• Lactose is a disaccharide of glucose and galactose which is connected by a β-galactoside bond.
• Lactose fermenting bacteria consist of both the permease and beta-galactosidase enzymes that are responsible for acid production during lactose fermentation.
• Permease permits the lactose to enter into the bacterial cell wall.
• Then, beta-galactosidase breaks down the lactose into glucose and galactose.
• But in some organisms, permease is absent and hence appear as late or non-lactose fermenters.
• O-Nitrophenyl-β-D-galactopyranoside (ONPG) resembles to lactose in structure, except that glucose is substituted by orthonitrophenyl group.
• In contrast to lactose, the substrate O-nitrophenyl-beta-D galactopyranoside (ONPG) can penetrate the bacterial cell wall even in absence of permease.
• ONPG test is a very sensitive test for lactose-fermentation.
• In this test, O-Nitrophenyl-β-D-galactopyranoside (ONPG) (artificial in nature) acts as a substrate for beta-galactosidase to ascertain the specific enzyme activity that serves in the identification and differentiation of organisms.
• If the organism consists of beta-galactosidase, then it will hydrolyse ONPG to yield galactose and o-nitrophenol.
• The positive test is confirmed by the yellow colour, which is given by ortho-nitrophenol.
• A positive ONPG test is mainly focused on providing the rapid identification of delayed lactose fermentation.

Requirements:

1. Sodium phosphate buffer, 1 M, pH 7.0
2. O-Nitrophenyl-β-D-galactopyranoside (ONPG), 0.75 M
3. Physiologic saline
4. Toluene
5. ONPG broth: Ingredients per 1000 mL
   • Na2HPO4 —————- (9.46 g)
   • phenylalanine ——— (4 g)
   • ONPG ———————– (2 g)
   • KH2PO4 ——————–(0.907 g)
   • pH 8.0
6. ONPG disk: 
   • ONPG Differentiation Disk is made by impregnating controlled concentrations of ONPG onto a 0.25 inch diameter filter paper disk.

Procedure of ONPG test:

• For ONPG disk method:
  • Keep an ONPG disk into a sterile tube.
  • Add 0.2 mL saline to it.
  • Use a loopful of test isolate to heavily inoculate the tube.
  • Incubate at 35-37°C for up to 4 hours.
  • Observe for colour change of the disk.
• For broth method:
  • Keep the test medium to room temperature.
  • Use heavy inoculum from a pure 18-24 hr culture to inoculate the test medium.
  • Incubate aerobically, at 35- 37ºC, with caps loosened.
  • Observe for a yellow colour development at 1 hour.
  • If no colour change is observed in the tube even after 1 hour of incubation, continue incubation for up to 24 hours.

Results interpretations:

• Positive result:
  • The positive test is indicated by the yellow colour of ortho-nitrophenol resulted by the hydrolysis of ONPG.
• Negative result:
  • The negative test is suggested by no change in colour, that means the absence of enzyme.

Limitations of ONPG test:

• Further biochemical, molecular, immunological, or mass spectrometry testing on colonies from pure culture is advised for complete identification.
• Cultures which naturally yield yellow colour cannot be tested in this media.
• Inoculation of test organism should be done only from lactose- containing medium.

O-Nitrophenyl-β-D-Galactopyranoside (ONPG) test: Principle, Procedure and Results

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