Beta-Galactosidase Test (ONPG)

Purpose of Beta-Galactosidase Test (ONPG)

The purpose/objective of the Beta-Galactosidase Test (ONPG) is to identify the ability of microorganisms (bacterium) to produce the enzyme beta-galactosidase. The beta-Galactosidase enzyme breaks down lactose for utilization.

This test is generally used for gram-negative bacteria as well as in the identification of Staphylococcus spp.

Introduction to Beta-Galactosidase Test (ONPG)

For any organism to utilize lactose, it must be able to synthesize a couple of enzymes – one of which is beta–galactosidase. The Beta-Galactosidase Test (ONPG) uses a molecular decoy called ONPG (Ortho-nitrophenyl-β -D-galactopyranoside) in which orthonitrophenyl has been substituted for glucose. The ONPG will turn yellow in color in the presence of the beta–galactosidase enzyme.

ONPG is an analog of lactose that has the capability to break down to produce O-nitrophenol – which is a yellow-colored end-product.

For the Beta-Galactosidase Test (ONPG), the substrate used for culture must contain lactose in high amounts so as to induce the production of beta–galactosidase by potential test organisms. Media such as  Triple Sugar Iron (TSI) agar, Kligler iron agar (KIA), or lactose broth can be used.

Principle of Beta-Galactosidase Test (ONPG)

Lactose fermenting bacteria produces both enzymes required for the production of acid in the lactose fermentation test - lactose permease and β-galactosidase. Permease functions by allowing lactose molecules to enter the bacterial cell while β-galactosidase cleaves the galactoside bond releasing glucose and galactose.

On the other hand, non-lactose fermenters lack both lactose permease and β-galactosidase and are unable to produce acid in the lactose fermentation test.

Some bacteria are able to produce β-galactosidase but lack permease. Such species appear to be non-lactose fermenters but do give a positive ONPG test. In such cases, lactose fermentation and production of acid are delayed due to sluggish permease activity.

Biochemistry of Beta-Galactosidase Test (ONPG)

Lactose is hydrolyzed by the action of the enzyme β-galactosidase which cleaves it into two residues - galactose and o-nitrophenol. While ONPG is a colorless compound, its cleaved product o-nitrophenol is yellow in color.

The biochemistry of the Beta-Galactosidase Test (ONPG) is as follows:

Reagents for Beta-Galactosidase Test (ONPG)

The reagents/materials required for Beta-Galactosidase Test (ONPG) include:

• ONPG discs

• O-Nitrophenyl-β-D-galactopyranoside (ONPG), 0.75 M

• Sodium phosphate buffer, 1 M, pH 7.0

• Toulene

• 1 ml of 0.85 - 0.9% NaCl

Procedure of Beta-Galactosidase Test (ONPG)

The procedure of the Beta-Galactosidase Test (ONPG) is as follows:

For liquid ONPG

1. a loop full of test bacterial culture is emulsified in 0.05mL of saline to produce a heavy suspension

2. a drop of toluene is added to the mixture and mixed vigorously for a few seconds

   * toluene lyse bacterial cells to release its contents including enzymes

3. an equal quantity of buffered ONPG solution is added to the suspension

4. the suspension is placed in a 37oC water bath

For ONPG discs

1. pipet 0.5ml of saline into a sterile test tube

2. take a loop full test organism that has been cultured in media containing a high amount of lactose

3. inoculate the test microorganism into the test tube

4. in a sterile manner, place the ONPG disc into the tube

5. incubate at 37º C for 4 hours

* since there is a very little amount of saline in the test tube used during Beta-Galactosidase Test (ONPG), it can dehydrate quickly. For the prevention of such cases, parafilm can be used to cover the test tube.

Result, Interpretation of Beta-Galactosidase Test (ONPG)

Positive

After four-hour incubation, yellow pigmentation develops in the test tube representing a positive result for Beta-Galactosidase Test (ONPG).

Negative

Absence of yellow color after four-hour incubation represents a negative result for the Beta-Galactosidase Test (ONPG).

Beta-Galactosidase Test (ONPG) positive organism

It produces both β-galactosidase and permease

• E.coli

• Klebsiella spp

• Enterobacter spp

Late Beta-Galactosidase Test (ONPG) positive organism

It produces only β-galactosidase but not permease

• Citrobacter spp

• Arizona spp

Beta-Galactosidase Test (ONPG) negative organism

They do not produce β-galactosidase and cannot ferment lactose

• Salmonella spp

• Shigella spp

• Proteus spp

• Providencia spp

• Morganella spp