Cat. no. Z52 Hippurate Test 20 tests/kit


Hardy Diagnostics Hippurate Test is to be used in the presumptive identification of Gardnerella vaginalis, Campylobacter jejuni , Listeria monocytogenes and group B streptococci, by detecting the ability of the organism to hydrolyze hippurate.


The ability of bacterial species to hydrolyze the compound hippurate was classically tested using ferric chloride indicator to detect benzoic acid, the first byproduct in the hippurate hydrolysis pathway. However, a 2½ hour rapid method as opposed to the 48 hour classical method for detecting hippurate hydrolysis has since been developed. The rapid test employs ninhydrin as the indicator, which detects glycine, the second byproduct of hippurate hydrolysis. The rapid hippurate hydrolysis test has been shown to be as specific and as sensitive as the classical method that detects the benzoic acid byproduct. (4,5,8,9)


Each tube contains 20.0gm of Sodium Hippurate per liter of distilled water when rehydrated.

Each 2ml Ninhydrin Indicator Solution bottle contains 45.0gm Ninhydrin Reagent per liter of distilled water when reconstituted.

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



Storage: Upon receipt store at 2-30ºC. Products should not be used if there are any signs of contamination, deterioration, if the expiration date has passed, or if the rehydrated mixtures do not appear clear and colorless. Do not use if the tube is cracked or the powder is not free-flowing. Do not expose to excessive heat or moisture.


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

1. To a Hippurate Test tube, add 0.2ml (3-4 drops) of distilled water at a pH of 6.8-7.2.

2. Using a heavy inoculum (a full 10ul loop is adequate) from an 18-24 hour culture, make a heavy suspension of the organism in the Hippurate Reagent with a standard inoculating loop.

3. Incubate the tube for two hours at 35-37ºC.

4. During the incubation period, reconstitute the Ninhydrin Indicator Solution in the dropper bottle by adding 2ml of distilled water at a pH of 6.8-7.2. Replace the cap tip and cap, and vigorously shake for one minute. Let stand at room temperature for 30 minutes or until all the substrate has dissolved.

5. After the two hour incubation period, add two drops of the Ninhydrin Indicator Solution to the Hippurate Reagent and organism mixture.

6. Reincubate at 35-37ºC. for 30 minutes. Observe the tubes at 10 minute intervals for the appearance of a deep blue color, which is a positive test. The color change will usually appear in 10 to 15 minutes after the Ninhydrin Indicator Solution has been added.


A positive test is indicated by the appearance of a deep blue/violet color in 30 minutes. A negative reaction is indicated by a faint blue color change, or no color change.


Insufficient inoculum may result in erroneous results.

When Streptococcus species are tested for their ability to hydrolyze hippurate, it must be remembered that not all group B streptococci are beta-hemolytic. In addition, a small number of group D streptococci are beta-hemolytic, and some hydrolyze hippurate as well.

Once rehydrated, the Ninhydrin Indicator Reagent should be clear and colorless and can be stored refrigerated for a period of up to 6 months. Check for signs of discoloration and discard the Reagent if it becomes discolored.


Standard microbiological supplies and equipment such as loops, incinerators, incubators, pipettes, test tube racks, and distilled water, etc., as well as serological and biochemical reagents, are not provided.


Test Organisms
Streptococcus agalactiae

ATCC ® 12386
Positive: Dark blue/violet color change
Streptococcus pyogenes

ATCC ® 19615
Negative: No color change or a faint blue seen

User Quality Control


1. The Hippurate powder should appear white in color.

2. The rehydrated Hippurate Reagent should appear clear and colorless.

3. The rehydrated Ninhydrin Indicator Reagent should appear clear and colorless.

Hippurate Test

Showing positive (left tube) and negative (right tube) hippurate reactions.
Four drops of deionized water were added to a Hippurate Test tube (Cat. no. Z52). Heavy suspensions from 24 hour cultures of Streptococcus agalactiae (ATCC ® 12386) and Streptococcus pyogenes (ATCC ® 19615) were made in the left and right tubes, respectively. The tubes were incubated aerobically for two hours at 35ºC. After the incubation process, two drops of reconstituted Ninhydrin Indicator Solution were added to each tube. A dark blue/violet color change was indicative of a positive hippurate 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. Jorgensen., et al. Manual of Clinical Microbiology, American Society for Microbiology, Washington, D.C.

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

4. Harvy, S.M. 1980. Hippurate hydrolysis by Campylobacter fetus. J. Clin. Microbiol.; 11:435-437.

5. Hwang, M. and G.M. Ederer. 1975. Rapid hippurate hydrolysis method for presumptive identification of group B streptococci. J. Clin. Microbiol.; 1:114-115.

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

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

8. Luechtefeld, N.W. and W.L. Wang. 1982. Hippurate hydrolysis by and triphenyltrazolium tolerance of Campylobacter fetus. J. Clin. Microbiol.; 15:137-140.

9. Piot, P., et al. 1982. Identification of Gardnerella (Haemophilus) vaginalis. J. Clin. Microbiol.; 19-24.

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