ONPG Test
A bench visual for the ONPG test, used to detect β-galactosidase activity to differentiate late lactose fermenters from true non-lactose fermenters.
ONPG Tubes A-B
An ONPG disk is added to a saline suspension of the organism. A yellow color shift indicates β-galactosidase hydrolyzed the substrate.
The suspension and disk turn yellow; β-galactosidase hydrolyzed ONPG to yield galactose and the yellow compound o-nitrophenol.
The suspension and disk remain colorless; the organism does not produce β-galactosidase.
The suspension and disk turn yellow; β-galactosidase hydrolyzed ONPG to yield galactose and the yellow compound o-nitrophenol.
The suspension and disk remain colorless; the organism does not produce β-galactosidase.
What to look for
| Visual clue | Meaning | Student shorthand |
|---|---|---|
| Yellow fluid or disk | o-nitrophenol was released from the ONPG substrate | Positive for β-galactosidase |
| Colorless fluid and disk | ONPG substrate remained intact | Negative for β-galactosidase |
| Read time | Usually read at 1 to 4 hours, up to 24 hours depending on the protocol | Delayed positives can occur |
ONPG Test Interpretive Matrix
| Pattern | What to call it | How to think about it |
|---|---|---|
| Yellow | Positive | Organism produces β-galactosidase. Correlates with late or rapid lactose fermentation. |
| Colorless | Negative | Organism does not produce β-galactosidase. Correlates with true non-lactose fermentation. |
- ONPG stands for o-Nitrophenyl-β-D-Galactopyranoside.
- The test bypasses the need for lactose permease by directly testing for the intracellular β-galactosidase enzyme.
- It is a high-yield test for differentiating Salmonella (ONPG-) from Shigella sonnei or Citrobacter (ONPG+).
If it turns yellow, it has the enzyme to break down lactose, even if it looked like a non-fermenter on your MAC plate yesterday.