ONPG or o-NITROPHENY-B-GALACTOPYRANOSIDE TEST Atlas page 68-9

The ONPG Test is used to identify bacteria capable of producing the enzyme B-galactosidase.  In order for bacteria to ferment lactose, they must possess two enzymes: B-galactoside permease (a membrane-bound transport protein) and B-galactosidase an intracellular enzyme that spilts the disaccharide into B-glucose and B-galactose. Bacteria possessing both enzymes are active B-lactose fermenters.  Enteric bacteria possessing neither enzyme do not ferment lactose and are often pathogenic.

 

Bacteria that possess B-galactosidase but no B-galactoside permease may mutate, and over a period of days or weeks, begin to produce the permease. This 3rd group of LATE LACTOSE FERMENTERS can be separated from the non-lactose fermenters (pathogens) using a compound ONPG.  This compound can enter the cell without the assistance of a permease and react with B-galactosidase producing a YELLOW color.

PROCEDURES:

B-galactosidase is an inducible enzyme and is only produced when the inducer (LACTOSE) is present.   To avoid false negatives, organisms are first cultured in lactose rich media (Kligler's Iron Agar or TSI Agar).

After the initial inoculum is obtained from a 2 lab old KIA agar it is placed into a sterile test tube containing sterile water containing a dissolved ONPG tablet and incubated at 37C.   The yellow positive color will develop overnight.

After the initial inoculum is obtained from a 2 lab old KIA agar it is placed into a sterile test tube containing sterile water containing a dissolved ONPG tablet.   The yellow positive color will appear overnight.