Capsule Staining: Principle, Methods, Procedure and Examples

Contents:

Principle:
(A) India ink method:
Procedure:
(B) Anthony’s Stain Method:
(C) Demonstration of Capsule by Maneval’s Method/Relief Staining:
Examples of Capsulated Bacteria:

Capsule Staining: Principle, Methods, Procedure and Examples

Some bacteria have a thin layer of polysaccharide capsule outside the cell wall. 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 protects the cell from dehydration. It harbours the Vi (virulence) antigen in pathogenic bacteria.

Capsule stain is a type of differential stain which uses acidic and basic dyes to stain background and bacterial cells respectively so that presence of capsule is easily visualized.

Principle:

Bacterial capsules are non-ionic, so neither acidic nor basic stains will adhere to their surfaces. Therefore, the simplest 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, Safranine, Basic fuchsin and Methylene blue).

Commonly used methods for capsule staining are as follows:

(A) India ink method:

In this method, two dyes, crystal violet and India ink are used. The capsule is seen as a clear halo around the microorganism against the black background. This method is used for demonstrating Cryptococcus.

Procedure:

Take one drop of India ink on a clean and grease free glass slide near the one end of slide.
Using a sterile nichrome wire loop, mix loopful of bacterial suspension with drop of India ink stain.
Using another clean and dry glass slide, spread out the drop and prepare thin film of it.
Allow the film to air dry for 5-7 minutes. Do not heat or blow dry because capsule dissolves due to the heat.
Flood the slide with crystal violet for 1 minute and rinse slightly and very gently with water.
Let the slide air dry for a few minutes.
Observe the slide under oil immersion lens.

(B) Anthony’s Stain Method:

In this type of capsule staining procedure, the primary stain is crystal violet, and all parts of the cell take up the purple crystal violet stain. There is no mordant in the capsule staining procedure. 20% copper sulphate solution serves a dual role as both the decolourizing agent and counter stain. It decolourizes the capsule by washing out the crystal violet but will not decolourize the cell. As the copper sulphate decolourizes the capsule, it also counter stains the capsule. Thus, the capsule appears as a faint blue halo around a purple cell.

Procedure:

Take one drop of crystal violet on a clean and grease free glass slide at the one end of slide.
Using a sterile nichrome wire loop, mix loopful of bacterial suspension with drop of India ink stain.
Using another clean and dry glass slide, spread out the drop and prepare thin film of it.
Allow the film to air dry for 5-7 minutes. Do not heat or blow dry because capsule dissolves due to the heat.
Rinse the slide with 20% copper sulphate solution.
Let the slide air dry for a few minutes.
Observe the slide under oil immersion lens.

(C) Demonstration of Capsule by Maneval’s Method/Relief Staining:

The capsule appears as a clear halo between the pink-stained bacterium and the bluish-grey stained background. The background stain is the acidic stain Congo red (which changes colour to bluish-grey due to the pH), and the pink bacterial stain is due to acid fuchsin.

Procedure:

Take a loopful of capsulated cell suspension on a clean grease free slide.
Add adrop of 1% Congo red solution in the suspension and then spread it gently on the slide to form a smear.
Allow the suspension to air dry and do not heat fix it.
Flood the slide with Maneval’s solution and keep it for 2 minutes. Maneval’s solution: 5% Phenol (accentuator), 25% acetic acid (changes colour of background i.e. Congo red to blue), 30% ferric chloride (mordant) 1% acid fuchsin (stains bacterial cell).
After 2 minutes discard excess stain and do not water wash the slide.
Allow the slide to air dry and observe under oil immersion lens.