Date:

Page No.
Experiment No. 3

Aim: Staining

a) Gram Staining
b) Endospore Staining
c) Capsule Staining

Theory: Microorganisms are very difficult to study as they are transparent and colorless and
hence become very problematic to observe when they are suspended in any aqueous medium.
Therefore, the main aim of staining is to increase visibility and reveal additional information
required to study the microorganisms and identify them. In recent times many stains and staining
techniques have been developed to study the various properties of microorganisms and also help
in differentiation into their specific groups/genera/species.

The chemical substances commonly used to color bacteria are known as stains. Stains may be
natural or synthetic. Usually all the stains has two important chemical constituent, i.e. a
chromophore (color imparting molecule) and auxochrome. Stains may be acidic, neutral and basic
depending on their chemical nature. Acidic dyes are anionic and stain the cytoplasmic
components of the cells which are more alkaline in nature. Example - Picric Acid, Acid Fuchsin
etc. Whereas, the basic dyes are cationic and they combine with those cellular elements which are
acidic in nature like nucleic acids. Example – Methylene Blue, Crystal Violet etc. Neutral stains
are formed by mixing both the aqueous solutions of acidic and basic dyes. Staining solutions are
usually prepared by dissolving the crystal compounds or powder form in distilled water or in
alcohol. The stain is applied to the glass slide containing the specimen (which may or may not be
heat dried) and then after some time washed, dried and examined under the compound
microscope.

There are two kinds of staining procedures, simple and differential. Simple stains employ a single
stain (eg. Methylene Blue) and cells and structure within each cell will attain the color of the
stain. Differential stain requires more than one stain and is used to distinguish between structures
within a cell or types of cells by staining them with different stains. Example: Gram Staining
technique where Crystal Violet is the primary stain used and the Saffranine is the secondary stain
used.

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Experiment No. 3

a. Gram Staining
Principle : The Gram stain (named after Christian Gram, Danish scientist and physician,
1853–1938) is the most useful and widely employed differential stain in bacteriology. It divides
bacteria into two groups— gram negative and gram positive. The first step in the procedure
involves staining with the basic dye crystal violet. This is the primary stain. It is followed by
treatment with an iodine solution, which functions as a mordant; that is, it increases the
interaction between the bacterial cell and the dye so that the dye is more tightly bound or the cell
is more strongly stained. The smear is then decolorized by washing with an agent such as 95%
ethanol or isopropanol-acetone. Gram-positive bacteria retain the crystal violet-iodine complex
when washed with the decolorizer, whereas gram-negative bacteria lose their crystal violet-iodine
complex and become colorless. Finally, the smear is counterstained with a basic dye, different in
color than crystal violet. This counterstain is usually safranine. The safranine will stain the
colorless, gram-negative bacteria pink but does not alter the dark purple color of the gram-
positive bacteria. The end result is that gram-positive bacteria are deep purple in color and gram-
negative bacteria are pinkish to red in color.

Requirements:

1) 24-hr broth cultures of Bacillus subtilis and Escherichia coli

2) Crystal Violet solution
3) Grams Iodine solution
4) Saffranine solution
5) Glass slides
6) Inoculating loop
7) Spirit Lamp
8) Tissue paper
9) Compound Microscope
10) Immersion Oil

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Experiment No. 3

11) Working Bench

12) Droppers
13) Staining Tray
14) Ethanol
15) Cotton
16) Wash Bottle of Distilled water

Procedure:

1) Heat-fixed smears of E. coli and B. subtilis was prepared.

2) The slides were placed on the staining tray.
3) The smears were flooded with crystal violet and let stand for 30 sec.
4) It was rinsed with water.
5) Two drops of Gram’s iodine was added and let stand for 1 min.
6) The Iodine was washed off by 95% ethanol for 15 to 30 sec. It was added drop by drop until
the crystal violet fails to wash from the slide.
7) The slide was washed with water.
8) It was counterstained with saffranine for about 60 sec.
9) It was washed with water.
10) The slide was air dried and examined under the compound microscope.

Comments/Remarks:
1) It is a very routine work in microbiology.
2) The principle and the techniques involved were thoroughly discussed.
3) The experiment was demonstrated and the Gram staining was understood.

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Experiment No. 3

b. Endospore Staining

Principle : Bacteria in genera such as Bacillus and Clostridium produce quite a resistant
structure capable of surviving for long periods in an unfavorable environment and then giving rise
to a new bacterial cell. This structure is called an endospore since it develops within the bacterial
cell. Endospores are spherical to elliptical in shape and may be either smaller or larger than the
parent bacterial cell. Endospore position within the cell is characteristic and may be central,
subterminal, or terminal. Endospores do not stain easily, but, once stained, they strongly resist
decolorization. This property is the basis of the Schaeffer-Fulton of staining endospores. The
endospores are stained with malachite green. Heat is used to provide stain penetration. The rest of
the cell is then decolorized and counterstained a light red with saffranine.

Requirements:

1) 48-50 hr broth cultures of Bacillus subtilis.

2) Malachite Green Solution (5%)
3) Saffranine solution (0.5%)
4) Water Bath
5) Wash Bottle of Distilled water
6) Glass slides
7) Inoculating loop
8) Spirit Lamp
9) Tissue paper
10) Compound Microscope
11) Immersion Oil
12) Working Bench
13) Droppers
14) Staining Tray
15) Cotton

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Experiment No. 3

Procedure:

1) Heat-fixed smears of Bacillus subtilis were prepared.

2) The slides were placed on the staining tray.
3) The smears were flooded with Malachite Green.
4) The slides were heat steamed in water bath for 5-10 min and from time to time more stain was
added to prevent the smear from becoming dry.
5) The slides were cooled and washed with water.
6) It was counterstained with saffranine for about 1 min.
7) It was washed with water.
8) The slide was air dried and examined under the compound microscope.

Comments/Remarks:
1) It is a very routine work in microbiology.
2) The principle and the techniques involved were thoroughly discussed.
3) The experiment was demonstrated and the Endospore staining was understood.

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Experiment No. 3

c. Capsule Staining

Principle : Many bacteria have a slimy layer surrounding them, which is usually referred to as a
capsule. The capsule’s composition, as well as its thickness, varies with individual bacterial
species. Polysaccharides, polypeptides, and glycoproteins have all been found in capsules. The
procedures for determining the presence of a capsule is the Anthony’s capsule staining method
Anthony’s procedure employs two reagents. The primary stain is crystal violet, which gives the
bacterial cell and its capsular material a dark purple color. Unlike the cell, the capsule is nonionic
and the primary stain cannot adhere. Copper sulfate is the decolorizing agent. It removes excess
primary stain as well as color from the capsule. At the same time, the copper sulfate acts as a
counter stains by being absorbed into the capsule and turning it a light blue or pink. In this
procedure, smears should not be heat-fixed since shrinkage is likely to occur and create a clear
zone around the bacterium, which can be mistaken for a capsule.

Requirements:
1) 48-hr broth cultures of Klebsiella pneumonia.
2) Crystal Violet solution (1% w/v)
3) Copper Sulfate (20%)
4) Wash Bottle of Distilled water
5) Glass slides
6) Inoculating loop
7) Spirit Lamp
8) Tissue paper
9) Compound Microscope
10) Immersion Oil
11) Working Bench
12) Droppers
13) Staining Tray

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Experiment No. 3

14) Cotton

Procedure:

1) Heavy smears of Klebsiella pneumonia were prepared. The slides were not heat fixed.
2) The slides were placed on the staining tray.
3) The smears were flooded with Crystal Violet for 2 min.
4) The stain was washed off by adding 20% Copper Sulfate solution.
5) The excess stain was drained off and the slide was blot dried and examined under the
compound microscope.
Comments/Remarks:
1) It is a very routine work in microbiology.
2) The principle and the techniques involved were thoroughly discussed.
3) The experiment was demonstrated and the Capsule staining was understood.

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Experiment No. 3

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