The document provides an extensive overview of microbial staining techniques used in microbiology, detailing types such as simple, differential, and special staining, along with specific methods like Gram staining and acid-fast staining. It explains the principles behind each technique, the necessary materials, and step-by-step procedures for effectively staining microorganisms to facilitate observation under a microscope. Various staining outcomes are described, indicating how different bacteria appear under different stains, which aids in their identification.

1. Madurai Kamaraj University
SCHOOL OF BIOLOGICAL SCIENCE
DEPARTMENT OF MICROBIAL TECHNOLOGY
MICROBIAL STAINING
SUBMITTED TO Dr. M. Murugan
1
SUBMITTED By Aniruddh Gupta
M.SC 1ST YEAR MICROBIOLOGY
MICROBIAL STAINING

2. Microbial Staining - giving color to microbes. Because
microbes are colorless and highly transparent
structures.
Staining process in which microbes are getting color.
INTRODUCTION :
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MICROBIAL STAINING

3. • Staines / dyes - organic compounds which carries either
positive charges or negative charges or both .
1. Based on the charges
• Basic stain / dyes :- stain with + ve charge .
• Acidic stain / dyes - stain with -ve charge .
• Neutral stain / dyes - stain with both charges .
STAINES / DYES
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MICROBIAL STAINING

4. • Simple staining only one dye is used differentiation among bacteria is
impossible Eg . Simple Staining.
• Differential staining- more than one dye is used- Differentiation
among bacteria is possible- Eg. Gram's staining, Acid - fast staining.
• Special staining - more than one dye used - Special structures are seen.
Eg. Capsule staining , Spore staining .
2. Based on function of stain :
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MICROBIAL STAINING

5. Each staining methods have own principles but the following steps
may be common :
PRINCIPLE OF STAINING
Basic stain :- + ve charge -
To stain -ve charged
molecules of bacteri.
Mostly used because cell
surface is -ve charge .
Acidic Stain ( -ve charge )
-To stain + ve charged
molecules of bacteria.
Used to stain the
bacterial capsules .
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MICROBIAL STAINING

6. • Clean grease - free slide .
• Bacteria to be stained.
• Inoculating loops- to transfer bacterial suspension to slide .
• Bunsen burner - to sterilize inoculating loops before and after smear
preparation .
• Pencil marker - to mark (particularly central portion of slide ) where
bacterial smear is applied.
Basic requirements for
staining :
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MICROBIAL STAINING

7. SIMPLE STAINING
PRINCIPLE :-
The Simple
Staining Is To
Visualize
Bacteria By
Proceeding
Contrast.
Dyes are
methylene blue
Carbon fuschin
Crystal violet
and many more.
Fig. simple staining Gram positive
Cocci shaped
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MICROBIAL STAINING

8. BASIC STEPS OF STAINING :
Air - dried , heat - fixed
The resultant preparation called bacterial smear- appears dull
white
Putting of bacterial suspension ( bacteria in liquid ) to be
stained on the central portion of slide in a circular fashion
Smear Preparation
Add stain like crystal violet for atleast 1 min
Observe under 100X magnification
Air dry the slide
Fig. simple staining
Gram positive Rod
shaped
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MICROBIAL STAINING

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MICROBIAL STAINING

10. GRAM STAINING DANISH BACTERIOLOGIST HANS CHRISTIAN GRAM ( 1880 )
PRINCIPLE: Gram staining depends on the structure and composition of the cell wall i.e
PEPTIDOGLYCAN
First , PRIMARY STAIN , crystal violet stains all the cells purple .
The function of a MORDANT in a Gram stain is to prevent the crystal violet from leaving the
Gram-positive cell. It increases the affinity of the cell wall for a stain by binding to the primary
stain thus forming an insoluble complex.
The DECOLOURISER used to decolorise the primary dye from gram negative bacteria. It
dehydrate the peptidoglycan layer that’s why the large crystal of crystal violet are kept inside the
gram positive bacteria. It also dissolve the lipid layer.
To observe the decolorized cells SECONDARY STAINS like Safranin or Basic fuchsin is added
which stains the gram negative organisms pink .
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MICROBIAL STAINING

11. 1. Prepare a thin bacterial smear , air dry & fix.
2. Flood the smear with crystal violet solution and allow standing for 2 minutes and washing with
tap water.
3. Add Gram's Iodine for 1 minute & wash with tap water .
4. Decolourize with 90 % Ethyl alcohol for 30 second , & wash with tap water .
5. Blot dry .
6. Counter stain with Dilute Carbol fuchsin or Safranin for 45 second.
7. Wash with tap water. Air dry and examine under oil immersion .
Bacterial Culture.
Slide and Microscope. .9% Normal saline, Spirit lamp, Platinum loop
Staining solution ( crystal violet , Gram's Iodine Solution , Decolourizer ( 90%
ethyl alcohol , Safranine ).
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MICROBIAL STAINING

12. Gram positive bacteria seems violet in color and gram negative bacteria
seems pink in color.
Gram positive Gram negative 12
EXAMPLES:
Gram positive cocci in clusters:
1. Staphylococci species.
Gram negative cocci in chains:
1. Streptococci species.
Gram negative cocci:
1. Neisseria species.
Gram negative bacilli:
1. Escherichia coli
2. Klebsiella pneumoniae
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ACID-FAST STAINING
• Also called Ziehl-Neelsen stain
Organisms such as
Mycobacteria are
extremely difficult
to stain by ordinary
methods like Gram
Stain because of the
high lipid content of
the cell wall.
The phenolic
compound carbol
fuchsin is used as
the primary stain
because it is lipid
soluble and
penetrates the waxy
cell wall
Acid alcohol can also
be used as
decolorizing solution,
resistant organisms
are referred to as
Acid Fast Bacilli
(AFB)
Note:- Acid-fast cells resist this decolorization. The ability of the bacteria to resist decolorization with acid confers
acid -fastness to the bacterium
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Broth culture of Mycobacterium tuberculosis(48-hour old).
Acid fast staining reagents (Conc.Carbol fuchsin, Acid alcohol (3% HCI + 95%
alcohol), Alkaline Methylene Blue).
Hot plate/water bath
Glass slides
Inoculating loop
Spirit Lamp, Blotting paper& Microscope
REQUIREMENTS
PROCEDURE
Prepare a smear of Mycobacterium cultures, air dry & fix it.
Flood the smear with Conc. Cabol fuchsin and gently heat the slide till the steam appears.
Continue heating for 3-5 minutes; avoid drying the stain during the process.
Wash the smear with tap water after cooling.
Decolorize the smear with acid alcohol for 20-30 seconds until the smear is faint pink.
Counter stain the smear with methylene Blue for 1 minute.
Wash the slide with water, air dry the smear observe under oil immersion
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Acid fast organism will appear red while non acid fast will appear blue in colour.
The Decolorizing agent :- Acid alcohol 3% HCL + 97% alcohol
20% H2So4 for Mycobacterium Tuberculosis
5% H2So4 for Mycobacterium Leprae
Mycobacterium
Tuberculosis
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Many bacteria are motile due to the presence of one or more very fine threadlike, filamentous
appendages, called flagella.
These are thin, proteinaceous structures which originate in the cytoplasm and project out from the
cell wall. Bacteria show four types of flagellation patterns.
FLAGELLA STAINING
Monotrichous = single flagellum.
Peritrichous = flagella all around.
Amphitrichous = flagella at both ends.
Lophotrichous = tuft of many flagella at one end or both ends.
Atrichous = without flagella, nonmotile.
REQUIREMENTS
18 hour nutrient agar culture of Esherichia coli, Loeffler's flagella mordant, Loeffler's flagella stain,
Specially clean glass slides, 1 ml distilled water blank, 95% alcohol, Inoculating loop, Wash bottle,
Bunsen burner, Microscope
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1. Take a clean grease free slide, pass it through the flames of Bunsen burner. Allow to cool.
2. Make suspension of the bacterium in distilled water.
3. Incubate the suspension at room temperature for 10-15 mins.
4. Place one loopful of the suspension toward on end of the slide that was heated on the Bunsen
burner.
5. Tilt the slide and allow the drop to spread to form a thin film on it.
6. Cover the slide with flagella mordant for 1 min.
7. Wash gently with distilled water.
8. Flood the slide with flagella stain for 2 to 1 min.
9. Wash the slide gently with distilled water.
10. Air dry the slide.
11. Observe the slide in oil immersion.
PROCEDURES
Loeffler's flagella mordant
20% aqueous tannic acid 100ml, Ferrous
sulphate 20g, 10% basic fuchsin in alcohol
10ml
Distilled water 40ml
Dissolve ferrous sulphate crystals in
distilled water by warming and add the
remaining ingredients.
Loeffler's flagella stain
1% basic fuchsin in alcohol 20ml
3% distilled water 80ml
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The cells appear pink, straight rods
surrounded by a deep stained rod,
outer coat that bears pink-stained
flagella. The flagella are
peritrichous (i.e. present all around
the cell), fine, wavy threads of
greater length than the cells.
Vibrio cholerae, flagella, stain
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CAPSULE STAINING
Capsule, when present is the outer most covering of the bacteria. The presence of capsule is considered
as the indication of the virulence and pathogenicity. It is antigenic in nature and made up to
polysaccharide. The capsule can be stained by Hiss copper surface method.
COMPOSITION of solutions
1. Saturated alcoholic soln. of Gentian violet 5 ml Distilled water and 95 ml. Mix the ingredient and filter.
2. Copper sulphate 20 ml and Distilled water 100 ml. Dissolve copper sulphate crystals in the distilled water by
gentle heating and filter.
REQUIREMENTS
Klebsiella culture, Blood
Serum , Staining
solutions (Gentian violet
or crystal violet)
Slides, Copper sulphate
solution, Inoculating
loop, Microscope, Spirit
lamp
PROCEDURE
Make smear in serum, air dry and fix it by gentle
heat.
Apply Gentian violet stain and heat gently until
steam rises.
Allow the dye to act for 15 - 20 sec.
Wash the dye with 20 % CuSO4 soln.
Do not wash with water, Blot dry and examine
under oil immersion. MICROBIAL STAINING

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Capsule appears as a blue halo around the dark purple cell body against a faint
purple back ground.
Capsulated bacterium
The capsule is a thick
polysaccharide layer around the
outside of the cell. It is nonionic,
so the dyes that we commonly
use will not bind to it. Two dyes,
one acidic and one basic, are
used to stain the background and
the cell wall
Note:-
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ENDOSPORE STAINING
Endospore staining is to differentiate bacterial spores from
other vegetative cells and to differentiate spore formers from
non-spore formers.
Primary stain-malachite green is forced into the spore by
steaming or heating the bacterial emulsion.
Malachite green is water soluble and has a low affinity for
cellular material, so vegetative cells may be decolourized with
water. In this technique heating acts as a mordant and water
is used as decolourizer.
Saffranine is then applied to counterstain any cells which
have been decolorized. At the end of the staining process,
vegetative cells will be pink, and endospores will be dark
green
Fig Bacillus spores
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REQUIREMENTS
Glass Slides, Specimen Bacterial culture at least 5 days old, Blotting paper, Inoculating loop, Spirit
lamp/Bunsen burner, Microscope, Distilled water, Primary Stain: Malachite green (0.5% (wt/vol),
Decolorizing: Tap water or Distilled Water, Counter Stain: Safranin.
Procedure
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Endospores are bright green and vegetative cells are brownish red to pink.
Gram-positive bacilli such as Bacillus
Bacillus
spores
What endospore is?
One example of an extreme survival
strategy employed by certain low G+C
Gram-positive bacteria is the formation
of endospores. This complex
developmental process is often initiated
in response to nutrient deprivation. It
allows the bacterium to produce a
dormant and highly resistant cell to
preserve the cell's genetic material in
times of extreme
Other method :-Dorner method
Carbol Fuchsin
primary stain
Acid-alcohol
decolouriser
Nigrosine
dye
Spores red Bacteria colorless Background Black
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Lactophenol Cotton Blue (LPCB) Staining
Lacto-phenol Cotton Blue staining method works on
the principle of aiding the identification of the fungal
cell walls.
➢ The fungal spore cell wall is made up of chitin of
which the components of the Lactophenol Cotton.
➢ The lactophenol cotton blue solution acts as a
mounting solution as well as a staining agent.
➢ The solution is clear and blue in color having three
main reagents:
• Phenol: It acts as a disinfectant by killing any
living organisms
• Lactic acid: To preserve the fungal structures
• Cotton blue: To stain or give color to the chitin on
the fungal cell wall and other fungal structures
The stain will give the fungi a blue-colored appearance
of the fungal spores and structures, such as hyphae. Image: Aspergillus
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Procedure
1. Clean the slide.
2. Add the fungal specimen to the drop of alcohol using a sterile mounter such as an
inoculation loop from solid medium.
3. Tease the fungal sample of the alcohol using a needle mounter, to ensure the
sample mixes well with the alcohol.
4. Using a dropper or pipette, add one or two drops of lactophenol cotton blue
solution (prepared above) before the ethanol dries off.
5. Carefully cover the stain with a clean sterile coverslip without making air bubbles
to the stain.
6. Examine the stain microscopically at 40x, to observe for fungal spores and other
fungal structures.
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Fungal spores, hyphae, and fruiting structures stain blue while the background stains pale blue.
Aspergillus niger stains the hyphae and fruiting structures a delicate blue with a pale blue background.
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Made and edited by Aniruddh MICROBIAL STAINING