Capsule staining



Principle

A capsule is a gelatinous outer layer that is secreted by the cells 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, a capsule is a polysaccharide, a glycoprotein, or a polypeptide. Capsule staining is more difficult than other types of differential staining procedures because the capsular material is water-soluble and may be dislodged and removed by vigorous washing. Smears should not be heated because resultant cell shrinkage may create a clear zone around the organism that is an artifact and may be mistaken as a capsule.

Primary stain

A violet stain is applied to the non-heat fixed smear. At this point, the cell and the capsular material will take on the dark color.

Decolorizing Agent 

Copper sulfate (20%): because the capsule is non-ionic, unlike the bacterial cell. The primary stain will only adhere to it but does not bind to it. Copper sulfate used as a decolorizing agent will wash the primary stain out of the capsular material without removing the stain bound to the cell. At the same time, the decolorized capsule will absorb the copper sulfate and will appear as blue in contrast to the deep purple colored cell. 

Materials

24 h broth culture of bacteria (Enterobacter aerogenes), 1% crystal violet, 20 % Copper sulfate, Bunsen burner, inoculation loop, staining tray, glass slides, microscope. 

Procedure 

  1. Obtain a clean glass slide by washing with acid alcohol.
  2. Place several drops of crystal violet stain on the slide.
  3. Add three loopful cultures of bacterial suspension and gently mix with inoculation loop.
  4. With the edge of the other slide spread the mixture to perform a smear. 
  5. Wait for 5-7 min. Allow the smear to air dry
  6. Wash the smear with 20% copper sulfate.
  7. Gently blot dry the slide and examine under oil immersion lens of a microscope

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