Cat. no. R92 ONPG Rapid Test Broth, 13x100mm Tube, 0.5ml 20 tubes/box


Hardy Diagnostics ONPG Rapid Test Broth is used in differentiating members of the Enterobacteriaceae and other microorganisms based on beta-D-galactosidase activity.


The ability of bacteria to ferment lactose depends on two enzymes, permease and beta-galactosidase. Permease allows lactose to enter the bacterial cell wall, where it is then broken down into glucose and galactose by beta-galactosidase. (3) The glucose and galactose can then be metabolized by the bacteria. However, some organisms lack permease and appear as late or non-lactose-fermenters. The ONPG test is considered to be a very sensitive test for lactose-fermentation.

O-nitrophenyl-beta-D-galactopyranoside (ONPG), an artificial substrate, is incorporated into this test and acts as the substrate for the beta-galactosidase. This substrate is capable of penetrating the bacterial cell without the presence of permease. The organism is taken from a medium containing a high concentration of lactose and is inoculated into the ONPG Broth. 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.


Ingredients per liter of deionized water:*

Disodium Phosphate 34.5gm
Proteose Peptone No. 3 7.5gm
Sodium Chloride 3.75gm
o-nitrophenyl-beta-D-galactopyranoside (ONPG) 1.5gm

Final pH 7.0 +/- 0.3 at 25ºC.

* Adjusted and/or supplemented as required to meet performance criteria.


Storage: Upon receipt store at less than -10ºC. (<-10ºC.) away from direct light. Product will arrive in the users laboratory non-frozen; the determination of shelf life has taken this transit period into consideration. Media should not be used if there are any signs of deterioration (shrinking, cracking, or discoloration), contamination, or if the expiration date has passed.

Product is light sensitive . Store frozen immediately and protect from light.



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

1. Bring test medium to room temperature.

2. Using a heavy inoculum from a pure 18-24 hour culture, inoculate the test medium.

3. Incubate aerobically, with loose caps, at 35ºC.

4. Examine for a yellow color development at 1 hour.

5. If the tube has not changed color after 1 hour of incubation, continue incubation for up to 24 hours.


A yellow color change is a positive test for ONPG. No color change is a negative test.


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

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


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* Incubation Results
Time Temperature Atmosphere
Escherichia coli
ATCC ® 25922**
D 1-24hr 35°C Aerobic ONPG positive; yellow color develops within 1-24 hours
Enterobacter aerogenes
ATCC ® 13048
D 1-24hr 35°C Aerobic ONPG positive; yellow color develops within 1-24 hours
Proteus mirabilis
ATCC ® 12453**
D 1-24hr 35°C Aerobic ONPG negative; no color development

** Recommended QC strains for User Quality Control according to the CLSI document M22 when applicable.

User Quality Control

Physical Appearance

ONPG Rapid Test Broth should appear clear, with no precipitate, and light amber in color.

Positive ONPG Test

Escherichia coli (ATCC ® 25922) growing in ONPG Rapid Test Broth (Cat. no. R92). 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) growing in ONPG Rapid Test Broth (Cat. no. R92). Incubated aerobically for 24 hours at 35ºC. No yellow color development was indicative of a negative ONPG reaction.


1. 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.

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

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

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

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

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

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