ONPG (o-Nitrophenyl-β-D-Galactopyranoside) test: Principle, Procedure, Result and Interpretation

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o-Nitrophenyl-β-D-Galactopyranoside (ONPG) is structurally similar to lactose except that o-nitrophenol has been substituted for glucose. On hydrolysis through the action of enzyme β-galactosidase, ONPG which is a colorless compound cleaves into two residues, galactose and o-nitrophenol which is yellow in color. Development yellow color provides visual evidence of hydrolysis of ONPG.

Lactose fermenting bacteria requires both lactose permease and β-galactosidase two enzymes required for utilization of lactose producing acid in lactose fermentation test. Permease is reqired for the lactose molecule to enter the bacterial cell whereas β-galactosidase cleaves the glycosidic bond producing glucose and galactose.

Non-lactose fermenting bacteria lack both of these enzymes and are incapable of producing acid from lactose.There are some bacterial species that appear to be non-lactose fermenters because they lack permease, but do possess β-galactosidase and give a positive ONPG test. So called, late lactose fermenters may be delayed in their production of acid from lactose because of slow permease activity. In these instances, a positive ONPG test may provide a rapid identification of delayed lactose fermentation.


  1. Sodium phosphate buffer, 1 M, pH 7.0
  2. O-Nitrophenyl-β-D-galactopyranoside (ONPG), 0.75 M
  3. Physiologic saline
  4. Toluene


  1. A loopful of bacterial growth from TSI slant is emulsified in 0.05mL of physiologic saline to produce a heavy suspension
  2.  A drop of toluene is added to the suspension and vigorously mixed for a few seconds to release the enzyme from the bacterial cells.
  3. An equal quantity of buffered ONPG solution is added to the suspension.
  4. The mixture is placed in a 37oC water bath


When Using ONPG Tablets,

  1. A loopful of bacterial suspension is added directly to the ONPG substrate resulting from adding 1mL of distilled water to a tablet in a test tube.
  2. The suspension is also placed in a 37oC water bath

NoteBacteria grown in medium containing lactose (to induce the production of the galactosidase enzyme) , such as Kligler iron agar (KIA)  or Triple sugar iron agar (TSI) , produces optimal results in ONPG Test.  β-galactosidase is an inducible enzyme which is produced only when lactose is present as substrate

Results and Interpretations:

Rate of hydrolysis of ONPG to o-nitrophenol may be rapid for some organisms; producing a visible yellow color reaction within 5 to 10 minutes. Most tests are positive within 1 hour; however, reactions should not be interpreted as negative before 24 hours of incubation.The yellow color is usually distinct and indicates that the organism has produced o-nitrophenol from the ONPG substrate through the action of β-galactosidase.


Quality control

Positive control: Escherichia coli

Negative control: Proteus spp

 Reference : Koneman’s Color Atlas and Textbook of Diagnostic Microbiology

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