Hippurate Hydrolysis Test: Introduction, Principle, Test Requirement

Introduction of Hippurate Hydrolysis Test

Table of Contents

The Hippurate Hydrolysis Test is applied to determine the ability of the organism to produce hippuricase which hydrolyzes the substrate hippurate. It is useful in the identification of Streptococcus agalactiae, Campylobacter jejuni, Listeria monocytogenes, and Gardnerella vaginalis.

Beta-haemolytic streptococci (Streptococcus agalactiae) growth on blood agar of High Vaginal Swab (HVS) — Fig. Beta-haemolytic streptococci (*Streptococcus agalactiae*) growth on blood agar of High Vaginal Swab (HVS)

Bacitracin resistant (0.04 U)-Streptococcus agalactiae — Fig. Bacitracin resistant (0.04 U)-*Streptococcus agalactiae* on blood agar

Streptococcus agalactiae showing beta-hemolytic colonies through bright transmitted light and bacitracin (0.04U) resistant — Fig. *Streptococcus agalactiae* showing beta-hemolytic colonies through bright transmitted light and bacitracin (0.04U) resistant

CAMP Test-Positive Streptococcus agalactiae — Fig. CAMP Test-Positive *Streptococcus agalactiae*

Listeria monocytogenes beta-haemolytic colony on Blood agar — Fig. *Listeria monocytogenes* beta-hemolytic colony on Blood agar

Fig. Campylobacter fetus growth on Chocolate agar

Principle of the Test

The end products of hydrolysis of the substrate hippurate by a constitutive enzyme hippuricase of the test organisms include glycine and benzoic acid. Glycine is deaminated by the oxidizing agent, ninhydrin, which is reduced during the process. The end products of ninhydrin oxidation react to form a purple-colored product that can be easily visualized.

Test Requirements for Hippurate Hydrolysis Test

Test organisms
Test tubes
Forceps
Hippurate disk
Sterile water
Inoculating wire
Bunsen burner
Water bath
Control strains (Streptococcus agalactiae-Positive control while Enterococcus species for Negative control)

Test Procedure

Add 0.1 mL of sterile water to a test tube (12 ×75 mm).
Prepare a heavy suspension of the organism to be tested.
Using sterile 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.
After that put 0.2 mL of ninhydrin reagent and reincubate for an additional 15 to 30 minutes.
Perform steps 1-5 if you are using control strains for positive and control tests.
Observe the solution of the test tube for the development of deep purple color.

Result and Interpretation of Hippurate Hydrolysis Test

Positive: Deep purple color

Negative: Slightly yellow-pink or colorless

Control strains-

Streptococcus agalactiae: Positive

Enterococcus species: Negative

Fig. Hippurate Hydrolysis Test-Positive and Negative Results

Limitations of the Test

A false-positive result may happen if incubated with ninhydrin for more than 30 minutes.
The assaying medium must contain only Hippurate since ninhydrin might react with any free amino acids present in using media or other broths.
The Hippurate hydrolysis test is also positive rather than above mention positive list like some species of other genera, Bacillus, Corynebacterium, Enterobacteriaceae, and so on.
The life span of Hippurate solution is short which may deteriorate in 7 days at 4°C. while the ninhydrin solution deteriorates in 6 months.
It is just a presumptive identification and therefore it is recommended that biochemical, immunological, molecular, or mass spectrometry testing be performed on colonies from pure culture for complete identification.
An insufficient amount of inoculum may result in erroneous results.

Keynotes on Hippurate Hydrolysis Test

This is only a presumptive test and thus it should be confirmed by further assaying.
A list of Hippurate Hydrolysis Test-positive organisms are- Gardnerella vaginalis, Campylobacter jejuni, Listeria monocytogenes, and group B streptococci (Streptococcus agalactiae).
1% Hippurate Broth devised by Hwang and Ederer is also an available alternative to the disk test.
This test is not feasible in growth media containing protein since ninhydrin may react with any free amino acids contained in the media.
Composition of Ninhydrin for Hippurate Hydrolysis Test: Formulation per 10 mL

Ingredients	Amount (for 10 mL)
Ninhydrin	0.35 g
Acetone	5.0 mL
Butanol-	5.0 mL

Table: Composition of Ninhydrin for Hippurate Hydrolysis Test Reagent

Hippurate Hydrolysis Test: Introduction, Principle, Test Requirements, Procedure, Result-Interpretation, Limitations, and Keynotes