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:
- Lactose fermenter (ONPG Positive): E.coli, Klebsiella spp, Enterobacter spp produce β-galactosidase and permease
- Late lactose fermenter (ONPG Positive): Citrobacter spp, Arizona spp produce only β-galactosidase so they slowly ferment lactose.
- 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
- Sodium phosphate buffer, 1 M, pH 7.0
- O-Nitrophenyl-β-D-galactopyranoside (ONPG), 0.75 M
- Physiologic saline
- Positive control: Escherichia coli
- 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
- A loopful of bacterial growth is emulsified in 0.05mL of physiologic saline to produce a heavy suspension
- One drop of toluene is added to the suspension and vigorously mixed for a few seconds to release the enzyme for bacterial cells.
- An equal quantity of buffered ONPG solution is added to the suspension.
- The mixture is placed in a 37oC water bath
When Using ONPG Tablets
- 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.
- 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.
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