Testing for ONPG
ONPG (Ortho-nitrophenyl beta-D-galactopyranoside) is a synthetic colourless compound (galactoside) structurally similar to lactose and serves as chromogenic substrate for detection of β-galactosidase enzyme (an enzyme found in lactose fermenting organisms). This test is routinely used for grouping of members of family Enterobacteriaceae are according to their lactose fermenting. Lactose utilization depends upon two enzymes: β-galactoside permease (absent in late lactose fermenters), which catalyzes transport of lactose inside the cell, and β- galactosidase, which breaks down lactose into galactose and glucose. Hydrolysis of ONPG results in the release of galactose and a yellow chromogenic compound, o-nitrophenol. ONPG hydrolysis does not depend on presence of permease enzyme to enter the cell, therefore reactions are rapid and occur within a 24-hour period even for late lactose fermenters. No colour change shows negative reaction, i.e., absence of β- galactosidase activity.