Fundamental Principles of

Microbiology
 Microbiology
 Microbiology is the study of living organisms that are
microscopic in size
 Medical microbiology is the study of microscopic
organism that infect man, his reaction to such
infections, the way in which the disease is produced
and the method for diagnosis, prevention and
treatment of such infectious disease.
 Microorganisms can be seen only with the help of
microscope.
 Bacteria, fungi, algae, viruses, protozoa,
mycoplasms, rickettsias all are microorganisms.
Classification of microorganism
 They are classified under kingdom protista.
 Depending on the cellular organisation and
biochemistry, the kingdom protista is divided into two
groups- prokaryotes and eukaryotes.

MICROORGANISMS

HAVING NO CELLULAR ORGANISATION

HAVING CELLULAR ORGANISATION Viruses
(KINGDOM PROTISTA) Viriods
prion

PROKARYOTES EUKARYOTES
Bacteria, Blue green Fungi, Algae, slime,
algae moulds, protozoa
Character Prokaryotes Eukaryotes
Cytoplasm
Cytoplasmic streaming Absent Present
Pinocytosis Absent Present
Mitochondria Absent Present
Lysosomes Absent Present
Golgi bodies Absent Present
Endoplasmic reticulum Absent Present
Nucleus
Nuclear membrane Absent Present
Nucleolus Absent Present
Deoxyribonucleoprotein Absent Present
Number of One More than one
chromosomes
Mitotic division Absent Present
Chemical variations
Sterols Absent Present
Muramic acid Present Absent
Bacteria
 A bacterium is a unicellular prokaryotic microorganism
which does not contain chlorophyll and does not
multiply by true branching.
 The unit of measurement used is micron (micrometer).
 Shape of Bacteria:
 Bacteria are classified into varieties based on their
shapes:
a) Cocci are oval or spherical bacterial cells
b) Bacilli are rod shaped cells
c) Vibrios are comma shaped curved rods. These
bacteria possess characteristic vibratory motility
d) Spirilla are rigid spiral shaped bacteria
e) Spirochaetes are spiral shaped cells look like
coiled hairs
f) Actinomycetes are branching filamentous bacteria.
In the tissue lesions they appear as if radiating
rays.
g) Mycoplasmas are bacteria that do not have cell
wall and therefore their morphology is not stable.
They occur as round or oval bodies and as
filaments
 Bacteria loose their cell wall and hence their shape can
change, due to defective formation of cell wall either
spontaneously or as a result of administration of drugs
such as penicillin. Such cells are called protoplasts,
spheroplasts or L forms
Arrangement of Bacteria:
 Cocci are arrange in pairs(diplococci), in chains
(streptococci), groups of four(tetrads) or eight(sarcina) or
as bunch of grapes (staphylococci).
 Bacilli can be arrange in chains (Anthrax bacilli) in
cluster, in groups of two(diplobacilli eg: pneumoniae)
and sometimes at angles to each other making a
cuneiform pattern(corynebacteria).
 Bacteria
Some of the algaes are unicellular whereas
others are multiceli
which may form branched or unbranched
filaments. The cell wa
composed of cellulose with which a number of
other compounos
associated. A definite nucleus is present inside
the e
reproduction may be sexual or asexual.
 BACTERIA
SPHERE SHAPESODS
(COCCI) SPIRAL
S Streptococci {BACILLI) S
(Streptococcu
s pyogenes)
Vibrios
Diplococci Chain ofbacilli
(Vibrio
(Streptococcu (Bacillus cholerae)
s anthracis)
pneumoniae) Tetrad

Spirilla
(He/icobacterpylori
Flagellate rods )
(Salmonella
typhi)

Staphylococci Sare/no Spore-former

(Staphylococcu (Sarcina (Clostridiu Spirochaetes
s ventricu/ m (Treponema
aureus) 1) botullnum) pollidum)
Anatomy of Bacteria:
 It consists of an outer layer cell envelop which is
differentiated into an outer rigid cell wall and beneath it a
cytoplasmic membrane which is also called plasma
membrane.
 Inside the cell envelop lies the protoplasm which contains
cytoplasm and cytoplasmic inclusions such as ribosomes,
mesosomes, granules, vacuoles and the nuclear body.
 Sometimes a bacteria is enclosed into a viscid layed which
may be a loose layer or organised as a capsule.
 Some bacteria also posses filamentous appendages at
their surface.
 These appendages can be flagella(organ of locomotion)
and fimbriae (organ of adhesion)
 Bacterial nucleus does not contain nuclear membrane or
nucleolus.
 The bacterial chromosome is haploid and replicate by
simple fission
Spores:
 Some bacteria(Bacilli and clostridium) have the ability
to form highly resistant resting stages called spores.
 a single spore is formed by one bacterium and one
spore gives a single bacterial cell germination.
 Spores which are formed inside the bacterial cells are
called as endospores.
 Bacterial spores are resistant to chemicals and heat.
 Spores of all medically important bacteria are
destroyed by autoclaving at 120°C for 15 minutes.
 Sporulation helps the bacteria to survive for long
period under unfavourable conditions.
 Spores germinate into bacterial cells when transferred
to favourable conditions
 It is oval or spherical in shape and is attached to the
parent cell
 It consists of a nuclear body, surrounded by spore
wall in the form of a delicate membrane.
 Outer to the spore wall lies a thick spore cortex ,
which in turn is enclosed in a multilayered tough
spore coat.
 Spores of some bacteria have an additional outer
covering called exosporium which have ridges and
grooves .
Fungi
 Fungi are eukaryotic protists.
 They have rigid cell wall containing polysaccharides-
chitin, mannan and others.
 The cytoplasmic membrane of fungi contains sterols.
 The nuclei contain nuclear membrane and paired
chromosomes.
 Fungi multiply sexually, asexually or both.
 They can be unicellular or multicellular.
 Elongation of the fungi cells produces a thin tubular
structure called hypha.
 A tangled mass of hyphae is called mycelium
 Fungi which can make mycelia are called filamentous
fungi or moulds
 Fungi
Types of fungi :
 Fungi are divided into 4 on the basis of cell morphology.
Yeasts:
 Are unicellular fungi
 Their cells are spherical or ellisoidal in shape.
 They multiply by budding.
 The pathogenic yeast is crytococcus neoformans.
Yeast like fungi:
 They partly grow as fungi and partly as elongated cells
like hyphae called pseudomycelium
 Eg: candida albicans
Moulds / filamentous fungi:
Can produce true mycelia
Multiply by spore formation
Eg: micosporum, trichophyton, epidermophyton
Dimorphic fungi:
 They occur as filaments or as yeasts.
In tissues or in culture at 37°C they occur as yeast,
while in the soil and in culture at 22°Cthey appear as
moulds.
 Eg: Blastomyces dermatitidis, Histoplasma capsulatum
etc
 Fungal infection (mycoses
4 Types
1. Superficial,
2. Sub cutaneous,
3. Deep seated (systematic)
4. Opportunistic
Superficial fungal infections:
 The fungi causing the disease are specialised
saprohytes which can digest keratin of the skin
including nails & hairs.
 It include various types of tinea and ringworm affecting
the skin, hair and nails.
 Causative fungi for superficial infections are
dermatophytes, candida albicans, pityrosporum
orbiculare, cladosporium, piedraia hortai.

Subcutaneous fungal infections(Subcutaneous mycosis):

 Caused by fungi such as rhinospordium, fonsecaea,
madurella norcardia, sporotrichum etc.
Deep seated fungal infections(systematic mycosis):
 Are caused by soil saprophytes.
 severity ranges from asymptomatic infections to fatal
diseases
 The causative agents include actinomycetes (produces
madura foot), rhinosporidium seeberi (produce
rhinosporidiosis), cryptococcus neoformans
(cryptococcosis), dimorphic fungi- blastomyces dermatitidis
(blastomycoses) and histoplasma capsulatum (produce
histoplasmoses)

Opportunistic fungal infections:

 Occur in patients suffering from diseases such as cancer,
AIDS or diabetes.
 The use of drugs like immunosuppresive agents, steroids,
board spectrum antibiotics and exposure to X-rays makes
the body vulnerable to these infections.
 Caused by mucor, penicillium, rhizopus and aspergillus
Mycotic poisoning:
 Many fungi can produce poisoning.
 Are of 2 types: mycetism and mycotoxicosis
Mycetism :
 Fungi which is eaten for itself and produces toxic
effects.
 Eg: some mushrooms after ingestion causes toxic
effects such as GIT disturbances, dermatitis or even
death.
Mycotoxicosis :
 The toxins produced by fungi ie, mycotoxins
contaminate the food material and produce side
effects.
 Eg: aflatoxin produced by Aspergillus flavus present in
mouldy foods such as ground nuts, corn and peas can
produce carcinogenic effects in humans
Ergotism:
 Poisoning caused by eating rye, infected with fungus
 ALGAE
Green algae are found in fresh water either as free floating or
attached to some support. They are also found in rivers,
ponds, ditches and other pools of stagnant water. Some grow
in the soil as well as on the surfaceof the soil. Still others grow
on the sides of the trees and on rocks
Generally algae are green in colour due to the presence of
chlorophyll hence they can manufacture their own food. In
some of the algaes the green chlorophyll is marked by other
pigments like blue, brown and red Some of the algaes are
unicellular whereas others are multicellular which may form
branched or unbranched filaments. The cell wall is
composed of cellulose with which a number of other
compounds are associated. A definite nucleus is present
inside the cell reproduction may be sexual or asexual.
.

Many of the sea weeds are used carbohydrates and

vitamins. They form an important food for fish and other
water animals .Brown algae called kelps are an
important source of iodine. Some sea weeds are used
as fertilizers because they are rich in potassium and
other mineral matters.
PROTOZOA
The protozoa is derived from Greek words protos- first and zoon
animal.
They are the lowest and simplest form of animal life .They are
unicellular organisms more lightly organised than bacteria.They
have protoplasm clearly differenciated into a nucleus and
cytoplasm.
The important human protozoal infections include malaria,
amobiasis, trypanosomiasis, leishmaniasis, trichomoniasis,
giardiasis etc
 MYCOPLASMAS
Mycoplasmas are the smallest micro organisms.The cell size ranges f
0.15 to 1µm in diameter.Thus in size mycoplasmas appears to be eve
smaller than some viruses.
Since they do not possess a true cell wall, thus they are characterised
marked pleomorphism. They give rise to coccoid, granular,filamentous
cluster like, ring shaped, filtrable forms etc. Pleomorphism is observed
In cultures and in the body of man and animals.The disease caused b
different mycoplasmas include primary atypical pneumonia and genita
infections Besides these diseases mycoplasmas are also associated
urethritis, cervicitis, pelvic inflammatory disease.
 RICKETTSIAS

These are simple, unicellular, gram negative microorganisms. They

may be rod shaped, spherical or pleomorphic in shape. According to
their properties rickettsiae are intermediate between bacteria and
viruses.
Pathogenic rickettsia invade different species of animals and man.The
disease caused by rickettsia e are known as rickettsiosis.Typhus fever
and related diseases are caused by rickettsiae.
 Viruses
 These microorganisms are much smaller than
bacteria.
 They do not have a cellular organisation.
 They contain only one type of nucleic acid , either
DNA or RNA.
 They do not contain enzymes necessary for protein
and nucleic acid synthesis.
 Viruses are dependent on the host cell for
replication.
 They are obligate intracellular parasites that are not
affected by the usual antibiotics.
 The extracellular infectious virus particle is called
virion
 Viruses
 Envelope protein

Envelope

Structure of virus
 Viruses produce diseases like AIDS, cancer, rabies or
yellow fever etc.
 The viral diseases can be sporadic like mumps,
endemic such as measles or pandemic such as
influenza.
Size:
 Viruses vary in size
 The largest virus such as pox virus is 300 nm in size.
 The smallest viruses such as those causing foot and
mouth diseases are 20 nm in size .
 Shape and Structure:
 The viruses vary in shape.
 Most viruses are spherical.
 The rabies virus is bullet shaped, the pox virus is brick
shaped and the tobacco virus is rod shaped.
 HIV Hepatitis B Ebola Virus

Adenovirus Influenza Bacteriophage

 Classification of viruses:
 Classified into two based on the presence of nucleic
acid present.
(1)The Deoxyriboviruses or DNA viruses:
 These viruses contain DNA in them.
 Eg: poxvirus, Herpes virus, Adenovirus, Papilloma
virus, Human Hepatitis Type B virus etc
(2) The Riboviruses or RNA viruses:
 These viruses contain RNA in them.
 Include Enteroviruses, Rhinovirus, Influenza virus,
Mumps virus, Cholera virus, Rabies virus, Corona
virus, Retrovirus etc
 Isolation of microorganisms
 For a proper diagnosis of infectious diseases , it is
necessary to know the type and nature of causative
microorganism.
 For this the specimen of the infected material are
collected.
 Mostly used specimens are swabs, pus, sputum,
urine, stool , blood, cerebrospinal fluids, pleural
fluids and aspiration material.
 The specimen should be collected before starting
the antimicrobial treatment otherwise the specimen
may become sterile.
 The specimen should be collected from the site
most likely to be infected by the microorganism
 Eg: sputum is examined in respiratory tract
infections, stool in diarrhoea , cerebrospinal fluid in
meningitis and blood in enteric fever is studied for the
presence of microbes.
 Stage of disease is an important factor for specimen
collection.
 Eg: in the early stage of enteric fever blood culture is
done, in the second week widal test is done and in
third week stool culture is performed.
 Timing of collection is also important for the
successful isolation of causative microorganism
 Eg: In UTI the first morning sample of urine is best for
culture.Sputum and conjunctival swabs should be
collected in the morning
 The specimen should be collected in sufficient
quantity in suitable containers.
 Urine sample is collected in sterile test tube.
 Swab from eye, throat, rectum or vagina should be
collected by sterile swab stick and that too should be
placed into sterilized test tube immediately after
taking the sample.
 Sputum should be collected in petridish.
 Cerebrospinal fluid is collected in sterilized vials.
 Blood for culture is collected in blood culture bottles.
 After collection the specimen should be delivered to
laboratory without any delay to avoid the overgrowth
of organisms.
 Sufficient clinical information should always be given
with the specimen
ISOLATION OF PURE CULTURE
For studying the morphological characteristics of pathogenic
micro organism of a particular disease · It is very important
to get a Pure culture The growth of micro-organism in or on
a laboratory medium is known as culture. When micro-
organisms are grown on a solid medium from a single cell or
spore it is called as colony'.
When culture contains only one type of microbes 1t is
termed as pure culture but when it contains several
species it is known as mixed culture' . It is very difficult to
obtain a pure culture of bacteria because generally exist as
mixed cultures. But for studying the morphological
characteristics of bacteria it is extremely important to have
pure culture of an organisms.
The pure culture of microorganisms can be obtained by
growing an aliquot portion of the specimen containing
bacteria on a suitable culture media which include:
(a)nutrient broth
(b) nutrient agar
(c) semisolid agar
(d) peptone water
{e) blood agar
(f) chocolate agar
(g) serum agar

For the isolation of pure bacterial culture, a number of

methods like
1.Direct transfer technique
2. Single cell isolation technique
3. Streak plate technique
4. Serial dilution technique
5. Pour plate technique etc. are used but generally pour
plate technique is used.
Pour plateTechnique –
The specimen containing the bacteria is first diluted in tubes of
agar medium a number of times so as to get well isolated colonies
of bacteria. The petriplates are thoroughly cleaned and sterilized.
The agar medium is maintained at a temperature of about 42°C so
as to keep in the liquid state.
The inoculum is added to this medium and shaken thoroughly so
as to distribute the inoculum in the medium. The inoculated
material is poured into previously cleaned and sterilized
petriplates under aseptic conditions. Allow the material to cool and
set. Then keep the petriplates in the incubator usually at a
temperature of 370c for 24 hours. Within this time the bacterial
colonies will develop. Remove the petriplates from the incubator
and watch the bacterial colonies which will be usually from single
cell and are pure culture . If the colonies are of mixed culture they
may be further grown on agar plate ,agar slants or on nutrient
broth .
Colonies on Agar Media-
The bacterial culture is mixed with sterilized agar media
which has been maintained at a temperature of 42°C to
keep it in the liquid state. The mixture is transferred under
aseptic conditions to sterilized petriplates, Allow the
petriplates to .incubate at a temperature of about 37°C for
24 hours. After 24 ·hours remove the petriplates from the
'incubator. The bacterial colonies of single cell or pure
culture will appear on the surface of agar medium
b) Colonies on Agar Slants
Agar slants are prepared by putting the molten agar· in
sterilized test tubes. The mouth of the test tube is covered
with sterilized cotton, then they are kept in a slanting
position to set the medium. The surface of the medium is
inoculated with bacterial culture by streaking the slanting
surface of the medium.in the tube with a stroke of an
inoculating needle. Then the tubes are incubated at a
temperature 37°C for 24 hours.
c)Colonies in Nutrient broth-
The sterilized nutrient broth is transferred to sterilized
tubes which are then inoculated with the help of a
transferring needle or loop. The tubes are then incubated
at a temperature of about 370C for 24 hours.
The colonies so developed will be pure cultures and it will
be easy to study various morphological characteristics of
isolated micro organisms.
 Staining techniques
 After the isolation of causative microorganism,from
infected tissue morphological detail is studied.
 For morphological study the bacteria are stained
properly.
 By staining the bacteria become clearly visible and
can be identified.Therefore staining of
microorganisms prior to microscopic examination is
of primary importance for the recognition of bacteria.
 DIFFERENT STAINING TECHNIQUES

Preparation of Film or Smear

The preparation of film or smear is the first step in routine

staining procedures. Film is usually prepared on 3" x 1" glass
slide or sometimes on cover slip. It is essential that the glass
slide or cover slip should be thoroughly clean, dry and
grease free otherwise the film will not be uniform.
For ordinary use wash the slide with soap and water, dry it
with clean dry cotton cloth and then hold it with forceps
and pass through bunsen's flame 6-12 times so as make the
slide free from grease. Coverslips are cleaned by dipping in
chromate sulphuric acid solution then they are first washed
with tap water and then with distilled water and stored in
stoppered jar in 50o/o alcohol.
A film is prepared by keeping a loopful of fluid
material on the surface of glass slide which is
spread thinly on the slide. The film is dried in the air
and then fixed by passing through a Bunsen flame
gently.
In the bacterial culture on agar, a loopful clean
water is placed on the slide with a sterilized loop.
Then a minute quantity of bacterial colony is
transferred to the drop with a loop and thoroughly
emulsified. The mixture is then spread uniformly as
a thin film on the slide. The film is dried in the air
and then heat fixed by passing through bunsens
flame gently.
 .
Smear preparation:
 First step in staining procedure
A loopful of liquid culture or fluid specimen or a
section of bacterial colony is taken and spread as a
thin film over the slide.
The smear is dried in air and heat fixed by passing
through a flame gently.
Simple Staining-
When the staining solution contains only one dye dissolved in
either dilute alcohol solution or water then the stains are
known as simple staining.
Simple staining is also known as monochrome staining. The
dyes commonly used for simple stains include crystal violet,
methylene blue, Fuchsin and safranin.
simple staining is used to study the size, shape. motility and
other morphological characteristics of micro-organisms. In this
type of staining, the simple stain is applied to the heat fixed
film and allowed to react 30 seconds to 3minutes (depending
on the type of stain used). Then the smear is washed with
water and dried. Bacterial cells will take up the colour of the
dye which will make the identification easier. Examine the
slide under oil emersion lens of the microscope either directly
or after mounting in glycerin.
 Differential Staining Methods – In differential staining
.
methods more than one dye is used which when properly
employed will differentiate nearly all types of bacteria.
These methods are also used to study the morphological
characteristics of bacterial cells, spores and capsules.
The various stains used for differential staining are Gram,
crystal violet, methyl violet, Ziehl-Neelsen. The various
staining methods used for differential staining include :
a) Gram's staining method
b) Acid fast staining technique
c) Ziehl-Neelsen method
d) Staining of spores
e) Staining of Capsules
Grams Staining Method-
This is the most commonly used method for differential staining
. It is very simple and useful method which was first
of bacteria.
used in 1884 by Gram and till now it has not lost its practical
significance.
All bacteria stained by Gram method can be grouped
according to colour as gram positive and Gram negative .The
procedure for staining is as follows
Reagents Used in Gram's Staining
a) Gentian violet - 0.5 gm.
Distilled water upto - 100ml
Dissolve in distilled water.
b) Iodine 1.0 gm.
Potassium iodide 2.0 gm
Distilled water upto 100ml
Dissolve potassium iodide in water and to this dissolve Iodine
Add sufficient water to make up the volume to I00 ml.
Store the solution in amber coloured glass bottles
 .
C) Basic fuchsin · 0. 1 gm .
Alcohol I 0.0 ml.
Distilled water upto 100 ml.
Dissolve basic fuchsin in alcohol and allow to stand for 24
hours . Add sufficient distilled water to make up the
volume to 100 ml.
Procedure
(a) Prepare a thin film or smear of a test bacterium on a clean
. aseptic precautions
slide using
(b) Heat fix the film by passing through bunsen's flame 2- 3
times . If heat fixation is contraindicated then dip the film in
alcohol or fixation . Heat fixation coagulates the proteins of
bacteria which disturbs-the morphological characters of micro-
organisms.
(c) Cover the fixed smear with gentian violet (crystal violet or
methyl violet) stain and allow· the stain to act for about one
minute .
(d) Remove the excess stain and wash the slide with excess of
Grams iodine solution thoroughly .
(e) Cover the whole slide with fresh Gram's iodine solution and
leave it as such for one minute. During this time compounds
are formed in the cytoplasm of the bacterial cell, which are
retained by some bacterial species during decoloration with
alcohol.
F ) Wash the slide with alcohol or acetone in order to
. slide. Go on washing the slide till no colour comes
decolorise
out.This process is very rapid and completes in 2-3 seconds.
G) After this .process wash the slide under running tap water
and Counterstain it with an aqueous solution of fuchsin for 30
seconds
(h)wash the slide with tap water, dry it and examine the slide
under oil immersion lens without mounting.Those bacteria
which cannot be decolorised with alcohol or acetone and retain
violet colour are known as Gram positive bacteria and those
bacteria which are decolorised by aicohol or acetone and
stains red due to fuchsin solution are known as -gram negative
bacteria.
The examples of gram positive bacteria are
.
staphylococci,streptococci,pneumococci,C.diphtheria,B.anthras
is,subtilus,CLtetani, Cl.welchi etc. The examples of gram
negative bacteria are gonococci, meningococci, E.coli, S. typhi,
Cholera vibrio.
The Gram staining method is commonly used for the
identification of mycobacterium, streptococci, staphylococci,
E.coli etc. but this method cannot be applied to capsules,
spores, flagella, fungi and protozoa. For this purpose other
techniques are used which are described under respective
categories.
Acid Fast Staining Technique
Acid fast. staining technique was first developed 1882 for
differential staining of microorganisms. In this method dyes like
melachite green and methylene blue are used. When the
smears are treated with these dyes and washed with acids and
alcohols they are not decolorised and retain the stain of the
dye. Such bacteria which are not decolorised are known as
acid fast bacteria but the bacteria which lose the stain and get
decolorised are known as non acid fast bacteria.
 Ziehl-Neelsen Method
The Ziehl-Neelson method is used for differentiating acid fast
bacteria .
mycobacterium tuberculosis, a causative organism of
tuberculosis (mycobacterium leprae a causative organism of
leprosy)

A. Z iehl-Neelsens (strong) carbol fuchsin solution

Basic fuchsin 10gm
Absolute alcohol 100ml
5% solution of phenol in water upto 1000ml
Dissolve the basic fuchsin in alcohol and add to the phenol
solution
B. Sulphuric acid 20% solution
C. Alcohol 95%
D. Counterstain methylene blue or malachite green
1. Prepare a smear of the sputum on a slide and fix it by passing
through bunsens flame.
.
2. Cover the slide with strong carbol fushin solution and heat until
steam rises. Allow the stain to remain in contact for 5 minutes
heat being applied at intervals to keep the stain hot but the stain
must not be allowed to evaporate dryness.
3. Wash the smear with water
4. Cover the slide with 20% sulphuric acid for one minute and
remove excess of the acid Wash the slide with water till the
colour of the smear ceases to come out
5.Counterstain the slide with methylene blue or dilute malachite
green for 30 seconds
6. Wash the slide thoroughly with water dry it and see under oil
immersion lens.
The slide will appear pink coloured and rod shaped tubercle bacilli
will be seen scattered in the film. The acid fast microorganisms are
stained pink or bright red wheres as back ground tissues cells and
other non acid fast bacilli are stained blue or green
Staining of Rickettsiae:
 Rickettsiae are gram negative but they are not stained
well wiith gram’s stain.
 They are stained with Giemsa or casteneda method
Staining of Yeast and Fungi:
 Fixed smear of yeast can be stained with crystal violet
or methylene blue.
 These dyes are put for 30 seconds to one minute.
 Wet mounts of yeast can be stained effectively with
methylene blue or Gram’s iodine.
 The cell can be emulsified in a drop of the either stain
and covered with a cover glass.
 Lactophenol cotton blue is excellent for staining fungi.
Staining of Spirochaetes

Spirochaetes are stained by fontana method.

Ammonia silver nitrate stain is used which
increases the apparent dimensions of the
spirochetes and these microorganisms are seen as
opaque black spiral hairs against a light back
ground
:
Simple staining of bacteria:
 Apply the stain to be used over the prepared smear.
 Crystal violet, methylene blue, fuchsin or safranin .
 Allow the stain to react for 30 seconds to 3 minutes
depending on the stain used.
 Wash the smear with gentle stream of cold water.
 Dry between bibulous paper and examine under oil
immersion lens of the compound microscope.
Special staining procedures:
Gr am’s staining
methods
 The most important staining method for bacteria
 The shape, size and structural details of
microorganisms are made visible.
 Helps to group organisms into gram positive and
gram negative
 Steps involved:
1. Prepare a thin film and dry it.
2. Stain the prepared smear with methyl violet for one
minute. Wash off the excess stain with Gram’s iodine
solution.
3. Cover the whole slide with fresh iodine solution for
one minute.
4. The smear is then decolorised with spirit.
5. Wash the smear quickly with running tap water.
6. Cover the smear with dilute carbol fuchsin for 30
seconds.
7. Wash it with tap water and then dry it in air
8. Examine the slide under oil immersion lens
 Gram positive bacteria retain the violet colour of methyl
violet.
 Gram negative bacteria are decolorized by spirit, alcohol
or acetone and are stained with a counter stain like
carbol fuchsin which imparts a pink color to them.
 Examples for Gram positive bacteria :
 Staphylococci, Corynebacterium diphtheria, Bacillus
anthracis, Clostridium tetani , Clostridium welchi ,
Streptococci, Pneumonococci etc
 Examples for Gram negative bacteria :
 Gonococci, Meningococci, E.coli, salmonella typhi etc
Acid Fast Staining:
 The acid fast stain is a differential stain used to
identify acid fast organism such as members of the
genus Mycobacterium.
 The technique was discovered by Ehrlich who
observed that after staining with aniline dyes, tubercle
bacilli resist decolourisation with acids.
Ziehl Neelsen method:
 Prepare a smear of the mucoid part of the sputum on
a slide and fix it.
 Put strong carbol fuchsin over the smear for five
minutes. Wash the smear with water.
 Put 20% sulphuric acid for one minute.
 Wash the slide again with water.
 Now put methylene blue for 30 seconds.
 Wash the slide with water.
 Dry the slide and observe it under oil immersion lens.
 The pink coloured, rod shaped tubercle bacilli will be
seen scattered in the sputum of open cases of
tuberculosis.
 The acid fast organism are stained pink or bright red.
 These organisms after being stained with carbol
fuchsin do not loose their red colour when washed
with acids