CAPSULE STAINING

This method of staining is specifically design for examination of structure of bacteria capsule. When an ordinary staining method is used, carbohydrate bacteria capsule remain unstained, in this case, they are seen as a clear zone or halo within the stained organism. But to explain capsule structure of bacteria, direct or negative staining method must me employed. On this employed method, there are;

1 Crystal violet capsule stain

2 Nigrosin-methylene blue capsule stains

3 India ink preparations

1 Crystal violet capsule stain: this staining method has two solutions

i Crystal violet 1.0% solution

ii Copper sulphate 2%aq solution

PROCEDURE

1 Prepare a thin smear and allowed to air dry.

2 Apply solutions 1 for 2 minutes without heat

3 Wash with a solution 2.

4 Blot carefully dry, and examine with microscope.

Result; capsule stain pale violet, the remaining bacteria cell stain deep violet

2 Nigrosin-methylene blue capsule stains: the method also has 2 solutions.

1 SOLUTIONS

Nigrosin                                 5.10g

Distilled water                       100ml

Formalin for preservative      0.5m

PREPARATION

Dissolved nigrosin in a warm distilled water and add formalin preservative.

2 SOLUTIONS

Loeffers alkaline methylene blue

PROCEDURE

1 Loopful of culture colony on a clean glass slide, add a loopful of a fresh filtered solution mix allowed to dry in air and fix with gentle heat.

2 Apply solutions 2 for 30 seconds

3 Rinse rapidly with water, blot carefully and dry by gentile heat.

Examine with microscope

Result; capsule unstained against dark grey background of nigrosin. Bacteria cell remain blue.

3 India ink solutions

This staining is also used for demonstration of bacteria capsule in a wet preparation by negative staining.

PROCEDURE

1 Place a loopful of a solid bacteria culture on a clean glass slide

2 Place 1 poopful of india ink solution mixed

3 Cover with cover glass and press down firmly to for thin film, care should be taken so that no liquid spill out to the edge of the slide

4 Seal round the edge of the cover slip with paraffin wax and allowed to strong

Examine with microscope with X100 objective.

Result; capsulated bacteria is surrounded with a clear halo set against a dark background of ink.