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




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

Principle of ONPG test:

  • 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