Mycobacteria have traditionally been treated as a separate group of bacteria in most clinical laboratories because of certain distinguishing characteristic. First, most strains are slow growing, having prolonged doubling time, ranging from 2-22 hours. This requires an ideal culture environment to be maintained for prolonged periods and the more rapidly growing contaminating bacterial species to be eliminated from the specimens. Determination of the enzyme arylsulphatase activity in Mycobacteria is helpful in identifying certain species, notably in differentiating members of the rapidly growing Mycobacteria fortuitism from group III non-photo chromogenic Mycobacteria. Some of the slower-growing species do not produce sufficient enzyme to give a consistently positive reaction. Wayne Sulphatase Agar was developed by Wayne (to enable recognition of M.fortuitum) using a 3-day phenolphthalein sulphatase test. Rapid growing and slow growing species of Mycobacterium can be differentiated, based on the 3 days test or 2 weeks test respectively. Some species of Mycobacteria produce arylsulphatase, an enzyme that attacks the substrate component viz. tripotassium phenolphthalein sulphate, with the resultant release of free phenolphthalein as indicated by a color change (red) in the medium after addition of sodium bicarbonate reagent.