STAINING TECHNIQUES USED IN MICROBIOLOGY

INTRODUCTION

Smear Preparation

Not only are most bacteria very small, they are also not very clear and difficult to view under
a microscope without first staining. You must firmly attach your bacteria to a glass slide
before you can stain them. There are two important things to consider when preparing a slide
for staining:

1. The bacteria must be evenly and lightly dispersed. If there are too many bacteria on
the slide they will form a big glob and you will not be able to see the morphology of
the individual cells. Large blobs of cells also do not stain properly and could yield
erroneous results from the improper staining.
2. The bacteria need to be firmly attached to the slide so they are not washed off during
the staining procedures. All procedures that attach the bacteria to the slide result in
some morphological changes. The cells typically shrink in size and will exhibit some
changes in shape and extra-cellular matrixes.

You can prepare slides for staining from both broth and agar surfaces. While the goals are the
same for both, evenly and lightly dispersed cells firmly adhered to the slide surface, the
techniques are slightly different. Staining is as much art as science. It will undoubtedly take
you several tries before you are successful.

Materials

 Clean glass slides

 Inoculating loops or needles
 Sterile normal saline
 Marking pen
 Assorted broth and plate cultures

Safety Considerations

Be careful of aerosols when transferring bacteria from your loop to the slide. The loop is very
flexible and it is easy to zing off a loop-full of organisms. Do not assume your organism is
dead. Heat or methanol fixation is not guaranteed to kill the organism. Dispose of your
completed slides in the disinfectant bucket at your bench.

General Considerations

You are striving for a light suspension of cells that will leave a faint cloudy deposit on your
slide. You have lots of room on your slide; use it! It helps to initially draw a circle on the
bottom of the slide so you know where to look for your smear. It is very easy to get confused
which side of the slide your smear is on. Be sure to label the far edge of the slide. Do this
consistently on the same end of the slide to help orient your slide.
Be patient and take the time to let your slide air dry before proceeding with adhering it to the
slide. If your slide is wet and you heat fix it, the bacteria will boil and the cellular
morphology will be lost. If your slide is wet and fix it in methanol, it will most likely wash
off the slide. Smears that are too thick will most likely wash off the slide regardless of the
fixation method.

Smear from Broth

Broth cultures are usually easier to work with because the cells are already diluted in the
broth. Be sure to carefully mix the culture tube to suspend the bacteria in the broth.

1. Label your slide. Aseptically transfer a loop-full of organism onto the centre of your
slide.
2. Use the flat part of the loop to smear the broth drop around the slide. Use a spiralling,
circular motion to spread out the drop. Because the broth is full of protein, the smear
will usually stay spread out and not bead up on the surface of the slide.
3. Set the slide aside to air dry. This will take several minutes at least. Do not rush this
step.

Smear from Plate

You can scoop a lot of organisms off with your loop. You may want to use an inoculating
needle to transfer your organism to the slide. Be sure to use sterile normal saline to dilute
your samples..

1. Label your slide and aseptically transfer a loop-full of sterile saline to the centre of the
slide.
o This serves to both dilute your bacteria and give you something to spread
around.
2. Pick a well-isolated colony with your sterile needle, or slightly scoop the edge of the
colony with your sterile loop.
3. Place your needle/loop in the centre of the drop and with a spiralling circular motion
spread the bacteria on the slide.
4. Set the slide aside to air dry. This will take several minutes at least. Do not rush this
step.
 Culture Smeared slide Smeared blood film

Staining methods are used to elevate the visibility and highlight specific
morphological structures of the microorganisms. It is also used to preserve them..
Before staining, the specimen is fixed by heat (by passing over a heat flam 3 times) or
by fixation using methanol.
Fixation is a method in which the complete structures of the cells or microorganisms are
fixed in the positions and are preserved. Usually, microorganisms are killed & attached
strongly to the slide during the fixation process.

1. Be sure your slide is totally dry. Set it on the staining rack over the sink.
2. Carefully flood the slide with 95% methanol. Let it sit for two minutes.
3. Tilt the slide and pour off the methanol. Touch the edge of the slide to a paper towel to wick
off the excess methanol.
4. Set the slide aside to air dry before staining.

DIFFEREN

T STAINING RACK

Safety precaution during staining

1. Ensure the use of a staining rack

2. Use clearly labelled reagents and stains

 3. Use dropper bottles to dispense stains alcohol and acetone

4. A particular staining and decolorizing technique must be followed to

ensure correct and reproducible staining reactions.

5. Wash bottles should be used when staining easily washable specimen like

CSF.

6. Place a stained slide on a draining rack for the smear to air dry

Types of Staining Techniques Used in Microbiology

 Gram staining.
 Acid-fast stain (Ziehl-Neelsen technique)
 Endospore stain.
 Capsule stain.
 Giemsa stain.
 Acridine orange Stain.
 Cytoplasmic inclusion stains.
 Field stain
 Leishman stain
 Rowmanowsky stain
 Wayson bipolar stain
 Albert staining of volutin granules
 Auramine-phenol stain
CLASSIFICATION OF DIFFERENT TYPES OF STAINING METHODS

Sample of Stained slides

SIMPLE STAINING
 Simple staining is used to stain prokaryotic cells for e.g., microorganisms, used
to determine the shape, size, and arrangements.
 In this staining method, only a single dye is needed.
 It’s simple with the procedure which includes covering fixed smears with stain for
a minute, and excess stain is washed off with water and blotted dry.
 In this case, basic dyes are used which are methylene blue, crystal violet, and
carbol fuchsin.
Examples of simple stain include Geimsa, Wright’s stain, leishman’s stain

GIEMSA STAIN

This is a romanowsky stain that is used mostly in parasitology to stain malaria and other
blood parasite

PRINCIPLE

Giemsa contains Methylene blue (AzureII) Eosin. Methylene blue on oxidation

produces colored compounds termed ‘Azure’ that have the ability to combine with
Eosin. Methylene blue azure is blue-violet and stains acidic cell components while
eosin is red and stains basic cell components.
Material

Giemsa stain

Buffered water PH 7.2

Method

- Fix the smear in methanol / methyl alcohol for 2-3 minutes and allow to air dry
- Dilute the stain in buffered water 1 in 20 i.e. 19ml of buffer and 1ml giemsa .
- Place the slide in a coupling jar to cover the smear and allow to stand for 30 minutes
- (At this stage dehaemoglobinization occur simultaneously as the slide is being stained
- Lift the slide off the staining solution with forceps and wash off the excess stain with
buffer.
- Drain and air dry and examine the slide.

THICK BLOOD SMEAR SHOWING PLASMODIUM MALARIAE SCHIZONT CONTAINING 10 MEROZOITE

DIFFERENTIAL STAINING
Gram staining

Christian Gram in 1894 described this method of staining which is the most important stain in
routine bacteriology. It serves as an aid in classifying bacteria into two categories positive
and negative depending on whether they can be decolorized with acetone, alcohol or aniline
oil after staining with crystal violet, methyl violet or gentian violet and treating with iodine.
Those that resist decolourization remain blue or violet in colour and are called Gram Positive
while those that are decolourized take up the red counter stain such as neutral red, safranin or
dilute Carbol fushin are called “Gram Negative”. The difference in gram reaction between
bacteria is thought to be due to difference in the permeability of the cell wall.

Examples of gram positive bacteria are staphylococcus, streptococcus clostridium,

corynebacterium. Examples of gram bacteria negative bacteria are Coliforms like E.coli
Salmonella, Klebsiella, Shiqella Vibro etc

Materials

Reagents :

Crystal violet/ Methyl red/ Gention violet

Lugols iodine

Absolute Ethyl alcohol or Acetone

Dilute Carbol fuchin / Safranin / Neutral red.

Procedure

1. Cover the already fixed slide with the primary stain crystal violet for 30 seconds wash
rapidly with clean water and drain off excess water.
2. Flood with iodine solution (mordant) for 1 minutes wash off with water
3. Decolorize rapidly with acetone and wash immediately with clean water. If ethanol is
used add drop wise on the tilted slide until all free purple colour has been removed
wash with water.
 4. Apply dilute Carbol fuchsin for 30 seconds (if neutral red or Saffranin is used allow
for 2-3minutes) wash with water and allow to air dry. (slide can also be blotted dry
over smooth filter or blotting paper and further dried with gentle heat) and view

Result

Gram Positive: Dark purple Gram Negative: Pale or dark red

The bacteria can take different morphological shapes such as cocci, streptococci Bacilli,
Bacilli in chains etc.

Gram positive and gram negative cocci Gram positive and gram negative rod

ZIEHL-NEELSEN (Acid Fast Stain)

Ziehl-Neelsen is a staining method used to demonstrate acid fast properly of bacteria

belonging to the genus mycobacterium which have the power of retaining stain even after
decolourization by mineral acids. This properly of acid fastness appears to be due to large
amount of lipid particularly a lipid fraction called mycolic acid and causes not only fastness
but a resistance to ordinary staining method. This is a diagnostic procedure employed for the
identification of tubercle bacilli mycobacterium tuberculosis is both Acid and Alcohol Fast
(ASFB) in sputum and other body fluids and the leprosy or Hansens bacilli mycobacterium
leprae which is Acid Fast (AFB) from skin slits and smears or affected body sites unlike most
other bacteria, do not stain well with gram technique. They can however be stained with
carbol fuchsin combined with phenol. The stain binds to the mycolic acid in the
mycobacterial cell wall. After staining an acid decolorizing solution is applied. This removes
the red dye from the background cells, tissue fibre and any organisms in the smear except
mycobacteria which retain the dye. Following decolorization, the smear is counter stained
with malachite green or methylene blue which stains the background material producing a
contrast colour against the red AFB can be seen.
Material

Strong Carbol funchsin stain,

3% acid alcohol,

Methylene blue/malachite green

1. Cover the smear with strong Carbol fuchsin stain and heat until steam rises about 60 0c
ensure the stain does not dry on the slide and do not over heat. Allow the stain to
remain on the slide for 5 minutes.
2. Wash off smear with 3% v/v acid alcohol for 5 minutes or until the smear is
sufficiently decolorized to pale pink
3. Wash thoroughly with clean water
4. Counter stain with 0.5% methylene blue or malachite green for 30 seconds. Wash in
water
5. Wipe the back of the slide clean and place it in a draining rack for the smear to air dry
(do not blot dry)
6. Examine the smear under the microscope.

Result

AFB Positive: Red straight or slightly curved rods, occurring singly or in small groups

SPECIAL STAINING
 This staining method is used to identifies or study special components of the
microorganisms.
Special Procedure Remarks
 Staining

 Used to reveal
the capsule
 Smear is prepared
present on the
and mixed with,
bacteria.
which Indian Ink or
 Dye particles are
Capsule Nigrosin, spread as a
not able to get
thin film on the slide.
Staining inside the
 Observation: Light
capsule
coloured bacterial
therefore they
cells are seen against
appear light in
the dark
colour against
background
the dark
background.

 Endospore is a
special resistant
structure, found
in the genera
of Bacillus &
 The Schaeffer- Clostridium.
Fulton  Helps bacteria to
procedure, first step: survive
Bacteria is heated unfavorable
with stain malachite conditions.
green, which gets  Endospores are
inside the endospore. present at
Endospore
 Excess stain is different
staining washed off and locations and
counterstained shapes, useful
with safranin. for their
 Observation: identification.
Endospore  Usually,
appears green, endospores
others are red to aren’t stained by
pink cells. the dye, after
receiving harsh
treatment, when
it is stained, then
it resists
decolorization.
  For taxonomical
 First, purposes, the
bacterial flagella are distribution of
coated with tannic flagella present
acid ( is a mordant) on prokaryotes
Flagella
& potassium alum, is considered
staining then stained valuable
by pararosaniline ( i information.
n Leifson method),  Flagella are
or basic fuchsin ( in thread-like
Gray Method). structures, used
for locomotion.

Endospore staining

Endospore Staining- Principle, Reagents, Procedure and Result

In 1922, Dorner published a method for staining endospores. Shaeffer and Fulton modified
Dorner’s method in 1933 to make the process faster The endospore stain is a differential stain
which selectively stains bacterial endospores. The main purpose of endospore staining is to
differentiate bacterial spores from other vegetative cells and to differentiate spore formers
from non-spore formers.
Principle of Endospore Staining

Bacterial endospores are metabolically inactive, highly resistant structures produced by

some bacteria as a defensive strategy against unfavorable environmental conditions. The
bacteria can remain in this suspended state until conditions become favorable and they can
germinate and return to their vegetative state.
In the Schaeffer-Fulton`s method, a primary stain-malachite green is forced into the spore
by steaming the bacterial emulsion. Malachite green is water soluble and has a low affinity
for cellular material, so vegetative cells may be decolorized with water. Safranin 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.
Spores may be located in the middle of the cell, at the end of the cell,
or between the end and middle of the cell. Spore shape may also be of diagnostic use.
Spores may be spherical or elliptical.
Reagents used for Endospore Staining

Primary Stain: Malachite green (0.5% (wt/vol) aqueous solution)

0.5 gm of malachite green
100 ml of distilled water
Decolorizing agent
Tap water or Distilled Water
Counter Stain: Safranin
Stock solution (2.5% (wt/vol) alcoholic solution)
2.5 gm of Safranin O
100 ml of 95% Ethanol
Procedure of Endospore Staining

1. Take a clean grease free slide and make smear using sterile technique.
2. Air dry and heat fix the organism on a glass slide and cover with a square of blotting
paper or towelling cut to fit the slide.
3. Saturate the blotting paper with Malachite green stain solution and steam for 5
minutes, keeping the paper moist and adding more dye as required. Alternatively, the
slide may be steamed over a container of boiling water.
4. Wash the slide in tap water.
5. Counterstain with 0.5% Safranin for 30 seconds. Wash with tap water; blot dry.
6. Examine the slide under microscope for the presence of endospores. Endospores are
bright green and vegetative cells are brownish red to pink.
Result of Endospore Staining

Endospores: Endospores are bright green.

Vegetative Cells: Vegetative cells are brownish red to pink.
Spores may be located in the middle of the cell, at the end of the cell, or between the end and

middle of the cell. Spore shape may also be of diagnostic use. Spores may be spherical or

elliptical.
Endospore Staining by Dorner’s Method

Carbolfuchsin stain
0.3 gm of basic fuchsin
10 ml of ethanol, 95% (vol/vol)
5 ml of phenol, heat-melted crystals
95 ml of distilled water
Dissolve the basic fuchsin in the ethanol; then add the phenol dissolved in the water.
Mix and let stand for several days. Filter before use.
Decolorizing solvent (acid-alcohol)
97 ml of ethanol, 95% (vol/vol)
3 ml of hydrochloric acid (concentrated)

Counterstain (Nigrosin solution)

10 gm of nigrosin
100 ml of distilled water

Procedure
1. Take a clean grease free slide and make smear using sterile technique.
2. Air dry and heat fix the organism on a glass slide and cover with a square of blotting
paper or towelling cut to fit the slide.
3. Saturate the blotting paper with Carbol fuchsin and steam for 5 to 10 minutes,
keeping the paper moist and adding more dye as required. Alternatively, the slides
may be steamed over a container of boiling water.
4. Remove the blotting paper and decolorize the film with acid-alcohol for 1 minute;
rinse with tap water and blot dry.
5. Further take a drop of nigrosine on one end of a slide and make a thin film of a stain
all over the smear with the help of other slide.
6. Allow the film of Nigrosin to air dry.
7. After air drying observe the slide under oil immersion.
Vegetative cells are Colourless, Endospores are red, and the background is black.
Examples of Endospore Staining
Capsule stain
Various types of methods are available for the demonstration of bacterial capsules. The
results (stain of the cells, background, and capsule) depend on the method used. In capsule
staining procedure “we do not heat fix and rinse the smear with water” as heat and water
may dislodge capsules from bacteria

Principle of Capsule Stain

Bacterial capsules are non-ionic, so neither acidic nor basic stains will adhere to their
surfaces. Therefore, the best way to visualize them is to stain the background using an
acidic stain (e.g., Nigrosine, congo red) and to stain the cell itself using a basic
stain (e.g.,Crystal violet, Safranin, Basic fuchsin, and Methylene blue).

Two commonly used methods are

A. India ink method

In this method, two dyes, crystal violet, and India ink are used. The capsule is seen as a clear
halo around the microorganism against the black background. This method is used for
demonstrating Cryptococcus.

 The background will be dark (colour of India ink).

 The bacterial cells will be stained purple (bacterial cells take crystal violet-basic dyes as
they are negatively charged).
 The capsule (if present) will appear clear against the dark background (capsule does not
take any stain).

B. Anthony’s stain method

In this type of capsule staining procedure, the primary stain is crystal violet, and all parts of
the cell take up the purple crystal violet stain. There is no mordant in the capsule staining
procedure. A 20% Copper sulphate solution serves a dual role as both the decolorizing
agent and counterstain. It decolorizes the capsule by washing out the crystal violet, but will
not decolorize the cell. As the copper sulphate decolorizes the capsule, it also counterstains
the capsule. Thus, the capsule appears as a faint blue halo around a purple cell.

Materials and Reagents

 Test bacteria: 36-48 hour culture of capsulated bacteria e.g. Klebsiella

pneumoniae growing on a slant of EMB agar or culture of other capsulated bacteria and
non-capsulated bacteria [Note: Growing Klebsiella pneumoniae in milk-based media (e.g.
Skim milk) increase its capsule size, making it easier to visualize.]
 Stain solutions: Depending on the method used (crystal violet, India ink, Nigrosin, copper
sulphate, Basic Carbol fuschin solution, methylene blue solution, etc).
 Microscopic slides
 Inoculating loop
 Light Microscope with 100x objective lens (oil immersion)
 Immersion oil
 Gas burner
 Tissue paper

Capsule Stain procedure

A. India Ink Method

1. Place a single drop of India ink on a clean microscope slide, adjacent to the frosted edge.
2. Using a flamed loop and sterile technique, remove some Klebsiella pneumoniae from
culture tube or plate and mix it into the drop of India ink. Be sure there are no large
clumps of organism, but try to avoid spreading the drop.
3. Place the end of another clean microscope slide at an angle to the end of the slide
containing the organism. Spread out the drop out into a film. This is done by contacting
the drop of India ink with the clean microscope slide and using the capillary action of the
dye/ slide to spread the India ink across the smear.
4. Allow the film to air dry (will take 5-7 minutes). DO NOT heat or blot dry! Heat will
melt the capsule!
5. Saturate the slide with Crystal violet for 1 minute and rinse slightly & very gently with
water. Be cautious water may remove the capsule from the cell.
6. Let the slide air dry for a few minutes. DO NOT blot the slide! Blotting will remove the
bacteria from the slide and/or distort the capsule.
7. Observe the slide under oil immersion.
Results: Look for purple cells surrounded by a clear halo on a dark background. The halo is
the capsule. You may need to decrease the amount of light in order to make the capsule easier
to see.

B. Anthony’s stain method

1. Place a single drop of crystal violet on a clean microscope slide, adjacent to the frosted
edge.
2. Using a flamed loop and sterile technique, add three loopful of test bacterium (any
capsulated bacteria such as Klebsiella pneumoniae, Streptococcus pneumoniae) from
broth culture. If you are adding bacteria from a culture plate make sure that there are no
large clumps of the organism, but try to avoid spreading the drop.
3. Place the end of another clean microscope slide at an angle to the end of the slide
containing the organism. Spread out the drop out into a film. This is done by contacting
the drop of crystal violet with the clean microscope slide and using the capillary action of
the dye/ slide to spread the crystal violet across the smear.
4. Allow the film to air dry (will take 5-7 minutes). DO NOT heat or blot dry! Heat will
melt the capsule!
5. Tilt the slide and rinse with 20% copper sulphate solution. DO NOT RINSE WITH
WATER! Water will remove the capsule from the cell.
6. Let the slide air dry for a few minutes. DO NOT blot the slide! Blotting will remove the
bacteria from the slide and/or distort the capsule.
7. Observe the slide under oil immersion.
Results: Look for purple cells surrounded by a clear or faint blue halo on transparent
background. The halo is the capsule. You may need to decrease the amount of light in order
to make the capsule easier to see.
FLAGELLA STAINING

Flagella are filamentous cytoplasmic structure which are projecting from the cell wall. These
are 12-30 nm in diameter and 5-16 µm in length, unbranched, thread-like structure, and made
of the protein flagellin.

Based on distribution they are classified into different classes such as Monotrichous (Single
polar flagellum), Amphitrichous (Single flagellum on both sides), Lophotrichous (Tufts of
flagella at one or both sides), and Peritrichous (Numerous flagella all over the bacterial
body).

Flagella is consists of different parts such as Filament, Hook and Basal Body. Flagella
involve in different activities such as Movements, Sensation, Signal transduction, Adhesion,
etc.

The staining of bacterial flagella is a very complex process that requires extraordinary care
for the slides, stain, and cells. For flagella staining, we use wet mount technique, for this
technique a stable stain and regular slides and coverslips are require. This method helps to
find the number and arrangement of flagella which is essential for the identification of
bacterial cells.

Principle

Flagella can not be seen under a bright-field microscope by using ordinary stains. A simple
and useful method has been used for visualization of flagella is known as wet mount
technique. This method is useful when the number and arrangement of flagella are critical to
the identification of species of motile bacteria. In this method, a mordant is used to adhere the
stain in flagella layer

1. Grow the organism to be stained at room temperature on blood agar for 16 to 24 hours.
2. Add a small drop of water to a microscope slide.
3. Dip a sterile inoculating loop into sterile water.
4. Touch the loopful of water to the colony margin briefly (this allows motile cells to swim
into the droplet of water).
5. Touch the loopful of motile cells to the drop of water on the slide. Note: Agitating the
loop in the droplet of water on the slide causes the flagella to shear off the cell.
6. Cover the faintly turbid drop of water on the slide with a coverslip. A proper wet mount
has barely enough liquid to fill the space under a coverslip. Small air spaces around the
edge are preferable.
7. Examine the slide immediately under 40× to 50× for motile cells. If motile cells are not
seen, do not proceed with the stain.
8. If motile cells are seen, leave the slide at room temperature for 5 to 10 minutes. This
allows the bacterial cells time to adhere either to the glass slide or to the coverslip.
9. Apply 2 drops of RYU flagella stain gently to the edge of the cover slip. The stain will
flow by capillary action and mix with the cell suspension. Small air pockets around the
edge of the wet mount are useful in aiding the capillary action. (Note: The RYU stain has
two components. Solution I, the mordant, contains 10 ml of 5% aqueous solution of
phenol, 2 g of tannic acid, and 10 ml of saturated aqueous solution of aluminum
potassium sulphate-12 hydrate. Solution II, the stain, is a saturated ethanolic solution of
crystal violet (12 g in 100 ml of 95% ethanol). The final stain was prepared by mixing 1
part solution Il with 10 parts solution I and then filtering the mixture through filter paper
to remove coarse precipitate)
10. After 5 to 10 minutes at room temperature, examine the cells for flagella.
11. Cells with flagella may be observed at 100× (oil) in the zone of optimum stain
concentration, about halfway from the edge of the coverslip to the centre of the mount.
12. Focusing the microscope on the cells attached to the coverslip rather than on the cells
attached to the slide facilitates visualization of the flagella. The precipitate from the stain
is primarily on the slide rather than the coverslip.
Result: Observe the slide under a microscope and write down the following characteristics;

 Presence or absence of flagella

 Number of flagella per cell
 Location of flagella per cell
 Peritrichous
 Lophotrichous
 Polar
Points to remember after viewing stained slide

 Clean your microscope with lens cleaner, removing all oil from lenses.
 Dispose of staining waste and slides in designated waste containers.
 Be cautious while handling the slide, since the organisms have not been killed.