ONPG Test Introduction

Biochemical tests helps us to determine the characteristics of the microbe in the medium. One such biochemical test is ONPG test. T

he ability of the bacteria to ferment the lactose is generally based on two enzymes named permease and the Beta-galactosidase.

Here permease allows the lactose to enter the cell wall of the bacteria easily then further it breaks down into glucose and the galactose molecules with the help of an enzyme Beta-galactosidase.

Glucose and the Galactose is further metabolized by the bacteria. On the other hand, some organisms lack the enzyme permease and it appears as a late or a non-lactose fermenter.

ONPG test is considered as one of the sensitive tests for fermenting the molecules like lactose. O-nitrophenyl-Beta-D-glucopyranoside is one of the artificial substrates which is incorporated into the medium used here which acts as the important substrate for the beta-galactosidase to ascertain the specific enzymatic activity and it also helps in aiding the identification and differentiation of the different organisms.

ONPG Test Objective

The main aim of the test is to determine the ability of the organism to produce an enzyme known as Beta-galactosidase.

ONPG Test Principle

O-nitrophenyl-beta-D-glucopyranoside, is an artificial substrate which is structurally similar to those of lactose, but this is exception to that of the glucose, substituted with an o-nitrophenyl group. Unlike substrate O-nitrophenyl-beta-D-glucopyranoside is capable of entering into the cell wall of the bacterium without the help of an enzyme permease.

While testing the organism into the broth medium, the organism is taken from a medium containing a high concentration of lactose and is inoculated into the ONPG broth.

If the organism contains the enzyme Beta-galactosidase, which helps in splitting the Beta-galactoside bond, which releases the O-nitrophenol an yellow-colored compound, which indicates the positive result.

Whereas in disk method, the organism that is being tested is taken from the medium containing a high concentration of the lactose. A dense suspension is prepared.

Further the ONPG disk is added to a 5.0 ml of the suspension. If the specific organisms’ posses the beta-galactosidase, the enzyme splits the Beta-galactosidase bond and results in the formation of a yellow color along with the change in suspension.

Organisms having a strong Beta-galactosidase activity helps in producing a positive reaction withing few minutes of inoculating the ONPG medium, where as the other organisms take up to 24 hours.

ONPG Test Reagents

ONPG Test Broth:

Ingredients	Gram per liter
Na₂HPO₄	9.46
Phenylalanine	4
ONPG	2
KH₂PO₄	0.907

ONPG Test Disk

ONPG differentiation disk is usually prepared by carefully impregnating the concentration of the ONPG into a 0.25 inch of diameter filter paper.

ONPG Test Procedure

ONPG test can be performed using two methods.

i. ONPG Test Disk Method

 Initially the ONPG disk is placed into a sterile tube and about 0.2ml of saline is added into it. Further the heavy inoculate is placed in the tube with a loopful of the test isolate.

 The inoculum is then incubated at a temperature of about 35 to 37ºC for about 4 hours.

 After incubation the disk is observed for any of the colour changes.

ii. ONPG Test Broth Method

 Initially the test medium is brought to a room temperature.

 Further the test medium is inoculated with the help of a heavy inoculum using a loop full of test isolate.

 Then the inoculum is incubated aerobically by loosening the caps and it is incubated at a temperature of about 35 to 37ºC.

 After incubation the development of yellow colour can be seen for positive results within one hour.

 If there is no change in colour in the medium after one hour of the incubation period, then the incubation is let to continue for next 24 hours.

ONPG Test Results

Positive ONPG Test: In case of positive test, the formation of yellow colour can be seen in the medium.

Negative ONPG Test: If the results are negative, then there will be no formation of the yellow colour.

ONPG Test Uses

 Generally, this test is used for differentiating the members of the Enterobacteriaceae form the other micro-organism depending on the activity of the enzyme Beta-D-galactosidase.

 This test also helps us to distinguish the lactose late fermenters from the non-lactose fermenters of the Enterobacteriaceae family.

ONPG Test Limitation

 Cultures which produce a natural yellow pigment cannot be tested using this media.

 For the complete identification of the biochemical, immunological and the molecular testing’s, the colonies that are taken from the pure culture is recommended.

 All the organisms that are tested is inoculated from the lactose that contains a medium.