What Is the Purpose of Acid Fast Staining?

In microbiology, staining a bacteria means to color it with a dye to see it more clearly under a microscope. If bacteria are not stained, they appear clear and colorless, and it is difficult to see their shapes. Aside from their shapes, how easily a bacteria is dyed by certain stains--if at all--often provides a clue to identify the bacteria. Acid-fast staining is a type of staining that is effective on some bacteria that cannot be stained by other methods.

Differential Staining

• To stain a normal bacteria, liquid stain is applied to the slide for about one minute, then rinsed off with water. Differential staining uses different stains for two different bacteria in the same slide. For example, in a Gram stain, the slide is first stained with crystal violet and iodine, then decolorized with alcohol. Cells that are Gram-positive will retain the dark purple color of the first stain even after the alcohol is used, while Gram-negative cells will return to colorless. Then a second stain is applied, in a contrasting color such as pink, and the Gram-negative cells will turn pink while the Gram-positive cells will hold their original stain.

History

• Some bacteria have thick cell walls made up of lipids, the building blocks of fats, which makes them waxy and nearly waterproof. This means a normal stain can't penetrate the cell. In the 1880s, scientists were trying to study Mycobacterium tuberculosis, the bacteria that causes tuberculosis. However, it would not hold a regular Gram stain unless it was immersed in the stain for up to 24 hours. Paul Ehrlich, who later developed the first drug effective against syphilis, developed acid-fast staining to force the stain into the bacteria. Ehrlich's method only took 45 minutes, while modern acid-fast staining technique takes only 5 minutes.

Method

• To force the dye, usually carbol-fuchsin, into the bacteria, it must be heated over a beaker of boiling water for 5 minutes, while the slide is constantly flooded with stain and not allowed to dry out. An alternative method to heat is to use modified Kinyoun carbol fuchsin stain, which does not need to be heated, but it is imperative that the slide does not dry out during 5 minutes of immersion in the stain. Whichever primary staining method is used, the slide is decolorized with acid alcohol--not regular alcohol--and stained with a secondary counterstain of a different color to show the other bacteria that may be present. Bacteria whose primary stains remain after decolorizing with acid alcohol are called acid-fast.

Organism precaution

• Mycobacterium tuberculosis is extremely hazardous, and is only examined in hospitals and clinical facilities that have separate rooms or specially designed transfer hoods to prevent contamination. It is illegal to use it in an academic laboratory. Instead, Mycobacterium smegmatis is used, but it has a much thinner cell wall than M. tuberculosis, so it's easier to accidentally over-decolorize the slide and remove the primary stain.

Procedural precaution

• Carbol-fuchsin fumes are toxic, so when heating them, use a fume hood to contain the fumes and vent them outside. Also, to avoid the dangers of heating slides altogether, Kinyoun carbol fuchsin can be used instead, which has a higher concentration of phenol and fuchsin, two active ingredients in the stain, and does not need to be heated to penetrate the bacteria.