ONPG Test : Introduction, Principle,Test Requirements,Procedure

Introduction of ONPG Test

ONPG test is useful in differentiating members of the family Enterobacteriaceae and genera Neisseria based on b-D-galactosidase activity. ONPG stands for o-Nitrophenyl-b-D-galactopyranoside. Here we discuss on ONPG Disk Test which is simple and easy to perform.

Fig. ONPG Test Negative (left) and Positive Bacteria (right)

Principle

The ability of an organism to ferment lactose depends on two enzymes and are permease and b-galactosidase. Permease allows lactose to enter the bacterial cell wall, where lactose is then broken down into glucose and galactose by the enzyme, b-galactosidase. Bacteria can metabolize glucose and galactose. The enzymes are inducible and are merely present when lactose, rather than glucose, is available to the bacterium for metabolism. Some organisms lack enzyme permease and appear as late lactose fermenters or non-lactose fermenters.

The ONPG test will detect true non-lactose fermenters (NLF) that have the b-galactosidase enzyme, even if they lack the permease enzyme. A lactose fermentation test will not detect bacteria lacking the permease. ONPG is a colorless substrate, similar in structure to lactose, applied in this test as the substrate for b-galactosidase. If the bacterium possesses b-galactosidase, the enzyme will split the b-galactoside bond, releasing galactose and o-nitrophenol, which is a yellow compound.

Requirements for ONPG Test

Test organisms-Gram-negative rods growing aerobically or Gram-negative diplococci growing aerobically
ONPG disks (Store at 4°C and store away from direct light since ONPG is light sensitive.)
Test tubes
Inoculating loops
Sterile physilogical saline
Incubator
Control strains to check quality control of test-Positive control (PC): Escherichia coli ATCC 25922, Negative Control (NC): Proteus mirabilis ATCC 12453 or Positive control (PC): Neisseria lactamica ATCC 23971, and Negative Control (NC): Neisseria gonorrhoeae ATCC 43069

Procedure of ONPG Disk Test

Inoculate test bacterial colonies into 0.5 ml of saline in a test tube to produce a heavy suspension (No. 2 McFarland standard).
Put the disk into the tube.
Incubate the tube aerobically at 35°C for up to 6 hours.
Observe for yellow color production.

Result- Interpretation of ONPG Test

Positive: Development of yellow color
Negative: No color change
Escherichia coli ATCC 25922—ONPG positive
Proteus mirabilis ATCC 12453—ONPG negative

Keynotes

An organism that produces a yellow color is an ONPG test positive and generally a lactose fermenter (LF).
An organism that does not produce a yellow color is ONPG test negative and non-lactose fermenting.
Among the Neisseria spp., Neisseria lactamica is ONPG test-positive.
ONPG test is used for β-galactosidase detection.

Limitations of the ONPG Test

For the rapid test i.e. disk method, colonies should be from a lactose-containing medium e.g., triple sugar iron(TSI) agar or MacConkey agar (MAC).
A culture that naturally produces a yellow pigment cannot be tested with this medium.
If the medium is not properly buffered, results may be inaccurate.
Do not use it if the growth medium is yellow.
Since glucose inhibits lactose fermentation by bacteria, organisms growing in a glucose-containing medium show less activity than they would in the presence of lactose.

ONPG Test: Introduction, Principle, Test Requirements, Procedure, Result -Interpretation, and Keynotes