NEGATIVE STAINING

 

NEGATIVE STAINING

AIM: To perform the negative staining for the given bacterial culture to study the morphological feature of the organism.

PRINCIPLE: Negative staining requires an acidic dye such as India Ink or Nigrosin. India Ink or Nigrosin is an acidic stain. This means that the stain readily gives up a hydrogen ion (proton) and the chromophore of the dye becomes negatively charged. Since the surface of most bacterial cells is negatively charged, the cell surface repels the stain. The glass of the slide will stain, but the bacterial cells will not. The bacteria will show up as clear spots against a dark background.

MATERIALS REQUIRED: Bacterial culture, Inoculation loop, Nigrosin or India ink, Glass slides,

PROCEDURE:

1.      Place a small drop of nigrosin close to one end of a clean slide.

2.      Using aseptic technique, place a loopful of inoculum from the bacterial culture in the drop of nigrosin and mix.

3.      Place a slide against the drop of suspended organisms at a 45° angle and allow the drop to spread along the edge of the applied slide.

4.      Push the slide away from the drop of suspended organisms to form a thin smear. Air-dry.

5.      Examine the slides under oil immersion.

RESULT:  When viewed with the microscope the bacterial cells appear light against the dark surrounding background.