ONPG Test (for β-galactosidase): Use, Principle, Procedure and Result Interpretation

ONPG test positive

Use 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.

Principle of ONPG Test

The ability of a bacterium to ferment lactose depends on two enzymes and they 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 b-galactosidase. Bacteria can metabolize glucose and galactose. The enzymes are inducible and are only present when lactose, rather than glucose, is available to the organism for metabolism. Some bacteria lack enzyme permease and appear as late lactose fermenters or non-lactose fermenters. The ONPG test will detect true non-lactose fermenters 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, used in this test as the substrate for b-galactosidase. If the organism 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 saline
  • Incubator
  • Control strains to check quality control of test

Positive control (PC):Escherichia coli ATCC 25922

Negative Control (NC): Proteus mirabilis ATCC 12453


Positive control (PC): Neisseria lactamica ATCC 23971

Negative Control (NC): Neisseria gonorrhoeae ATCC 43069

Procedure of ONPG Disk Test

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

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

Keynotes on ONPG Test

  • An organism that produces a yellow color is an ONPG test positive and generally a lactose fermenter.
  •  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.

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 if the growth medium is yellow.
  5. Since glucose inhibits lactose fermentation by bacteria, organisms growing on glucose-containing medium show less activity than they would in the presence of lactose.

Further Readings

  1. Cowan & Steel’s Manual for identification of Medical Bacteria. Editors: G.I. Barron & R.K. Felthani, 3rd ed 1993, Publisher Cambridge University Press.
  2. Bailey & Scott’s Diagnostic Microbiology. Editors: Bettey A. Forbes, Daniel F. Sahm & Alice S. Weissfeld, 12th ed 2007, Publisher Elsevier.
  3. 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.
  4. Colour Atlas and Textbook of Diagnostic Microbiology. Editors: Koneman E.W., Allen D.D., Dowell V.R. Jr and Sommers H.M.
  5. 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.
  6. 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.
  7.  Textbook of Diagnostic Microbiology. Editors: Connie R. Mahon, Donald G. Lehman & George Manuselis, 3rd edition2007, Publisher Elsevier.
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