Mycobacterium smegmatis is generally a non-pathogenic bacterium related Mycobacterium tuberculosis, one of the most important worldwide bacterial pathogens. Both organisms, as well as another genus named Nocardia, are considered Acid-Fast. Mycobacterium smegmatis is the organism you will stain today in order to learn the procedure for the Acid-Fast stain.
Hopefully you noticed while learning the Gram stain, that Mycobacterium smegmatis does not Gram stain well. This is because of the unique cell wall structure in the genera Mycobacterium and Nocardia. Their cell walls contain a waxy lipid called mycolic acid, which resists staining and decolorizing once it is stained. In the Acid-Fast stain a very concentrated stain solution of Carbolfuchsin (pink) is used in order to penetrate and stain the cell walls. Then, an acid based decolorizer (as opposed to the acetone based used in the Gram stain) is used to decolorize the cells. Mycobacterium and Nocardia are “acid-fast” because even the acid decolorizer will not remove the Carbolfuchsin from the cells. You can think of it as the Carbolfuchsin “holding fast” to the cell wall. Therefore, acid-fast bacteria will be pink, non-acid-fast will decolorize (lose the pink color) and can be counterstained. You will use methylene blue for the counterstain; so non-acid-fast bacteria will be blue after the procedure. There are very few acid-fast bacteria, so this stain is a valuable diagnostic tool for finding tuberculosis and nocardial diseases in patient specimens.3
Contributors and Attributions
Kelly C. Burke (College of the Canyons)