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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
- Place an ONPG disk into a sterile tube and add 0.2 mL saline.
- Heavily inoculate the tube with a loopful of the test isolate.
- Incubate at 35-37°C for up to 4 hours.
- Examine for color change of the disk.
For broth method
- Bring test medium to room temperature.
- Inoculate the test medium with heavy inoculum from a pure 18-24 hour culture.
- Incubate aerobically, with loose caps, at 35- 37ºC.
- Examine for a yellow color development at 1 hour.
- If the tube has not changed color after 1 hour of incubation, continue incubation for up to 24 hours.
Result Interpretation of ONPG Test
Positive test: Yellow coloration (presence of β-galactosidase)
Negative test: Colorless (absence of enzyme)
Limitations of ONPG Test
- All organisms tested must be inoculated from a lactose-containing medium (e.g., TSI or MacConkey).
- Cultures that naturally produce a yellow pigment cannot be tested with this media.
- 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
- 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
- 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
- Reynolds J. 2018. Beta (β)-GALACTOSIDASE (ONPG) TEST. Richland College http://delrio.dcccd.edu/jreynolds/microbiology/2421/lab_manual/ONPG.pdf