Gram’s stain (mixture)

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Presentation transcript:

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

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

Gram Staining

Crystal violet ↓ Iodine Acetone-alcohol Safranin

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

Acid-Fast Staining

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 acid-alcohol→ 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.

Materials:- Acid Fast Staining 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 fuchsin MB 30-60 sec alcohol 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

Spore Stain

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.

Materials :- The Spore Stain Culture of B. subtilis Spore-staining kit: 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

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