Indirect (negative) staining Most biological materials show little contrast with their surroundings unless they are stained. In the case of light microscopy, contrast can be enhanced by using colored stains which selectively absorb certain wavelengths.

Indirect (negative) staining To heighten the contrast between bacteria and the background, use is made of electron-dense "stains". These are usually compounds of heavy metals of high atomic number, that serve to scatter the electrons from regions covered with the stain. If bacterial particles are coated with stain (positive staining), fine detail may be obscured.

Indirect (negative) staining Negative staining overcomes this problem by staining the background and leaving the bacteria relatively untouched. The negative stain is molded round the bacterial particle, outlining its structure, and is also able to penetrate between small surface projections and to delineate them.

Indirect (negative) staining The acidic dye nigrosine will be used to visualize the capsule or sheath that surrounds some bacteria in a process called negative staining. Capsules are composed primarily of polysaccharides or glycoproteins and are gelatinous in texture.

Indirect (negative) staining They are readily destroyed by heating and hence direct staining methods cannot be utilized. In general, the size and shape of microorganisms is often less distorted with indirect staining procedures, especially when sampled from a broth culture

Indirect (negative) staining Therefore negative staining is useful whenever the morphology of individual bacteria is in question. Morphology can often be determined with confidence with only the high dry lens. Consider that this procedure does not necessarily kill the organism, so be careful.

Indirect (negative) staining After preparing a clean, greaseless slide, a small drop of nigrosine is mixed with a small drop from a broth culture or with a quantity of dry material. The drop is spread across the slide using the edge of another slide as a spreader. This same procedure is used for blood smears.