ONPG Test: How to Catch the Lactose Fermenters That the Lactose Test Misses

The organism that could ferment lactose but got filed as one that couldn't

A Gram-negative rod from a stool sample is streaked onto MacConkey agar. After overnight incubation the colonies are pale. Non-lactose fermenter, the technician notes, which in a stool workup pushes the organism toward the Salmonella and Shigella end of the bench, the pathogens worth chasing.

But this organism is not a non-fermenter. It carries a perfectly good β-galactosidase, the enzyme that splits lactose. What it lacks is an efficient permease, the membrane transporter that carries lactose into the cell in the first place. The enzyme is inside, waiting, with no substrate reaching it. On MacConkey, which reads acid produced from lactose, the organism looks negative because the lactose never got in to be fermented.

This is a late or cryptic lactose fermenter, and if you stop at the MacConkey plate you will misclassify it.

The ONPG test exists precisely to catch this organism. ONPG is a synthetic molecule built to look like lactose to the enzyme, but small and permeable enough to slip into the cell without needing the permease. Once inside, β-galactosidase cleaves it and releases a bright yellow compound. The organism that looked negative on lactose turns the ONPG tube yellow, revealing the enzyme that was there all along.

One test reads the transporter and the enzyme together. The other reads the enzyme alone. Knowing the difference is what keeps a late lactose fermenter from being mistaken for a pathogen it is not. This article is about that difference.

Principle of ONPG Test

O-Nitrophenyl-β-D-galactopyranoside (ONPG) is structurally similar to lactose (i.e. O-Nitrophenyl-β-D-galactopyranoside 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 itself is colorless; the o-nitrophenol released when it is cleaved is yellow, and that yellow is the visual evidence of hydrolysis.

Figure: ONPG Test

Lactose fermenting bacteria possess 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 test may provide rapid identification of delayed lactose fermentation.

Why lactose fermentation needs two proteins, and ONPG needs only one

Fermenting lactose is a two-step problem, and each step has its own protein.

Step one: get lactose into the cell. Lactose is a disaccharide and cannot cross the bacterial membrane on its own. It needs a dedicated transporter, lactose permease, to carry it inside.

Step two: split it once inside. Within the cell, the enzyme β-galactosidase cleaves lactose into glucose and galactose, which the organism then ferments to acid.

A standard lactose test, such as MacConkey agar or the lactose reaction in TSI, detects the acid produced at the end of step two. To make that acid, the organism needs both proteins: the permease to admit the lactose and the enzyme to split it. Miss either one and the test reads negative.

This creates a blind spot. An organism that has β-galactosidase but lacks the permease, or has a slow, sluggish permease, cannot get enough lactose inside to make detectable acid quickly. On MacConkey it looks like a non-fermenter or a slow, late fermenter. But it is not enzyme-deficient. It is transporter-deficient.

ONPG removes the transporter from the question. ONPG (o-nitrophenyl-β-D-galactopyranoside) is a synthetic analog of lactose. To β-galactosidase it looks enough like lactose to be cleaved. But unlike lactose, ONPG is small and lipophilic enough to diffuse into the cell without a permease. It reaches the intracellular enzyme on its own.

So ONPG tests β-galactosidase in isolation, independent of whether the organism can transport lactose. If the enzyme is present, ONPG is cleaved and the tube turns yellow, whether or not the organism has a working permease.

Put the two tests side by side and they answer different questions:

The reason ONPG is worth running is entirely in that bottom row. It rescues the organisms the lactose test wrongly files as negative.

Media and Reagents

1. Sodium phosphate buffer, 1 M, pH 7.0
2. O-Nitrophenyl-β-D-galactopyranoside, 0.75 M
3. Physiologic saline
4. Toluene

Quality control

1. Positive control: Escherichia coli ATCC 25922, yellow (β-galactosidase present)
2. Negative control: Proteus vulgaris ATCC 13315, colorless (no β-galactosidase)

Run controls with each new lot of ONPG substrate or tablets, since the substrate degrades and can give false negatives if old.

Procedure

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 this test.

Note: β-galactosidase  enzyme (inducible enzyme)  is made ONLY in the presence of the lactose substrate

1. A loopful of bacterial growth is emulsified in 0.05mL of physiologic saline to produce a heavy suspension
2. One drop of toluene is added to the suspension and vigorously mixed for a few seconds to release the enzyme from bacterial cells.
3. An equal quantity of buffered ONPG solution is added to the suspension.
4. The mixture is placed in a 37°C water bath

When Using ONPG Tablets

1. 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.
2. This suspension is also placed in a 37°C water bath

Why toluene?
Toluene is a solvent that permeabilizes the bacterial membrane, releasing β-galactosidase from inside the cell so it can act on the ONPG in solution directly. ONPG can diffuse into an intact cell on its own, but permeabilizing the cell speeds the reaction by removing even that small barrier and letting a large amount of enzyme meet the substrate at once. It makes a slow reaction fast, which is why the conventional method reads within minutes to an hour. The tablet method skips the toluene step and relies on ONPG's own ability to enter intact cells, which is why it can be slower.

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.

Figure: ONPG Test results

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.

ONPG test results vs. lactose fermentation

1. Lactose fermenter (ONPG Positive): E. coli, Klebsiella spp, Enterobacter spp produce β-galactosidase and permease

2. Late lactose fermenter (ONPG Positive): Citrobacter spp, Arizona spp produce only β-galactosidase so they slowly ferment lactose.

3. Non-lactose fermenter (ONPG Negative): most Salmonella serotypes, Shigella species (except some S. sonnei), Proteus, Providencia, and Morganella lack β-galactosidase and cannot ferment lactose by any route.

   A useful exception: Salmonella enterica subsp. arizonae (the old "Arizona") is ONPG positive and a late lactose fermenter, unlike the common Salmonella serotypes. And Shigella sonnei is often ONPG positive despite being a non-lactose fermenter on MacConkey, a classic demonstration of the enzyme-present, permease-poor pattern. These exceptions are exactly the organisms ONPG was designed to reveal.

How to remember

Two locks, one key that skips a lock.

Lactose fermentation has two locks: the permease lock (getting in) and the enzyme lock (getting split). A standard lactose test needs both locks opened. ONPG is a skeleton key that ignores the permease lock entirely, walks into the cell on its own, and tests only the enzyme lock. So when the lactose test says no but ONPG says yes, you have found an organism whose permease lock was stuck, not one that lacks the enzyme.

ONPG rescues the "shy" fermenters.

Picture a late lactose fermenter as an organism that can do the job but is slow to let the substrate in. MacConkey gives up on it too early and calls it negative. ONPG is patient in a different way: it hands the enzyme its substrate directly, so the shy fermenter finally shows its yellow. Ask yourself when you see a MacConkey-negative, ONPG-positive result: is this really a non-fermenter, or just a slow one that got mislabeled?

Yellow means the enzyme is home.

ONPG is colorless; cleaving it releases yellow o-nitrophenol. Yellow tube equals β-galactosidase present, full stop, regardless of what the lactose plate said. The two exceptions worth naming, Shigella sonnei and Salmonella Arizonae, are precisely the organisms this test was built to catch.

Key exam facts

Where students get confused

"ONPG positive means the organism ferments lactose." Not exactly. ONPG positive means the organism has β-galactosidase. Whether it ferments lactose in practice also depends on the permease. An organism can be ONPG positive and still read as a non-fermenter on MacConkey if its permease is absent or slow. That mismatch is not an error; it is the entire diagnostic point of the test.

Confusing what ONPG and the lactose test each measure. The lactose test measures acid production, which needs the transporter and the enzyme together. ONPG measures the enzyme alone. When the two disagree, the organism has the enzyme but a transport problem. Reading them as interchangeable throws away the one piece of information ONPG uniquely provides.

Not understanding why toluene is added. It looks contradictory: ONPG can enter an intact cell, so why break the cell open? Because permeabilizing the cell releases a large amount of enzyme to meet the substrate at once, turning a slow reaction into a fast one. Toluene is about speed, not about getting ONPG in.

Reading a negative too early. Some organisms hydrolyze ONPG slowly. A tube that is colorless at one hour is not yet negative. The result should not be called negative before 24 hours of incubation.

Forgetting to induce the enzyme. β-galactosidase is an inducible enzyme, made only when lactose is present. If the organism was grown on a medium without lactose, the enzyme may not be expressed and a true positive can read negative. Grow the organism on a lactose-containing medium such as KIA or TSI first.

Missing the Shigella sonnei and Salmonella Arizonae exceptions. These are the organisms that look like non-fermenters but are ONPG positive, and they are exactly what the test was built to reveal. In a stool workup, remembering that some Shigella sonnei are ONPG positive prevents both confusion and misidentification.

Using degraded substrate. ONPG substrate and tablets deteriorate over time and with light exposure. Old substrate gives false negatives. Run a positive control with each new lot.

References

1. Lowe GH. The rapid detection of lactose fermentation in paracolon organisms by the demonstration of β-D-galactosidase. J Med Lab Technol. 1962;19:21-25.
2. Le Minor L, Ben Hamida F. Advantages of the ONPG test in Salmonella and Arizona identification. Ann Inst Pasteur.
3. LaPage SP, Efstratiou A, Hill LR. The ortho-nitrophenol (ONPG) test and acid from lactose in Gram-negative genera. J Clin Pathol. 1973;26(11):821-825. doi:10.1136/jcp.26.11.821
4. Buelow P. The ONPG test in diagnostic bacteriology: methodological investigations. Acta Pathol Microbiol Scand. 1964;60:376-386. doi:10.1111/apm.1964.60.3.376
5. Procop GW, Church DL, Hall GS, Janda WM, Koneman EW, Schreckenberger PC, Woods GL. Koneman's Color Atlas and Textbook of Diagnostic Microbiology. 7th ed. Philadelphia: Wolters Kluwer; 2017.
6. Tille PM. Bailey and Scott's Diagnostic Microbiology. 15th ed. St. Louis: Elsevier; 2022.