Liofilchem® ONPG Test Discs

Liofilchem® ONPG Test is a rapid in vitro diagnostic (IVD) assay for the detection of bacterial β-galactosidase activity. The test consists of filter paper discs impregnated with ortho-nitrophenyl-β-D-galactopyranoside (ONPG), enabling differentiation and identification of bacteria—particularly Gram-negative organisms—based on their ability to produce β-galactosidase.

This test serves as an important aid in microbial identification workflows and should be used in conjunction with additional biochemical and diagnostic tests to complete identification.

Intended Purpose

The ONPG Test is intended for the qualitative detection of β-galactosidase activity in bacterial isolates. It is recommended for use with pure cultures as part of a diagnostic workflow.

Principle of the Method

Lactose fermentation depends on two enzymes:

• Permease – transports lactose into the bacterial cell
• β-Galactosidase – hydrolyzes lactose into glucose and galactose

Some Enterobacteriaceae (e.g., Escherichia coli, Klebsiella spp., Enterobacter spp.) produce both enzymes and are true lactose fermenters.

Late lactose fermenters (e.g., Citrobacter spp.) may produce β-galactosidase but lack permease.

True non-fermenters (e.g., Salmonella, Shigella, Proteus, Providencia, Morganella) lack both enzymes.

ONPG is structurally similar to lactose but does not require permease to enter the bacterial cell. When hydrolyzed by β-galactosidase, ONPG produces o-nitrophenol, a yellow compound.

Positive reaction: Yellow color development

Negative reaction: No color change

Kit Contents

• 5 cartridges containing 50 discs each
• Total: 250 ONPG test discs

Order Reference: 88105

Packaging: 5 × 50 discs

Test Procedure Summary

1. Allow discs to reach room temperature.
2. Place one disc into a sterile tube.
3. Add 0.2 mL sterile physiological solution (0.85% saline).
4. Emulsify a well-isolated colony (18–24 h culture) in the tube.
5. Incubate at 35 ± 2°C for up to 24 hours.
6. Observe color change at 4 hours and up to 24 hours.

Positive and negative controls should be tested simultaneously.

Quality Control Strains

Performance testing met established acceptance criteria.

Storage and Shelf Life

• Store at 2–8°C
• Protect from light
• Shelf life: 1 year
• Do not use beyond expiry date

Applications

• Differentiation of lactose fermenters and non-fermenters
• Identification of Enterobacteriaceae
• Detection of β-galactosidase activity
• Clinical microbiology laboratories
• Biochemical identification workflows

Limitations

• Cultures that naturally produce yellow pigment cannot be tested.
• Organisms should be inoculated from lactose-containing media.
• Reaction speed depends on inoculum size.

Safety and Regulatory Information

• For in vitro diagnostic (IVD) use
• For professional laboratory use only
• Compliant with IVDR 2017/746
• Follow laboratory biosafety procedures
• Refer to SDS for hazard information

Liofilchem ONPG Test provides a reliable and rapid method for detecting β-galactosidase activity in bacterial isolates. Its disc-based format, clear yellow color endpoint, and compatibility with standard microbiology workflows make it a valuable tool for differentiation of lactose-fermenting and non-fermenting Gram-negative bacteria in clinical and diagnostic laboratories.