ONPG Test (for β-galactosidase): Use, Principle, Procedure and Result Interpretation
Use of ONPG Test
ONPG test is useful in differentiating members of the family Enterobacteriaceae and genera Neisseria based on b-D-galactosidase activity. ONPG stands for o-Nitrophenyl-b-D-galactopyranoside.
Principle of ONPG Test
The ability of a bacterium to ferment lactose depends on two enzymes and they are permease and b-galactosidase. Permease allows lactose to enter the bacterial cell wall, where lactose is then broken down into glucose and galactose by b-galactosidase. Bacteria can metabolize glucose and galactose. The enzymes are inducible and are only present when lactose, rather than glucose, is available to the organism for metabolism. Some bacteria lack enzyme permease and appear as late lactose fermenters or non-lactose fermenters. The ONPG test will detect true non-lactose fermenters that have the b-galactosidase enzyme, even if they lack the permease enzyme. A lactose fermentation test will not detect bacteria lacking the permease. ONPG is a colorless substrate, similar in structure to lactose, used in this test as the substrate for b-galactosidase. If the organism possesses b-galactosidase, the enzyme will split the b-galactoside bond, releasing galactose and o-nitrophenol, which is a yellow compound.
Requirements for ONPG Test
Test organisms-Gram-negative rods growing aerobically or Gram-negative diplococci growing aerobically
ONPG disks ( Store at 4°C and store away from direct light since ONPG is light sensitive.)