2.4.1: Acid-Fast Stain

The acid-fast stain, while not as commonly used as the Gram stain, is an important diagnostic tool. The acid-fast stain is used to detect the presence of bacteria that contain waxy lipids known as mycolic acids. Mycolic acids are waxes found embedded in the thick cell walls of bacteria in the genera Mycobacteria and Nocardia. The waxes prevent the cells from desiccating and protect them from many chemical cleaners.

Some clinically noteworthy examples of species that contain mycolic acids are Mycobacterium tuberculosis and Mycobacterium leprae, which are known to cause tuberculosis and leprosy respectively.

The mycolic acids not only protect the bacteria from harsh environments but also make it more challenging to stain cells within these two genera. Many of the stains employed in microbiology are aqueous, meaning that they are mixed with water, thus causing the solution to be a polar solution. These do not interact well with the non-polar nature of the mycolic acids, and thus are not easily taken into the cells. The staining procedure you will employ to stain acid-fast cells is known as the Ziehl-Neelsen protocol. The acid-fast staining procedure uses a primary stain known as carbolfuchsin. Carbolfuchsin is a dark red stain that is lipid soluble, thus allowing it to penetrate the waxy mycolic acid found in the cell walls. In addition to using a lipid soluble stain, heat is applied at the same time the primary dye is added to the smear. This is done by placing the slide over a steam bath and then applying the stain. The heat melts the waxy lipid, thereby facilitating the entry of the stain into the cell. The slide is then removed from the heat and allowed to cool, and the wax then solidifies trapping the primary stain in cells containing mycolic acid.

After rinsing with water, a decolorizer is applied to the smear. The decolorizer used in this protocol is acid-alcohol, a combination of ethyl alcohol and hydrochloric acid. The decolorizer is repelled by cells containing mycolic acid, because of its polar nature, thereby allowing the primary stain to remain in the cells. These cells are referred to as acid-fast cells because they have held fast or tightly to the primary stain. Cells that do not have mycolic acid are left colorless after the application of the decolorizer because the stain is removed from the cell, and thus are referred to as non-acid fast. The counter-stain methylene blue is then applied. Methylene blue cannot enter the cells of acid-fast bacteria because it is repelled by the mycolic acid. Non-acid-fast cells, lacking mycolic acid, do not repel the stain, and thus are readily stained blue. Upon completion of the staining procedure, acid-fast bacteria will appear red, while non-acid-fast bacteria appear blue.

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