ONPG: β-galactosidase Test


Last updated on June 11th, 2021

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 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 a 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

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. 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

About Acharya Tankeshwar 476 Articles
Hello, thank you for visiting my blog. I am Tankeshwar Acharya. Blogging is my passion. I am working as an Asst. Professor and Microbiologist at Department of Microbiology and Immunology, Patan Academy of Health Sciences, Nepal. If you want me to write about any posts that you found confusing/difficult, please mention in the comments below.