➢ Golden Age of Microbiology (1857-1914): Rapid advances in the science of microbiology were made
between 1857 and 1914.
K KAILASH INSTITUTE OF PHARMACY & MANAGEMENT, GIDA, GORAKHPUR 1

• Louis Pasteur (1822-1895): Beginning with Pasteur’s work, discoveries included the relationship between
microbes and disease, immunity, and antimicrobial drugs.
• Fermentation and Pasteurization: Pasteur showed that microbes are responsible for fermentation.
Fermentation is the conversation of sugar to alcohol to make beer and wine. Microbial growth is also
responsible for spoilage of food. Pasteur demonstrated that these spoilage bacteria could be killed by heat that
was not hot enough to evaporate the alcohol in wine. This application of a high heat for a short time is called
pasteurization. Now a days pasteurization is used to kill bacteria in milk.
➢ The Germ theory of disease:
• Agostino Bassi (1835) showed a casual relationship between microorganisms and disease.
• Pasteur (1865) believed that another silkworm disease was caused by a protozoan.
• Ignaz Semmelwise (1840) said hand washing to prevent transmission of puerperal fever from one obstetric
patient to another.
• Joseph Lister (1860) introduced the use of disinfectant to clean surgical dressings in order to control infection
in humans.
• Robert Koch (1876) proved that microorganisms transmit disease. Koch’s postulates to prove that a particular
microorganism causes a particular disease. He introduced pure cultures. Koch also developed techniques for
isolating organisms. He identified the bacillus that causes tuberculosis and anthrax, developed tuberculin and
studied various diseases in Africa and Asia. His studies on Tuberculosis won him Nobel Prize for philosophy
and medicine in 1905.
➢ Vaccination:
• Edward Jenner (1796) inoculated a person with cowpox virus. The person was then protected from smallpox.
• Called vaccination from vacca for cow.
• The protection is called immunity.
➢ The Birth of Modern Chemotherapy
• Treatment with chemicals is chemotherapy.
• Chemotherapeutic agents are used to treat infectious disease can be synthetic drugs or antibiotics.
• Antibiotics are chemicals produced by bacteria and fungi that inhibit or kill other microbes.
• Quinine from tree bark was long used to treat malaria.
• Paul Ehrlich (1910) developed a synthetic arsenic drug, Salvarsan, to treat syphilis.
• 1930s Sulfonamides were synthesized.
• Alexander Fleming (1928) discovered the first antibiotic. He observed that Penicillium fungus made an
antibiotic, penicillin that killed S. aureus.
• Penicillin (1940s) was tested clinically and mass produced.

BRANCHES OF MICROBIOLOGY
➢ The entire microbiology may be sub-divided into followings:
A. Pure Microbiology:
1. Bacteriology: The study of organism (bacteria).
2. Mycology: The study of fungi.
3. Phycology/Algology: The study of algae.
4. Protozoology: The study of protozoans.
5. Virology: The study of viruses.
6. Parasitology: The study of parasites
7. Immunology: The study of the immune system
8. Nematology: The study of nematodes
9. Microbial cytology: The study of microscopic details of microorganisms
10. Microbial physiology: The study of microbial growth, microbial metabolism and microbial cell structure
11. Microbial Ecology: The relationship between microorganisms and their environment
12. Microbial Genetics: The study of how genes are organized and regulated in microbes.
13. Evolutionary microbiology: The study of the evolution of microbes.
14. Generation microbiology: The study of those microorganisms that have the same characters as their parents
15. Systems microbiology: A discipline bridging systems biology and microbiology.
16. Phylogeny: The study of the genetic relationships between different organisms.
B. Other
1. Astro microbiology: The study of microorganisms in outer space
2. Biological agent: The study of those microorganisms which are being used in weapon industries.
3. Nano microbiology: The study of those organisms on nano level.
C. Applied Microbiology
1. Medical microbiology: The study of the pathogenic microbes and the role of microbes in human illness. This
area of microbiology also covers the study of disease pathology and immunology, the study of human
microbiota, cancer, and the tumor microenvironment.
2. Pharmaceutical microbiology: The study of microorganisms that are related to the production of antibiotics,
enzymes, vitamins, vaccines, and other pharmaceutical products.
3. Industrial microbiology: The exploitation of microbes for use in industrial processes. Closely linked to
the biotechnology industry.
4. Microbial biotechnology: The manipulation of microorganisms at the genetic and molecular level to
generate useful products.
5. Food microbiology: The study of microorganisms causing food spoilage and food borne illness.

6. Agricultural microbiology: The study of agriculturally relevant microorganisms. Further classified into:
a) Plant microbiology and Plant pathology: The study of the interactions between microorganisms and
plants and plant pathogens.
b) Soil microbiology: The study of those microorganisms that are found in soil.
7. Veterinary microbiology: The study of the role of microbes in veterinary medicine or animal taxonomy.
8. Environmental microbiology: Study of the function and diversity of microbes in their natural environments.
9. Water microbiology (or aquatic microbiology): The study of those microorganisms that are found in water.
10. Aeromicrobiology (or air microbiology): The study of airborne microorganisms.
11. Biotechnology: related to recombinant DNA technology or genetic engineering
SCOPE & ADVANTAGES OF MICROBIOLOGY

1. Aero-Microbiology helps in the overall preservation and preparation of food, food-prone diseases and their
ultimate prevention.
2. Beverage Microbiology helps in making of beer wine and a variety of alcoholic beverages e.g., whisky,
brandy, rum etc.
3. Exomicrobiology help in the exploration of life in the outer space.
4. Food Microbiology help in making of cheese, yogurt.
5. Geochemical Microbiology to help in the study of coal, mineral deposits, and gas formation, prospecting the
deposits of gas and oil, coal, recovery of minerals from low-grade ores.
6. Industrial Microbiology helps in making of ethanol, acetic acid, citric acid, glucose & high-fructose syrup.
7. Medical Microbiology helps in the diagnostic protocol for identification of causative agents of various
human ailments and subsequent preventive measures.
8. Pharmaceutical Microbiology helps in making of life-saving drugs, ‘antibiotics’ e.g., penicillins, ampicillin,
chloramphenicol, ciprofloxacin, tetracyclines, streptomycin.
9. Soil and Agricultural Microbiology helps in the maintenance of a good farm land by keeping and sustaining
a reasonable and regular presence of microbes in it.
10. Waste-Treatment Microbiology help in treatment of domestic and industrial effluents or wastes by lowering
the BOD (Biological Oxygen Demand) and COD (Chemical Oxygen Demand).
11. Genetics: Mainly involves engineered microbes to make hormones, vaccine, antibiotics and many other
useful products for human being.
12. Food science: It involves the prevention of spoilage of food and food borne diseases and the uses of microbes
to produce cheese, yoghurt, pickles and beer.
13. Immunology: The study of immune system which protect the body from pathogens.
Medicine: It deals with the identification of plans and measures to cure diseases of human and animals which are
infectious to them.

INTRODUCTION TO PROKARYOTES & EUKARYOTES

➢ Microorganisms and all other living organisms are classified as prokaryotes or eukaryotes.
➢ Prokaryotes and eukaryotes are distinguished on the basis of their cellular characteristics.
➢ For example, prokaryotic cells lack a nucleus and other membrane‐bound structures known as organelles,
while eukaryotic cells have both a nucleus and organelles.
➢ Prokaryotic and eukaryotic cells are similar in several ways. Both types of cells are enclosed by cell
membranes (plasma membranes), and both use DNA for their genetic information.
➢ Prokaryotes include several kinds of microorganisms, such as bacteria and cyanobacteria.
➢ Eukaryotes include such microorganisms as fungi, protozoa, and simple algae.
➢ Viruses are considered neither prokaryotes nor eukaryotes because they lack the characteristics of living
things, except the ability to replicate (which they accomplish only in living cells).
The important cellular features of (a) a prokaryotic cell (a bacterium) and (b) a eukaryotic cell.

Characteristic Distinguishing Features of Prokaryotic & Eukaryotic Cells
Prokaryotic Cell Eukaryotic Cell
These are organisms made up of cells that These organisms are made up of cells
Definition lack a cell nucleus or any membrane-bound that have a membrane-bound nucleus as
organelles. well as membrane-bound organelles.
It has a true nucleus, bounded by a

Nucleus It has no nucleus.
double membrane.
DNA arrangement It has a circular loop. It is linear.
Size Small cells ( < 5 µm) Large cells ( < 10 µm)
Cell Always unicellular Mostly multi-cellular
Usually present; chemically complex in When present, chemically simple in

Cell wall
nature nature
It contains proteins in the DNA to form

Protein It does not contain protein in its DNA.
chromatin.
Ribosome It contains small ribosomes. It contains large ribosomes.
Cytoplasm No cytoskeleton Always have cytoskeleton
Cell division Cell division is by binary fission Cell division is by mitosis
Reproduction Reproduction is always asexual Reproduction is asexual or sexual
It is complex in nature and consists of

Flagella Consist of two protein building blocks
multiple microtubules
Multi-cellular
Rare Common with extensive tissue formation
forms
They perform functions of Golgi-bodies and

Mesosomes mitochondria, and also help in separation of Not present
chromosomes.
Sterols and carbohydrates are both

Plasma membrane No carbohydrates and lacks sterols
present
Example Bacteria and Archaea Animal cells and plant cells

BACTERIA
➢ Prokaryotic organisms.
➢ Vary in sizes, measure approximately 0.1 to 10.0 μm
➢ Widely distributed. It can be found in soil, air, water, and living bodies.
➢ Some bacteria can cause diseases for human, animals and plants. Some bacteria are harmless (i.e. live in
human bodies as normal flora)
STRUCTURE
➢ The outer layer or cell envelope consists of two components, a rigid cell wall and beneath it a cytoplasmic or
plasma membrane. The cell envelope encloses the protoplasm, comprising the cytoplasm, cytoplasmic
inclusions such as ribosomes and mesosomes, granules, vacuoles and the nuclear body.
1. Cell Wall: It is very rigid & gives shape to the cell. Its main function is to prevent the cell from swelling and
rupturing due to water uptake. Chemically the cell wall is composed of peptidoglycan (also known as Murein
or Mucopeptide). Mucopeptide formed by repeating disaccharide units of N acetyl glucosamine (NAG) & N
acetyl muramic acid (NAM) alternating in chains and cross linked by peptide chains. Peptide chain is
composed of amino acids, L-alanine, D-glutamic acid, L-lysine and D-alanine. Polyalcohols called Teichoic
acid are embedded in it to form a carbohydrate backbone. Some are linked to Lipids & called Lipoteichoic
acid. Lipotechoic acid link peptidoglycan to cytoplasmic membrane and the peptidoglycan give rigidity.
Teichoic acids are responsible for negative charge, major antigenic transport ions, anchoring and external
permeability barrier.

• On the basis of cell wall structure bacteria can be classified as Gram positive bacteria (G +) and Gram negative
bacteria (G-). The names originate from the reaction of cells to the Gram stain, a test long-employed for the
classification of bacterial species. Gram-positive bacteria possess a thick cell wall containing many layers of
peptidoglycan and teichoic acids. Gram-negative bacteria have a relatively thin cell wall consisting of a few
layers of peptidoglycan surrounded by anouter membrane (second lipid membrane containing
lipopolysaccharides and lipoproteins). These differences in structure can produce differences in property as
antibiotic susceptibility.
• Characteristics of Gram Positive &Gram Negative
Characteristics Gram Positive Gram Negative

Thickness Thicker Thinner
Variety of amino acids Few Several
Lipids Absent Present
Teichoic acid Present Absent
2. Outer Membrane:Outer membrane is found only in Gram-negative bacteria. It actas an initial barrier to the
environment and is composed of lipopolysaccharide (LPS) and phospholipidsLipopolysaccharide (LPS)are
endotoxic and antigen specific.
3. Cytoplasmic membrane:Cytoplasmic membrane is present immediately beneath the cell wall, found in
both Gram positive & negative bacteria.It is a thin layer lining of inner surface of cell wall and separating it
from cytoplasm. It acts as a semipermeable membrane controlling the flow of metabolites to and from the
protoplasm.
4. Cytoplasm:It is a colloidal system contains a variety of organic and inorganic solutes containing 80% Water
and 20% Salts, Proteins. They are rich in ribosomes, DNA & fluid. DNA is circular and haploid. Plasmids are
extra circular DNA.
5. Ribosomes:They are the centers of protein synthesis. They are slightly smaller than the ribosomes of
eukaryotic cells.
6. Mesosomes:They are vesicularand formed by invagination of plasma membrane into the cytoplasm. They
are principal sites of respiratory enzymes and help with cell reproduction.
7. Cytoplasmic Inclusions:The Inclusion bodies are aggregates of polymers produced when there is excess of
nutrients in the environment and they are the storage reserve for granules, phosphates and other substances.
Volutin granules are polymetaphosphates which are reserves of energy and phosphate for cell metabolism and
they are also known as metachromatic granules.
8. Nucleus:The Nucleus is not distinct and has no nuclear membrane or nucleolus and the genetic material
consist of DNA. The cytoplasmic carriers of genetic information are termed as plasmids or episomes.

9. Capsule: Capsule is the outer most layer of the bacteria (extra cellular). It is a condensed well defined layer
closely surrounding the cell. They are usually polysaccharide. They are secreted by the cell into the external
environment and are highly impermeable. When masses of polymer t appear to be totally detached from the
cell and if the cells are seen entrapped in it are described as slime layer. The Capsule protects against drying
or harsh chemicals.
10. Flagella: Flagella are long hair like helical filaments extending from cytoplasmic membrane to exterior of
the cell. Flagellin is highly antigenic and helps in cell motility. They hel[s to move the bacteria towards
nutrients and other attractants and this process or movement is known as Chemotaxis.
• Positive chemotaxis is movement of bacteria towards chemicals.
• Negative chemotaxis is movement of bacteria away from chemicals.
• Phototaxis is movement of bacteria towards light.
• The parts of flagella are the filament, hook and the basal body.
a) Filament is a hollow, rigid cylinder and external to cell wall and is connected to the hook at cell surface.
Hook & basal body are embedded in the cell envelope. Hook & filament is composed of protein subunits
called as flagellin. Flagellin is synthesized within the cell and passes through the hollow centre of flagella.
b) Basal body anchors the flagellum to the cell wall and plasma membrane. It is most complex part and inserted
into a series of rings. In E. coli and most gram negative bacteria there are four rings present named as L,P,S
and M ring. Gram positive bacteria have only two basal body ring. Movement of prokaryotic flagellum is due
to the rotation of basal body either clockwise or anti-clockwise.
• On the basis of flagella bacteria are divided into following types:

i. Atrichous: flagellum is absent. Eg. Pastulella
ii. Monotrichous: single flagella on one side. Eg. Pseudomonas
iii. Lophotrichous: tuft of flagella on one side. Eg. Vibrio
iv. Amphitrichous: single or tuft on both sides. Eg. Nitrosomonas
v. Peritrichous: surrounded by lateral flagella. Eg. Escherichia

11. Pili / Fimbriae: Hair-like proteineous structures that extend from the cell membrane to external
environment are pili which are otherwise known as fimbriae. They are thinner, shorter and more numerous
than flagella and they do not function in motility. There are two types pili namely Non-sex pili (Common pili)
eg. fimbriae or type IV and the sex pili. The fimbriae are antigenic and mediate their adhesion which inhibits
phagocytosis. The sex pili help in conjugation.
MORPHOLOGICAL CLASSIFICATION OF BACTERIA

➢ Depending on their shape, bacteria are classified into several varieties
1. Cocci:Spherical or oval cells. They are further subdivided into:
i. Monococcus: They occur singly. E.g. Micrococcus
ii. Diplococcus: They occur in pair. E.g. Diplococcus pneumonia.
iii. Tetracoccus: They occur in group of four.
iv. Streptococcus: They occur in the chain form. E.g. Streptococcus lactis.
v. Staphylococcus: They occur in group. E.g. Staphylococcus aureus
2. Bacilli:Rod shaped cells.They are further subdivided into:
i. Monobacillus: They occur singly.
ii. Diplobacillus: They occur in pair.
iii. Streptobacillus: They occur in chain. E.g. Bacillus antracis
3. Vibrios:Comma shaped curved rods and derives their name from their characteristics vibratory motility. E.g.
Vibrio cholerae
4. Spirilla:They are rigid spiral forms. E.g. Spirillum ruprem
5. Spirochetes:They are flexuousspiral forms
6. Actinomycetes are branching filamentous bacteria.
7. Mycoplasmas are bacteria that are cell wall deficient and hence do not possess a stable morphology. They
occur as round or oval bodies and asinterlacing filaments.

GRAM-NEGATIVE BACTERIA V/S GRAM-POSITIVE BACTERIA

Gram-Negative Bacteria Gram-Positive Bacteria
Gram reaction They don't retain the Gram stain when Retain crystal violet dye and stain dark
washed with alcohol and acetone. They violet or purple, they remain coloured
decolourized to accept counter stain blue or purple with gram stain when
(Safranin or Fuchsine). Stain red/pink. washed with absolute alcohol and water.
Cell wall The cell wall is 70-120 A0 thick; two The cell wall is 100-120 A0 thick; single
composition layered. Lipid content is 20-30% (high), layered. Lipid content of the cell wall is
Murein content is 10-20% (low). low, Murein content is 70-80% (higher).
Peptidoglycan layer Thin (single-layered) Thick (multilayered)
Teichoic acids Absent Present in many
Periplasmic space Present Absent
Outer membrane Present Absent
Lipopolysaccharide High Virtually none

(LPS) content
Lipid and High (due to presence of outer Low (acid-fast bacteria have lipids linked
lipoprotein content membrane) to peptidoglycan)
Flagellar structure 4 rings in basal body 2 rings in basal body
Toxins produced Primarily Endotoxins Primarily Exotoxins
Resistance to Low High

physical disruption
Inhibition by basic Low High

dyes
Susceptibility to Low High

anionic detergents
Resistance to Low High

sodium azide
Resistance to drying Low High
Mesosome Mesosome is less prominent. Mesosome is more prominent.
Antibiotic More resistant to antibiotics. More susceptible to antibiotics

Resistance

NUTRITIONAL REQUIREMENTS FOR BACTERIA

➢ Bacterial requirements for growth includes:
A. Physical or Environmental Requirements:
1. Energy: Bacteria can be classified nutritionally based on their energy requirements.
a) Bacteria which derive energyfrom sunlight are called phototrophs.
b) Those that obtain energy from chemicalreactions are called chemotrophs.
c) Bacteria that can synthesise all their organicompounds from inorganic substances are called autotrophs.
d) Some bacteria are unable to synthesise their own metabolites. They depend on preformed organic
compounds. They are called heterotrophs.
2. pH: The range of pH over which an organism grows is defined as the minimum pH, below which the
organism cannot grow, the maximum pH, above which the organism cannot grow, and the optimum pH, at
which the organism grows best.
a) Acidophiles: Microorganisms which grow at pH (3-5).
b) Neutrophiles: Microorganisms which grow best at neutral pH (6-8).
c) Alkaliphiles: Microorganisms which grow best under alkaline conditions pH as high 10.5 are called.
3. Temperature:The temperature range at which organism grow best is called optimum temperature. In human
parasitic organism optimum temperature ranges between 30°C and 37°C. There are three groups of bacteria as
regard to the temperature:
a) Psychrophilic:The bacteria are growing between 0° C and 25°C. They are mostly soil and water bacteria.
b) Mesophilic:Some bacteria grow between 20°C and 44°C. This group includes bacteria producing disease.
c) Thermophilic: The bacteria can grow between 50 and 80°C. These bacteria will survive after
pasteurization processes of milk.
4. Moisture: Water is needed for the growth and reaction of metabolism like glycolysis and protein synthesis. In
the absence of the water some bacteria will form a spore for continue its survival.
B. Nutrients Requirements: Some common nutrients are Macro elements (macronutrients) which required
in relatively large amounts such as H, O, C, N, S, P & Micronutrients (trace elements) which required in
trace amounts such as K, Ca, Mg, Fe, Cu, Mn, Zn, Co, Mo and Ni.
1. Carbon:It is needed for skeleton or backbone of all microorganisms. Source of carbon are CO 2 and organic
compounds.
2. Nitrogen: It is a major component of protein and nucleic acid. It is needed for synthesis of important
molecules (e.g., amino acids, nucleic acids). Most organisms obtain N fro ammonia and oxidized form of
nitrate such as reduce nitrate (NO3-) and Nitrite (NO2-).
3. Hydrogen:Hydrogen is usually obtained from water, and oxygen is obtained from atmosphere or from water.

4. Oxygen: Oxygen can be obtained from various forms such as water etc. Microorganisms are classified with
respect to the effect of oxygen on their growth and metabolism:
a) Obligate aerobes: Theyuse and require oxygen as electron acceptor. They have respiratory enzymes and
lack the capacity for fermentations. Examples: Pseudomonas, some Bacillus
b) Obligate anaerobes: They do not need or use O2 as a nutrient. In fact, O2 is a toxic substance, which
either kills or inhibits their growth. They may live by fermentation, anaerobic. Examples: Clostridium,
Bacteroides.
c) Facultative organisms: They are organisms that can switch between aerobic and anaerobic types of
metabolism. Under anaerobic conditions (no O2) they grow by fermentation or anaerobic respiration, but
in the presence of O2 they switch to aerobic respiration. Examples: all Enterobacteriaceae (E.coli), some
Bacillus.
5. Sulphur: It is needed for synthesis of some amino acids such as cysteine and methinine. Sulphate is the
principal source of sulphur.
6. Other elements:Inorganic compounds present in the environment and those released in decomposition of
organic substrates are principal sources of other major nutrient elements and micronutrients. Phosphorus that
is bound in organic compounds is releases as phosphoric acid during decomposition.
7. Growth Factors: Growth factors are required in small amounts by cells because they fulfill specific roles in
biosynthesis. The need for a growth factor results from either a blocked or missing metabolic pathway in the
cells. Growth factors are organized into three categories.
a) Purines and pyrimidines: Required for synthesis of nucleic acids (DNA and RNA).
b) Amino acids: Required for the synthesis of proteins.
c) Vitamins: Needed as coenzymes and functional groups of certain enzymes.
CULTURE MEDIA
➢ A culture medium is a solid, liquid or semi-solid designed to support the growth of microorganisms or cells.
➢ A culture medium contains water, a source of carbon & energy, source of nitrogen, trace elements and some
physical growth parameters or factors necessary for microbial growth.The pH of the medium must be set
accordingly.
➢ Basic Ingredients:
1. Protein source: Peptone (a complex mixture of partially digested proteins). E.g. are Neopeptone& proteose
peptone. Digest broths can also be used such as meat extract.
2. Mineral salt: Sodium chloride.
3. Agar: Long chain polysaccharides. Prepared for using solid media. Obtained from sea weeds. Also contains
varying amounts of inorganic salts and small quantities of a protein like substance. Hydrolized at high

temperature at high acid or alkaline pH. Melts at 98ºC and usually sets at 42ºC depending on agar
concentration. Approximately 2% agar is used to prepare solid media.
4. Water: Source of oxygen and hydrogen.
➢ Classification of Culture Media

A. On The Basis of Consistency
1. Solid medium: Solid medium contains agar at a concentration of 1.5-2.0%. Solid medium is useful
for isolating bacteria or for determining the colony characteristics of the isolate.
2. Semisolid media: They are prepared with agar at concentrations of 0.5% or less. They have soft custard like
consistency and are useful for determination of bacterial motility.
3. Liquid (Broth) medium: These media contains specific amounts of nutrients but don’t have gelling agents
such as gelatin or agar. Broth medium serves various purposes such as propagation of large number of
organisms, fermentation studies, and various other tests.
B. On the basis of composition:

1. Natural Medium: It includes milk, urine, blood, meat extract, vegetable juices, peptone etc.
2. Synthetic or chemically defined medium: They are prepared from purified ingredients and there exact
composition is known. E.g. Richard’s solution.
3. Non synthetic or chemically undefined medium: Their chemical composition is partially known. They
contain agar as a component. E.g. Potato dextrose agar.
C. On the basis of purpose/ functional use/ application:

1. General purpose media/ Basic media: Basal media are basically simple media. Peptone water, nutrient
broth and nutrient agar are considered as basal medium. These media are generally used for the primary
isolation of microorganisms.
2. Enriched medium (Added growth factors):Addition of extra nutrients in the form of blood, serum, egg yolk
etc, to basal medium makes them enriched media. E.g. Blood agar, chocolate agar, Loeffler’s serum.
3. Selective and enrichment media: Both these media serve the same purpose. They selectively allow the
growth of particular type of microorganism and prevent the growth of other type of microorganism.
a) Selective media are agar based (solid). E.g. MacConkey’s agar
b) Enrichment media are liquid in consistency. E.g. Selenite F broth
4. Differential/ indicator medium: Theses media are designed in such a way (by the incorporation of dyes) that
different bacteria can be recognized on the basis of their colony colour. E.g. Mannitol salts agar, Blood agar
etc.

GROWTH AND MULTIPLICATION OF BACTERIA

➢ Bacteria divide by binary fission and cell divides to form two daughter cells.
➢ Bacterial growth may be considered as two levels, increase in the size of individual cells and increase in
number of cells.
➢ Growth in numbers can be studied by bacterial counts that of total and viable counts.
➢ The total count gives the number of cells either living or not and the viable count measures the number of
living cells that are capable of multiplication.
➢ Bacterial Growth Curve: When bacteria is grown in a suitable liquid medium and incubated its
growthfollows a definite process. If bacterial counts are carried out at intervals afterinnoculation and plotted
in relation to time, a growth curve is obtained. Thecurve shows the following phase:
1. Lag phase: Immediately afterinoculation there is no appreciable increase in number.There may be an increase
in the size of the cells. This initial period is the time required for adaptation to the new environment and this
lag phase varies with species, nature of culture medium and temperature.
2. Log or exponential phase: After the lag phase, the cell starts dividing and their numbers increase
exponentially with time.
3. Stationary phase: After a period of exponential growth, cell division stops due to depletion of nutrient and
accumulation of toxic products. The viable count remains stationary as equilibrium exists between the dying
cells and the newly formed cells.
4. Phase of decline: This is the phase when the population decreased due to cell death.
PHYSICAL PARAMETERS OR FACTORS THAT AFFECT THE GROWTH OF BACTERIA

➢ Many factors affect the generation time of the organism like temperature, oxygen, carbon dioxide, light, pH,
moisture, salt concentration.
1. Nutrition: The principal constituents of the cells are water, proteins, polysaccharides, lipids, nucleic acid and
mucopeptides. For growth and multiplication of bacteria, the minimum nutritional requirement is water, a
source of carbon, nitrogen and some inorganic salts. Some bacteria require certain organic compounds in
minute quantities. These are known as growth factors or bacterial vitamins. Growth factors areessential
because growth does not occur in their absence, or they are necessary for it.

2. Oxygen: Depending on oxygen for growth and viability, bacteria are divided into aerobes and anaerobes.
Aerobic bacteria require oxygen for growth. They may be obligate aerobes like vibrio cholera, which will
grow only in the presence of oxygen or facultative anaerobes which are ordinarily aerobic but can grow in the
absence of oxygen. Most bacterial of medical importance are facultative anaerobes. Anaerobic bacteria, such
as clostridia, grow in the absence of oxygen and the obligate anaerobes may even die on exposure to oxygen.
Microaerophilic bacteria are those that grow best in the presence of low oxygen.
3. Carbon Dioxide: All bacteria require small amounts of carbon dioxide for growth. This requirement is
usually met by the carbon dioxide present in the atmosphere. Some bacteria like Brucella abortus require
much higher levels of carbon dioxide.
4. Temperature: Bacteria vary in their requirement of temperature for growth. The temperature at which
growth occurs best is known as the optimum temperature. Bacteria which grow best at temperatures of 25-
40°C are called mesophilic. Psychrophilic bacteria are those that grow best at temperatures below 20°C.
Another group of non pathogenic bacteria, thermophiles, grow best at high temperatures, 55-80°C. The lowest
temperature that kills a bacterium under standard conditions in a given time is known as thermal death point.
5. Moisture and Drying: Water is an essential ingredient of bacterial protoplasm and hence drying is lethal to
cells. The effect of drying varies in different species.
6. Light: Bacteria except phototrophic species grow well in the dark. They are sensitive to ultraviolet light and
other radiations. Cultures die if exposed to light.
7. H-ion concentration: Bacteria are sensitive to variations in pH. Each species has a pH range, above or below
which it cannot survive and an optimum pH at which it grows best. Majority of pathogenic bacteria grow best
at neutral or slightly alkaline pH (7.2 – 7.6)
8. Osmotic Effect: Bacteria are more tolerant to osmotic variation than most other cells due to the mechanical
strength of their cell wall. Sudden exposure to hypertonic solutions may cause osmotic withdrawal of water
and shrinkage of protoplasm called plasmolysis.
ISOLATION METHODS FOR PURE CULTURES

➢ Pure culture can be defined as a population of cells arising from a single cell.
➢ The three most commonly cultures of microorganisms are:
1. Spread Plate Method:
• It is an easy and direct method.
• In this technique microorganisms are spread over the solidified agar medium with a sterile L-shaped glass rod
to separate the microorganisms from each other. Procedure is as follow:
i. Dip the glass rod in 95% alcohol.
ii. Transfer the loop full culture on nutrient agar plate aseptically and cover it with the help of another petri dish.

iii. Take out the glass rod from the beaker and sterile the bent portion in the Bunsen burner flame. Cool the rod.
iv. Remove the cover of petri dish and spread the culture evenly with the help of glass rod over the agar surface.
v. Replace the petri dish cover and dip the glass rod in alcohol and reflame to sterilize it.
vi. Incubate the plate in an incubator in an inverted position at 35ºC for 24-48 hours.
Spread Plate Method Streak Plate Method
2. Streak Plate Method:

• Most practical method of obtaining discrete colonies and pure culture.
• In this method sterilized loop or transfer needle is dipped into a suitable dilute suspension of organisms which
is then streaked on the surface of solidified agar plate to make a series of parallel, non-overlapping streaks.
• Procedure is as follows:
i. Hold the tube containing the broth in the left hand.
ii. Sterilize the loop holding in the right hand, remove the cotton wool plug, using the little figure of right hand
and immediately flame the mouth of testy tube.
iii. Introduce the loop into the broth and withdraw one loop full of culture.
iv. Flame the mouth of test tube, replace the cotton wool plug and place it in the test tube rack.
v. Lift the petri dish cover with the left hand and hold it.
vi. Place the innoculum (loop containing the droplet of broth) on agar surface edge which is on far end and streak
the innoculum from side to side in parallel lines across the surface of agar.
vii. Reflame and cool the loop and turn the petri dish at 90º. Touch the loop to a corner of the previous area streak
the innoculum across area.
viii. Again flame and cool the loop & then make another streak from the second are by turning the petridish at 90º.
ix. After completing the streak sterilize the loop by flaming.
x. Incubate the plate in an inverted position for 48-72 hours.
3. Pour Plate Method: In this method successive dilutions of the inoculums (serial dilution of original
specimen) are added into sterile petri dish which contain molten agar medium (at 42-45ºC) and thoroughly
mixed by rotating the plates which is then allowed to solidify.Procedure is as follow:

i. Liquefy the nutrient agar in the tubes by heating them in water bath.
ii. Cool the tubes to 45°C and hold at this temperature until ready to pour into the plate.
iii. Label or mark the tubes and corresponding petri dishes with increasing order of number.
iv. Aseptically transfer 1 ml of the bacterial suspension from stock to tube number 1 with the use of sterile
pipette.
v. Serially dilute the culture aseptically from the test tube no. 1 to the test tube no. 6 by taking 1 ml of the
sample from each test tube to the next test tube.
vi. Mix the each sample with agar medium by gently rotating the tubes between palms.
vii. Pour the contents of tubes into the corresponding labeled petri dish and allow to solidify.
viii. Incubate the plates in inverted position at 37°C for 24-48 hours.
Pour Plate Method
PRESERVATION METHODS FOR PURE CULTURES

➢ Once a microorganism has been isolated and grown in pure culture, it becomes necessary to maintain the
viability and purity of the microorganism by keeping the pure culture free from contamination.
➢ Preservation includes:
1. Periodic Transfer to Fresh Media: Normally in laboratories, the pure cultures are transferred periodically
onto or into a fresh medium (subculturing) from the previous stock culture to allow continuous growth and
viability of microorganisms. The transfer is always done in aseptic conditions to avoid contamination.
2. Refrigeration: Pure cultures can be successfully stored at 0-4°C either in refrigerators or in cold-rooms. This
method is applied for short duration (2-3 weeks for bacteria and 3-4 months for fungi) because the metabolic
activities of the microorganisms are greatly slowed down but not stopped. Thus their growth continues
slowly, nutrients are utilized and waste products released in medium. This results in, finally, the death of the
microbes after sometime.

3. Paraffin Method/ preservation by overlaying cultures with mineral oil: This is a simple and most
economical method of maintaining pure cultures of bacteria and fungi. In this method, sterile liquid paraffin is
poured over the slant (slope) of culture and stored upright at room temperature. The layer of paraffin ensures
anaerobic conditions and prevents dehydration of the medium. This condition helps microorganisms or pure
culture to remain in a dormant state and, therefore, the culture can be preserved form months to years (varies
with species).
4. Cryopreservation: Cryopreservation (i.e., freezing in liquid nitrogen at -196°C or in the gas phase above the
liquid nitrogen at -150°C) helps survival of pure cultures for long storage times (10 to 30 years). In this
method, the microorganisms of culture are rapidly frozen in liquid nitrogen at -196°C in the presence of
stabilizing agents such as glycerol or Dimethyl Sulfoxide (DMSO) that prevent the cell damage due to
formation of ice crystals and promote cell survival.
5. Lyophilization (Freeze-Drying): Freeze-drying is a process where water and other solvents are removed
from a frozen product via sublimation. Sublimation occurs when a frozen liquid goes directly to a gaseous
state without entering a liquid phase. Freeze-dried products are hygroscopic and must be protected from
moisture during storage. Under these conditions, the microbial cells are dehydrated and their metabolic
activities are stopped; as a result, the microbes go into dormant state and retain viability for years (more than
30 years). Lyophilized or freeze-dried pure cultures and then sealed and stored in the dark at 4°C in
refrigerators.
CULTIVATION OF ANAEROBIC BACTERIA

1. Special Anaerobic Culture Media (Prereduced Media):Removal of oxygen from the medium is the
simplest method of all the methods available for the cultivation of anaerobic bacteria. During preparation, the
liquid culture medium is boiled by holding in boiling water both for 10 minutes to remove most of the
dissolved oxygen. Then a reducing agent (e.g., cysteine 0.1%, ascorbic acid 0.1%, sodium thioglycollate
0.1%), is added to further lower the oxygen content.Oxygen-free N2 is bubbled through the medium to
maintain anaerobic condition. The medium is then dispensed into tubes, which are stoppered tightly and
sterilized by autoclaving. Such tubes can be stored for many months before being used. During inoculation,
the tubes are continuously flushed with oxygen free CO2 by means of gas cannula, re-stoppered, and
incubated.
2. Anaerobic Chamber: It is a plastic anaerobic glove box that contains an atmosphere of H2, CO2 and N2.
Rubber gloves are used by the operator to perform work within the chamber. There is an air-lock with inner
and outer doors.Culture media are first placed inside air-lock. Air of the chamber is removed by a vacuum
pump and replaced with N2. The culture media are now transferred from air-lock to the main chamber, which
contains H2, CO2 and N2. A palladium catalyst circulator is used to remove any residual O2. When the culture

media become completely anaerobic they are inoculated with bacterial culture and placed in an incubator
fitted within the chamber.
Prereduced Media Anaerobic Chamber
3. Anaerobic Bags or Pouches:Anaerobic bags or pouches make convenient containers when only a few
samples are to be incubated anaerobically. They are available commercially. Bags or pouches have an oxygen
removal system consisting of a catalyst and calcium carbonate to produce an anaerobic, CO 2-rich
atmosphere.One or two inoculated plates are placed into the bag and the oxygen removal system is activated
and the bag is sealed and incubated.
4. Anaerobic Jars (or Gas Pak Anaerobic System):When an oxygen-free or anaerobic atmosphere is required
for obtaining surface growth of anaerobic bacteria, anaerobic jars are the best suited. The most reliable and
widely used anaerobic jar is the Melntosh-Fildes’ anaerobic jar. It is a cylindrical vessel made of glass or
metal with a metal lid, which is held firmly in place by a clamp. It contains one gas inlet and one gas outlet. It
carries palladium pelletssachet which convert hydrogen and oxygen into water at room temperature.
Inoculated culture plates are placed inside the jar. The outlet tube is connected to a vacuum pump and the air
inside is evacuated. The outlet tap is then closed and the gas inlet tube connected to a hydrogen supply.
Hydrogen is drawn inside the chamber until oxygen present inside is converted into water by palladium
catalyst.
Anaerobic Jar
QUANTITATIVE MEASUREMENT OF BACTERIAL GROWTH (TOTAL & VIABLE

COUNT)
➢ Micro-organisms can be counted.
➢ Most methods of counting are based on indirect or direct counts of tiny samples.
➢ DenotedNumber of Cells per milliliter of liquidas cells/ml.
➢ Bacterial cultures contain millions of cells. They can be counted either by Direct count or Indirect count
method.
➢ Direct Counts mean actually counting cells. It is more accurate but takes a lot longer.
➢ Indirect Counts mean estimating the number of cells based on turbidity in liquid or dry weight. It is less
accurate but quick.
➢ Total count means all cells in a culture.
➢ Viable count means only live cells.
A. Total Count: This technique involves the count of all bacteria in a given suspension either viable or dead.
This can be performed by following methods:
1. Turbidimetry Method:it is indirect method of counting. Total number of bacteria is calculated from a
standard calibration curve which is obtained by plotting log bacterial count of a suspension against
absorbance of this suspension. Turbidity is measured by Spectrophotometer which measure % transmittance
i.e. amount of light going through culture. Transmittance is inversely proportional to turbidity (cell
concentration). As turbidity increases cell number increases and transmittance decrease.
Turbidity Method of Total Count

2. Counting Chamber Method: The most common method of enumerating the totalmicrobial cells is the direct
counting of cell suspension ina counting chamber of known volume using amicroscope. The most common
type of counting chamberis calledhaemocytometer (originally designed for performingblood cell counts). It
consists of a thick glass microscopicslide with a rectangular groove that creates a chamberof known
dimensions.This chamber is marked with a counting grid (network)of perpendicular lines divided into nine
large squares (1mm x 1mm). The center square is divided into 25 smaller squares (0.2mm x 0.2mm). Depth of
the counting chamber is 0.1 mm.Therefore it is possible to count the number of cells ina specific volume of
fluid (10-20 µl in each slide notch),and thereby calculate the concentration of cells in the original fluid
volume.
Counting Chamber Method of Total Count
3. Electronic Counters:An electronic instrument called the Coulter counter can also be used for the direct
enumeration of cells in a suspension. The instrument is capable of accurately counting thousands of cells in a
few seconds.The suspending fluid, however, must be free of inanimate particles (e.g. dust).
B. Viable Count: Viable cont involves counting of colonies produced by only viable cells under favorable
growth conditions. The count represents the number of colony forming units (cfu) per gm (or per ml) of the
sample. This can be accomplished by following methods:
1. Plate Count Method: This is the most frequently used method. In the method culture is evenly spread across
a plate and counts the colonies. Each colony represents a cell in the original culture.This can be done by
techniques like pour plating or spread plating or serial dilution. In this procedure a diluted cell suspension is
introduced into a petridish. An appropriate melted agar medium is poured into the petri dish and is thoroughly
mixed with the inoculum by rotating the plate. After the solidification of the medium, the plates are inverted
and incubated for 18 to 24 hours. A plate having 30 to 300 colonies is selected for counting the number of
organisms.

Plate Count Method of Viable Count
2. Membrane Filters Count:This method is the same in principle as that of a plate count. A suspension of
micro-organisms, such as in water or air, if filtered through a millipore filter membrane. The organisms are
retained on the filter disc. The disc is then placed in a petridish containing a suitable medium. The plates are
incubated and the colonies are observed on the membrane surface.

MICROSCOPY
➢ Microscope are instruments that help to enlarge minute (micro = very small) organisms or their parts.
➢ The first microscope was constructed by Anton Van Leeuwenhoek (1632-1723). This, microscope consisted
of a single biconvex lens fitted in a small window of a “board” and the object was viewed through it.
➢ Resolving Power: It is the ability of a microscope to show two closely lying points as two distinct points.
➢ Magnification: It is the ratio of the size of the image to that of the object.
Types of Microscopes
A. Optical (or Light) Microscope
1. Bright Field Microscope (Simple & Compound Microscope)
2. Dark Field Microscope
3. Phase Contrast Microscope
4. Fluorescence Microscope
B. Electron Microscope
1. Transmission Electron Microscope (TEM)
2. Scanning Electron Microscope (SEM)
A. Optical (or Light) Microscope: The most common microscope is the optical microscope, which
uses light to pass through a sample to produce an image. Visible light transmitted or reflected from
the sample through a single or multiple lenses to allow a magnified view of sample. The resulting
image can be detected directly by the eyes or on a photographic plate.
1. Bright Field Microscope: Bright-field microscopy is the simplest of all the optical microscopy
illumination techniques. The typical appearance of a bright-field microscopy image is a dark sample on a
bright background, hence the name. Light path is as follows:
• A transillumination light source, commonly a halogen lamp in the microscope stand.
• A condenser lens, which focuses light from the light source onto the sample.
• An objective lens, which collects light from the sample and magnifies the image.
• Oculars and/or a camera to view the sample image.
a) Simple Microscopes: It consists of a biconvex lens which is moved up and down by an adjustment
screw to bring the object in sharp focus. The object is placed on the platform and light is focused
with the help of a concave mirror fitted below. In simple microscope, convex lens of short focal
length is used to see magnified image of a small object.

b) Compound Microscope: A compound microscope consists of two set of convex lenses. A lens of
short aperture and short focal length facing the object is called objective. Another set of lens of
relatively moderate focal length and large aperture facing the eye is called the eye piece. The
objective and the eye piece are placed at the two end of a tube. It forms real, inverted and magnified
image. This image acts as an object for the eye piece which is then produced as virtual, erect and
magnified image.
Simple Microscope Compound Microscope
• Basic parts of the microscope:

i. Eyepiece Lens: The lens at the top from where we look through. They are usually 10X or 15X power.
ii. Tube: Connects the eyepiece to the objective lenses.
iii. Arm: Supports the tube and connects it to the base
iv. Base: The bottom of the microscope, used for support.
v. Illuminator: A steady light source used in place of a mirror.
vi. Stage: The flat platform where slides are placed.
vii. Revolving Nosepiece: It is the part that holds two or more objective lenses and can be rotated to change
power.
viii. Objective Lenses: Usually 3 or 4 objective lenses are present on a microscope.
ix. Condenser Lens: The purpose of the condenser lens is to focus the light onto the specimen.

Bright Field Microscope Dark Field Microscope
Light
Path in Dark Field Microscope Difference between Light Path in Bright & Dark Field Microscope

2. Dark Field Microscope: It produces a bright image of the object against a dark background. In dark field
microscopy a special condenser is used so that only light reflected by the specimen enters the objective lens.
Unreflected or unrefracted light do not enter to the objective lens hence a bright image of specimen against a
dark background with better resolution than bright field microscope produces. The light's path is as follow:
• Light enters the microscope for illumination of the sample.
• A specially sized disc called patch stop or wide phase annulus, blocks some light from the light source,
leaving an outer ring of illumination.
• The condenser lens focuses the light towards the sample.
• The light enters the sample. Some is scattered from the sample while some light are unreflected.
• The scattered light enters the objective lens, while the unreflected light misses the lens and is not
collected.
• Only the scattered light goes on to produce the image, while the directly transmitted light is omitted.
3. Phase Contrast Microscope: It produces high-contrast images of transparent specimens or unstained

specimen, such as living cells, microorganisms, thin tissue slices, fibers etc. This is used to study the
behavior of living cells, observe the nuclear and cytoplasmic changes taking place during mitosis and the
effect of different chemicals inside the living cells.
❖ The phase contrast microscopy is based on the principle that small phase changes in the light rays,
induced by differences in the thickness and refractive index of the different parts of an object, can be
transformed into differences in brightness or light intensity.
❖ The phase changes are not visible to human eye whereas the brightness or light intensity can be easily
detected by human eye.
❖ The phase contrast microscopy is similar to an ordinary compound microscopy in except two additional
components known as annular stop and Phase plate.
❖ The annular stop produces a narrow or hollow cone of light that is focused on the specimen before
reaching the objective lens.
❖ Phase plate is located at the back plane of objective lens. It is a transparent disk coated with phase
retarding materials. Some part of the plate (circularly just below the outer periphery) is not coated with
phase retarding materials which is known as phase ring.
❖ Light path is as follow:
• The annular stop produces a hollow cone of light that is focused on the specimen.
• Light pass through a specimen who have different region of different refractive index and thickness.
• When light passes through the area of less refractive index (thinner region) remain undeviated (no
retardation in phase) and directly enters into objective lens.

• When light passes through an area of high refractive index (thicker region), it deviates from its normal
path i.e. phase change or phase retardation occurs. Hence they have a delayed phase than the undeviated
rays.
• The retardation of the phase of the light is ¼ of the λ of the incident light.
• The both rays deviated and undeviated enter into Phase plate.
• Phase plate is designed and positioned in such a way that un-retarted rays will pass through phase ring so
that their phase is not altered by the phase plate.
• But retarted light rays will pass through the area of phase plate which is coated with phase retarding
material.
• When the ¼ (or λ/4) retarted light is pass through this region of phase plate, it is further retarted by ¼ (or
λ/4). With this, the final change or retardation will be λ/4 + λ/4 = λ/2.
• When the unretarted and λ/2 retarted lights are recombined a negative or destructive interference is
created because crest and trough of the unretarted and retarted light will cancel each other. With this
destructive interference, the image of the specimen appears darker against a bright background.
• And if the unretarted and λ/2 retarted will be in the same phase a positive or constructive interference is
created. In this case the image of the specimen becomes brighter against dark background.
• Thus in phase contrast microscopy, the combination of destructive and constructive interferences crates
high contrast in the final image.

4. Fluorescence Microscope: The fluorescence microscope uses fluorescence to generate an image.

Fluorescence is the property of some substances which, when illuminated by light of a certain wavelength,
will re-emit the light at a longer wavelength. Such substances are called fluorophores. In general, a
fluorophore will be excited by high frequency light (wavelengths in the ultraviolet, violet, or blue region of
the spectrum), and emit light at slightly lower frequencies (wavelengths in the green or red region of the
spectrum). A typical fluorescence microscope consists of three filters: excitation filter, emission filter, and
the dichroic beam splitter. Sample is stained with fluorescent satins. Light path is as follow:
• A high intensity mercury or xenon lamp is used as a light source which passes through exciter filter.
• Exciter filter transmits only blue colour light to the specimen and block all other colours light.
• This blue light is reflected downward to the specimen by a dichroic mirror or beam splitter. The dichroic
filter or beam splitter is placed in between the excitation filter and emission filter, at a 45° angle. It
reflects the excitation signal towards the fluorophore (sample) under inspection, and to transmit the
emission signal towards the detector.
• The blue light then pass through the objective lens to specimen which then emits green light.
• The green light then passes through dichroic mirror and reaches to emission filter.
• The emission filter is placed within the imaging path of a fluorescence microscope. Its purpose is to filter
out the entire excitation range and to transmit only green light to detector and blocks any residual light
from specimen.
• Then detector receives and produces an image of stained portion of the specimen.
Fluorescence Microscope

B. The Electron Microscope: The electron microscope was developed in 1932 by M. Knoll and Ruska in
Germany. It consist of a source of supplying a beam of electron of uniform velocity, a condenser lens for
concentrating the electron on the specimen, a specimen stage for displacing the specimen which transmits
the electron beam, an objective lens, a projector lens and a fluorescent screen on which final image is
observed. There are two types of electron microscope.
1. Transmission Electron Microscope (TEM): It is complex and sophisticated. It forms an image of
radiation that has passed through a specimen.
• It uses a high voltage electron beam to create an image. The electron beam is produced by an electron
gun, commonly fitted with a tungsten filament cathode as the electron source. The electron beam is
accelerated by an anode.
• Since electron cannot pass through a glass lens so that a magnetic lens (electromagnetic) are used to
focus the electron beam.
• The column containing lenses and specimen must be under high vacuum because electrons are deflected
by collision with air molecules.
• Specimen to be examined is prepared as an extremely thin dry film on small screens and is introduced
into the instrument between magnetic lens (condenser) and magnetic objective (comparable to stage).
• As the incident electron beam falls on the specimen, it (specimen) scatters some electron beam which
carries information about the structure of the specimen that is magnified by the objective lens system of
the microscope.
• The spatial variation in this information (the “image”) may be viewed by projecting the magnified
electron image onto a fluorescent viewing screen coated with a phosphorous or zinc sulfide.
• Alternatively, the image can be photographically recorded by exposing a photographic film or plate
directly to the electron beam.

2. Scanning Electron Microscope (SEM): It is capable of producing high resolution images of a sample. It
creates 3D image of the specimen. It produces an image from electron released from the atoms on an object
surface. It is used to examine the surfaces of microorganisms and differentiate the different structures.
• Specimen used in SEM is produced by fixing, dehydrating and drying. Dried sample is coated with a thin
layer of metal to prevent the formation of electric charge on the surface.
• A beam of electrons is produced at the top of the microscope by an electron gun. The electron beam
follows a vertical path through the microscope, which is held within a vacuum.
• The beam travels through electromagnetic fields and lenses, which focus the beam down toward the
sample.
• Once the beam hits the sample, electrons and X-rays are ejected from the sample.
• Detectors collect these X-rays, backscattered electrons, and secondary electrons and convert them into a
signal that is sent to a screen similar to a television screen. This produces the final image.
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AMIT KUMAR RAI PHARMACEUTICAL MICROBIOLOGY

K KAILASH INSTITUTE OF PHARMACY & MANAGEMENT, GIDA, GORAKHPUR 1

K KAILASH INSTITUTE OF PHARMACY & MANAGEMENT, GIDA, GORAKHPUR 2

K KAILASH INSTITUTE OF PHARMACY & MANAGEMENT, GIDA, GORAKHPUR 3

SCOPE & ADVANTAGES OF MICROBIOLOGY

K KAILASH INSTITUTE OF PHARMACY & MANAGEMENT, GIDA, GORAKHPUR 4

INTRODUCTION TO PROKARYOTES & EUKARYOTES

K KAILASH INSTITUTE OF PHARMACY & MANAGEMENT, GIDA, GORAKHPUR 5

Characteristic Distinguishing Features of Prokaryotic & Eukaryotic Cells

Prokaryotic Cell Eukaryotic Cell

It has a true nucleus, bounded by a

DNA arrangement It has a circular loop. It is linear.

Size Small cells ( < 5 µm) Large cells ( < 10 µm)

Cell Always unicellular Mostly multi-cellular

Usually present; chemically complex in When present, chemically simple in

It contains proteins in the DNA to form

Ribosome It contains small ribosomes. It contains large ribosomes.

Cytoplasm No cytoskeleton Always have cytoskeleton

Cell division Cell division is by binary fission Cell division is by mitosis

Reproduction Reproduction is always asexual Reproduction is asexual or sexual

It is complex in nature and consists of

They perform functions of Golgi-bodies and

Sterols and carbohydrates are both

Example Bacteria and Archaea Animal cells and plant cells

K KAILASH INSTITUTE OF PHARMACY & MANAGEMENT, GIDA, GORAKHPUR 6

K KAILASH INSTITUTE OF PHARMACY & MANAGEMENT, GIDA, GORAKHPUR 7

Characteristics Gram Positive Gram Negative

K KAILASH INSTITUTE OF PHARMACY & MANAGEMENT, GIDA, GORAKHPUR 8

• On the basis of flagella bacteria are divided into following types:

K KAILASH INSTITUTE OF PHARMACY & MANAGEMENT, GIDA, GORAKHPUR 9

MORPHOLOGICAL CLASSIFICATION OF BACTERIA

K KAILASH INSTITUTE OF PHARMACY & MANAGEMENT, GIDA, GORAKHPUR 10

GRAM-NEGATIVE BACTERIA V/S GRAM-POSITIVE BACTERIA

Peptidoglycan layer Thin (single-layered) Thick (multilayered)

Teichoic acids Absent Present in many

Periplasmic space Present Absent

Outer membrane Present Absent

Lipopolysaccharide High Virtually none

Flagellar structure 4 rings in basal body 2 rings in basal body

Toxins produced Primarily Endotoxins Primarily Exotoxins

Resistance to Low High

Inhibition by basic Low High

Susceptibility to Low High

Resistance to Low High

Resistance to drying Low High

Mesosome Mesosome is less prominent. Mesosome is more prominent.

Antibiotic More resistant to antibiotics. More susceptible to antibiotics

K KAILASH INSTITUTE OF PHARMACY & MANAGEMENT, GIDA, GORAKHPUR 11

NUTRITIONAL REQUIREMENTS FOR BACTERIA

K KAILASH INSTITUTE OF PHARMACY & MANAGEMENT, GIDA, GORAKHPUR 12

K KAILASH INSTITUTE OF PHARMACY & MANAGEMENT, GIDA, GORAKHPUR 13

➢ Classification of Culture Media

B. On the basis of composition:

C. On the basis of purpose/ functional use/ application:

K KAILASH INSTITUTE OF PHARMACY & MANAGEMENT, GIDA, GORAKHPUR 14

GROWTH AND MULTIPLICATION OF BACTERIA

PHYSICAL PARAMETERS OR FACTORS THAT AFFECT THE GROWTH OF BACTERIA

K KAILASH INSTITUTE OF PHARMACY & MANAGEMENT, GIDA, GORAKHPUR 15

ISOLATION METHODS FOR PURE CULTURES