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PHT 381
Lab # 3
Staining of Bacteria

Types of staining technique:-
Simple staining
(use of
a single basic stain)
Differential staining
(use of two contrasting stains
separated by a decolorizing agent)
Identification
For visualization of
morphological
shape & arrangement.
Gram
stain
Visualization
of structure
Acid fast
stain Spore
stain
Capsule
stain
Crystal violet
↓
Iodine
↓
Acetone-alcohol
↓
Safranin
Gram Stain [mixture]

Procedure:
CV
safranin
alcohol
iodine
mo
30-60
sec
1 sec
min
10
30
Acid Fast Staining
 Mycobacteria have a 3rd type of cell
envelope (rather than the basic gram- related
properties).

The cell wall of bacteria in this genus
contain → considerable amounts of lipids
→ form an extremely hydrophobic external
layer.
Acid Fast Staining
These organisms are not readily stainable with
ordinary stains.
 Staining of these bacteria needs exposure to a
strong stain e.g., concentrated carbol fucsin
With application of heat.


Once they are properly stained, they resist
decolorization by strong mineral acids or acidalcohol→ so they are said to be
Acid-fast.
Acid Fast Staining
AFS is an important diagnostic value in
identifying pathogenic members of genus
Mycobacterium such as M. tuberculosis
and M. leprae.
Acid Fast Staining
Materials:Culture of M. phelei
Acid-fast staining kit:
Carbol fuchsin (primary dye)
Acid-alcohol (decolorizing agent)
Methylene blue (counter stain)
Acid Fast Stain

Procedure:-
5 min
Carbol
alcohol
MB
fuchsin
\\\\
30-60
sec
1 min
Results
Type of Staining: Acid fast stain
Shape: beaded bacilli
Arrangement: Tree shaped
Colour: red
Name of microorganism:
M.phelei
The Spore Stain

Some bacteria (e.g., Bacillus and Clostridia) form
resistant bodies in the cell known as endospors.

Bacterial spores are highly resistant to physical &
chemical agents (primarily due to a thick tough
spore coat).

They are not easily stained by routine staining.
Heat is required in spore staining to promote the
penetration of the dye into the spore.
Once the spores stained they resist decolorization.


The Spore Stain
Materials :of B. subtilis
 Spore-staining kit:
 Culture
 Malachite
green (primary stain)
 Safranine (counter stain)
Spore Stain of
Bacillus subtilis
Type of Staining: Spore stain
Shape: bacilli
Arrangement: Chains
Colour of spores: green
Colour of vegetative cells: red
Name of microorganism:
B. subtilis