Cat. no. Z7321 ONPG Differentiation Disk 50 disks/cartridge


HardyDisk™ ONPG Differentiation Disk is used to differentiate members of the Enterobacteriaceae and other microorganisms based on beta-galactosidase activity.


Lactose is a disaccharide composed of molecules of galactose and glucose. The ability of bacteria to ferment lactose depends on two enzymes; permease and beta-galactosidase. Permease regulates the movement of lactose across the bacterial cell wall. Once lactose is inside the cell, it is broken down into the individual components, glucose and galactose, by beta-galactosidase.(1)

However, some organisms lack permease and consequently appear as late or non-lactose-fermenters. The ONPG test is valuable for the detection of beta-galactosidase activity in late lactose-fermenting organism like Shigella sonnei and some strains of Escherichia coli.(6) The ONPG test detects the enzyme beta-galactosidase with greater speed and sensitively than lactose-fermentation tests.

O-nitrophenyl-beta-D-galactopyranoside (ONPG) is an artificial substrate structurally similar to lactose with the exception that glucose is substituted with an o-nitrophenyl group. ONPG is able to enter the bacterial cell more easily than lactose as it is not dependent on the presence of the permease enzyme. If the organism possesses beta-galactosidase, the enzyme will split the beta-galactoside bond, releasing o-nitrophenol, which is a yellow-colored compound. The activity of the galactosidase enzyme is increased in the presence of sodium ions.(6)

The organism to be tested is taken from a medium containing a high concentration of lactose. A dense suspension (turbidity equivalent to a McFarland 3) is prepared. An ONPG disk is added to 0.5ml of the suspension. If the organism possesses beta-galactosidase, the enzyme will split the beta-galactoside bond, creating a yellow color change in the suspension. Organisms with strong beta-galactosidase activity can produce a positive reaction a few minutes after inoculation of the ONPG medium; other organisms may take up to 24 hours.


Each HardyDisk™ ONPG Differentiation Disk is prepared by impregnating carefully controlled concentrations of ONPG onto a 0.25 inch diameter filter paper disk.


Storage: Upon receipt store at -20 to +8ºC. away from direct light. Product should not be used if there are any signs of deterioration, discoloration, or if the expiration date has passed. Protect the product from light, excessive heat, and moisture.



Specimen Collection: This product is not intended for primary isolation of patient specimens. It should be used only with cultures of isolated organism. This product is used in conjunction with other biochemical tests to identify cultures of isolated organism.

1. Use a loop to transfer bacteria from pure 18-24 hour culture to a test tube containing 0.5ml of 0.85% sterile saline. The resulting suspension should be approximately equivalent in density to a McFarland 3 opacity standard.

2. Add a single ONPG disk to the dense bacterial suspension. Upon the addition of the disk the bacterial suspension will be clear.

3. Incubate at 37ºC. and check hourly, for up to 4 hours, for the development of a yellow color change.

4. Incubate any negative reactions (colorless) for 24 hours. Observe after 24 hours for possible delayed reactions of late lactose-fermenters.


A yellow color change is a positive reaction; indicating beta-galactosidase activity. No color change is a negative reaction; indicating the absence of beta-galactosidase activity.


All organisms to be tested must be inoculated from a lactose-containing medium (e.g., TSI, KIA, or MacConkey Agar).

Cultures that naturally produce a yellow pigment cannot be tested with this media.

The ONPG test is not a substitute for the determination of lactose-fermentation since only the beta-galactosidase enzyme is evaluated.(6)


Standard microbiological supplies and equipment such as loops, other culture media, swabs, applicator sticks, incinerators, and incubators, etc., as well as serological and biochemical reagents, are not provided.


Test Organisms
Inoculation Method*
Escherichia coli

ATCC ® 25922
ONPG positive; yellow color develops within 1-24 hours
Proteus mirabilis

ATCC ® 12453
ONPG negative; no color development in 24 hours

User Quality Control


HardyDisk™ ONPG Differentiation Disks are 0.25 inch (in diameter) filter paper disks imprinted with the letters ONPG on both sides and should appear white in color.

Positive ONPG Test

Escherichia coli (ATCC ® 25922) suspension in 0.85% sterile saline with a HardyDisk™ ONPG Differentiation Disks (Cat. no. Z7321). Incubated aerobically for one hour at 35ºC. The yellow color development was indicative of a positive ONPG reaction.

Negative ONPG Test

Proteus mirabilis (ATCC ® 12453) suspension in 0.85% sterile saline with a HardyDisk™ ONPG Differentiation Disks (Cat. no. Z7321). Incubated aerobically for 24 hours at 35ºC. No yellow color development was indicative of a negative ONPG reaction.


1. Gottschalk, G. 1986; p.97-98, 178-179. Bacterial Metabolism, 2nd ed. Springer-Verlag, New York.

2. Anderson, N.L., et al. Cumitech 3B; Quality Systems in the Clinical Microbiology Laboratory, Coordinating ed., A.S. Weissfeld. American Society for Microbiology, Washington, D.C.

3. Jorgensen., et al. Manual of Clinical Microbiology, American Society for Microbiology, Washington, D.C.

4. Tille, P., et al. Bailey and Scott's Diagnostic Microbiology, C.V. Mosby Company, St. Louis, MO.

5. Isenberg, H.D. Clinical Microbiology Procedures Handbook, Vol. I, II & III. American Society for Microbiology, Washington, D.C.

6. Koneman, E.W., et al. Color Atlas and Textbook of Diagnostic Microbiology, J.B. Lippincott Company, Philadelphia, PA.

7. Lowe, G.H. 1962. The rapid detection of lactose fermentation in paracolon organisms by the demonstrations of B-D-galactosidase. Journal of Medical Lab Technology; 19:21-25.

8. Commission on Laboratory Accreditation, Laboratory Accreditation Program Microbiology Checklist. College of American Pathologists. Rev. 9/30/2004.

9. Centers for Medicare and Medicaid, Appendix C, Survey Procedures and Interpretive Guidelines for Laboratories and Laboratory Services. Subpart K - Quality System for Non-Waived Testing. 493;1200-1265. www.cms.hhs.gov/clia.

ATCC is a registered trademark of the American Type Culture Collection.