Hippurate Hydrolysis Test: Definition, Principle, Procedure, and Results

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Hippurate Hydrolysis Test

Hippurate hydrolysis test is one of the biochemical tests, which is used to differentiate the micro-organisms on the basis of their ability to hydrolyze Hippurate into benzoic acid and glycine by the action of the enzyme hippuricase, present in the bacteria.

Hippuricase is one of the constitutive enzymes which helps in hydrolyzing the Hippurate and helps in producing amino acid, glycine.

Glycine can be detected by the oxidizing the Ninhydrin reagent, that results in the production of a deep purple color.

Hippurate hydrolysis test is used in the identification of Gardnerella vaginalis, campylobacter jejuni, Listeria monocytogenes and group B streptococci; to detect the ability of the organism to hydrolyze Hippurate.

What is Hippurate Hydrolysis Test?

In olden days this test was performed using a ferric chloride indicator to identify the benzoic acid, but those traditional methods would take a longer time to resolve.

But now a days with the modern techniques, this test has been modified and it is used as a rapid test by detecting the glycine by adding ninhydrin as an indicator.

This test can also be used to distinguish the Group B streptococci from other groups A, C, F and G which cannot hydrolyze sodium Hippurate.

Other group of viridians like Group D can be also detected by using Hippurate sodium hydrolyze.

This test is considered as one of the class of test which differentiates the bovine-β-hemolytic Group B streptococci from human β-hemolytic Group B species of streptococcus.

Hippurate Hydrolysis Test Objective

Hippurate hydrolysis test is generally used to detect the production of the enzyme hippuricase; for identifying the presumptive of different microorganisms. Its main aim is to differentiate bacteria based on their ability to produce hydrolyzed Hippurate.

Hippurate Hydrolysis Test Principle

Hippurate test is based on the ability of the organism to hydrolyze sodium Hippurate into glycine and benzoic acid with the action of an enzyme hippuricase.

This test is primarily used in identifying the campylobacter jejuni, Gardnerella vaginalis, Listeria monocytogenes and streptococci’s agalactiae.

This ability of the few bacterial species to hydrolyze the Hippurate was classically tested using a ferric chloride indicator which helps in detecting the benzoic acid.

However, now a days a 2-hour rapid method is opposed to 48-hour tractional method, for detecting the Hippurate hydrolysis.

The rapid is done by using ninhydrin as an indicator, which on reaction with protein or amino acids detects glycine.

As glycine is deaminated by oxidizing action of ninhydrin, which results in the reduction of ninhydrin and the substance resembles in purple color.

The test medium used here should contain only Hippurate as a source of protein as ninhydrin acts with the free amino acids that is present.

Thus, rapid Hippurate hydrolysis test helps in detecting the by- product of the benzoic acid which is seen as a sensitive and classical method.

Hippurate → Glycine + Benzoic acid

Glycine + Ninhydrin → Purple colored complex

The two important reagents used in this test are Hippurate solution and Ninhydrin.

1. Hippurate Solution

Hippurate reagent can be found commercially in the form of dehydrated tubes or in the form of disks or tablets. This can also be prepared in the laboratory in the form of 1% of Hippurate solution, for preparing in the laboratory, one gram of sodium Hippurate is added into 100 ml of distilled water.

2. Ninhydrin

Ninhydrin can also to purchased commercially. But mostly it is prepared in the laboratories while performing the diagnosis, for laboratory preparation, of the ninhydrin, 50ml of 1-butanol is added into a dark colored glass bottle. Then 3.5 grams of ninhydrin is added to the bottle and it is mixed well.

Hippurate Hydrolysis Test Materials

 Sterilised wooden stick and inoculating loops

 Incubator

 Test tubes

 Distilled water

Hippurate Hydrolysis Test Procedure
1. Preparation of Hippurate Solution

If dehydrated Hippurate is used, 0.2ml of distilled water is added at a pH of 6.8 to 7.2 is added with the test reagent. Then the two drops of distilled water are added to an empty tube for disk or tablets If Hippurate solution is prepared in the laboratory, 0.4ml of the reagent is added to the tube for each test.

2. Hippurate Hydrolysis Test

A heavy suspension is prepared in a tube from an 18 to 24-hour culture. The colony is then picked up carefully where the agar which contains protein should not be taken. The tube is then incubated for 30 minutes, at 35ºC to 37ºC.

Then the tube is observed for every for 10 minutes intervals till the deep blue colour; The change in colour appears usually appears within 15 minutes, after the addition of ninhydrin solution.

Hippurate Hydrolysis Test Result

 A positive Hippurate hydrolysis test results in the appearance deep blue which almost looks like a crystal violet within 30 minutes.

 A negative Hippurate hydrolysis test, results in the appearance of a faint blue colour or there will be no colour change.

Control Organism in Hippurate Hydrolysis Test

 Streptococcus agalactiae: it indicates Hippurate positive

 Streptococcus pyogenes: It indicates Hippurate negative

Hippurate Hydrolysis Test Citations


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