Staining is a method used to impart color to cells and tissues under a microscope. There are various staining techniques classified by chemical nature and method. Simple staining uses one stain like methylene blue. Differential staining distinguishes characteristics like Gram positive and negative bacteria. Special staining identifies structures like capsules, endospores, and flagella. Staining enhances contrast and highlights specific cellular components for examination and analysis in biological and biochemical research applications.

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*Staining is a method of imparting colour to cells, tissues or microscopic components,
so they are highlighted and visualized better under a microscope.*
 There are a variety of staining methods used for various purposes ranging from the
study of microscopic organisms to cellular structures, metabolic processes, etc.,
 Simple
 Differential
 Special staining
 Staining is carried out with the help of a reagent termed as “stain“.
 It can be done in two ways, namely in-vitro and in-vivo.
The protocol of staining generally involves three sequential stages:
1.Smear preparation: This is the primary stage, which involves the mixing of the
inoculum with a drop of sterile water and spreading it until a thin film is formed over
theglassslide.
2.Fixation of smear: It is the second stage, which involves drying and heat fixing the
thin microbial layer formed on the glass slide.
3.Staining of the specimen: This is the final stage where the stain is applied onto the
dried smear, which imparts colour to the microscopic matter. This procedure is
carried out prior to microscopic examination and biochemical tests.
*Stains are chemical reagents or dye that imparts colour to cells and tissue sections of
the biological specimens and aids in its visualization under a microscope.*
Stains work by increasing the contrast between different cellular components, thereby
highlighting specific cell structures.

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Stains can be classified into the following types, depending upon its chemical nature and
the type of staining methods.
Based on chemical nature: There are three kinds of stain, acidic, basic and neutral,
depending upon the chemical nature of the stain.
Based on the staining method: There are four kinds of stain, viz. direct, indirect,
differential and selective stains.

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Mechanism
 Stains are organic compound composed of a benzene ring, a chromophore group
and an auxochrome group.
 Now benzene is a colourless solvent, and the chromophore group is a molecule that
imparts colour to the benzene. As a result, the compound formed is called
a ‘Chromogen’. Now, this chromogen is not a stain in itself, it is just a coloured
compound.
 The second part of the stain, the auxochrome, is a chemical group that ionizes the
chromogen i.e. it imparts a positive or negative charge to the chromogen group. As a
result, the auxochrome enables the ionized chromogen to bind to cells or tissue
fibres of opposite charge and thereby colour it.
Types of Staining
1) Simple Staining
It determines the cell shape, size and arrangement of the microorganisms. It is a very quick
or simple method to perform and it makes the use of a single stain only. These are of two
types, namely direct and indirect staining.

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Characteristic Differences between Direct and Indirect Staining:
Characteristics Direct staining Indirect staining
Stain used Basic stain Acidic stain
Charge of stain Positive Negative
Examples Methylene blue, crystal violet,
carbol fuschin
Nigrosine, india ink, congo red
Outcome Stains the specimen Stains the background
General view
after staining
Principle for
discoloration
Because of the positively charged
stain, it gets attracted towards the
negatively charged cell, hence it get
fixed to the cell that retain the color
of stain results in colorless
background with colored cell.
Because of the negatively charged
stain, it gets repelled by the
negatively charged cell, hence it
does not fixed to the cell, results in
colorless cell with colored
background.
1) Differential Staining
It differentiates between the physical and chemical properties of two different groups of an
organism, depending on the cell-wall characteristics. It makes the use of multiple or more
than one stains. It can be categorised into two types that are given below:

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A. Gram staining : It provides an important tool to differentiate the two major groups
of bacteria, i.e. gram-positive and gram-negative. Dr Hans Christian Joachim Gram
introduced this method in 1884. It is carried out by the use of differential stain
known as Gram’s stain.
Procedure:
Gram staining Protocol
Gram
positive bacteria
Gram
negative bacteria
Primary
staining
Heat fixed smear is
flooded by crystal violet
and allowed to stand for
1min.
Mordanting After washing, iodine is
then flooded and allowed
to stand for 1min.
Decolourization After washing, alcohol is
added that is washed
immediately
Counter
staining
At last, safranin is
flooded over the smear
and allowed to stand for
30sec, then washed by
water.
Observation After air drying, place
one drop of oil
immersion over the
smear and adjust the
microscope to identify
the specimen, whether it
is gram negative or gram
positive.
Appear purple in
colour because of
teichoic acid that
resist the primary
stain.
Appear pink in color
due to lack of teichoic
acid,alcohol creates
pore in the cell which
decolourizes the
primary stain

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B. Acid fast Staining: It differentiates species of mycobacterium from the other groups
of bacteria. Paul Ehrlich first developed it in 1882. And later, this technique was
modified by a scientist named Ziehl Neelson.
Procedure:
Acid fast
staining
Protocol Acid fast bacteria
Non acid fast
bacteria
Primary
staining
Heat fixed smear is
flooded with carbol
fuschin and allowed to
stand for 1 min.
Decolourization After washing, acid
alcohol is added.
Counter
staining
At last, methylene
blue is flooded over
the smear and allowed
to stand for 30 sec,
then wash it with
water
Observation After air drying, place
one drop of oil
immersion over the
smear and adjust the
microscope to identify
the specimen, whether
specimen is acid fast
or not.
Appears red in colour
due to presence of
mycolic acid that
resist the color of
primary stain and
does not decolourize.
Appears blue in
colour, as they lack
mycolic acid, alcohol
creates pore in the cell
that decolourizes the
primary stain.
2) Special Staining
It helps in the identification of particular internal and external structural components of
the specimen. It includes capsule, endospore and flagella staining.

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A. Capsule staining: It differentiates the capsule from the rest of the cell body. This is
carried out by the use of both positive and negative dyes.
Procedure:
Capsule
staining
Protocol Diagram
Primary
staining
Drop of India ink is placed on a
clean slide.
Smearing Inoculum is then smeared in a dye.
Dragging Use another slide to drag the
mixture into thin film, and then air
dried.
Secondary
staining
Crystal violet is flooded over the
thin film, and then air dried.
Observation Examine the cells whether they are
encapsulated or not.
Interpretation of result
Positive: Zone formation occurs
against dark background
Negative: Zone formation does not
occur

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B. Endospore Staining: It differentiates the endospore from the vegetative cell and
makes the use of both acidic and basic stains.
Procedure:
Endospore
staining
Protocol Diagram
Primary staining Malachite green is flooded
over the smear
Heat fixing Then the mixture is heat
fixed
Decolourization Decolourized by water
Counter staining Safranin is then flooded over
the mixture and then air
dried
Observation Examine the slide under the
microscope, whether
endospore is present or not
Interpretation of result:
Positive: If Endospore present, it will
appear green in color whereas
vegetative cell appears as pink
Negative: And if endospore is absent
then only vegetative cells will appear
pink in color

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C. Flagella Staining: It helps in the identification of the bacterial motility through the
presence or absence of flagella. It makes the use of acidic and neutral stain.
Procedure:
Flagella
staining
Protocol Diagram
Primary
staining
One drop of leifson’s stain is
flooded over the smear
Secondary
staining
After that methylene blue is
added, and allowed to stand for
one minute
Observation Examine the appearance of
flagella to know whether the
bacteria is motile or not
Interpretation of result:
Positive: If flagella is present, then it will
appear red in color while cell appears
blue
Negative: And if not present, only cell
will appear blue in color

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Examples of Bacteria in different Staining Methods
Simple
staining
Direct stain positive organism:
Staphylococcus sp. , E.coli etc
Indirect stain positive organism:
Staphylococcus sp. ,Micrococcus
luteus etc
Differential
staining
Gram positive organisms:
Streptococcus sp. , Enterococcus sp. ,
Listeria sp. , Bacillus sp. etc.
Gram negative organisms:
Pseudomonas sp. , Salmonella sp. ,
Klebsiella sp. , Yersinia sp. etc.
Acid fast organisms:
Mycobacterium sp.
Non acid fast organisms:
Enterobacter sp.
Special
staining
Capsule stain positive bacteria:
B.anthracis, K.pneumoniae etc.
Capsule stain negative bacteria:
Neisseria gonorrhoreae
Endospore stain positive bacteria:
Clostridium sp. , bacillus sp. etc.
Endospore stain negative bacteria:
E.coli , Salmonella sp. etc
Flagella stain positive organisms:
B.subtilis, pseudomonas sp. , E.coli
etc
Flagella stain negative organisms:
shigella sp. , M.tuberculosis,
C.diphtheriae
Applications
 Staining methods have wide applicability in both biological and biochemical research.
 It is used in staining of metal.
 Used in staining of the wood.
References:
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