Hippurate Hydrolysis Test: Principle, Procedure, Results

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

Hippurate hydrolysis occurs in an organism that can form glycine and benzoic acid.

In the classical test (48 hours), the production of benzoic acid was detected using ferric chloride as an indicator. The gas-liquid chromatography (GLC) method is also available to detect benzoic acid.

In contrast, the rapid test (2 hours) detects glycine, the second byproduct of Hippurate hydrolysis. It uses ninhydrin as the indicator. Rapid Hippurate hydrolysis is more specific and as sensitive as the classical method.

Principle of Hippurate Hydrolysis

Hippuric acid is hydrolyzed to benzoic acid and glycine by the enzymatic action of hippuricase. The addition of a ninhydrin reagent detects glycine. Ninhydrin reacts with glycine to form a deep blue or purple color (Ruhemann’s purple). Hwang and Ederer described this rapid test for detecting glycine.

Similarly, the traditional method detects benzoic acid using ferric chloride. The ferric chloride dissolves the benzoic acid present as residue after centrifugation of hippurate solution with the test organism.

Uses of Hippurate Hydrolysis

The hippurate test identifies Campylobacter jejuni, Listeria monocytogenes, Gardnerella vaginalis, and Streptococcus agalactiae.

  1. The hippurate hydrolysis test is critical for separating Campylobacter jejuni (hydrolyzes hippurate) and Campylobacter coli (negative) strains.
  2. It also aids in differentiating β-hemolytic Streptococcus agalactiae from other β-hemolytic streptococci.

Test Procedure

Classical Method

  • Firstly, take or prepare sterile sodium hippurate broth and inoculate it with the test organism.
  • After that incubate it overnight at 35°C.
  • Then, centrifuge the broth and remove the sediment.
  • Finally, add ferric chloride reagent to the supernatant.
  • If the residue remains after 10 minutes, benzoic acid is present. The test is positive for hippurate hydrolysis.

Ninhydrin Method

  • Firstly, to a hippurate test tube, add 0.2ml (3-4 drops) of distilled water at a pH of 6.8-7.2.
  • Then, 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).
  • After that, incubate the tube for two hours at 35-37°C.
  • During the incubation period, reconstitute the ninhydrin indicator solution in the dropper bottle by adding 2ml of distilled water at pH 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.
  • After the two-hour incubation period, add two drops of the ninhydrin indicator solution to the hippurate reagent and organism mixture.
  • Then re-incubate at 35-37°C for 30 minutes.
  • After that, 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 10 to 15 minutes after the added ninhydrin indicator solution.

Interpretations of Hippurate Hydrolysis Test

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

Quality Control used in Hippurate Hydrolysis Test

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

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

Hippurate Hydrolysis Test Positive Organisms :

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

  • Gardnerella vaginalis,
  • Campylobacter jejuni,
  • Listeria monocytogenes and
  • Group B streptococci (Streptococcus agalactiae)


  • 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; performing other tests to confirm the identification of non-hemolytic colonies is essential.
  • A small number of enterococci are beta-hemolytic and may hydrolyze hippurate, but they are pyrrolidinyl-β-naphthylamide (PYR) positive (S. agalactiae is PYR negative)
  • Furthermore a small percentage of C. jejuni organisms are hippurate negative and use of other methods for complete identification is necessary.

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

Acharya Tankeshwar

Hello, thank you for visiting my blog. I am Tankeshwar Acharya. Blogging is my passion. As an asst. professor, I am teaching microbiology and immunology to medical and nursing students at PAHS, Nepal. I have been working as a microbiologist at Patan hospital for more than 10 years.

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