CAPSULE STAINING

 

CAPSULE STAINING

AIM: To prepare a smear of an encapsulated bacterium and stain its capsule and to visualize the capsule and differentiate it from the cell body.

PRNCIPLE: Capsules stain very poorly with reagents used in simple staining and a capsule stain can be, depending on the method, a misnomer because the capsule may or may not be stained. Negative staining methods contrast a translucent, darker colored, background with stained cells but an unstained capsule. The background is formed with india ink or nigrosin or congo red. India ink is difficult to obtain nowadays; however, nigrosin is easily acquired. A positive capsule stain requires a mordant that precipitates the capsule. By counterstaining with dyes like crystal violet or methylene blue, bacterial cell wall takes up the dye. Capsules appear colourless with stained cells against dark background. Capsules are fragile and can be diminished, desiccated, distorted, or destroyed by heating. A drop of serum can be used during smearing to enhance the size of the capsule and make it more easily observed with a typical compound light microscope.

MATERIALS REQUIRED: Crystal violet or Nigrosin, Bacterial culture, Glass slides, Inoculation loop, Microscope

PROCEDURE: 

1. Place a small drop of a negative stain on the slide. 
2. Using sterile technique, add a loopful of bacterial culture to slide, smearing it in the dye.
3. Use the other slide to drag the ink-cell mixture into a thin film along the first slide and let stand for 5-7 minutes.
4. Allow to air dry (do not heat fix).
5. Flood the smear with crystal violet stain (this will stain the cells but not the capsules) for about 1 minutes. Drain the crystal violet by tilting the slide at a 45 degree angle and let stain run off until it air dries .
6. Examine the smear 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