ONPG test principles,Composition and Cultural response

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Ortho-nitrophenyl-b-D-galactopyranoside abbreviated ONPG is a microbiological biochemical test used for the rapid detection of  the enzyme b-galactosidase activity in some microorganisms, especially those late lactose fermenters (cryptic lactose fermenters).

Lactose fermentation by microorganisms requires a couple of enzymes and one of which is beta-galactosidase

ONPG  is a synthetic colourless compound (galactoside) which is structurally similar to lactose hence used as a molecular decoy which is broken down by the enzymes Beta-galactosidase to release a yellow colored en point substance called O-nitrophenol by those beta-galactosidase positive microorganisms.

In Order to demonstrate lactose fermentation,the presence of two enzymes is required to in a conventional test. The first enzyme is permease which facilitates the entry of lactose molecules into the bacterial cell while the second enzyme required is b-galactosidase which hydrolyzes the lactose to yield glucose and galactose.

These enzymes are not found in true non-lactose fermenters microorganisms.There are some organisms that lack  the enzyme permease but possesses beta-galactosidase.These organisms are called Lates lactose fermenters (cryptic lactose fermenters).

This test is mostly used to differentiate enterobacteriaceae since they are routinely grouped according to their lactose fermenting ability and some some Staphylococcus species

Principle of ONPG test

  • Casein peptone serves as a source of nitrogenous compounds and other growth factors.
  • Phosphate is responsible in buffering the medium.
  • Sodium chloride maintains the osmotic balance.
  • Ortho-nitrophenyl beta-D-galactopyranoside serves as a substrate for the presence of beta galactosidase enzyme.

Composition of ONPG Broth

Casein Peptone7.500
Sodium Phosphate Dibasic0.350
Sodium chloride3.750
Final pH at Temperature 25°C7.5±0.2
Also read  Difference Between Fat-Soluble and Water-Soluble Vitamins

Preparation of ONPG Broth

  • Suspend 13.10 grams in 1000 ml distilled water.
  • Heat to boiling to dissolve the medium completely.
  • Cool to 45-50°C and dispense into sterile tubes.


Appearance of ONPG Broth

  • It’s appears Light amber coloured clear solution in tubes after preparation
ONPG broth
ONPG broth

Procedure for Use of ONPG Broth

  • Using a sterile wire loop or glass rod,Heavily inoculate on ONPG Broth the isolated colonies from a pure, 18-24 hour culture.
  • Now aerobically incubate the broth at 33-37°C for 1 to 24 hours
  • Examine for a yellow color development at 1 hour and up to 24 hours.

Interpretation of ONPG Test

  • Positive Test – Yellow color development
  • Negative Test – No color change
ONPG Negative(Left) Positive(Right)
ONPG Negative(Left) Positive(Right)

Cultural Response

OrganismONPG Test response
Citrobacter freundii yellow colour
Enterobacter aerogenesyellow colour
Escherichia coliyellow colour
Proteus vulgarisno colour change
Salmonella Typhimuriumno colour change

Reference .

  • Lowe G.H., 1962., J. Med. Lab. Technol., 19:21
  • Seidman, M. and K.P. Link. 1950. J. Am. Chem. Soc. 72:4324.
  • Lederberg, J. 1950. J. Bacteriol. 60:381.
  • LeMinor, L. and F. Ben Hamida. 1962. Ann. Inst. Pasteur. 102:267.
  • W.H. Ewing. 1986. Edwards and Ewing’s Identification of Enterobacteriaceae. 4th ed. Elsevier, New York, N.Y.
  • Winn, W., S. Allen, J. William, E. Koneman, G. Procop, P. Schreckenberger, and G.
  • Woods. 2006. Koneman’s Color Atlas and Textbook of Diagnostic Microbiology. 6th ed. Lippincott Williams and Wilkins, Baltimore, MD.

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About the Author: Arthur Westmann

DEFFE ARTHUR (AMOEBAMANN) is the founder and author of MLTGEEKS and MLTEXPO.He’s from Cameroon and is currently a Final year State Medical Laboratory Technician (MLT MA). Beyond lab works, he’s a passionate internet user with a keen interest in web design and blogging. Furthermore He likes traveling, hanging around with friends and social networking to do in his spare time.

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