* The main purpose of negative staining is to study the Morphological shape, size and arrangement of the bacteria cells that is difficult to stain e.g. Spirilla.
* It can also be used to stain cells that are too delicate to be heat-fixed.
* As heat fixation is not required in negative staining, the cells are not subjected to the distorting effect of chemicals and heat, due to which their natural size and shape can be seen.
* 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 -
Negative staining is based upon the principle that there is no reaction between the stain and the specimen. This is accomplished when the stain is used at a pH at which the interaction between stain and biological materials is negligible.
Mechanism -
* The acidic stain, with its negatively charged chromogen is used in negative staining.
* Since the surface of most bacterial cells is negatively charged, the cell surface repels the acidic stain.
* The glass of slide i.e. background will stain, but the bacterial cells will not.
* The bacteria will show up as clear spot against a dark background.
Stains -
Acidic stains such as 10% Nigrosin, 2% Congo red, India ink etc.
Procedure -
* 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.
* 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.
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* Use another slide against the drop of suspended organisms at a 45° angle and allow the drop to spread along the edge of the applied slide.
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* Push the slide away from the drop of suspended organisms to form a thin smear Air dry.
* Note: Do not heat fix the slide.
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* Focus a thin area under oil immersion and observe the unstained cells surrounded by the grey stain.
Observation -
Dark background with a light specimen.
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