VANITA VISHRAM WOMEN’S UNIVERSITY

Managed By Vanita Vishram, Surat

School of Science and Technology

F.Y. B.Sc. Microbiology

Semester –I
Microbiology Practicals
Laboratory Report File
MBM201-1C-Fundamentals of Microbiology
CERTIFICATE

This is to certify that Ms./Mrs._____________________________________________

has satisfactorily performed _________ experiments recorded in this report out of _________

experiments in _______________________ laboratory during the academic year

__________.

Roll No. __________ Date: ______________

Remarks___________________

Laboratory Coordinator Head of Department

Seal of School/College
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Index

Sr.No Experiment Date of the Signature Grade

Experiment
Monochrome staining using basic stain &
1. acidic stain. (Positive & Negative
Staining)
Diffential staining- Gram Staining by
2. Hucker’s Modification method & Acid fast
staining –Study using permanent slide.

3. Capsule Staining (Maneval’s method)

4. Endospore staining

5. Spirochetes staining

6. Cell wall staining (Dyar’s method)

7. Metachromatic granules staining.

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Microbiology good laboratory practices and bio-safety.

All hazards that are commom to all laboratory work can be encountered in the microbiology
laboratory ‘biohazards’ are the biggest and most constant problem. A biohazard is a
potentially dangerous living microbe. Hence , it is essential that students of microbiology
learn and consistently practice the fundamental of laboratory safety.

LABORATORY SAFETY RULES

 Note carefully all instruction given by your instructor.

 Laboratory doors are kept closed when experiments are in progress.
 Dyes and chemicals used in laboratory may spoil cloths. Hence, a knee-length, cloth
laboratory coat [apron or gown] must be worn in the laboratory. Laboratory clothing
must not be worn in non-laboratory areas.
 This is necessary to avoid carrying infectious agents out of the laboratory. Outside
wraps are not be brought into the laboratory at any time. Keep your apron clean by
regular laundering.
 Do not put anything in your mouth while you are in laboratory. This includes pencils,
fingers, cigarettes, food, drink and gum. Do not moisten labels with the tongue. Do
not bring food or drink into the laboratory or drink water from or in the laboratory.
 Smoking is absolutely prohibited in the laboratory hair must not fall into face, the
work, or lighted Bunsen burner. To prevent this, must be kept short or long should be
tied back or pinned back securely.
 Place all extra clothing, unnecessary books, backpacks, purses, and paraphernalia in
an appropriate place. Drawers or racks are provided for these materials. The
laboratory work area must be kept free of articles not in use.
 Regularly decontaminate your bench top working area with a phenolic disinfectant
such as 5 persent Lysol or phenol or quarernary compound such as cetylpyridinium
chloride before and after each laboratory period. This standard procedure lessens the
chance for accidental infection as well as for contamination of cultures.
 Get in the habit of washing your hands often. They should always be washed before
and after each experiment, as well as whenever you suspect that they may have
become contaminated.
 Culture are never to be taken out of the laboratory. Mechanical pipetting devices are
to be used; mouth pipetting is prohibited. All procedures are conducted carefully to
minimize the creation of aerosols.
 The gas burner is a very dangerous device and should be treated with care. All burner
must be turned off to pilot when not in use.
 All contaminated materials including cultures should be placed in proper in
receptacles for decontamination. Culture tube are not be discarded in baskets and
petri dishes in pails which are supplied in each laboratory . No culture should ever be
kept or stored in drawer or on the desk.
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 Discard pipettes and microscope slides in proper disinfectant receptacles.

 Put all supplies and equipments in proper place and turn off all ultilities at the end of
each laboratory period.
 Always kept the containers of alcohol from flames.
 Report all accidents, broken glassware, injuries, etc. to the instructor immediately.
You will be given exact instruction to follow. Do not hide accidents, no matter how
inconsequential they may seem. You will not be penalized for reporting accidents.
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Practical: 1

Aim: Monochrome staining using basic stain. (Positive Staining)

INTRODUCTION
Staining procedures that use only one stain are called simple stains. It is usually an single step
process. A simple staining that stains (imparts color to) the bacteria is called monochrome
staining, and a simple stain that stains the background, but leaves the bacteria unstained is a
negative staining. In such simple staining all the cells are stained in the similar way. however,
differences in the size, shape and the arrangement can be easily visualized in few cases
structural features (like colorless spores) can also be observed.

MONOCHROME STAINING METHOD

PRINCIPLE
It is believed that the process of staining involves ion-exchange reactions between the stain
and active sites at the surface of or within the cell. For example, the colored ions of the dye
(chromophore) ions of the dye may replace other ions on cellular components. Certain
chemical groupings of cell proteins or nucleic acids may be involved in salt formation with
positively charged ions such as Na+ or K+.
It it is assumed that the surface of a bacterial cell has an overall acidic characteristic because
of large amount of carboxyl groups located on the cell surface due to acidic amino acids.
Therefore, when ionization of carboxyl groups takes place it impans negative chargeto the
cell surface as per the following equation.

COOH ionization COO- + H+

In nature, H is replaced by another positive charged ion e.g.. Na+, or K+, and H+ bonds with
oxygen to form water.
Basic dye are usually salt of acids, eg, methylene blue; the dye which is actually methylene
blue chloride, (it has the positively charged colored ion (a cation), and if this ion is
represented by the symbol Mb, may be represented as Mb+ Cl-). When methylene blue
rehydrates, it ionizes as follows.

Mb.Cl ionization Mb+ + Cl-

The ionic exchange taking place during staining may be represented by the following
equation, in which the Mb+ cation replaces the Na+ cation in the cell:
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Thus, when coloring agent forms ionic bond with cell or cell components, it will result into
the staining of cell.
Other basic stains like, crystal violet, safranin, malachite green, gention violet, basic
fuchsin or carbol fuchsin can also be used for staining bacterial cells which have net
negative charge.

REQUIREMENT
1. Young culture of Escherichia coli, Bacillus subtilis and Staphylococcus aureus.
2. Methylene blue staining solution.

PROCEDURE
1. Prepare a heat fixed smears of the above culture.
2. Cover the smear with methylene blue stain for 4-5 minutes.
3. Wash the slide in tap water ( without draining the stain ), drain, blot and air dry the smear.
4. Examine under oil-immersion objective.
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Aim: Monochrome staining using acidic stain. (Negative Staining)

INTRODUCTION
Negative staining is a type of simple staining and is also called because the dye does not stain
cells , it imparts contrasting background , thereby making organism visible.

NEGATIVE STAINING METHOD

Negative staining use acidic dyes like nigrosine, eosin or Congo red. Acidic dyes have
negetively charged chromogen which will not bind (react) with bacterial cells because of the
similar acidic(negative) charge on the surface of bacterial cells. The stain does not stain the
bacteria because of ionic repulsion. Therefore cells remain unstained and can be easily
visualised against the contrasting background. Though, this staining technique is not very
popular, it has advantage over the direct or positive staining method for study of morphology
of cells.this is because of fact that the cells do not receive vigorous physical or chemical
treatments . Practical appliances of negative staining are :
1. Natural shape and size of the cell is not distorted because the heat fixation is not required.
2. It is possible to observe those structures (capsule) and bacteria (spirochetes) which are
difficult to stain.

REQUIREMENTS:
1. Young culture of Escherichia coli , Bacillus subtilis and Staphylococcus aureus.
2. Nigrosine staining solution (10%).

PROCEDURE:
1. Place a small drop of nigrosine at the end of the slide. For cultures on solid media, add a
loopful of distilled water and emulsify a small amount of the culture in the nigrosine –
water drop. For both cultures , mix a loopful of the culture into the drop of nigrosine . Do
not spread the drop or let dry.
2. Using the end edge of another slide , spread the drop out to produce a smear varying from
opaque black to gray . The angle of spreading slide will determine the thickness of the
smear .
3. Let the smear air dry aand examine (do not heat fix the slide).
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Practical: 2

Aim: Gram Staining by Hucker’s Modification method.

INTRODUCTION

The gram stain was developed in 1884 by Hans Christian Gram. It is a very important
differential staining because it separates bacteria into two broad categories namely gram-
positive and gram-negative. It is almost always the first step formed for the identification of
bacteria.

PRINCIPLE

 Details of the chemical mechanism of the gram stain were determined in 1983 by
Beveridge and Davies. It is most widely accepted mechanism of gram’s staining.
 Crystal violet (CV) dissociates in aqueous solution into CV and chloride (Cl) ions.
These ions penetrate through the cell wall and cell membrane of both gram-positive
and gram-negative cells. The CV ions interacts with negatively charged of bacterial
cell and stains the cells purple.
 Addition of negatively charged iodine (in the mordant) binds to the positively charged
dye and forms dye-iodine complex within the cell. Crystal violet (hexamethyl-para-
rosaniline chloride) interacts with aqueous KI-I2 via a simple anion exchange to
produce a chemical precipitate. The small chloride anion is replaced by the bulkier
iodide, and the complex thus formed becomes insoluble in water.
 When a decolorizer such as alcohol or acetone is added, it interacts and extracts lipid
from the gram negative wall and increase its porosity. A gram negative cell will lose
its outer membrane and the lipopolysaccharide layer is left exposed. The cv
complexes are washed from the gram- negative cell along with the outer membrane.
In contrast, gram-positive cells become dehydrated from the ethanol treatment,
closing pores as the cell wall shrinks dehydration. The large cv complexes are trapped
within the gram-positive cell due to multilayered nature of its peptidoglycan.
 After decolorization, the gram-positive cell remains purple and the gram-negative cell
loses its purple colour. Counterstai, which is usually positively charged safranin or
basic fuchsin, is applied last to give decolorized gram-negative bacteria a pink or red
colour.

Gram's Staining Method

Requirements

1. Young cultures of Escherichia coli. Bacillus subtilis & Staphylococcus aureus

2. Crystal violet stain, Gram's iodine. 95 % ethanol and safranin stain

Procedure

1. Prepare a heat fixed smear of the culture.

2. Gently flood smear with crystal violet and let stand for 1 minute.
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3. Tilt the slide slightly and gently rinse with tap water or distilled water using a wash bottle.
4. Gently flood the smear with Gram’s iodine and let stand for 1 minute.
5. Tilt the slide slightly and gently rinse with tap water or distilled water using a wash bottle
The smear will appear as a purple circle on the slide.

6. Decolorize using 95% ethyl alcohol or acetone. Tilt the slide slightly and apply the alcohol
drop by drop for 30 seconds until decolorizing agent running from the slide runs clear.
Be careful not to over-decolorize.
7. Immediately rinse with water.
8. Gently flood with safranin to counter-stain and let stand for 5-7 minutes.
9. Tilt the slide slightly and gently rinse with tap water or distilled water using a water bottle.
10. Drain the water, blot, air dry and examine under oil immersion objective.

Examples of Gram-Positive Bacteria Examples of Gram-Negative Bacteria

• Bacillus megaterium • Escherichia coli
• Bacillus subtilis • Azospirillium lipoferum
• Bacillus cereus • Azotobacter spp.,
• Bacillus stearothermophilus • Desulphococcus spp.
• Bacillus thuingiensis • Pseudomonas aeruginosa
• Streptocuccus pneumonia • Salmonella typhosa
• Micrococcus leutus • Neisseria gonorrhoeae
• Clostridium botulinum • Veillonella ssp.
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Aim: Acid fast staining –Study using permanent slide.

Introduction
Colour fastness is a term used in the dyeing of textile materials, meaning resistance of the
material's colour to fading. Some bacteria once stained are not decolorized even with the
strong decolorizing agent like acid alcohol. Because of this property, these organisms are
known as acid-fast, while other organisms which are easily decolorized by acid alcohol are
called non-acid-fast (This makes acid fast staining a differential staining). This property was
first discovered by Ehrlich in 1882 during the staining of tubercle bacilli from sputum. This
staining technique is of great importance for the diagnosis of diseases like tuberculosis and
leprosy.

Cell wall of Acid Fast Bacteria

In addition to peptidoglycan, the acid-fast cell wall of Mycobacterium contains a large
amount of glycolipids, especially mycolic acids. The peptidoglycan layer is linked to
arabinogalactan (D-arabinose and D- galactose) which is then linked to high- molecular
weight mycolic acids. The arabinogalactan/mycolic acid layer is overlaid with a layer of
polypeptides and mycolic acids consisting of free lipids, glycolipids. I and
peptidoglycolipids. Other glycolipids include lipoarabinomannan andphosphatidyinositol
mannosides (PIM).
Like the outer membrane of the gram-negative cell wall, lipoarabinomannan and porins are
required to transport small hydrophilic molecules through the outer membrane of the acid-
fast cell wall. Bacteria of the genera Mycobacterium and Nocardia have unusual cell walls
that are waxy and nearly impermeable due to the presence of the waxy molecules.
Moreover, there are far fewer porins in the acid-fast cellwall compared to the gram-negative
cell wall and the pores are much longer. These unusual waxy substances impede (obstruct)
the entry of chemicals causing the organisms to grow slowly and be more resistant to
disinfectants, desiccation and are difficult to stain with water-based stains such as the Gram
staining.

Ziehel -Neelsen Staining Method

Principle
The Ziehl-Neelsen stain, also known as the acid-fast stain, was first described by two
German doctors: the bacteriologist Franz Ziehl and the pathologist Friedrich Neelsen.
Explanation of acid fastness lie in the impermeability of bacterial surface to the stain rather
than in the ability to bind to a particular bacterial component. Substances involved in
preventing removal of the dye by acid alcohol are lipids and mycolic acid esters. The cell
wall of acid-fast bacteria have high lipoidal content (25-60%) as compared to gram-positive
(0.5%) and gram-negative (3%) bacteria.
Acid fastness property of the intact cell depends on trapping of intracellular mycolate-fuchsin
complexes formed in the cell wall during staining. The dye fuchsin is more soluble in phenol
than in water or acid alcohol. Phenol in turn, is more soluble in lipids present in the cell wall
of acid-fast bacteria. During staining fuchsin enters the cell (phenol and heat acts as
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intensifiers). On application of decolorizer (acid-alcohol), acid-fast cells resist

decolorization,since the primary stain is more soluble in cellular waxes than in the
decolorizing reagent. Non acid-fast bacteria get easily decolorized and are counter stained
with the dye of different color like methylene blue or malachite green.

Requirements
1. Culture of Mycobacterium spp. or sputum from tuberculous patients.
2. ZNCF (Ziehl-Neelsen carbol fuchsin) stain, acid-alcohol and malachite green or methylene
blue stain.

Procedure
(Ziehl-NeelsenMethod)
1. Prepare a heat fixed smear from the sputum or culture.
2. Place the slide on tripod stand and place strip of blotting paper over the slide (marked area
of smear)
3. Flood the smear with carbol fuchsin (ZNCF stain) and Gently heat the slide from below till
it begins to steam; allow the slide to steam for 3-5 minutes. (Don't allow stain to evaporate
but replenish the stain as and when needed. Also prevent the stain from boiling by
adjusting the temperature during heating). Slide may also be heated indirectly by placing it
over the steaming waterbath.
4. Allow the slides to cool, remove the filterpaper and wash well with tap water.
5. Decolorize with acid-alcohol for 10-20 seconds, until no color comes out of the slide.
Decolorization should not be attempted in one stage; there should be intermittent washing
in water and reapplication of alcohol (20 % H2 SO4, can be used in place of acid-alcohol).
6. Rinse well with tap water.
7. Counter stain with methylene blue or malachite green for 3 minutes.
8. Rinse with water, allow it to air dry and examine.

Examples of Acid Fast Bacteria

Mycobacterium tuberculosis: causes tuberculosis in humans.
Mycobacterium bovis: causes tuberculosis in animals.
Mycobacterium microti: causes tuberculosis in mammals.
Mycobacterium avium: causes tuberculosis in birds.
Mycobacterium scrofulaceum: causes lymphadentis in children.
Mycobacterium leprae: causes leprosy in humans.
Mycobacterium phlei: a saprophyte found on grass.
Mycobacterium smegmatis: a saprophyte found in milk and grass.
Nocardia asteroides: causes nocardiasis in humans.
Corynebacterium spp., Gordona spp
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Practical: 3

Aim: Capsule Staining (Maneval’s method)

INTRODUCTION
Glycocalyx is the general term used for substances that surrounds cells. The bacterial
glyocalyx is a viscous (sticky), gelatious polymers composed of polysaccharide,polypeptide
or both.When this layer is well organized and not easily washed off, it is referred to as a
capsule; while a slime layer medium and remain as a loose undermarcated secretion,
which can easily removed.
Other Methods for Capsule Staining are :
=> Anthony's Method
=> White Method
=> Mollers Method
=> Butt's India ink Method
=> Grahm & Evans india ink Method

PRINCIPLE
It is a combination of positive and negative staining. Due to the non-ioninon-ionic of Capsule
it has a very little affinity for the dye .Therefore ,when dye solution is applied,it stain cell
amd the capsule is left colourless,while the background stain the dye as the smear is wash.

REQUIREMENTS
1. Young culture of ccapsulated bacteria
2. Maneval's Stain and 1% congo red stain

PROCEDURE
1. Place 1 drops of congo red stain on glass slide.
2. Pick up loopful of capsule growth and emulsify it in congo red on the slide.
3. Prepare a smear and allow to air dry.
4. Treat the smear with maneval's stain for 4-5 minutes.
5. Drain the stain, allow it to air dry & Examine.

EXAMPLES OF CAPSULATED BACTERIA

-Streptococcus pneumonia - Streptococcus mutans
- Clostridium welchil - Acetobactor xylium
-Cryptococcus pneumonia - Rhizobium spp.
-Azotobacter spp. - Enterbacterer spp.
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Practical: 4
Aim: Endospore staining

Introduction
When environmental conditions become unfavorable, certain gram-positive bacteria undergo
sporogenesis and give rise to resistant, metabolically Inactive ," resting" intracellular
structure called endospore. When the favorite conditions return, the spore may revert to a
metabolically active vegetative cell through germination. Then it should be noted that
sporogenesis and germination are notem means of reproduction, but merely a mechanism
which ensures the survival of cells under difficult environment conditions.
Other method for endospore staining are :
– Schaeffer and Fulton's method
– Eleming's method
– snyder's modification of Dorner's method.

Principle
It is a Cold method of spore staining and avoids heating. However spores are stained due to
A) longer heat treatment during fixation
B) prolonged staining period (10 minutes)
C) high concentration of stain (7.6% Malachite green)
Use of tap water as weak decolorlizing agent will decolorize the cytoplasm only while the
spore is unaffected. Cytoplasm is then counter stained with a stain of a different color from
that of primary stain.

Requirements
1. Young sporulating culture of Bacillus megaterium/Bacteria cereus (12-14 hours old).
2. Malachite green (7.6% aqueous solution) and safranin (0.25%)

Procedure
1. Prepare a thick smear and fix it with heat by passing the slide through flame for 20
times.
2. Allow the slide to cool, and flood it with 7.6% malachite green stain for 10 minutes.
3. Rinse with tap water for 10 seconds.
4. Counter stain with 0.25% safranin for 5 minutes.
5. Rinse in tap water, blot, air dry and examine.

Examples of endospore formers

– Bacillus cereus
– Bacillus subtilis
– Bacillus megaterium
– Bacillus polymyxa
– desulfotomaculum
– sporosarcina ureae
– Bacillus thuringiensis
– Bacillus anthracis
– Clostridium botulinum
– Sporolactobacillus opp
– Thermoactinomyces vulgaris
– Coxiella burnetii
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Practical: 5

Aim: Spirochetes staining

INTRODUCTION
Spirochetes or (spirochaetes) are Gram-negative, flexible, spiral shaped organism who have
distinct morphology and mechanism of locomotion. Length of spirochetes varies from 3-500
micrometer and width ranges from 0.1-3 micrometer. Instead of having flagella projecting
from the cell, the flagella of Spirochetes form Central axial fibrils (endoflagella) that runs
through the periplasm.

PRINCIPLE
Due to delicate nature of spirochetes, heat fixation is avoided as it destroy the shape of
organism. Chemical fixatives like formalin and glacial acetic acid are used for fixation.
Moreover, the use of mordant containing tannic acid increases the affinity of stain towards
the cell. Finally when stain in containing ammoniacal silver nitrate is heated up, Silver oxide
is formed with precipitates on the organism thereby the apparent diameter is adequately
increase an organism are visible.

REQUIREMENTS
1. Fontana’s fixative, Fontana’s mordant, Fontana’s stain, 95% ethanol, sterilized pin
and distilled water.

PROCEDURE
1. Sterilize a pin by dipping in alcohol and flaming in it Bunsen’s burner
2. Allow the pin to cool down, and scrap out tartar from interspaces between teeth.
3. Emulsify tartar in a drop of distilled water water placed in center of the slide.
4. Spread and prepare a smear over a small area. Allow it to air dry; don’t heat fix the smear.
5. Immerse the slide in coplin jar containing Fontana’s fixative for 30 seconds. Repeat the
process for three times at an interval of 1 minute each.
6. Wash off the fixative with alcohol and allow the alcohol to act for 3 minutes.
7. After 3 minutes the excess alcohol is washed off and slide is hold with the help of forcep
and passed through flame to remove excess alcohol.
8. Add Fontana’s mordant on the smear, and heat till steam rises for 30 seconds.
9. Wash well in distilled water and allow the slide to dry.
10.Add Fontana’s stain, and heat till steam rises for 30 seconds.
11. Allow the slide to cool. Wash well in distilled water, drain, blot dry and examine.

EXAMPLE
Spirochaeta plicatilis Treponema denticola Treponema macrodenticum
Treponema oralis Traponema pallidum Borrelia bucalis
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Practical: 6

Aim: Cell wall staining (Dyar’s method)

INTRODUCTION
Most species of bacteria (except Mycoplasma, Spiroplasma and certain Archaebacteria) have
the outermost rigid layer known as the cell wall. Bacteria can be divided into two groups on
the basis of their differences in cell walls. The gram positive cell wall consists of a single 20-
80 nm thick homogeneous layer made up of peptidoglycan(murein) surrounded by teichoic
acids. In contrast, the gram negative cell wall has 1-3 nm peptidoglycan as the inner layer,
surrounded by 7-8 nm thick outer membrane which is made up of lipopolysaccharides.

Other methods for cell wall staining are Bouin’s method and Webb’s method.

PRINCIPLE

Cetylpyridinium chloride(C₂₁H₃₈NCl.H₂O) ionize in water to form positively charged

cetylpyridinium and negatively charged chloride ions. As the cell wall is negatively charged,
the positively charged ions of cetylpyridinium are absorbed on it making it a positively
charged surface. Subsequent treatment with acidic dye like Congo Red will stain the cell
wall. The cytoplasm may later be stained with basic dyes like Methylene blue.

REQUIREMENTS

1) Young culture of bacteria (Bacillus megaterium).

2) 0.34% cetylpyridinium chloride solution, saturated Congo Red and 0.5% methylene blue
stain.

PROCEDURE

1) Prepare a heat fixed smear, and place 3-4 drops of cetylpyridinium chloride on the smear.
2) Add 1 drop of Congo Red and mix it with the drop of cetylpyridinium chloride by rotating
the slide slowly.
3) Allow it to act for 2 minutes. Rinse the slide with tap water.
4) Counterstain with methylene blue for 3 minutes.
5) Rinse with water, air dry and examine.
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Practical: 7

Aim: Cytoplasmic membrane staining-Demonstration.

The outermost layer of the cytoplasm is called the cytoplasmic membrane. This layer is 75A
(Angstrom) (7.5 nm = 0.0075 um) units thick. The cell membrane in bacteria is a
phospholipid-protein bilayer similar to that present in eucaryotic cells. The major difference
is that there are no sterols in the cell membranes of most procaryotes. Major disruptions in
the membrane result in the spilling of the cytoplasm from the cell and the death of the
organism. A plasmolysis technique is used to separate cell wall from the cytoplasm and the
membrane is then stained.

Requirements

1. Bacillus cereus culture

2. Potassium nitrate solution,
3. Bouin's fixative solution
4. Victoria blue solution

Procedure
1. Prepare a smear of the bacterium on a clean slide.
2. Heat fix the smear.
3. Immerse the slide in potassiunm nitrate solution for 15 minutes for plasmolysis to take
place.
4. Keep the slide in Bouin's fixative solution for 15 minutes.
5. Wash the slide in tap water.
6. Apply victoria blue solution for 3 seconds.
7. Wash the slide in tap water.
8. Blot dry the smear.

Observations

Examine the preparation microscopically under oil-immersion objective.

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Practical: 8

Aim: Metachromatic granules staining.

INTRODUCTION
Metachromatic granular are the type of cytoplasmic inclusions found in many bacteria and
some higher organisms like algae, fungi, protozoa. The team metachromatic was first
introduced by Ehrlich as a meaning of staining of granules in a tone different from that
possessed by a dye. Metachromatism was originally described in a bacterium called
S𝘱𝘪𝘳𝘪𝘭𝘭𝘶𝘮 𝘷𝘰𝘭𝘶𝘵𝘢𝘯𝘴. Hence they are also known as volutin granules. These granules are also
known as Babes- Ersnt granules because they were the first to study them as a strong
basophilic substance. Other method for metachromatic granules staining are gohr’s method
and Neisser’s method.

PRINCPLE
The cationic dye (basic dye) has good affinity for their substrate with lower pH than the pH
of the solution in which it is dissolved. Due to high acidic nature of the granule, it can be
selectively stained by acidified basic dyes. The pH of the dye solution is around 2.8 which is
acidic to cytoplasm and basic to the acidic granules. Toludine blue O preferentially stain
metachromatic granules while malachite green stain the cytoplasm. Later on due to the
application of iodine the dye molecules are fixed by precipitation, thus preventing change in
orbital paths of electrons, hence metachromatism is inhibited.

REQUIRMENTS
1. Curd sample (about 48 hours old )
2. Chloroform
3. Albert’s stain
4. Lugol’s iodine solution

PROCEDURE
1. Prepare the heat fix smear from the curd sample.
2. Treat with chloroform to remove fats by immersing the slide in coplin jar for three
times for 30 seconds at an interval of 30 seconds each.
3. Cover the smear with Albert ‘s stain for 4-5 minutes.
4. Drain the stain. Do not wash with tap water.
5. Cover the smear with Lugol’s podine for 1 minute.
6. Rinse with tap water; drain, blot, air dry and examine.

Examples of organisms containing metachromatic granules.

• 𝑪𝒐𝒓𝒚𝒏𝒆𝒃𝒂𝒄𝒕𝒆𝒓𝒊𝒖𝒎 𝒅𝒊𝒑𝒉𝒕𝒉𝒆𝒓𝒊𝒂𝒆
• S𝒑𝒊𝒓𝒐𝒄𝒉𝒂𝒆𝒕𝒂 𝒑𝒍𝒊𝒄𝒂𝒕𝒊𝒍𝒊𝒔
• C𝒂𝒖𝒍𝒐𝒃𝒂𝒄𝒕𝒆𝒓 𝒗𝒊𝒃𝒓𝒊𝒐𝒊𝒅𝒆𝒔
• 𝒍𝒂𝒄𝒕𝒐𝒃𝒂𝒄𝒊𝒍𝒍𝒖𝒔 𝒔𝒑𝒑.