Hippurate hydrolysis test: Principle, Procedure, Result interpretations and Limitations

Hippurate hydrolysis test is a biochemical experiment used to distinguish between specific bacteria species, particularly those from the Streptococcus and Campylobacter genera. This test is mostly used to identify Group B Streptococcus (Streptococcus agalactiae) and Campylobacter jejuni.

Hippurate Hydrolysis Test

The hippurate hydrolysis test is a biochemical experiment that distinguishes and identifies certain bacterial species by their capacity to hydrolyze hippurate into glycine and benzoic acid. This test is frequently used in clinical microbiology to diagnose Group B Streptococcus (Streptococcus agalactiae) and Campylobacter jejuni, among others.

Principle

The hippurate hydrolysis test relies on the ability of particular microorganisms to hydrolyze hippurate into glycine and benzoic acid. Hippuricase is the enzyme responsible for this hydrolysis. When bacteria that generate hippuricase are treated with sodium hippurate, the enzyme degrades it into glycine and benzoic acid. The presence of glycine can then be identified using the ninhydrin reagent, which reacts with free amino acids (including glycine) to give a purple color.

Procedure

Materials Needed:

• Sodium hippurate solution.
• Bacterial inoculum and ninhydrin reagent.
• Test or microcentrifuge tubes
• Set the incubator to 35-37°C. 
• Use a water bath or heat block.

Steps

Preparation:

Make a sodium hippurate solution by dissolving it in sterile water.

Inoculation:

Inoculate the sodium hippurate solution with a high concentration of the bacterial culture to be examined.

Incubation:

Incubate the infected solution at 35–37°C for 2 hours. Some procedures may recommend an overnight incubation for best results.

Addition of Ninhydrin:

Following incubation, add the ninhydrin reagent to the solution. Typically, a few drops are plenty.

Heating:

Heat the mixture in a water bath or heat block at 80-100°C for around 10 minutes. This phase is necessary for color development.

Observation:

After heating, note the color change in the solution.

Result Interpretations

Positive Result:

• Appearance: The solution takes on a deep purple tint.
• Interpretation: The presence of glycine from hippurate hydrolysis suggests that the bacterium makes hippuricase. This finding is typical of Group B Streptococcus (Streptococcus agalactiae) and Campylobacter jejuni.

Negative Result:

• Appearance: There is no or only a minor color change, and the solution remains clear or slightly muddy.
• Interpretation: The absence of glycine implies that the bacterium does not hydrolyze hippurate. This outcome is common among bacteria that do not make hippuricase, such as Group A Streptococcus (Streptococcus pyogenes).

Limitations

Specificity and sensitivity:

While the test is generally reliable for detecting Group B Streptococcus and Campylobacter jejuni, it may not be completely accurate for all strains or species. False positives or negatives may occur.

Quality of reagents:

The purity and freshness of the ninhydrin reagent are crucial for obtaining accurate outcomes. Degraded ninhydrin may produce weak or false-negative reactions.

Incubation Conditions:

The incubation period and temperature must be strictly managed. Deviations have the potential to influence enzyme activity and the resultant color response.

Interfering Substance:

The presence of additional amino acids or pollutants in the sample may interfere with the ninhydrin reaction, yielding misleading findings.

Bacterial concentrations:

A adequate inoculum is required for the test to function. A little inoculum may not offer enough enzyme to produce measurable levels of glycine.

Technical variability:

Variations in the procedure steps, such as heating time and temperature, can have an impact on the outcome, demanding rigorous protocol adherence to achieve consistent results.

Frequently Asked Question

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