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The objective of Hippurate Hydrolysis Test
Production of the enzyme hippuricase is used for the presumptive identification of a variety of microorganisms. The hippurate test is used in the identification of Campylobacter jejuni, Listeria monocytogenes, Gardnerella vaginalis and Streptococcus agalactiae by detecting the ability of the organism to hydrolyze sodium hippurate to benzoic acid and glycine by the action of the enzyme hippuricase.
Principle of Hippurate Hydrolysis Test
The end products of hydrolysis of hippuric acid by hippuricase include glycine and benzoic acid. Glycine is deaminated by the oxidizing agent ninhydrin, which is reduced during the process. The end products of the ninhydrin oxidation react to form a purple-colored product. The test medium must contain the only hippurate because ninhydrin might react with any free amino acids present in growth media or other broths.
The procedure of Hippurate Hydrolysis Test
- Add 0.1 mL of sterile water to a 12 ×75 mm plastic test tube.
- Make a heavy suspension of the organism to be tested.
- Using heated forceps, place a rapid hippurate disk in the mixture.
- Cap and incubate the tube for 2 hours at 35°C; the use of a water bath is preferred.
- Add 0.2 mL ninhydrin reagent and reincubate for an additional 15 to 30 minutes.
- Observe the solution for the development of deep purple color.
Result Interpretation of Hippurate Hydrolysis Test
Positive: Deep purple color. Example: L. monocytogenes, S. agalactiae, C. jejuni, G. vaginalis, Trueperella pyogenes.
Negative: Colorless or slightly yellow-pink color. Example: Arcanobacterium haemolyticum.
Limitation of Hippurate Hydrolysis Test
- Not all S. agalactiae organisms are beta-hemolytic. Viridans group streptococci can be hippurate positive; another test must be done on nonhemolytic colonies to confirm the identification.
- A small number of enterococci are beta-hemolytic and may hydrolyze hippurate, but they are pyrrolidinyl-β-naphthylamide (PYR) positive.
- A small percentage of C. jejuni organisms are hippurate negative and must be identified by other methods.
- A negative test does not rule out the identification of G. vaginalis since the biotypes that cause bacterial vaginosis can be hippurate negative.
- False-positive results can occur if incubation with ninhydrin exceeds 30 min.
Positive: Streptococcus agalactiae (ATCC12386)
Negative: Streptococcus pyogenes (ATCC19615)