Capsule staining

 Capsule Staining  (Negative Staining)

Aim:

To demonstrate the presence of capsule in microorganisms  .

Introduction:

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, but some are composed of glycoproteins and polypeptides. The capsule differs from the slime layer that most bacterial cells produce in that it is a thick, detectable, discrete layer outside the cell wall. Not all bacteria have the ability to form capsules even when the required genes are present. various environmental factors influence production of capsule.

Capsule facilitate the attachment of bacteria to surface, retard the loss of nutrients from the cell and help to scavenge or concentrate nutrients from the environments. Capsule staining is more difficult than other types of differential staining procedures because the capsular materials are water soluble and may be dislodges and removed with vigorous washing. Smears must 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.  

          To visualize the bacterial capsules is by using negative staining Technique. During staining the non heat fixed bacterial smear with the acidic stains such as Nigrosin will not penetrate the bacterial cells (since both acidic stain and bacterial surface has negative charge). Instead the acidic stain deposits around the bacterial cells and create a dark back ground and the bacteria appear as unstained with a clear area around them, capsule.

Materials Required:

Bacterial Cultures  (Klebsiella pnemoniae, Bacillus Sp.,)

Glass Slides

Nigrosin

Inoculation loop. 

  

Procedure: 

1.   

1.      Placed a small drop of a Nigrosin, on the clean glass slide. 
Using sterile technique, added a loopful of bacterial culture to slide, smearing it in the dye.

2.      Used the other slide to drag the ink-cell mixture into a thin film along the first slide.

3.      The slide was allowed to air dry.

4.      The slide was air dried for few minutes and  examined microscopically (100X) for the presence of encapsulated cells as indicated by clear zones surrounding the cells.

 

 Result:

Capsule: Clear halos zone against dark background
No Capsule: No Clear halos zone