The second differential stain you will perform is an Acid-fast stain. This stain is used in the identification of bacteria of the genera Mycobacterium, which are bacilli. Mycobacterium tuberculosis is the causative agent of tuberculosis and Mycobacterium leprae causes leprosy. The acid-fast stain is most commonly used on the clinical sample sputum when tuberculosis is suspected. In lab we will use Mycobacterium smegmatis (M. smeg), which is a non-pathogenic bacterium in this genus. Mycobacteria have a cell wall that is different from both Gram-negative and Gram-positive bacteria. Mycobacteria can stain either Gram-positive or Gram-negative depending on the age of the culture. They are said to be “Gram variable.” The cell wall of Mycobacteria contains a waxy substance called mycolic acid. The presence of this waxy material means that it is difficult to get stains into and out of Mycobacteria. In an acid-fast stain, the primary stain carbolfuchsin is forced into Mycobacteria with heat. Mycobacteria are not easily decolorized with acid-alcohol after staining with hot carbolfuchsin; they are said to be acid fast. (An old term for “won’t fade in the wash” was color-fast.) Non-acid-fast bacteria will decolorize under this rigorous regime. After the decolorization process, the cells are counterstained with crystal violet. All cells that were decolorized will now stain blue/purple. Cells that decolorize with the acid alcohol are known as ‘non acid fast’ bacilli/cocci. The most important clinical use of acid-fast staining is in the diagnosis of tuberculosis.