There are different types of bacteria and each has different abilities and reactions to certain organisms. The lactose-fermenting ability of bacteria’s ability primarily depends on the enzyme’s permease and beta-galactosidase.

What permease does is it enables lactose to get through the cell wall of the microorganism and allowing beta-galactosidase to break it down into galactose and sugar. The bacteria will metabolize galactose and glucose. Some bacteria do not have permease and classified as non-lactose fermenters.

To find out the lactose fermenting ability of bacteria, a test has to be performed – the ONPG Test. It is an extremely sensitive test used to check the ability of the bacteria to ferment lactose.

It uses an artificial substrate called O-nitrophenyl-beta-D-galactopyranoside to ascertain enzymatic activity which helps in the identifying and differentiating various types of organisms. (1, 2, 3, 4, and 5)

Two tubes

Image 1: Two tubes – the one on the left with no obvious changes in color while the one on the right turned yellow, which is an indicator of the presence of enzymes.
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What is the purpose of the ONPG test?

The purpose of the ONPG test is to check for the organism’s ability to produce the β-galactosidase enzyme.

What are the uses of the ONPG test?

  •  It is performed to help differentiate the members of Enterobacteriaceae and other microorganisms according to their beta-D-galactosidase activity.
  • It helps in distinguishing Enterobacteriaceae that are late lactose fermenters non-lactose fermenters. (4, 5, and 6)
ONPG test of Streptococcus bacteria as seen on two test tubes

Image 2: An ONPG test of Streptococcus bacteria as seen on two test tubes.
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What is the principle of the ONPG test?

The test uses an artificial substrate called O-nitrophenyl-beta-D-galactopyranoside, which is similar in structure to that of lactose. The sugar is replaced with the o-nitrophenyl group, which has the ability to penetrate the cell in the absence of permease.

When using the broth way, the organism to be tested is taken out from a medium that contains a high level of lactose. The organism is inoculated using the ONPG broth. You will know if it contains beta-galactosidase if the beta-galactoside bond splits allowing the release of o-nitrophenol; a compound characterized by its yellow color.

Once you notice these changes, it means that the organism being tested produces the β-galactosidase enzyme.

If the disk method is used, the subject of the test is taken out from a medium that contains a high level of lactose. A suspension is prepared wherein an ONPG disk is added to about 0.5 ml of suspension.

Watch out for changes in color, specifically yellow color as it indicates that the organism being tested has beta-galactosidase.

The changes in color usually take place within 24 hours. If the organism has a high level of beta-galactosidase, the changes in color is rapid. It would only take a few minutes’ post ONPG medium inoculation. (4, 5, 6, 7, and 8)

How is the ONPG test done?

There are two methods of performing the ONPG test: the broth method and the disk method.

Broth Method

Media – ONPF broth which consists of ONPG, Na2HPO4, KH2PO4 per 1 liter and a pH of 8.0.


  1. The test medium should be brought into room temperature.
  2. The test medium should be inoculated with heavy inoculum from a pure culture 18 hour to 24 hours.
  3. Incubate with a loose cap at the desired temperature of 35 to 37-degree centigrade.
  4. After an hour, look for any changes in color.
  5. If after an hour and no changes in color are detected, then continue the incubation for 24 hours.

Disk Method

Media – To prepare the disk, you have to impregnate a controlled concentration of ONPG onto a filter paper disk, which is around0.25-inch in diameter.


  1. Put the ONPG disk into the test tube and slowly add a saline solution (0.2 ml).
  2. Inoculate the tube using a loop that is full of test isolate.
  3. Incubate for 4 hours at a temperature of 35 to 37 degrees Celsius.
  4. Examine the disk for any changes in color. (8, 9, and 10)

Interpreting Results

  • Positive Results – if the color changes to yellow, it is an indicator of the presence of β-galactosidase. It is easy to tell the changes in color as the disc and the fluid turn yellow.
  • Negative Results – If the color remains the same, it means that the organism being tested does not contain the β-galactosidase enzyme.

Are there any limitations?

  1. It is not ideal to use in cultures that have naturally producing yellow pigment.
  2. ONPG test alone is not reliable for complete identification of microorganisms. Other tests have to be done such as biochemical, molecular, immunological, and mass spectrometry.
  3. The organism subject for testing should be inoculated from a lactose-containing medium. (2, 5, and 9)



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