MCB 202: PRINCIPLES OF STAINING

Lecture Notes
By
Dr. Yakubu, A. Ajang
Department of Microbiology,
Faculty of Natural Sciences
University of Jos.

PREPARATION AND STAINING OF SPECIMENS

The importance of staining is to increase visibility, accentuate specific

morphological features, and preserve them for future study.

Therefore, in preparation of a specimen for examination, a smear is made and

fixed before staining.

FIXATION

Is the process by which the internal and external structures of cells and
microorganisms are preserved and fixed in position.

The principle of fixation is to inactivate enzymes that might disrupt cell

morphology and toughen cell structures so that they do not change during
staining and observation. A microorganism usually is killed and attached firmly
to the microscope slide during fixation.

TYPES OF FIXATIONS

1. Heat-fixing: - This is done by gently flaming and air-dried film or smearing

3 times through the flame of a Bunsen burner or spirit lamp the smear is
allowed to cool before staining, this preserves morphology but not
structures within cells e.g microwaving cryopreservation (freeze-drying).
2. Chemical-Fixing: Protects fine cellular structures, it penetrates cells and
reacts with cellular components, usually protein, and lipids to render them
insoluble, immobile, and active e.g. formaldehyde, ethanol, acetic acid. The
procedure involves adding one to 2 drops of the chemical fixatives in an air-

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 dried smear and allowed to stand for a minimum of 2 minutes or until the
chemical evaporates).

STAINING

Staining is an auxiliary technique used in microscopy to enhance contrast of

microscopic structures. It is easier to observe microbes by placing them on a
glass slide in a drop of water with a cover slip. This is termed wet preparation or
wet mount. This method is of advantage in that, microbes are viewed in as
natural a state as possible without any chemical treatment and some behaviors
of the organisms such as motility can be observed. However, owing that most
living cells are transparent and therefore show little contrast with the external
medium is a set-back to this method, hence, making it hard to differentiate cells
from their surroundings.

Staining enhances cells contrast under a microscope; however, the procedures

usually kill the cells. Stains and dyes are frequently use in biology and medicine
to highlight structures in biological tissues for viewing often with the aid of a
microscope.
Stains are chemicals containing chromophores, that is, groups that impact
colour, and their specificity is determine by their chemical structures. Stains are
generally salts in which one of the ions is usually colored. For instance,
methylene blue is actually methylene chloride which dissociate in water into a
positively charge methylene blue which is blue in colour and a negatively charge
chloride ion which is colourless.

Stains can be divided into three (3) groups;

1. Basic Dyes
Basic dyes are stains that are cationic and will therefore react with materials
that are negatively charged (e.g Bacterial walls). Some basic dyes are; Crystal
violet, Safranin, Basic Fuchsin, Methylene blue, Malachite green.

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 2. Acid Dyes

Acid dyes have negatively charged chromophores and are thereby repelled by
negatively charge bacterial walls, making them negative stains. Negative stains
are usually absorbed by the background but not the cells and produce a
silhouette of the organism against a colourful background. Common acid dyes
include; Acid Fuchsin, Eosin, Rose Bengal, Nigrosine, Congo red.

3. Neutral Dyes

These dyes result from combining basic and acidic dyes. Since basic dyes stain
the nuclei of cells whereas acid dyes stain the cytoplasm, neutral dyes would
therefore stain both the nuclei and cytoplasm of cells. Examples of neutral dyes
include eosinate of methylene blue, Giemsa stain.

Microbial Staining Techniques

A. Simple Stain
B. Differential Stain
i. Gram staining
ii. Acid fast staining
C. Special Stain
i. Capsule staining
ii. Spore staining
iii. Flagella staining
iv. Metachromatic Granules staining

A. Simple Stain

Simple staining technique is a general staining technique that employs one of

the basic dyes to stain the cells. Acidic dyes are sometimes used to stain
backgrounds, against which colorless cells can be seen, a technique called
negative staining. Simple stains are useful in revealing the morphology and
arrangements of bacterial cells.

Simple staining Procedures

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 ✓ A liquid containing the organism is drop on a glass slide and allow to air-
dry
✓ The resulting specimen forms a film or smear.
✓ The specimens are then heat fixed by passing over a flame
✓ The specimens are flooded with basic dyes and allowed for 1min
✓ Excess dyes are removed by rinsing with water and slide blotted
✓ Specimens are allowed to air-dry before viewing.

B. Differential Stains

Differential staining techniques are used to distinguish one group of bacteria

from another. The two most frequently used differential staining techniques are
the Gram stain and the acid-fast stain.

1. Gram Stain

The Gram stain is a differential stain commonly used in the microbiology

laboratory and the most important Staining technique in bacteriology. The
technique differentiates bacteria on the basis of their cell wall structure. Most
bacteria can be divided into Gram-positive and Gram-negative.
Gram-positive cell walls have a thick peptidoglycan layer beyond the plasma
membrane. Characteristic polymers called teichoic and lipoteichoic acids stick
out above the peptidoglycan and it is because of their negative charge that the
cell wall is overall negative. These acids are also very important in the body’s
ability to recognize foreign bacteria. Gram-positive cell walls stain blue/purple
with the Gram stain.
Gram-negative cell walls are more complex. They have a thin peptidoglycan layer
and an outer membrane beyond the plasma membrane. The space between the
plasma membrane and the outer membrane is called the periplasmic space. The
outer leaflet of the outer membrane is composed largely of a molecule called
lipopolysaccharide (LPS). LPS is an endotoxin that is important in triggering the
body’s immune response and contributing to the overall negative charge of the
cell. Spanning the outer membrane are porin proteins that enable the passage

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of small molecules. Lipoproteins join the outer membrane and the thin
peptidoglycan layer. Gram-negative cells will stain pink with the Gram stain
Basic Steps of Gram Staining

There are four basic steps of the Gram stain, which include;
✓ applying a primary stain (crystal violet) to a heat-fixed smear of a bacterial
culture,
✓ the addition of a mordant (Gram's iodine),
✓ rapid decolorization with alcohol or acetone,
✓ and counterstaining with safranin.

Principle of Gram Staining

The structure of the organism's cell wall determines whether the organism is
Gram-positive or Gram-negative. When staining with a 1st or primary stain and
fixed by a mordant, some bacteria can retain the 1 st stain by resisting
decolorization, and therefore cannot get decolorized by a decolorizer. Those that
retain the 1st or primary stain are called Gram-positive and those that get
decolorized and then counter stained are called Gram-negative.

Crystal violet (CV) dissociates in aqueous solutions into CV+ and chloride Cl–
ions. These ions penetrate through the cell wall and cell membrane of both
Gram-positive and Gram-negative cells. The CV+ ion interacts with negatively
charged components of bacterial cells and stains the cells purple.

Iodine (I – or I3 –) used as a mordant interacts with and forms large complexes of

crystal violet and iodine (CV-1) within the inner and outer of the cell.

When a decolorizer such as alcohol or acetone is added, it interacts with the

lipids of the cell membrane. A Gram-negative cell will lose its outer membrane
and the peptidoglycan layer is left exposed. The CV – I complex are washed from
the Gram-negative cell along with the outer membrane.

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In contrast, a Gram-positive cell becomes dehydrated from an ethanol treatment.
The large CV – I complex become trapped within the Gram-positive cell due to
the multilayered nature of its peptidoglycan. The decolorization step is critical
and must be timed correctly; the crystal violet stain will be removed from both
Gram-positive and negative cells if the decolorizing agent is left on too long (a
matter of seconds). After decolorization, the Gram-positive cell remains purple
and the Gram-negative cell loses its purple color. Counter stain, which is usually
positively-charged safranin or basic fuchsin, is applied last to give decolorized
Gram-negative bacteria a pink or red color.

OBSERVATION

E.g. of Gram-positive

- Bacillus - Enterococcus
- Nocardia - Mycoplasma
- Clostridium -Staphylococcus
- Streptococcus -Streptomyces
- Corynebacterium -Gardnerella

E.g. of Gram-negative

- Escherichia - Hemophilus
- Klebsiella - Neisseria
- Enterobacter - Chlamydia
- Salmonella - Shigella
- -Vibrio

PROCEDURE

- Take a grease-free dry slide

- Sterilize an inoculating loop on a flame 2-3 times

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 - Transfer a loopful of culture (or the specimen) by the sterile loop and
make a smear at a Centre
- Allow to air dry
- Fix the smear by the slide 3-4 times through the flame quickly with the
smear side facing up.

Gram procedure
- The heat-fixed smear is stained with crystal violet (1st or primary stain)
and left for 30 sec and a max of 1 min
- Wash carefully under running tap water
- Flood the smear with Lugol’s iodine solution functioning as a mordant
of 1min
- Drain off the iodine with clean water
- Flood the smear with ethanol or acetone-alcohol (decolorizing agent) for
30 seconds. This generates the different aspects of the gram stain
- Gently wash the slide under running tap water and drain completely
- Counterstain by staining any of the basic dyes different in colour form
crystal violet for about 30 seconds to 1 min
- Wash the slide gently until no colour appears
- Wipe the back of the slide or blot dry with absorbent paper and observe
microscopically under x100 oil immersion objective

2. Acid-fast Stain

Acid-fast stain is another widely use differential staining procedure developed by

Paul Ehrlich in 1882 during his work on etiology of tuberculosis. It is a procedure
used to stain a small group of organisms that do not readily take up stains.
Among these are members of the genus Mycobacterium, including a species that
causes tuberculosis and one that causes Hansen’s disease (leprosy). The cell wall
of these bacteria contains high concentrations of lipid (mycolic acid), preventing
the uptake of dyes. Therefore, harsher methods are needed to stain these

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organisms. Once stained, however, these same cells are very resistant to
decolorization. Because mycobacteria are among the few organisms that retain
the stain in this procedure, the acid-fast stain can be used to presumptively
identify them in clinical specimens that might contain a variety of different
bacteria.

To stain these bacteria, penetration of primary dye (carbol fuchsin) is facilitated

with the use of 5% aqueous phenol which acts as chemical intensifier and heat
as physical intensifier. Though Paul Erhlich was the first to described this
technique, Ziehl-Neelson modify it and the reagents used were much more stable
than that of Erhlich, hence, sometimes the technique is called Ziehl-Neelson
staining.

Here's a breakdown of the principle:

1. Primary Staining:

o The smear is stained with carbol fuchsin, a red dye.

o The carbol fuchsin penetrates the waxy cell walls of AFB due to its
lipid solubility.

2. Decolorization:

o The slide is washed with acid alcohol (a mixture of hydrochloric acid

and ethanol).

o This step is crucial as it decolorizes non-acid-fast bacteria, which

lack the waxy layer.

o The waxy layer of AFB prevents the decolorization of the carbol

fuchsin, so they retain the red stain.

3. Counterstaining:

o The slide is then stained with methylene blue or malachite green.

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 o This step stains the non-acid-fast bacteria blue or green, providing
contrast.

Result:

• Acid-fast bacilli appear as red rods against a blue or green background.

• Non-acid-fast bacteria appear blue or green.

Key points:

• The Ziehl-Nelson staining technique is used to diagnose diseases caused

by AFB, such as tuberculosis and leprosy.

• The technique is based on the differential staining properties of AFB due

to their waxy cell walls.

• The carbol fuchsin stain penetrates the waxy layer, while acid alcohol
decolorizes non-acid-fast bacteria.

• The counterstain provides contrast for visualization of non-acid-fast

bacteria.

Procedure

✓ Prepare a smear and heat fixed specimen

✓ Flood the smear with carbol fuchsin
✓ Heat the slide from below for about 5minutes until steam rises
✓ Allow the slide to cool, to prevent slide breakage then wash with water
✓ Decolorize the specimen with 20% sulphuric acid until the red color is no
longer seen
✓ Wash with water
✓ Counter stain with 1% methylene blue for 15 seconds
✓ Wash, blot-dry and then view under oil immersion objective lens.

Result:

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 Acid-fast bacteria appear reddish-pink while non-acid-fast take up the blue
coloration of the counter stain.

C. Special Staining

Dyes can also be used to stain specific structures inside or outside the cell. The
staining procedure for each component of the cell is different, being geared to
the chemical composition and properties of that structure.

1. Capsule staining

Capsules are form by organisms such as Klebsiella pneumonia. Capsule protects

bacteria from the phagocytic action of leucocytes and allows pathogens to invade
the body and it is sometimes correlated with an organism’s ability to cause
disease. Bacteria capsules are non-ionic, so, neither basic nor acidic stain will
adhere to their surfaces. The best way to visualize them is to stain the
background using acidic dyes and stain the cell itself using basic dyes leaving
the capsule as a clear hallow surrounding a purple cell in a field of black.

Staining Procedures

✓ Prepare thin smears of bacterial culture on a microscope slide.

✓ Allow the smear to only air-dry. Do not heat-fix as this will cause the
capsule to shrink or be destroyed.
✓ Apply 1% crystal violet and allow it to remain on the slide for 2 minutes.
✓ Gently wash off the crystal violet with 20% copper sulfate. Caution: Do not
wash the copper sulfate and stain directly into the sink.
✓ Blot the slide dry with bibulous paper.
✓ Observe with the oil immersion lens
Result; capsule appears as a hallo object around a dark background.

2. Endospore

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Members of certain Gram-positive genera including Bacillus and Clostridium
form a special type of dormant cell, an endospore, that are metabolically inactive
and highly resistant against harsh environmental conditions. Bacteria can
remain in this state until condition becomes favorable and they germinate and
return to their vegetative state.

Endospore Staining Procedure

✓ A smear is prepared
✓ the primary stain, malachite green applied followed by a gentle heating
✓ rinse with water (water removes malachite green from vegetative cells but
not endospores)
✓ Counter stain with safranin. Safranin will stain the vegetative cells.
✓ At the end of the staining process vegetative cells will be pink (Safranin
color) and endospores dark green.

3. Staining of Metachromatic Granules

Metachromatic granules staining employ the use of Albert stain. The application
of Albert stain is aimed at identifying bacteria that contain special structures
known as metachromatic granules which are found in Corynebacterium
diphtheria a Gram positive, non-spore forming, non-motile bacilli.
Metachromatic granules are intracellular inclusion bodies found in the
cytoplasmic membrane of some bacterial cells for storage of complex inorganic
polyphosphate (poly-P) and enzymes.

Basically, Corynebacterium diphtheria is initially cultured on a selective media

either a Loeffler agar or Mueller-Miller tellurite agar and its colony are used for
liquid culture preparation before used for staining.

Procedure for Metachromatic Staining

1. Aseptically take a loopful culture of the organism and prepare a smear.

2. Heat fix the smear gently,

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3. Add Albert stain A and allow for 3-5 minutes

4. Rinse the slide with water,

5. Add Albert staining solution B allow for 1 minute

6. Rinse the slide with water

7. Blot dry

8. Apply oil immersion and view using X100 objective lens.

NEGATIVE STAINING

Microorganisms that are not stained early by ordinary stains may be made
visible by the process known as negative staining. It is also a technique that has
been developed to study specific bacteria structures with the light microscope.
In this method, the background, but not the microorganisms are stained.

The procedures involve mixing microorganisms with Indian ink or

nigrosine and spread out in a thin film on a appear as a colorless object against
a black background (like blackboard and a chalk). E.g. applied in the
identification of spirochetes (T. pallidum).

Negative staining is also applied to reveal the presence of the diffuse

capsules and other structures surrounding many bacteria e.g. streptococcus
pneumonia, clostridium leuconostoc spp. Perfringens, klebsiella pneumonia. This
is because Indian ink or Nigrosine particles cannot penetrate either the bacteria
cell or its capsule.

STAINING FOR CELL STRUCTURES

Spore staining: (Shaeffer and Fulton method)

End spores are dormant structures within the cell that enable the bacteria to
survive unfavorable conditions for long periods. The location and morphology
vary with species and often are valuable in identification; endospores may be
spherical to elliptical and either smaller or larger than the diameter of the parent

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bacterium. Examples of endospores formers are Bacillus and Clostridium
butyricum.

Procedure

- Prepare and tie a smear of a bacteria culture or sample on a clean slide.

- Cover the smear with malachite green.
- Heat the slide gently until it steams allow to stand for 2- 3 mins
- Wash slide with tap water and then apply safranin solution for 30 sec- 1
min.
- Wash smear with tap water, drain, blot dry, and examine using X100 or
oil immersion objective.

OBSERVATION

The endospores stain green, while the vegetative cells stain pink to red.

FLAGELLA STAINING

Flagella are fine thread-like organisms of locomotion, that cannot be seen

directly with the electronic microscope

However, to observe them with the Light microscope the thickness of the flagella
is increased by coating them with Mordants like tannic acid and potassium
alum\they are stained with ammonia silver nitrate solution which has been pre-
heated near boiling point for 3-5 minutes

This will reveal the presence and distribution pattern g the flagella (by absorbing
the silver nitrate)

For example, the organism may be

1. Monotrichus – possess a single polar flagellum

E.g. Vitro cholera
2. Lopotrichus –two or more flagella at one polar
E.g. E. coli
3. Petrichus- flagella distributed all around the cell

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 E.g. E. coli

4. Amphitrichus –one or several flagella at each end

E.g. Aquaspillium bengal

ALBERT’S STAINING

The bacteria met achromatic (volition) inclusion granules may stained with
Albert stain

(toluidine blue and methylene blue)

Procedure

- Place the smear on the staining rack and flood it with Alberts stain
(toluidine blue and methylene blue) for 3-5 minutes
- Wash under tap water
- Flood with lugholes iodine solution for 1 minute
- Wash under tap water, air dry, and examine under x100 or immersion
objective

Observation

Volutin granules appear blue-black while cytoplasm appears light green.

e.g. Corynebacterium diphtheriae used in identifying diphtheria ----------

CAPSULES

Certain bacteria saccade is a polysaccharide that adheres and forms an external

layer to the cell wall. Such a search is called a capsule in four cases the capsule
can be polypeptide. If the search is in fluid form is called the slime layer

Bacteria that possess a capsule

Diplococcal pneumonia klebsiella

1. Prepare a thin smear of bacteria as voluntary

2. Flood slide with crystal stain for smear
3. Stem and flame for 40 mins.
4. Wash off the stain with a supplicate

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 5. Blot dry without using
6. Examine under oil-immersion lens

Observation

Capsule smear as faint blue-violet tones and dark-blue bacterial cells

Flagella

- protein pneumonia
- smear preparation of
- fixation
- staining

Staining fungal hyphae

E.g. rhino pop, spp penicillium

- Place a drop of lactophenol cotton blue on a clean slide

- Using an inoculating needle in either hand, remove a small piece of
mycelium.
- Transfer mycelium to stain on the slide and using both needles tease not
the mycelium carefully.
- Cover with color ship;
- Examine slide under low power x then water high power of microscope (x
10 x 40).

FIELD STAINING TECHNIQUE

Thin film

- Fix the smear with methanol for 1- 2 minutes.

- Add 0.5ml of field’s stain B acid mixed with an equal volume of field stain
A and leave to stain for 1 minute.
- Wash with clean water. Wipe the back of the slide clear and place it in a
draining rack of dry.

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 Observe under X100 objective.

Thick film (Recommends because of sensitivity 30x more sensitive than thin
film.

- Do not fix, dip the slide into the field’s stain A for 5 seconds drain off the
excess stain by touching a corner of the slide against the side of the
container.
- Wash gently in clean water
- Dip the slide in fields stan B for 5 seconds. Drain off the excess stain.
- Wash in clean water.
- Wipe the back of the slide clean and place it upright in a draining rack for
the film to air-dry.

Observe under the microscope using x 100 objective.

In both thin and thick films, the chromatin appears dark red while the cytoplasm
of the parasite is blue.

FIELD’S STAIN A AND FIELD’S STAIN B

Field’s Stain is a histological method for staining blood smears. The definitive
diagnosis of malaria infection and other parasites is still based on finding such
organisms in blood films. In thin films, the red blood cells are fixed and
parasitized cells can be identified by their morphology.

Field’s Stain method for thick blood films

Red blood cells are lysed during this procedure diagnosis is based on the
appearance of the parasite. The microscope) in thick films than thin blood films.

METHOD/ PROCEDURES

1. Place a drop of blood on a microscope slide and spread to make an area of

approximately 1cm2.
2. Air dry the film (do not fix it in methanol)

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 3. Dip the slide into field stain A for 3 – 5 minutes and drain off the excess
by touching a corner of the slide against the side of the container.
4. Wash the slide in water for 3 sec, and agitate gently.
5. Dip the slide in the field’s stain B for 3 – 5 sec. and drain off the excess.
6. Wash in clean water.
7. Wipe the black of the slide clean and place it upright in the draining rack
for the film to air dry.
8. Observe microscopically under X100 oil immersion objectives.

Observation

In both thin and thick blood films the chromatin appears dark red while
the cytoplasm appears pale blue leucocyte – purple nuclei and pale blue
background.

Field’s stain method for thin blood films

This is a modification of the original field’s stain to enable rapid staining of fixed
thin films, it’s a useful method for rapid presumptive species identification of
MP. This method is suitable for MP, Babesia spp, and leishmania spp.

Procedure:

1. Air-dry the already prepared film.

2. Fix in methanol for 1 – 2 mins
3. Add 0.5ml of field’s stain B and mix with equal volume of field’s stain A
and allow staining for 1 min.
4. Rinse well in tap water and wipe the back of the slide clean and place it in
a draining rack to air- dry.

Observe under x 100 objective.

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 Thin Film thick film

GIEMSA STAIN

This is used mainly for staining malaria parasites, trypanosomes,

leishmanial parasites, and microfilariae. It gives the best staining of malaria
parasites in both thin and thick film. Before staining, the stain is diluted to the
required concentration depending on the staining, time. That is a 3% Giemsa
solution for 30-minute staining. (Measure 50ml of buffered water pH 7.1, 7.2
and add to 1.5ml Giemsa) or 10% Giemsa solution for 10 minutes of staining.

The staining technique involves fixing the smear in methanol for 1 minute
dipping it and into a shallow tray containing the diluted stains. It is allowed to
stain based on the concentration of the Giemsa stain.

- The after staining, the slide is washed with clean water. To remove excess
stain.
- Wipe the back of each slide clean and place it in a draining rack for the
preparation to air dry.
- When the films are completely dry the area is examined microscopically
using x 100 oil immersion objectives for the presence of malaria parasite.
- Chromatin of the parasite appears dark red while the cytoplasm of
the parasite will appear blue- in color.

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