ONPG Test: Introduction, Principle, Test Requirements, Procedure, Result -Interpretation, and Keynotes

Introduction of ONPG Test

ONPG test is useful in differentiating members of the family  Enterobacteriaceae and genera Neisseria based on b-D-galactosidase activity. ONPG stands for o-Nitrophenyl-b-D-galactopyranoside. Here we discuss on ONPG Disk Test which is simple and easy to perform.

ONPG Test Negative (left) and Positive Bacteria (right)
Fig. ONPG Test Negative (left) and Positive Bacteria (right)


The ability of an organism to ferment lactose depends on two enzymes and are permease and b-galactosidase. Permease allows lactose to enter the bacterial cell wall, where lactose is then broken down into glucose and galactose by the enzyme, b-galactosidase. Bacteria can metabolize glucose and galactose. The enzymes are inducible and are merely present when lactose, rather than glucose, is available to the bacterium for metabolism. Some organisms lack enzyme permease and appear as late lactose fermenters or non-lactose fermenters.

The ONPG test will detect true non-lactose fermenters (NLF) that have the b-galactosidase enzyme, even if they lack the permease enzyme. A lactose fermentation test will not detect bacteria lacking the permease. ONPG is a colorless substrate, similar in structure to lactose, applied in this test as the substrate for b-galactosidase. If the bacterium possesses b-galactosidase, the enzyme will split the b-galactoside bond, releasing galactose and o-nitrophenol, which is a yellow compound.

Requirements for ONPG Test

  • Test organisms-Gram-negative rods growing aerobically or Gram-negative diplococci growing aerobically
  • ONPG disks (Store at 4°C and store away from direct light since ONPG is light sensitive.)
  • Test tubes
  • Inoculating loops
  • Sterile physilogical saline
  • Incubator
  • Control strains to check quality control of test-Positive control (PC): Escherichia coli ATCC 25922, Negative Control (NC): Proteus mirabilis ATCC 12453 or Positive control (PC): Neisseria lactamica ATCC 23971, and Negative Control (NC): Neisseria gonorrhoeae ATCC 43069

Procedure of ONPG Disk Test

  1. Inoculate test bacterial colonies into 0.5 ml of saline in a test tube to produce a heavy suspension (No. 2 McFarland standard).
  2. Put the disk into the tube.
  3. Incubate the tube aerobically at 35°C for up to 6 hours.
  4. Observe for yellow color production.

Result- Interpretation of ONPG Test

  • Positive: Development of yellow color
  • Negative: No color change
  • Escherichia coli ATCC 25922—ONPG positive
  • Proteus mirabilis ATCC 12453—ONPG negative


  • An organism that produces a yellow color is an ONPG test positive and generally a lactose fermenter (LF).
  •  An organism that does not produce a yellow color is ONPG test negative and non-lactose fermenting.
  • Among the Neisseria spp., Neisseria lactamica is ONPG test-positive.
  • ONPG test is used for β-galactosidase detection.

Limitations of the ONPG Test

  1. For the rapid test i.e. disk method, colonies should be from a lactose-containing medium e.g., triple sugar iron(TSI) agar or MacConkey agar (MAC).
  2. A culture that naturally produces a yellow pigment cannot be tested with this medium.
  3. If the medium is not properly buffered, results may be inaccurate.
  4. Do not use it if the growth medium is yellow.
  5. Since glucose inhibits lactose fermentation by bacteria, organisms growing in a glucose-containing medium show less activity than they would in the presence of lactose.

Further Readings

  • Bailey & Scott’s Diagnostic Microbiology. Editors: Bettey A. Forbes, Daniel F. Sahm & Alice S. Weissfeld, 12th ed 2007, Publisher Elsevier.
  • Clinical Microbiology Procedure Handbook, Chief in editor H.D. Isenberg, Albert Einstein College of Medicine, New York, Publisher ASM (American Society for Microbiology), Washington DC.
  • Cowan & Steel’s Manual for identification of Medical Bacteria. Editors: G.I. Barron & R.K. Felthani, 3rd ed 1993, Publisher Cambridge University Press.
  • Colour Atlas and Textbook of Diagnostic Microbiology. Editors: Koneman E.W., Allen D.D., Dowell V.R. Jr, and Sommers H.M.
  • Jawetz, Melnick and Adelberg’s Medical Microbiology. Editors: Geo. F. Brook, Janet S. Butel & Stephen A. Morse, 21st ed 1998, Publisher Appleton & Lance, Co Stamford Connecticut.
  • Mackie and Mc Cartney Practical Medical Microbiology. Editors: J.G. Colle, A.G. Fraser, B.P. Marmion, A. Simmous, 4th ed, Publisher Churchill Living Stone, New York, Melborne, Sans Franscisco 1996.
  •  Textbook of Diagnostic Microbiology. Editors: Connie R. Mahon, Donald G. Lehman & George Manuselis, 3rd edition2007, Publisher Elsevier.

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