Capsule Staining

 Capsule Staining

Aim

To perform capsule staining to observe whether the given bacterium possesses capsule.

Principle

A capsule is a gelatinous outer layer secreted by bacterial cell and that surrounds and adheres to the cell wall. Most capsules are composed of polysaccharides, or polypeptides. Capsule is synthesized in the cytoplasm and secreted to the outside of the cell where it surrounds the bacterium.  Most of the capsulated bacteria have a capsule made up of polysaccharide layer but some bacteria have capsule made up of polypeptide, or glycoprotein.

Capsule stain is a type of differential stain which uses acidic and basic dyes to stain background & bacterial cells respectively so that presence of capsule is easily visualized.  Bacterial capsules are non-ionic and thus acidic or basic stains will not adhere to it. Therefore, the best way to visualize them is to stain the background using an acidic stain (e.g., Nigrosine, congo red) and to stain the cell itself using a basic stain (e.g.,Crystal violet, safranin, basic fuchsin and methylene blue).

Materials required

1. Cultures - 24 hour old Bacillus species culture

2. Reagents – Crystal violet, 20% Copper Sulphate

3. Equipments - Bunsen burner, inoculating needle, staining tray, microscope

Procedure

1.  A drop of crystal violet was placed on a clean glass slide.  The bacterial smear was prepared in the crystal violet drop on the slide and allowed to air dry. The smear was not heat fixed

2. The smear was washed gently using 20% Copper Sulphate solution.

3. The smear was air dried and observed under oil immersion objective

Observation

The vegetative cells appeared dark blue against a blue back ground and capsule was observed as a halo surrounding the bacterial cells.

Results

Capsule was observed as a light halo surrounding the bacterial cells

                                           

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