ONPG Test- Principle, Procedure, Results, Uses


The ONPG test is a biochemical test that can be used to determine the ability of bacteria to produce the enzyme β-galactosidase. This enzyme is responsible for breaking down lactose, a sugar found in milk and dairy products, into glucose and galactose. The ONPG test can help to differentiate between lactose fermenters and non-lactose fermenters among Gram-negative bacteria, especially the members of the Enterobacteriaceae family and some Neisseria species.

Lactose fermenters are bacteria that can utilize lactose as a source of energy and produce acid and gas as by-products. Non-lactose fermenters are bacteria that cannot metabolize lactose and instead use other sugars or organic compounds. The ability to ferment lactose is an important characteristic for the identification and classification of many bacterial pathogens, such as Escherichia coli, Salmonella, Shigella, Klebsiella, Vibrio and others.

The ONPG test is based on the use of a synthetic substrate called o-nitrophenyl-β-D-galactopyranoside (ONPG), which mimics the structure of lactose but can enter the bacterial cell without the need of a transport protein called permease. Permease is another enzyme that facilitates the uptake of lactose into the cell. Some bacteria can produce β-galactosidase but not permease, which makes them slow lactose fermenters. The ONPG test can detect these bacteria by measuring their β-galactosidase activity independently of permease.

The ONPG test is simple, rapid and inexpensive to perform. It can be done by using either a liquid broth or a paper disk impregnated with ONPG. The test is positive when the bacteria produce β-galactosidase and cleave ONPG into galactose and o-nitrophenol, which is a yellow compound that changes the color of the medium or the disk. The test is negative when the bacteria do not produce β-galactosidase and no color change occurs.

The ONPG test has several applications in microbiology, such as:

  • Differentiating lactose fermenters and non-lactose fermenters among Gram-negative bacteria
  • Determining the presence or absence of permease in lactose fermenting bacteria
  • Characterizing and identifying bacterial isolates based on their biochemical profiles
  • Screening for potential mutants or recombinants that have altered β-galactosidase activity