Staining Techniques

Because microbial cytoplasm is usually transparent, it is necessary to stain microorganisms before they can be viewed with the light microscope. In some cases, staining is unnecessary, for example when microorganisms are very large or when motility is to be studied, and a drop of the microorganisms can be placed directly on the slide and observed. A preparation such as this is called a wet mount. A wet mount can also be prepared by placing a drop of culture on a cover‐slip (a glass cover for a slide) and then inverting it over a hollowed‐out slide. This procedure is called the hanging drop.

In preparation for staining, a small sample of microorganisms is placed on a slide and permitted to air dry. The smear is heat fixed by quickly passing it over a flame. Heat fixing kills the organisms, makes them adhere to the slide, and permits them to accept the stain.

Simple stain techniques. Staining can be performed with basic dyes such as crystal violet or methylene blue, positively charged dyes that are attracted to the negatively charged materials of the microbial cytoplasm. Such a procedure is the simple stain procedure. An alternative is to use a dye such as nigrosin or Congo red, acidic, negatively charged dyes. They are repelled by the negatively charged cytoplasm and gather around the cells, leaving the cells clear and unstained. This technique is called the negative stain technique.

Differential stain techniques. The differential stain technique distinguishes two kinds of organisms. An example is the Gram stain technique. This differential technique separates bacteria into two groups, Gram‐positive bacteria and Gram‐negative bacteria. Crystal violet is first applied, followed by the mordant iodine, which fixes the stain (Figure ). Then the slide is washed with alcohol, and the Gram‐positive bacteria retain the crystal‐violet iodine stain; however, the Gram‐negative bacteria lose the stain. The Gram‐negative bacteria subsequently stain with the safranin dye, the counterstain, used next. These bacteria appear red under the oil‐immersion lens, while Gram‐positive bacteria appear blue or purple, reflecting the crystal violet retained during the washing step.

Another differential stain technique is the acid‐fast technique. This technique differentiates species of Mycobacterium from other bacteria. Heat or a lipid solvent is used to carry the first stain, carbolfuchsin, into the cells. Then the cells are washed with a dilute acid‐alcohol solution. Mycobacterium species resist the effect of the acid‐alcohol and retain the carbolfuchsin stain (bright red). Other bacteria lose the stain and take on the subsequent methylene blue stain (blue). Thus, the acid‐fast bacteria appear bright red, while the nonacid‐fast bacteria appear blue when observed under oil‐immersion microscopy.

Other stain techniques seek to identify various bacterial structures of importance. For instance, a special stain technique highlights the flagella of bacteria by coating the flagella with dyes or metals to increase their width. Flagella so stained can then be observed.

A special stain technique is used to examine bacterial spores. Malachite green is used with heat to force the stain into the cells and give them color. A counterstain, safranin, is then used to give color to the nonsporeforming bacteria. At the end of the procedure, spores stain green and other cells stain red.

The Gram stain procedure used for differentiating bacteria into two groups.

Another differential stain technique is the acid-fast technique. This technique differentiates species of Mycobacterium from other bacteria. Heat or a lipid solvent is used to carry the first stain, carbolfuchsin, into the cells. Then the cells are washed with a dilute acid-alcohol solution. Mycobacterium species resist the effect of the acid-alcohol and retain the carbolfuchsin stain (bright red). Other bacteria lose the stain and take on the subsequent methylene blue stain (blue). Thus, the acid-fast bacteria appear bright red, while the nonacid-fast bacteria appear blue when observed under oil-immersion microscopy.

Other stain techniques seek to identify various bacterial structures of importance. For instance, a special stain technique highlights the flagella of bacteria by coating the flagella with dyes or metals to increase their width. Flagella so stained can then be observed.

A special stain technique is used to examine bacterial spores. Malachite green is used with heat to force the stain into the cells and give them color. A counterstain, safranin, is then used to give color to the nonsporeforming bacteria. At the end of the procedure, spores stain green and other cells stain red.