Hippurate Hydrolysis Test: Principle, Procedure, Results

Last updated on June 23rd, 2021

Hippurate is the glycine conjugate of benzoic acid. When hippurate is hydrolyzed by an organism glycine and benzoic acid are formed.

Hippurate hydrolysis test-Left (positive tube), Right (Negative tube)
Hippurate hydrolysis test-Left (positive tube), Right (Negative tube)

In the classical test (48 hours), the benzoic acid produced was detected using a ferric chloride indicator, by its precipitation in an excess of ferric chloride. The gas-liquid chromatography (GLC) method for the detection of benzoic acid is also available nowadays.

In the rapid test (2 hours), ninhydrin is used as the indicator to detect 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.


Hippuric acid is hydrolyzed to benzoic acid and glycine by the enzymatic action of hippuricase. The glycine end product is detected by the addition of ninhydrin reagent. Ninhydrin reacts with glycine to form a deep blue or purple color (Ruhemann’s purple). This rapid test used in most laboratories is one of several variations of the ninhydrin tube test described by Hwang and Ederer for detection of glycine.


The hippurate test is used in the identification of Campylobacter jejuni, Listeria monocytogenes, Gardnerella vaginalis, and Streptococcus agalactiae.

  1. Hippurate hydrolysis test is critical for the separation of Campylobacter jejuni (hydrolyzes hippurate) and Campylobacter coli (negative) strains.
  2. Aids in the differentiation of β-hemolytic Streptococcus agalactiae from other β-hemolytic streptococci.

Test Procedure

A. Classical Method:

  1. Take or prepare sterile sodium hippurate broth and inoculate it with the test organism.
  2. Incubate overnight at 35°C.
  3. Centrifuge the broth and remove the precipitate.
  4. Add ferric chloride reagent in the supernatant.
  5. If the precipitate remains after 10 minutes, benzoic acid is present and the test is positive for hippurate hydrolysis

B.  Ninhydrin Method:

  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 from an 18-24 hour culture, make a heavy suspension of the organism in the hippurate reagent with a standard inoculating loop (the tube should be cloudy looking after inoculation, turbid).
  3. Incubate the tube for two hours at 35-37°C.
  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. Re-incubate at 35-37°C 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.


  1. A positive reaction is indicated by the appearance of a deep blue color (about the color of crystal violet) within 30 min.
  2. A negative reaction is indicated by a faint purple color or no color change.

Quality Control

Test each new lot or shipment of reagent with known positive and negative controls, and retest at least monthly thereafter. Discard all reagents and prepare new ones if the reagents do not pass QC. For quality control grow the ATCC strains in sodium hippurate broth and look for the results.

  1. Streptococcus agalactiae (ATCC 4768): Moderate to heavy growth and hydrolysis of hippurate
  2. Streptoccus pyogenes (ATCC 19615): Moderate to heavy growth; hippurate not hydrolyzed.

Hippurate Hydrolysis Test Positive Organisms :

Hippurate hydrolysis test is used in the presumptive identification of various bacteria. Organisms giving positive (+ve) test are:


  • False-positive results can occur if incubation with ninhydrin exceeds 30 min.
  • A negative test does not rule out the identification of G. vaginalis, since the biotypes that cause bacterial vaginosis can be hippurate negative.
  • Viridans group streptococci can be hippurate positive; another test must be done on nonhemolytic colonies to confirm the identification as some S. agalactiae organisms may be non-hemolytic.
  • A small number of enterococci are beta-hemolytic and may hydrolyze hippurate, but they are pyrrolidonyl-β-naphthylamide (PYR) positive (S. agalactiae is PYR negative)
  • A small percentage of C. jejuni organisms are hippurate negative and must be identified by other methods.

References and further readings

  1. Clinical Microbiology Procedures Handbook, Fourth Edition. (2016). In Clinical Microbiology Procedures Handbook, Fourth Edition. American Society of Microbiology. https://doi.org/10.1128/9781555818814
  2. Color Atlas and Textbook of Diagnostic Microbiology, Koneman, 5th edition
About Acharya Tankeshwar 474 Articles
Hello, thank you for visiting my blog. I am Tankeshwar Acharya. Blogging is my passion. I am working as an Asst. Professor and Microbiologist at Department of Microbiology and Immunology, Patan Academy of Health Sciences, Nepal. If you want me to write about any posts that you found confusing/difficult, please mention in the comments below.