The ONPG (ortho-Nitrophenyl-beta-D-Galactopyranoside) test determines if an organism can hydrolyze lactose.
Lactose is a disaccharide composed of galactose and glucose. It is hydrolyzed by beta-galactosidase to galactose and glucose.
ONPG is structurally similar to lactose with orthonitrophenol substituted for glucose. Hydrolysis of ONPG (which is colorless) releases orthonitrophenol (which has a yellow color).
A second enzyme permease allows lactose to enter bacteria. Lactose-fermenting bacteria with permease develop a positive ONPG result more rapidly than bacteria that do not have permease.
The test is performed on a Gram-negative bacteria.
An isolate grown on a media containing lactose (KIA, TSI) produce optimal results.
Testing:
(1) An emulsion is prepared by mixing isolate in physiologic saline.
(2) A small amount of toluene is added to the mixture followed by mixing, which breaks down the bacterial wall and releases beta-galactosidase.
(3) ONPG is then added, either as a solution or from a tablet.
(4) The mixture is incubated in a 37°C water bath for 24 hours.
Interpretation:
• A positive result is indicated by a yellow color.
• Most lactose-fermenting bacteria are positive within an hour, but a test is not deemed negative until after 24 hour incubation.
Causes of a false negative:
(1) reading the test as negative too soon
Causes of a false positive:
(1) mixed isolate
