Studyaid4microbiologists in Unical

NEGATIVE STAINING

main purpose of Negative staining is to study the morphological shape, size and arrangement of the bacteria cells that is difficult to stain. eg: Spirilla. It can also be used to stain cells that are too delicate to be heat-fixed.

It is also used to prepare biological samples for electron microscopy. It is used to view viruses, bacteria, bacterial flagella, biological membrane structures and proteins or protein aggregates, which all have a low electron-scattering power

Principle of Negative Staining

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.

Reagents of Negative Staining

India ink :Nigrosin (Nigrosin 100 gm/L, Formalin 5 ml/L in water)

Procedure of Negative Staining

1. Place a very small drop (more than a loop full, less than a free falling drop from the dropper) of nigrosin near one end of a well-cleaned and flamed slide.

2. Remove a small amount of the culture from the slant with an inoculating loop and disperse it in the drop of stain without spreading the drop.

3. Use another clean slide to spread the drop of stain containing the organism using the following technique.

4. Rest one end of the clean slide on the center of the slide with the stain. Allow the stain to spread across the edge of the spreader and swiftly move the spreader forward to create a thin smear.

5 Air-dry the thin smear.
6. Focus a thin area under oil immersion and View unstained cells which are gray in colour via the electron microscope....

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Good news for microbiologist in Nigeria

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