A capsule is a gelatinous outer layer that is secreted by the cell and that surrounds and adheres to the cell wall. It is not common to all organisms. Cells that have a heavy capsule are generally virulent and capable of producing disease, since the structure protects bacteria against the normal phagocytic activities of host cells. Chemically, the capsular material is composed mainly of complex polysaccharides such as levans, dextrans, and celluloses.
Capsule staining is more difficult than other types of differential staining procedures because the capsular materials are water-soluble and may be dislodged and removed with vigorous washing. Smears should not be heated because the resultant cell shrinkage may create a clear zone around the organism that is an artifact that can be mistaken for the capsule.
Objectives of Capsule Stain
- To prepare a smear of an encapsulated bacterium and stain its capsule using the Anthony capsule stain.
- To visualize the capsule and differentiate it from the cell body.
Principle of Capsule Stain
Primary Stain: Crystal Violet (1% aqueous)
A violet stain is applied to a non–heat-fixed smear. At this point, the cell and the capsular material will take on the
Decolorizing Agent: Copper Sulfate (20%)
Because the capsule is nonionic, unlike the bacterial cell, the primary stain adheres to the capsule but does not bind to it. In the capsule staining method, copper sulfate is used as a decolorizing agent rather than water. The copper sulfate washes the purple primary stain out of the capsular material without removing the stain bound to the cell wall. At the same time, the decolorized capsule absorbs the copper sulfate, and the capsule will now appear blue in contrast to the deep purple color of the cell.
Procedure of Capsule Stain
- Prepare thin smears of bacterial culture on a microscope slide.
- Allow the smear to only air-dry. Do not heat-fix as this will cause the capsule to shrink or be destroyed.
- Apply 1% crystal violet and allow it to remain on the slide for 2 minutes.
- With the slide over the proper waste container provided, gently wash off the crystal violet with 20% copper sulfate. Caution: Do not wash the copper sulfate and stain directly into the sink.
- Blot the slide dry with bibulous paper.
- Observe with the oil immersion lens.
Result Interpretation of Capsule Stain
Capsule: Clear halos zone against dark background
No Capsule: No Clear halos zone
Capsulated: Bacillus anthracis, Klebsiella pneumoniae, Streptococcus pneumonia, Neisseria meningitidis Clostridium spp, etc.
Non-Capsulated: Neisseria gonorrhoreae, etc.