LECTURECOURSE MLSC 417

TOPIC: STAINING METHODS

IN
BACTERIOLOGY

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Introduction

Stained preparations are needed to examine micro-

organisms microscopically in order to study their
morphology and observe their cellular constituents.

Smears (or tissue sections) are made and stained by

any one of the required staining methods.

Smears can be made from liquid or solid cultures or

..from the clinical specimen
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The staining result is dependent on the quality •
.of the smear

From the liquid culture, smears should be •

evenly spread on the slide and allow to dry in
.air

Smears from solid culture should not be thick •

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Types of staining

Indirect staining: This is when an organism is

stained only in the presence of a mordant. e.g.
Gram's stain.

Direct Staining: This a simple one-step staining

procedure in which the presence and morphology of
bacteria are demonstrated.

Some of the stains used for this technique are also

used as counterstains in some other staining
procedures.
4 e.g.. Methylene blue
Negative staining: This is when the organism.
remains unstained against a stained background. This
is one of the few methods where acid stains such as
nigrosin, are used.
 
Metachromatic staining: In this staining method the
organism or part of the organism is stained a different
shade of colour from that of the stain.

Commonly used staining methods

 
In Bacteriology, staining methods are divided into
three5 categories
:
Simple stains: This makes use of the direct
staining method.

Differential stains: This staining method divides

bacteria into two groups

Special stains: These are specialised staining

methods to demonstrate certain bacterial
.components, e.g. spore

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Simple staining methods

Simple staining methods are used to

demonstrate the presence of and the morphology
of bacteria and cells.

They employ simple stains most of which are

also used as counterstains.

Few of these methods may contain a mordant

.e.g. phenol in carbol thionin

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Methylene blue stains

Neutral methylene blue, Loeffler's alkaline

methylene blue and Polychrome methylene blue
solutions are all used in like manner and with
almost similar results.

The polychrome methylene blue is particularly

useful for the identification of anthrax bacilli by
Mcfadyean reaction

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Staining procedure

Stain the heat-fixed smear for 1 minute.

Wash with water.
Blot carefully and dry.

Results

:Bacteria and cells

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Differential staining methods .1

Gram's stain

In 1884, Christian Gram, a Danish bacteriologist,

described this staining method which is the most
important stain in routine bacteriology.

It divides bacteria into two groups - the Gram

.positive and Gram negative bacteria

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The Gram positive organism must have an intact cell wall. A
damaged cell wall inevitably results in Gram negative
reaction.

This shows the importance of cell wall in Gram stain reaction.

The Gram's stain reaction is based on the ability of the

organism to resist decolourisation with acetone, alcohol or
aniline oil after the initial staining with one of the rosaniline
basic dyes and then treating with a mordant.

The rosaniline dyes commonly used are crystal violet, methyl

violet and gentian violet.

.Iodine
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is the mordant used
A counterstain, contrasting in colour with the primary
stain, is used to stain Gram negative bacteria which are
decolourised.

Gram positive bacteria: In a Gram stained smear Gram

positive bacteria which retain the primary stain, appear
violet or blue-black or deep purple in colour.

The Gram positive reaction is due to the presence of

techoic acid in the cell wall.

The teichoic acid forms a mesh which prevents the

primary stain-mordant complex from being washed away
.by12the decolouring agent
Gram negative bacteria: These appear red. •
Gram negative bacteria have a high lipid content
.which dissolves in the decolourising agent

This allows the primary stain-mordant complex to •

.be washed off
o •
The red counterstain makes the decolourised •
Gram negative bacteria visible in a contrast
.colour

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Solutions

Crystal Violet: 0.5 to 1% in distilled water.

Lugol's iodine:
Iodine 10g
Potassium iodide 20g
Distilled water 1000mll

Dissolve the potassium iodide in about 50 ml of the

distilled water, add iodine.

Dissolve the iodine by shaking and make up the volume to

.1000 ml
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Decolouriser:

Solution: Absolute ethyl alcohol or acetone or

acetone and alcohol mixture (1:1)

Counterstain

Aqueous solution of neutral red or safranin 0.5%,

or dilute carbol fuchsin. (1:10 dilution of strong
carbol fuchin in distilled water)

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Procedure

Make a smear, allow to dry and then fix with a gentle

heat by passing the slide 2 or 3 times over a bunsen
flame or placing the slide on a slide warmer.

Stain with crystal violet for 1 minute.

Wash with tap water.

Apply Lugol's iodine and leave for 1 minute.

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.Wash with tap water
Decolourise with acetone or alcohol until no more colour •
appears to ooze out of the smear (about 1-2 seconds for
acetone and 1-2 minutes for alcohol and 10 seconds for
acetone/alcohol mixture)

.Wash immediately with tap water •

Counterstain with neutral red or safranin or dilute carbol

.fuchsin for 1 minute

.Wash with tap water

.Blot dry with a blotting or filter paper, and dry

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 .7

Examine microscopically using x100 objective.

Results

Gram positive bacteria blue/black

Gram negative bacteria red

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Acid-fast stain )Zehiel-Nelseen method(

Acid-fast staining is another example of a differential stain

used in Bacteriology.

It divides bacteria into two groups, acid fast-and non acid

fast. Members of the genus Mycobacterium are acid-fast in
nature. Like the Gram reaction, the acid-fastness is also
called cell wall dependent.

Mycobacteria have a high lipid content, especially mycolic

acid, in their cell wall. The ordinary aniline dye solutions
cannot penetrate the mycobacterial cell
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wall. Strong staining solutions containing phenol
preferably with application of heat, are used for staining
them. Once stained, they resist decolourisation with
mineral acid. Therefore they are called acid-fast bacilli
(AFB).

The degree of acid-fastness varies with species.

Mycobacterium tuberculosis can resist decolourization
with 20% sulphuric acid, or 3% hydrochloric acid in 95%
ethanol. Hence they are also called acid-alcohol-fast
(AAFB).

M.leprae resist 5% sulphuric acid while Nocardia

.species are acid-fast only with 1% sulphuric acid
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Ziehl-Neelsen (ZN) method
Solutions
Strong carbol fuchsin

Powdered basic fuchsin 5g

Phenol 25g
Alcohol 95% 50ml
Distilled
Water 50ml

Dissolve the fuchsin in the solution of phenol with a little

water over a boiling water bath. Add the alcohol and
.mix.
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Add the rest of the water
sulphuric acid (H2SO4) or 3% acid alcohol (i.e 3% 20%
hydrochloric acid in 95% alcohol).

0.5% methylene blue or 0.5% malachite green.

Procedure

Make and fix smear by heat

Flood the slide with the carbol fuchsin solution
and heat gently until steam rises. Do not allow the stain
to boil.
Stain for 5-10 minutes and reheat. Leave for another 10
minutes.
.Wash with tap water
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Decolourise with 20% sulphuric acid or 3% acid alcohol for
about 5 minutes. (with 20% H2SO4, until the film becomes
yellowish).
Wash well in water.

Counterstain with 0.5% methylene blue or malachite green

for 30 seconds.
Wash well with tap water.
Blot , dry and examine under oil immersion laws.

Results
Acid-alcohol fast bacteria : red
.Background and other bacteria :blue or green
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Kinyoun's method

This is a radical modification of the Ziehl-

Neelsen's method in which no heating is
.required

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Auramine phenol fluorescent stain

This is a fluorescent staining method for the

detection of acid fast bacilli.

Solutions
1 Auramine phenol
2 3% Acid alcohol
3 0.1% Potassium permanganate

Result
Acid fast bacilli: Bright luminous rods against
dark
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bachground
Special staining methods
 
Staining of metachromatic granules
Certain bacteria possess granules which are seen in
the cytoplasm. The presence of these granules help
in the identification of such bacteria. e.g.
Corynebacterium species. Special stains such as
Albert's, Neisser's and Pugh's stains are used to
demonstrate these granules

Solutions
Albert’s 1
Albert’s 2
Result:
Granules: bluish black; Organism: green
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Special staining methods
 
Staining of metachromatic granules

Certain bacteria possess granules which are seen in the

cytoplasm.

The presence of these granules help in the

identification of such bacteria. e.g. Corynebacterium
species. Special stains such as Albert's, Neisser's and
Pugh's stains are used to demonstrate these granules

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Staining of capsules

Capsules are not stained by ordinary stains due to their

low affinity for stains.

They are demonstrated by either negative staining or

one of the direct staining methods such as the Hiss
method.
 
1. Nigrosin (or India ink) negative staining

2.Nigrosine-methylene blue

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Staining of spores
The spore wall is resistant to stains but spores
can be demonstrated with the application of
heat to the preparation.
 
Malachite green stain for spores

5% malachite green
0.5% safranin

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Staining of bacterial cell wall

The composition of cell wall differs from bacteria to

bacteria, therefore there are variations in the length of
time for the stain and cell wall to interact.

The staining method uses tannic acid as mordant and

Congo red as a selective decolouriser while crystal violet
.stains the cell wall, leaving the cytoplasm unstained

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Staining methods for flagella

Young cultures are used to demonstrate flagella.

Chemically clean slides must be used and the smear
made with proper care. All the methods are of silver
impregnation techniques.
 
Fontana's silver method for flagella.
1. 1% Osmic acid
2. Fontana's mordant
10% Tannic acid 10 ml
Saturated potassium alum 10 ml

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 10% Ferric chloride 1 ml.
Mix and stand for 3 minutes before use. The
solution does not keep.

3. Fontana's silver solution

Dissolve 5g silver nitrate in 100 ml distilled

water.
Dilute 5 ml of concentrated ammonia with 45
ml distilled water.
Add 35 ml of the diluted ammonia solution to
90 ml of silver nitrate.

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 Continue to add drop by drop until the
precipitate which is formed is redissolved.
Continue to add more until a faint opalescence
is seen.
Procedure
1 .Place one drop of 1% osmic acid towards
one end of a very clean slide.

2 .With a loop, gently transfer one drop of

the water of condensation of an 18 hour old agar
slope culture to the drop of Osmic acid and mix
gently.

Allow the drop to run the whole length

of the slide.
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3. 3. Allow to dry in air.

4. Add Fontana’s mordant for 3 minutes.

5. Wash in distilled water.

6. Heat Fontana's silver solution in a test tube and

flood the slide. Leave for 2 minutes.

7. Wash in distilled water.

9. Dry in air and mount immediately.

Results: Organisms : black

34 Flagella : light brown.
Staining method for spirochaetes
The usual and easier way of examining spirochaetes is with
the darkfield microscope.
They do not stain well with aniline dyes and because the
spirochaetes spirals are so thin, they are not easily seen by
most other staining methods.
However, they have the capacity to reduce silver nitrate to
metallic silver, thus the demonstration of spirochaetes in
.smears can be achieved by the silver impregnation method
 
.

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Fontana's method for spirochaetes

Solutions:
1. Fontana's fixative
Glacial acetic acid 1 ml
Formalin 2 ml
Distilled water 100 ml

2. Absolute alcohol

3 Fontana's mordant: refer to Fontana’s silver

method for flagella

4.Fontana's silver solution: refer to Fontana’s silver

method
36 for flagella
Procedure
1. Make a thin smear
2. Treat smear with Fontana's fixative 3 times for
30 seconds each.
3. Wash off the fixative with absolute alcohol
and cover with absolute alcohol for 3 minutes.
4. Drain off the alcohol and carefully burn off
the remainder until the film is dry.
5. Apply Fontana's mordant, heat until steam
rises and leave for 30 seconds.
6. Wash well in distilled water and dry the slide.

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7. Apply the Fontana's silver solution, heat until
steam rises and leave for about ½ minute (the film
turns brown in colour).
8. Wash in distilled water, dry and mount in
neutral synthetic medium.
 
Results
Spirochaetes : brownish black
Background : brownish yellow.

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