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ONPG DISCS

Code: DD0013

For the rapid detection of beta-galactosidase activity in microorganisms.

Contents
1 cartridge. containing 50 discs.

Disc Contents
Each disc is impregnated with phosphate buffered O-nitrophenyl-b -D-galacto-pyranoside (ONPG).

Description
Lactose fermentation is a classical identification test for many microorganisms. It is normally demonstrated by acid production after the disaccharide has been cleaved into galactose and glucose by the enzyme beta-galactosidase. However, it is essential for the lactose to be conveyed into the cell by a specific galactoside-permease enzyme before such cleavage.
The role of these two essential enzymes is important in the classification of organisms into:
1 Active lactose fermenters (taking 18-24 hours) possessing both permease and galactosidase P+G+.
2 Delayed lactose fermenters (taking longer than 24 hours) lacking permease but possessing galactosidase: P- G+.
3 Non-lactose fermenters lacking both permease and galactosidase: P- G-.
For the ONPG test¹ a synthetic galactoside (ortho-nitrophenyl-b-D-galacto-pyranose) is substituted for lactose. It is hydrolysed in the same way as lactose but the ortho-nitrophenol is chromogenic and when cleaved off in alkaline solution it produces a yellow solution:

This test is independent of an induced or constitutive permease enzyme and can be very rapid.

The ONPG test can be used to:
1 Differentiate late lactose fermenters (P- G+) from non-lactose fermenters (P- G-).
(a) Citrobacter (+) and Salmonella arizonae (+) from Salmonella (-).
(b) Escherichia coli (+) from Shigella sonnei (-).
2 Aid in species differentiation.
(a) Burkholderia cepacia (+) and Stenotrophomonas maltophila (+) from other Pseudomonas species (-).
(b) Neisseria lactamica (+) from other Neisseria species (-).

Technique
1 Place one disc into a sterile tube.
2 Add 0.1 ml of sterile 0.85% sodium chloride (physiological saline).
3 Pick the colony under test with a sterile loop and emulsify it in the tube containing the disc and physiological saline.
4 Incubate at 35°C.
5 Examine at hourly intervals for up to 6 hours to detect active lactose fermenters.
6 Organisms that are negative after 6 hours should be incubated for up to 24 hours to detect the late lactose fermenters.

Interpretation of Results

The reaction speed depends on the size of inoculum.

Quality Control
Use known positive and negative beta-galactosidase producing organisms to monitor the disc reactions.

Reference
1 Lowe G. H. (1962) J. Med. Lab. Technol. 19. 21-25.