1. Gram’s stain (mixture)
PHT 381 Lab # 3
Gram’s stain (mixture)
Acid fast stain
Spore stain

2. Staining of Bacteria Types of staining technique:-
Differential staining
(use of two contrasting stains
separated by a decolorizing agent)
Simple staining
(use of
a single basic stain)
Identification
Visualization
of structure
For visualization of morphological
shape & arrangement.
Gram
stain
Acid fast
stain
Spore
stain
Capsule
stain

3. Gram Staining

4. Crystal violet ↓
Iodine
Acetone-alcohol
Safranin

5. Gram Stain [mixture] Procedure: CV mo 30-60 sec 10 sec 2min 60sec
safranin CV
iodine
alcohol mo
30-60 sec
10 sec
2min
60sec

7. Acid-Fast
Staining

8. 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.

9. 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 acid-alcohol→ so they are said to be
Acid-fast.

10. Acid Fast Staining
AFS is an important diagnostic value in identifying pathogenic members of genus Mycobacterium such as M. tuberculosis and M. leprae.

11. Materials:- Acid Fast Staining Culture of M. phelei
Acid-fast staining kit:
Carbol fuchsin (primary dye)
Acid-alcohol (decolorizing agent)
Methylene blue (counter stain)

12. Acid Fast Stain \\\\ Procedure:- 5 min Carbol fuchsin MB 30-60 sec
alcohol
30-60 sec
1 min

13. Results Type of Staining: Acid fast stain Shape: beaded bacilli
Arrangement: Tree shaped
Colour: red
Name of microorganism: M.phelei

14. Spore Stain

15. The to Spore Stain
Some bacteria (e.g., Bacillus and Clostridia) form resistant bodies in the cell known as endospors.
Bacterial spores are highly resistant 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.

16. Materials :- The Spore Stain Culture of B. subtilis
Spore-staining kit:
Malachite green (primary stain)
Safranine (counter stain)

17. 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

21. Thank You