Capsule Staining - Introduction, Principle, Procedure, Result, Interpretation

Introduction to Capsule Staining

The main purpose of capsule staining is to differentiate bacterial cells from capsular material if present around the bacteria.

Capsules, which are limited to certain bacteria, are a gelatinous outer layer that surrounds and adheres to the bacterial cell wall after being secreted. Depending upon the genera and species, the capsule is chemically made up of polysaccharides, glycoproteins, or polypeptides.

The presence or absence of a capsule in a microorganism can be directly correlated to the virulence of the microbe. Microbial cells that produce heavy capsules are generally more virulent and likely to be a pathogen and cause diseases. The virulence is increased as the capsule protects the pathogen from host phagocytosis as well as from bacteriophages by preventing the adherence of the viruses.

Principle of Capsule Staining

Since capsular materials are water-soluble and may be dislodged or removed during vigorous washing, thus careful measures should be taken to prevent it. Before staining the smears should not be heated as the heat shrinks the cells, a false clear zone may be seen which may be misinterpreted as a capsule.

The principle of capsule staining is based on the bacterial capsule - since capsules are non-ionic, the primary stain (India ink) used cannot bind with the capsule - as it does with bacterial cells and instead just adheres to the capsule. Crystal violet is used to wash away the primary stain in place of water as capsules are water-soluble. Moreover, crystal violet is a basic dye that removes the primary stain and it is able to bind with bacterial cells.

Capsules seem like transparent areas surrounding the bacterial cells when viewed under a microscope.

Procedure of Capsule Staining

The procedure of capsule staining is as follows:

1. Take a clean, grease-free slide and make a heavy smear from a fresh culture of the test organism

2. Allow the smear to air dry. (do not use heat or water)

3. Flood the smear with primary stain (India ink) for 5-7 minutes

4. Wash the smear with a secondary stain (crystal violet) and let it stand for one minute.

5. Gently blot dry and observe under the microscope.

Result, Interpretation of Capsule Staining

The result and interpretation of capsule staining can be done as:

Positive

Microbial cells surrounded by a transparent area against a deep blue background are seen under the microscope.

Negative

No transparent area around microbial cells is seen when viewed under the microscope.