3. 3
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..
4. 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
4
5. 5
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. e.g.. Methylene blue
6. 6
.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
three categories
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:
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.
8. 8
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.
9. 9
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
10. 10
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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1. Differential staining methods
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 is the mordant used.
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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
by the decolouring agent.
14. 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.
The red counterstain makes the decolourised Gram
negative bacteria visible in a contrast colour.
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15. 15
Solutions
Crystal Violet: 0.5 to 1% in distilled water.
Lugol's iodine:
10g
Iodine
20g
Potassium iodide
1000ml
Distilled water
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.
Wash with tap water.
18. 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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20. 20
(Zehiel-Nelseen method)
Acid-fast stain
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
21. 21
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. Add the rest of the water.
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20% sulphuric acid (H2SO4) or 3% acid alcohol
(i.e 3% 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
: red/pink
Acid-alcohol fast bacteria
Background and other bacteria :blue/violet.
25. 25
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
26. 26
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
