SIMPLE STAINING (NEGATIVE STAINING) INTRODUTION Negative staining is type of simple staining and is so called because the dye

dose not stain the cell instead it imparts contrasting background, thereby making the organism visible. NEGATIVE STAINING METHOD PRINCIPLE Negative staining use acidic dyes like nigrosine, eosin or congo red. Acidic dyes have negatively charged chromogen, which will not bind (react) with bacterial cells because of the similar acidic (negative) charge on the surface of bacterial cell. The stein does not stein the bacteria because of ionic repulsion. Therefore cells are unstained and can be easily visualized against the background. Practical applications of negative staining are 1. Natural shape and size of the cell is not distorted because the heat fixation is not required during the staining. 2. It is possible to observe those bacteria, which are difficult to stein, such as spirochetes. Requirements 1.Young cultures of Escherichia coli , Bacillus subtilis &Stapyloccocus aureus. 3. Nigrosine staining solution (10%). Procedure 1.plase a small drop of nigrosine at the end of slide. For cultures on solid media, add a loopful of distilled water and emulsify a small amount of the culture in the nigrosine water drop. For broth cultures, mix a loopful of the culture into the drop of nigrosine. Do not spread the drop or let dry. 2. Using the end edge of another slide, spread the drop out to produce a smear varying from opaque black to gray. The angle of spreading slide will determine the thickness of the smear. 4. Let the smear air dry, and examine, (do not heat fix the slide).

Results Organisms are observed as transparent objects against the dark background.