HISTORY –

LOUIS PASTEUR –
Louis Pasteur is also known as father of microbiology. He has many contributions to microbiology:

1. He has proposed the principles of fermentation for preservation of food

2. He introduced sterilization techniques and developed steam sterilizer, hot air oven and autoclave.

3. He described the method of pasteurization of milk

4. He had also contributed for designing the vaccines against several diseases such as anthrax, fowl
cholera and rabies

5. He disproved the theory of spontaneous generation of disease and postulated the ‘germ theory of
disease’. He stated that disease cannot be caused by bad air or vapor, but it is produced by the
microorganisms present in air.6. Liquid media concept- He used nutrient broth to grow
microorganisms. 7. He was the founder of the Pasteur Institute, Paris.

ROBERT KOCH –

Robert Koch provided remarkable contributions to the field of microbiology:

 He used of solid media for culture of bacteria-Eilshemius Hesse, the wife of Walther
Hesse, one of Koch’s assistants had suggested the use of agar as solidifying agent.

 He also introduced methods for isolation of bacteria in pure culture.

 Described hanging drop method for testing motility.

 Discovered bacteria such as the anthrax bacilli, tubercle bacilli and cholera bacilli.

 Introduced staining techniques by using aniline dye.

 Koch’s phenomenon: Robert Koch observed that guinea pigs already infected with
tubercle bacillus developed a hypersensitivity reaction when injected with
tubercle bacilli or its protein. This reaction is called Koch’s phenomenon.

 Koch’s Postulates:

 According to Koch’s postulates, a microorganism can be accepted as the

causative agent of an infectious disease only if the following conditions are
fulfilled:

The microorganism should be constantly associated with the lesions of the disease.

ii. It should be possible to isolate the organism in pure culture from the lesions of the disease.

iii. The same disease must result when the isolated microorganism is inoculated into a
 suitable laboratory animal.

iv. It should be possible to re-isolate the organism in pure culture from the lesions produced
in the experimental animals.

An additional fifth criterion was introduced subsequently which states that antibody to the
causative organism should be demonstrable in the patient’s serum.

Exceptions to Koch’s postulates: It is observed that it is not always possible to apply these
postulates to study all the human diseases. There are some bacteria that do not satisfy all the
four criteria of Koch’s postulates. Those organisms are:

• Mycobacterium leprae and Treponema pallidum: They cannot be grown in vitro; however can
be maintained in animals.• Neisseria gonorrhoeae: There is no animal model; however, bacteria
can be grown in vitro.Molecular Koch’s postulates: It was a modification of Koch’s postulates
(by Stanley Falkow). He stated that gene (coding for virulence) of a microorganism should satisfy
all the criteria of Koch’s postulates rather than the microorganism itself.

PAUL EHRLICH –

 He was the first to report the acid-fast nature of tubercle bacillus.

 He developed techniques to stain tissues and blood cells.

 He proposed a toxin antitoxin interaction called as Ehrlich phenomenon and

also introduced methods of standardising toxin and antitoxin

 He proposed the ‘side chain theory for antibody production’.

 He discovered ‘salvarsan’, an arsenical compound (magic bullet) for

treatment of syphilis, hence known as father of chemotherapy.

 The bacteria ‘Ehrlichia’was named after him.

BACTERIAL TAXONOMY –
It divides organisms into 6 kingdoms Bacteria, Protozoa, Chromista, Plantae, Fungi
and Animalia.

Principle used for Bacterial Classification

1. Phylogenetic classification: This is a hierarchical classification representing a
branching tree-like arrangement; one characteristic (or trait) is being employed for
division at each node of the tree

2.Adansonian (or phonetic) classification: To avoid the use of weighted

characteristics, Michel Adanson proposed a scheme that classifies organisms
based on giving equal weight to every character of the organism. This principle is
used in numeric taxonomy.

3.Molecular classification: Based on genetic relatedness (guanine+ cytosine

content) of different organisms.

STAINING TECHNIQUES –
Staining is necessary to produce color contrast and thereby increase the visibility of
the object. Before staining, the fixation of the smear to the slide is done:

Heat fixation is usually done for bacterial smears by gently flame heating
an air-dried film of bacteria

Chemical fixation such as ethanol, acetic acid, mercuric chloride,

formaldehyde, methanol and glutaraldehyde. This is useful for
examination of blood smears.

STAINING TECHNIQUES USED IN MICROBIOLOGY—

 Simple stain: Basic dyes such as methylene blue or basic fuchsin are used
as simple stains. They provide the color contrast, but impart the same color
to all the bacteria in a smear.
 Negative staining, e.g. India ink or nigrosin. The background gets stained
black whereas unstained bacteria stand out in contrast. This is very useful
in the demonstration of bacterial capsules which do not take up simple
stains.
 Impregnation methods (e.g. silver): Used for demonstration of thin
structures like bacterial flagella and spirochetes

 Differential stain: Two stains are used which impart different colors which
help in differentiating bacteria, e.g.

o Gram stain: Differentiates bacteria into Gram-positive (appear violet)

and Gram- negative (appear pink) groups

 Primary stain by crystal violet (or gentian violet or methyl

violet) for one min- ute.

 Mordant by Gram’s iodine for one minute.

  Decolorization by acetone (for 1–2 sec) or ethanol (20–30 sec)

with immediate wash. Decolorizer removes the primary stain
from Gram-negative bacteria while the Gram-positive bacteria
retain the primary stain.

 Counter stain or Secondary stains by safranin or diluted

carbol fuchsin; is added for 30 sec.

o Acid-fast stain: Acid-fast organisms (table below) resist decolorization

to mineral acids. This is due to presence of mycolic acids in the cell
wall. (details in chapter 3.6).
 o Albert stain: Differentiates bacteria having metachromatic granules
(e.g. Corynebacterium diphtheriae) from other bacteria that do not have

 Other Special Staining Methods:

1. Spore staining: Acid fast stain (using 0.25% sulfuric acid) and Malachite
green stain (Schaeffer and fulton method modified by Ashby) methods are
used; however, phase contrast microscope of unstained wet film is the best
method.
2. Lipids stained by: Sudan Black stain
3. Carbohydrate (Glycogen) stained by Iodine stain
4. Flagellar stain: Tannic acid staining (Leifson method)

 Microscopy of Bacteria in Living State

Unstained (wet) preparations: Used for:○ Checking bacterial motility (e.g.

in hanging drop and wet mount preparations) ○ For demonstration of
spirochetes (e.g. in dark field or phase contrast micros- copy).

Vital stains: Differentiate the living cells from dead cells:

○  In supravital staining, living cells that have been

removed from an organism are stained; whereas intravital
staining is done by injecting stain into the body.

○  Examples of vital stains are eosin, propidium iodide,

trypan blue, erythrosine and neutral red.

MORPHOLOGY OF BACTERIA –
The cell wall is a tough and rigid structure, surrounding the bacterium.

certain parts of cell wall (e.g. LPS) are immunogenic and act as virulence factor.

 Peptidoglycan is main component of the cell wall which makes it rigid. It is

composed of alternate units of N-acetyl muramic acid (NAM) and N-acetyl
glucosamine (NAG) molecules; cross linked to each other via tetrapeptide
side chains and pentaglycine bridges.

 Gram-positive bacteria has a thicker peptidoglycan and contains teichoic acid

OTHER PARTS OF BACTERIAL CELL WALL

Intracytoplasmic Inclusions
They are the storage sites of nutrients/bacteria, formed in nutritional deficiency
conditions:

 Organic inclusion bodies, examples include glycogen granules and poly-hydroxyl

butyrate granules.

 Inorganic inclusion bodies, examples include:○ Polymetaphosphate or volutin or

metachromatic granules in C.diphtheriae. ○ Suflur granules found in
Actinomyces.

Nucleoid
 Bacteria do not have a true nucleus; but the genetic material is located in an
irregularly shaped region called the nucleoid.

 There is no nuclear membrane or nucleolus and lacks basic proteins.

 Possess a single haploid chromosome, comprising of super coiled circular ds DNA

 divides by simple binary fission.

 nucleoid can be seen by electron microscopy or on staining with the Feulgen stain

Capsule and Slime Layer

 possess a layer of amorphous viscid material lying outside the cell wall called
glycocalyx

 may be well organized (capsule) or unorganized loose material (slime layer).

 may possess both capsule and slime layer as in Streptococcus salivarius.

The capsule has various functions

  Acts by inhibiting phagocytosis and complement-mediated lysis

 Biofilm formation and thereby helps in adherence to damaged tissues and

plastic surfaces

 Source of nutrients and energy for the bacteria

 Capsules as vaccine, e.g. Pneumococcus, Meningococcus and Haemophilus

influenzae serotype-b and S. Typhi Vi Vaccine.

Demonstration of capsule by

 • Negative staining by India ink and nigrosin stain:

 M’Faydean capsule stain for Bacillus anthracis by using polychrome methylene

blue stain.

 Serological test: Capsular material is antigenic and can be demonstrated by mixing

it with a specific anticapsular serum: ○ Quellung reaction for Streptococcus
pneumoniae○ Latex agglutination test by using specific anticapsular antibodies
coated on latex.

Flagella
 thread-like appendages,

 omposed of protein subunits called flagellin.

 has three parts: filament, kook and basal body.

 organs of locomotion, confer motility to the bacteria.

ARRANGEMENT –

 Monotrichous (single polar flagellum)

 Lophotrichous (multiple polar flagella)

 Peritrichous over the entire cell surface)

 Amphitrichous (single flagellum at both the ends)

Flagella can be demonstrated by

 Direct demonstration of flagella○ Tannic acid staining (Leifson’s method and Ryu’s
method) ○ Dark ground, phase contrast or electron microscope
 Indirect means by demonstrating the motility:○ Cragie tube method and Hanging
drop method○ Semisolid medium, e.g. mannitol motility medium.