Negative Stain

The **negative stain** is an example of an indirect stain, using **nigrosine** as the staining agent. This stain is negatively charged, which prevents its penetration into cell walls due to the repulsive forces between like charges. Instead, it colors the background, leaving the cells unstained, which enhances their visibility. The primary purpose of negative staining is to allow for the observation of living cells without heat fixing, a method that can distort their shape. To begin the negative staining process, aseptically transfer a small drop of 10% nigrosine onto one end of a sterilized glass microscope slide. Next, using a sterile wire inoculating loop, mix a small amount of bacteria with the nigrosine on the slide. Then, use a second sterile slide at a 45-degree angle to spread the mixture across the slide, forming a thin film. Allow the smear to air dry fully before examining it under oil immersion using a microscope. Utilizing the negative staining method, the stain surrounds the bacteria, making them considerably easier to identify.