O-Nitrophenyl-β-D-Galactopyranoside (ONPG) Test

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Last Updated on January 10, 2020 by Sagar Aryal

Objectives of ONPG Test

  • To determine the ability of an organism to produce β-galactosidase, an enzyme that hydrolyzes the substrate ONPG to form a visible (yellow) product, orthonitrophenol
  • To distinguish late lactose fermenters from non–lactose fermenters of Enterobacteriaceae

Principle of ONPG Test

The sugar lactose is a disaccharide of galactose and glucose joined by a β-galactoside bond. The ability of bacteria to ferment lactose depends on two enzymes, permease and beta-galactosidase. Permease allows lactose to enter the bacterial cell wall, where it is then broken down into glucose and galactose by beta-galactosidase. The enzyme β-galactosidase, the product of the lacZ gene, breaks the β-galactoside bond to release the galactose and glucose monosaccharides. The glucose and galactose can then be metabolized by the bacteria. A compound known as ONPG (o-nitrophenol-β-d-galactopyranoside) has a structure that mimics lactose and can be cleaved by β-galactosidase.  ONPG, however, carries an o-nitrophenol ring group rather than glucose. O-nitrophenyl-beta-D-galactopyranoside (ONPG), an artificial substrate, is incorporated in the test acts as the substrate for the beta-galactosidase. This substrate is capable of penetrating the bacterial cell without the presence of permease. When ONPG, a colorless compound, is cleaved, o-nitrophenol (ONP), a yellow compound, is released which will turn medium to a yellow color in the presence of the enzyme β galactosidase. ONPG is an analog of lactose that the enzyme can break down to produce a yellow colored end-product, O-nitrophenol.

Media Used in ONPG Test

  • ONPG broth: Na2HPO4 (9.46 g), phenylalanine (4 g), ONPG (2 g), KH2PO4 (0.907 g), per 1000 mL, pH 8.0
  • ONPG disk

Procedure of ONPG Test

For ONPG disk method

  1. Place an ONPG disk into a sterile tube and add 0.2 mL saline.
  2. Heavily inoculate the tube with a loopful of the test isolate.
  3. Incubate at 35-37°C for up to 4 hours.
  4. Examine for color change of the disk.

For broth method    

  1. Bring test medium to room temperature.
  2. Inoculate the test medium with heavy inoculum from a pure 18-24 hour culture.
  3. Incubate aerobically, with loose caps, at 35- 37ºC.
  4. Examine for a yellow color development at 1 hour.
  5. If the tube has not changed color after 1 hour of incubation, continue incubation for up to 24 hours.

Result Interpretation of ONPG Test

O-Nitrophenyl-β-D-Galactopyranoside (ONPG) Test

Positive test: Yellow coloration (presence of β-galactosidase)

Negative test: Colorless (absence of enzyme)

Limitations of ONPG Test

  1. All organisms tested must be inoculated from a lactose-containing medium (e.g., TSI or MacConkey).
  2. Cultures that naturally produce a yellow pigment cannot be tested with this media.
  3. For complete identification biochemical, immunological, molecular, or mass spectrometry testing on colonies from pure culture is recommended.

Quality control of ONPG Test

Escherichia coli (ATCC25922) – ONPG positive, yellow color development

Proteus mirabilis (ATCC 12453) – ONPG negative, no color development


  1. Tille P.M. 2014. Bailey and Scott’s diagnostic microbiology. Thirteen edition. Mosby, Inc., an affiliate of Elsevier Inc. 3251 Riverport Lane. St. Louis. Missouri 63043
  2. UK Standards for Microbiology Investigations. 2014. ONPG (ß-Galactosidase) Test https://assets.publishing.service.gov.uk/government/uploads/system/uploads/attachment_data/file/377880/TP_24i3.pdf
  1. Reynolds J. 2018. Beta (β)-GALACTOSIDASE (ONPG) TEST. Richland College http://delrio.dcccd.edu/jreynolds/microbiology/2421/lab_manual/ONPG.pdf

O-Nitrophenyl-β-D-Galactopyranoside (ONPG) Test

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