Hydrolysis Test- Principle, Uses, Procedure, Result Interpretation with Limitations

Hippurate Hydrolysis test

Hippurate hydrolysis test is used in the presumptive identification of Gardnerella vaginalis, Campylobacter jejuni, Listeria monocytogenes and group B streptococci, by detecting the ability of the organism to hydrolyze hippurate.

Principle

Hippurate hydrolysis positive organisms can hydrolyze 1% aqueous sodium hippurate to produce glycine and sodium benzoate. Glycine is deaminated by the oxidizing agent ninhydrin which gets reduced and becomes purple. The test medium must contain only hippurate, since ninhydrin reacts with any free amino acids present.

Significance

Used in the differentiation of β-hemolytic Streptococcus agalactiae from other β-hemolytic streptococci.
Aids in the separation of Campylobacter jejuni and Campylobacter coli strains.

Procedure

1. Take a test tube and add 0.2ml (3-4 drops) of sterile distilled water.
2. Make a heavy suspension of the organism to be tested from an 18-24 hour culture.
3. Place a rapid hippurate disk in the mixture.
4. Cap and incubate the tube for 2 hours at 35-37⁰
5. After the 2 hours of incubation, add 0.2 ml of the ninhydrin reagent to the mixture.
6. Reincubate at 35-37^oC for 15-30 minutes.
7. Observe the tubes at 10 minute intervals for the appearance of a deep purple color.

Result interpretation

Positive: Development of a deep purple color within 5-10 minutes is a positive result.

Negative: No development of color within 5-10 minutes is a negative result.

Quality Control

Cultural Response

 The Hippurate hydrolysis reaction is observed after an incubation at 35-37°C for 24-48 hours, of various bacteria with Hippurate differentiation discs, tested using Brain Heart Infusion Broth.

Limitations

1. This test is only useful when distinguishing β-hemolytic streptococcus of human or bovine origin.
2. A faint purple color is considered a negative result.
3. Hippurate hydrolysis is also found in some species of other genera, such as Bacillus, Corneybacterium, Enterobacteriaceae, and others.
4. The hippurate solution deteriorates in 7 days at 4°C.
5. The ninhydrin solution deteriorates in 6 months.

Hydrolysis Test- Principle, Uses, Procedure, Result Interpretation with Limitations

Tags: Hippurate Hydrolysis test, Hippurate Hydrolysis test Limitation, Principle of Hippurate Hydrolysis test, Procedure of Hippurate Hydrolysis test, Result Interpretation of Hippurate Hydrolysis test, Significance of Hippurate Hydrolysis test