Staining of Microorganisms

one of the most widely used microbiological techniques, involving the staining of fixed cells of microorganisms with special dyes. The cells are applied with a loop to a drop of water on a slide or cover glass. After the cell suspension dries, the preparation is fixed with special fluids. To highlight the morphology of cells, alcohol solutions of the basic dyes methylene blue, gentian violet, or fuchsine or the acidic dyes erythrosine or eosin are used. The spores or flagella of bacteria and the mucous capsule of some microorganisms are stained using special techniques. Gram’s method of diagnostic staining is widely used. Coccal and spore forms of bacteria and yeasts are Gram-positive and stain blue. Many bacteria that do not form spores are Gram-negative and stain red.

Certain dyes or chemical reagents are used for microscopic chemical analysis, that is, to detect organic compounds in cells. For example, intracellular lipids are stained black by osmic acid anhydride and red by Sudan. Red metachromatin granules precipitate in vacuoles with neutral red stain. Lugol’s solution stains starch cinnamon-brown and granulose blue. Acid-resistant bacteria, for example, mycobacteria (specifically, the causative agents of leprosy and tuberculosis in man and animals), are stained red by fuchsine and do not lose their color when treated with sulfuric acid. A common procedure is vital staining, that is, the staining of nonfixed, living microbial cells. Fluorescent dyes, for example, acridine orange, in combination with fluorescence microscopy are used to distinguish living cells, which stain green, from dead ones, which stain red.

Another widely used technique is to combine fluorescent dyes with serum that contains antibodies to a certain microbial species: only the cells of the given species will fluoresce under the microscope. This method permits the researcher to identify a microorganism in the soil or a pathogenic microbe in the intestine, blood, or sputum of a patient without resorting to culture techniques, which are considerably more time-consuming.