phenotypic testing of mycobacteria phenotypic testing of mycobacteria in microbiology, the phenotypic testing of mycobacteria uses a number of methods. the most-commonly used phenotypic tests to identify and distinguish mycobacterium strains and species from each other are described below. tests acetamide as sole c and n sources media: kh2po4 (0.5 g), mgso>4*7h20 (0.5 g), purified agar (20 g), distilled water (1000 ml). the medium is supplemented with acetamide to a final concentration of 0.02m, adjusted to a ph of 7.0 and sterilized by autoclaving at 115°c for 30 minutes. after sloping, the medium is inoculated with one loop of the cultures and incubated. growth is read after incubation for two weeks (rapid growers) or four weeks (slow growers). arylsulfatase test arylsulfatase enzyme is present in most mycobacteria. the rate by which arylsulfatase enzyme breaks down phenolphthalein disulfate into phenolphthalein (which forms a red color in the presence of sodium bicarbonate) and other salts is used to differentiate certain strains of mycobacteria. 3 day arylsulfatase test is used to identify potentially pathogenic rapid growers such as m. fortuitum and m. chelonae. slow growing m. marinum and m. szulgai are positive in the 14-day arylsulfatase test. catalase, semiquantitative activity most mycobacteria produce the enzyme catalase, but they vary in the quantity produced. also, some forms of catalase are inactivated by heating at 68°c for 20 minutes (others are stable). organisms producing the enzyme catalase have the ability to decompose hydrogen peroxide into water and free oxygen. the test differs from that used to detect catalase in other types of bacteria by using 30% hydrogen peroxide in a strong detergent solution (10% polysorbate 80). citrate sole carbon source egg medium growth on löwenstein–jensen medium (lj medium) l-glutamate sole carbon and nitrogen source growth rate the growth rate is the length of time required to form mature colonies visible without magnification on solid media. mycobacteria forming colonies visible to the naked eye within seven days on subculture are known as rapid growers, while those requiring longer periods are termed slow growers. iron uptake the ability to take up iron from an inorganic iron containing reagent helps differentiate some species of mycobacteria. lebek medium lebek is a semisolid medium used to test the oxygen preferences of mycobacterial isolates. aerophilic growth is indicated by growth on (and above) the surface of the glass wall of the tube; microaerophilic growth is indicated by growth below the surface. macconkey agar without crystal violet niacin accumulation (paper strip method)