Microbial Staining

SYED MUHAMMAD KHAN (BS HONS. ZOOLOGY)
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Microbial Staining
Staining refers to the use of colored dyes/stains to make cells and/or cellular structures
visible or to produce contrast between different types of cells / cellular components,
i.e. it enhances the contrast of a microscopic image.
Staining allows visualization of cells and cellular components, it also allows
differentiation of different cell types or different cellular organelles. It is important in the
detection of microbes in a medium, i.e. blood. It is also very vital in the medical
diagnosis of infections. Staining techniques may either involve a single stain, intended
to point out cells / cellular components (simple staining) or multiple stains to
differentiate between different cells / cellular components (differential staining).
Stains
Stains are colored organic compounds (salts) used for staining cells, tissues,
microorganisms, etc. Stains contain ions which impart them color, these ions are
called chromophore. If the chromophore is a positive ion, then the dye/stain is called
a basic stain and if it is negative, then the dye/stain is known as acidic stain. According
to pH, stains can be classified into:
Acidic Stains: These stains have a negative charge, hence they bind to positively
charged cellular structures like some proteins. Acidic dyes are not very often used in
a microbiology lab, except to provide background staining like in negative staining.
Examples include nigrosine, picric acid, eosin, carbol fuchsin, etc.
Basic Stains: These stains have a positive charge, hence they bind to negatively
charged molecules such as nucleic acids and acids in bacterial cell walls (teichoic
acids in gram-positive cells and phospholipids in gram-negative cells). Since the
surface of bacterial cells are negatively charged, basic dyes are most commonly used
in bacteriology. Examples include: crystal violet, methylene blue, safranine, basic
fuchsin, etc.
Neutral Stains: These stains are usually formed from precipitation, when aqueous
acidic and basic stains are combined. Neutral dyes stain nucleic acids and cytoplasm.
Examples include: eosinate of methylene blue, Giemsa stain, etc.
Staining Techniques in Microbiology
Microorganisms are small, transparent, and motile in fluids hence it is very difficult to
observe under a microscope unless these are immobilized (fixed) and stained with a
suitable stain. Some of the staining techniques used in microbiology are as follows:
 Simple staining
 Acid-fast staining
 Negative staining
 Endospore staining
 Gram staining
 Flagellar staining

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Some Steps Associated With Staining
1. Preparation of Smear
Smear is a sample of cells or tissue or other material taken from a biological context,
spread thinly on a microscope slide for examination.
It is prepared as follows:
 Place a small drop of the sample (i.e., blood, microbial culture, etc.) one side of a
clean slide, about 1-2 cm from one end.
 Place another slide (spreader) at an angle of 45° from the slide and move it back
to make contact with the drop. The drop should spread out quickly along the line
of contact of the spreader with the slide.
 Spread the film by rapid smooth forward movement of the spreader. The film
should be 3-4 cm in length.
Figure: Preparation of smear.
2. Fixation of Smear
Fixation is a process that kills the bacteria, firmly attaches the smear to the microscope
slide, and allows the sample to more readily take up the stain.
Before fixation, the smear is air-dried so that it fixes to the glass to avoid it being
washed when treated with liquid stain. Fixation denatures the bacterial enzymes and
prevents autolysis and ensures bacterial adherence to a microscopic slide. Soon after
fixing, the slide can be stained. Fixation can be done in one of two ways:
 Heat Fixation: The smear is passed through the top part of the Bunsen burner
flame 2-4 times taking care that the glass slide is quite hot but bearable to one’s
hand skin.

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 Chemical Fixation: The smear can be chemically fixed by covering the smear with
95% methanol for 1-5 minutes.
Materials Used In Staining
Some of the materials used in microbial staining techniques are as follows:
Stains & Reagents Staining Rack
Glass Slides & Cover Slips Inoculation Loop Dropper
Wash Bottle Bunsen Burner Compound Microscope
Simple Staining
Simple staining is a staining procedure that uses only one stain. A simple stain is
usually an aqueous or alcoholic solution of dye applied for 1-2 minutes to the smear
and then washed off. The most common simple stains are Loeffler's alkaline
methylene blue, methylene blue, safranine, carbol fuchsin, and gentian violet.

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Most bacteria stain easily and quickly with simple stains while the capsules and spores
remain unstained. Simple stain that stains bacteria is a direct stain and which does not
stain bacteria but stains background is called a negative stain. Simple staining is easy,
cost-effective, and very useful in studying the morphology, size, and arrangement of
microorganisms.
Requirements
 Methylene blue
 Coverslips
 Wash bottle
 Staining rack
 Inoculation loop
 Bunsen burner
 Glass slides
 Dropper
 Microscope
Bacterial cultures: Staphylococcus epidermidis (slant), Bacillus megaterium
(broth), Escherichia coli (broth), etc.
Procedure
Clean a microscopic glass slide, rinse it with water, and air dry (hold the slide from its
edges). Place one loopful (inoculation loop) or sterile distilled water in the center of
the slide. The loop must be sterilized before and after use by making it red hot in
Bunsen burner flame, and it should be allowed to cool.
Transfer a small amount of culture from solid medium/broth to the distilled water drop
and spread it evenly as a thin smear. Allow the smear to air dry at room temperature.
Fix the smear by passing the slide through the burner flame 2-4 times or covering it
with methanol for 5 minutes, air-dry before staining.
Place the slide on the staining rack and cover the smears with any stain (i.e.,
methylene blue) and let it stain for 30-60 seconds. Then drain off the stain and wash
the slides under running tap water or water from a wash bottle. Gently blot dry the
smear with absorbent/filter paper and air dry. Apply coverslip, examine the slide under
oil immersion objective, and record observations.
Figure: Steps involved in simple staining.

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Negative Staining
Negative staining is done to elucidate colorless capsules against a colored
background. A negative stain does not stain bacteria but imparts color to the
background. The bacteria and capsule appear colorless against colored background.
If the culture is treated with crystal violet and negative stain mixture, the capsule
appears unstained sandwiched between colored bacterial cells and background. In
negative staining, smears are not heat-fixed; hence, bacterial cells don’t distort.
Capsule is a gelatinous and slimy extracellular material formed by bacteria, which
remains adhered to and covers the cell as a layer. This is called a capsule when it is
thick and regular, round or oval; and slime layer when it is irregular and loosely bound
to bacterium. The ability to form capsules is inherent but the thickness depends on
cultural conditions.
The majority of the capsules are water-soluble, uncharged polysaccharides hence
they do not imbibe simple stain. Some capsules are protein in nature as in Bacillus
anthracis. Capsular organisms usually make the broth viscous and stringy and the
colonies produced on solid media are generally moist, glistening, mucoid, and sticky.
Capsules are antiphagocytic and play an important role in virulence. Some capsule
producing organisms are Streptococcus pneumoniae, Klebsiella pneumonia,
Haemophillus influenzae, etc. Capsule bearing strains produce smooth colonies.
Rough strains of Streptococcus pneumoniae (lack capsule) are avirulent.
Capsule producing organisms are also troublesome for sugar and paper industries
resulting in clogging of pipes in sugar industries and pores in the paper. Serum treated
capsules visualized using negative staining appear bigger, this is called Quellung
reaction (Quellung reaction / Neufeld reaction is a biochemical reaction in which
antibodies bind to a bacterial capsule. Quellung is the German word for swelling, i.e.
the capsules become opaque and appear to be enlarged after their polysaccharide
antigens have combined with specific antibodies, from the serum of an animal).
Requirements
 Nigrosine (negative stain)
 Coverslips
 Wash bottle
 Staining rack
 Crystal violet (direct stain)
 Bunsen burner
 Glass slides
 Dropper
 Microscope
Bacterial cultures: Klebsiella pneumoniae, Bacillus subtilis, Staphylococcus albus,
etc.
Procedure
Place a small drop of nigrosine at the end of a clean grease-free slide. Transfer a small
amount of the bacterial culture to the drop and mix, in the case of capsular bacteria,
add crystal violet as well.

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Spread the drop to produce a smear of desired thickness (opaque black to gray). The
angle of the spreading slide determines the thickness of the smear. Let the smear air-
dry (do not heat fix). Apply coverslip, examine the slide under oil immersion objective,
and record observations.
Figure: Procedure of negative staining.
Gram Staining
Gram staining is a differential staining technique as it differentiates bacteria into two
groups: Gram-positive and Gram-negative. Hans Christian Gram introduced this
technique in 1884. Gram staining is highly useful for the identification of
microorganisms, because bacteria stain differently because of chemical and physical
differences in their cell walls:
In the case of the Gram-positive bacteria, the crystal violet-iodine complex (CVI)
formed because of crystal violet and iodine penetrating the bacterial cell does not
leach out on treatment with alcohol because of the thick peptidoglycan layer. In the
case of the Gram-negative bacteria, the alcohol dissolves the outer lipopolysaccharide
layer; consequently, the CVI complex leaches out through the thin layer of
peptidoglycan.
Gram stain is more consistent with young cultures (8-16 hours old). Old cultures may
show variations in Gram staining. It is the first step in the identification of
microorganisms. The technique makes use of:

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 Primary Stain (Crystal Violet): It stains all bacteria purple.
 Mordant (Iodine): It combines with crystal violet to form a CVI complex (mordant
is a substance that combines with a dye or stain and thereby fixes it in a material).
 Decolorizing Agent (95% Alcohol / Acetone): It washes out the CVI complex
from some bacteria (Gram-negative bacteria).
 Counter Stain / Secondary Stain (Safranine): It stains the decolorized bacteria.
Requirements
 Crystal violet (Primary)
 Safranine (Secondary)
 Coverslips
 Wash bottle
 Dropper
 Gram's iodine
(Mordant)
 Staining rack
 Bunsen burner
 95% Ethanol / Acetone
(Clearing agent)
 Glass slides
 Microscope
Bacterial culture: Staphylococcus aureus, Escherichia coli, and Bacillus
megaterium.
Procedure
Prepare and heat fix a smear on a clean glass slide. Place the slide on a staining rack
and cover the smear with crystal violet and leave it for one minute. Wash the slide with
tap water / via wash bottle, gently. Cover the smear with Gram's iodine for one minute.
Wash off the iodine by tilting the slide and pouring water over the smear.
Decolorize with ethyl alcohol or acetone for about 10-15 seconds, or until no more
color comes off, do not over or under decolorize. Immediately wash with water. Cover
the smear with safranine for 30 seconds. Wash with water, blot dry, apply coverslip,
examine the slide under oil immersion objective, and record observations.
Figure: Procedure of gram staining.

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Precautions
The factors that may affect the reliability of Gram staining are:
 Culture age: The culture must not be older than 24 hours, to avoid false results.
 Decolorization: Over decolorizing is one of the most common errors.
Heat fixation: If the smear is overheated it may char the organisms or create
artifacts, which will absorb and retain crystal violet that may be mistaken as Gram-
positive organisms.
Acid Fast Staining (Ziehl – Neelsen Carbol Fuchsin Method)
Acid-fast staining is a differential staining technique used to differentiate between acid-
fast bacteria such as Mycobacterium spp. (which possess mycolic acid in their cell
walls) and non-acid fast bacteria. In 1882, Paul Ehrlich discovered that in contrast to
most bacteria, Mycobacterium tuberculosis did not stain readily with primary stain but
once stained, did not lose the stain even after washing with an acid alcohol mixture,
hence, they are called "acid-fast bacteria".
The technique is diagnostically important in the identification of acid-fast
Mycobacterium species and Nocardia species. Acid-fast organisms contain mycolic
acid that renders the cell wall impermeable to most stains and detergents. Therefore
these organisms remain alive in clinical specimens treated with 4% NaOH. This
feature is exploited in the inactivation of "non-acid fast organisms” in clinical samples
for culturing acid-fast bacilli.
Currently, the Ziehl-Neelsen procedure is one of the most widely used techniques of
acid-fast fasting, although there are many other derived techniques. In the Ziehl-
Neelsen procedure, the smear is flooded with hot carbol fuchsin, or the stain is heated
on the slide from underneath to facilitate stain penetration into bacteria. Stained
smears are washed with an acid alcohol mixture that decolorizes non-acid fast
bacteria. Methylene blue is used as a counterstain for staining non-acid fast
organisms. Carbol fuchsin has more affinity for lipids than acid alcohol hence remains
bound to the cell wall of acid-fast bacteria when washed with acid alcohol.
Requirements
 Ziehl – Neelsen carbol
fuchsin stain (Primary)
 Coverslips
 Wash bottle
 Staining rack
 Bunsen burner
 Acid alcohol OR 20 %
H2SO4 solution
(Clearing agent)
 Glass slides
 Methylene blue OR
Malachite green
(Secondary)
 Dropper
 Microscope
Bacterial culture: Mycobacterium phlei and Escherichia coli.

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Procedure
Prepare and heat fix the smear of the culture on a clean grease-free glass slide. Cover
the smears with boiled carbol fuchsin and leave it for 5-10 minutes. Alternatively, put
the slide on a beaker containing boiling water and stain for 10 minutes.
Gently wash with water and then with decolorizer (acid alcohol) for one minute, or until
no more color comes out. Wash the slide with water. Counterstain for one minute, with
methylene blue or malachite green. Wash the slide with water, blot dry, apply
coverslip, examine the slide under oil immersion objective, and record observations.
Figure: Procedure of acid-fast staining.
Endospore Staining (Schaeffer – Fulton Stain)
Endospore staining is a differential staining technique that differentiates between
endospores and vegetative cells. Out of the ten genera of bacteria that form
endospores, two genera: Bacillus and Clostridium are the most common. The spores
of bacteria do not stain as easily as vegetative cells.
With ordinary stains, spores remain unstained or slightly tinged with stain. Endospores
are metabolically inactive and are resistant to heat, chemicals, and harsh
environmental conditions. Spores contain dipicolinic acid which complexes with
calcium ions and thus imparts heat resistance to the spores. The cell wall disintegrates
soon after endospore formation.
The Schaeffer-Fulton stain procedure of endospore staining was designed by Alice B.
Schaeffer and MacDonald Fulton, two microbiologists at Middlebury College, during
the 1930s. The procedure also goes by the name Wirtz-Conklin method, referring to
two bacteriologists during the 1900s. Spores after staining, resist decolorization. The
information regarding spores (shape, diameter, and position) is very useful for
taxonomy.

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Requirements
 Malachite green (Primary)
 Coverslips
 Wash bottle
 Staining rack
 Safranine (Secondary)
 Bunsen burner
 Dropper
 Distilled water
(Clearing agent)
 Glass slides
 Microscope
Bacterial culture: Bacillus megaterium and Bacillus subtilis (I6-72 hours old).
Procedure
Make a smear from the culture on a clean glass slide, then air dry and heat fix it. Boil
malachite green in a test tube and pour it on the smear for 5-10 minutes. Alternatively,
put the slide on a beaker containing boiling water and stain for 10 minutes with
malachite green.
Wash with distilled water and counterstain with safranine for 30 seconds. Wash with
water, air dry, apply coverslip, observe under oil immersion objective, and record
observations.
Figure: Procedure for endospore staining.
Flagellar Staining
Flagellar staining highlights the presence, number, and position of flagella or their
absence in a cell. Flagella are a fine thread-like appendages arising from the
cytoplasm of motile bacteria. Most motile bacteria possess flagella but other forms of
motility are also seen in bacteria. Myxobacteria spp. exhibit gliding motion and
spirochetes exhibit screw-like motion using axial filament. Flagella are protein in nature

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and project out of the cell wall. They are very fragile and break on mere shaking,
heating, and treating with acid or detergent.
Flagella are not visible with a light microscope being very thin, much below the
resolving power of a bright field microscope. Hence, special staining methods are
employed to increase the thickness of the flagella by depositing coats of mordant that
increase their diameter. The presence and location of flagella are also helpful in the
identification and classification of bacteria, based on flagellation the bacteria have
been grouped as:
 Monotrichous Bacteria: These bacteria have a single flagellum at one end of
the cell. Examples include: Vibrio cholera, Pseudomonas aerogenosa, etc.
 Lophotrichous Bacteria: These bacteria have a bundle of flagella at one end of
the cell. Example: Pseudomonas fluroscens.
 Amphitrichous Bacteria: These bacteria either have a single flagellum or a
cluster of flagella at both ends of the cell. Example: Aquaspirillium spp.
 Peritrichous Bacteria: These bacteria have flagella all over the cell surface.
Examples include Escherichia coli, Salmonella spp., Klebsiella spp., etc.
 Atrichous Bacteria: These bacteria don’t possess any flagella. Example:
Shigella spp.
Requirements
 Ziehl – Neelsen carbol
fuchsin stain
 Coverslips
 Wash bottle
 Staining rack
 10% Tannic acid in 5%
NaCl solution
(Mordant)
 Bacterial culture
 Bunsen burner
 Glass slides
 Dropper
 Microscope
Bacterial culture: Proteus vulgaris (8-16 hours old), Pseudomonas aeruginosa (8
hours old), and Escherichia coli (8-16 hours old).
Procedure
Take a clean grease-free slide and pass it through Bunsen burner blue flame. Add 2-
3 ml sterile saline solution to the slant (bacterial culture in a test tube) and keep it for
an hour. With a sterile pipette or sterile loop transfer a drop of culture on one end of
the slide, tilt the slide, and allow the drop to trickle down slowly.
Air-dry the film, but do not heat fix. Cover the smear with mordant (10% tannic acid in
5% NaCl solution) for 10-30 minutes. Then rinse it gently with water. Add stain (Ziehl
– Neelsen carbol fuchsin stain) over the smear and let it remain for 5-15 minutes.
Rinse off the stain with water, air dry, apply coverslip, observe under microscope using
oil immersion objective, and record observations.

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Stained Microbial Slides
The following are the micrographs of stained microbial slides:
Simple Staining via Methylene Blue
Stain: blue-stained bacterial cells.
Negative staining: unstained capsules
sandwiched between stained cell bodies
and background.
Gram staining: gram-positive bacteria
stained violet/purple; gram-negative
bacteria stained as reddish-pink.
Acid Fast staining: Mycobacterium
spp. cells stained reddish pink in
contrast to blue-stained human cells
and other bacteria.
Endospore staining: Endospores
appear green colored, whereas the
bacterial cells are stained reddish pink.
Flagellar staining: Flagella of
Escherichia coli were thickened by
mordant and stained with the dye.

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