ONPG Test – ( o-nitrophenyl-β-D-galactoside)


  • this test is used to demonstrate the presence or absence of the enzyme B-galactosidase using the substrate Ortho-nitrophenyl-D-galactopyranoside in order to differentiate Lactose-fermenting from non lactose-fermenting organisms.

Principle :

  • ONPG is a rapid test that determine if an organisms ferment lactose by identifying the presence Beta-Galactosidase. This enzyme splits the B-Galactosidase bond in lactose, producingglucose and galactose.
  • In order for lactose to be break down, it must first be transported across the cell membrane by another enzyme Permease.Organisms that posses both enzymes ferment lactose quickly,and some organisms lack the enzyme permease and appear as late or non lactose fermenters.
  • ONPG test reagent is an artificial substrate, colorless in nature,in which one molecule of o-nitrophenol is combined with galactose by B-Galactoside bond. The substrate is capable of penetrating the bacterial cell with out the presence of permease.

Procedure :

  1. Place an ONPG disk into a sterile tube and add 0.2 mL saline.
  2. Heavily inoculate the tube with a loopful of the test isolate.
  3. Incubate at 35 C for up to 4 hours.


  • The substrate diffuse ONPG into the cell, if organism posses B-Galactosidase, the enzyme will splits the B-Galactosidase bond, releasing of o-nitrophenol yellow color compound. If organism lacks enzyme. The Galactoside bond remains intact, the medium remains colorless.

Positive: yellow color within 4 hours
Negative: colorless at 4 hours

Postive and Negative ONPG Test

Postive and Negative ONPG Test

The ONPG Test

The ONPG Test

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