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LECTURE TOPIC:
STAINING METHODS IN
BACTERIOLOGY
1
Introduction
3
Types of staining
6
Simple staining methods
7
Methylene blue stains
8
Staining procedure
Results
9
1. Differential staining methods
Gram's stain
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The Gram positive organism must have an intact cell wall. A
damaged cell wall inevitably results in Gram negative
reaction.
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.
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Solutions
Lugol's iodine:
Iodine 10g
Potassium iodide 20g
Distilled water 1000mll
Counterstain
15
Procedure
Results
18
Acid-fast stain (Zehiel-Nelseen method)
Procedure
Results
Acid-alcohol fast bacteria : red
Background
23
and other bacteria :blue or green.
Kinyoun's method
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Auramine phenol fluorescent stain
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
Solutions
Albert’s 1
Albert’s 2
Result:
Granules:
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bluish black; Organism: green
Special staining methods
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Staining of capsules
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.
5% malachite green
0.5% safranin
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Staining of bacterial cell wall
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Staining methods for flagella
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%10Ferric chloride 1ml.
Mix and stand for 3 minutes before use. The
solution does not keep.
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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.
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Fontana's method for spirochaetes
Solutions:
.1 Fontana's fixative
Glacial acetic acid 1ml
Formalin 2ml
Distilled water 100ml
.2 Absolute alcohol
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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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