ONPG Test (for β-galactosidase): Principle, procedure and results

O-Nitrophenyl-β-D-galactopyranoside (ONPG) is structurally similar to lactose (i.e. ONPG is an analog of lactose), except that orthonitrophenyl has been substituted for glucose.

On hydrolysis, through the action of the enzyme β-galactosidase, ONPG cleaves into two residues, galactose and o-nitrophenol. ONPG is colorless compound: O-nitrophenol is yellow, providing visual evidence of hydrolysis.


Lactose fermenting bacteria posses both lactose permease and β-galactosidase, two enzymes required for the production of acid in the lactose fermentation test. The permease is required for the lactose molecule to penetrate the bacterial cell where the β-galactosidase can cleave the galactoside bond, producing glucose and galactose.

Non-lactose fermenting bacteria are devoid of both enzymes and are incapable of producing acid from lactose.

Some bacterial species 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 sluggish permease activity. In these instances, a positive ONPG test may provide a rapid identification of delayed lactose fermentation.

ONPG Test results Vs. Lactose fermentation:  

  1. Lactose fermenter (ONPG Positive): E.coli, Klebsiella spp, Enterobacter spp produce β-galactosidase and permease
  2. Late lactose fermenter (ONPG Positive): Citrobacter spp, Arizona spp produce only β-galactosidase so they slowly ferment lactose.
  3. Non lactose fermenter (ONPG Negative): Salmonella spp; Shigella spp; Proteus spp; Providencia spp and Morganella spp do not produce β-galactosidase so can not ferment lactose.

Media and Reagents

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

Quality control

  1. Positive control: Escherichia coli
  2. Negative control: Proteus vulgaris


Bacteria grown in medium containing lactose (to induce the production of the galactosidase enzyme) , such as Kligler iron agar (KIA) or Triple sugar Iron (TSI) agar, produces optimal results in ONPG Test.

Note: β-galactosidase  enzyme (inducible enzyme)  is made ONLY in the presence of the lactose substrate

  1. A loopful of bacterial growth is emulsified in 0.05mL of physiologic saline to produce a heavy suspension
  2. One drop of toluene is added to the suspension and vigorously mixed for a few seconds to release the enzyme for 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
  5. When Using ONPG Tablets
  6. 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.
  7. This suspension is also placed in a 37oC water bath

Results and Interpretations:

The 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.

ONPG Test results
ONPG Test results

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


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