Hippurate Hydrolysis Test – Principle, Procedure, Result

Hippurate Hydrolysis Test is a biochemical test used for detecting the enzyme hippuricase(hippurate hydrolase) in microorganism. It is used for checking whether the organism can hydrolyze sodium hippurate or not.

This process occurs when bacteria having hippuricase is exposed to sodium hippurate. The substrate is hydrolyzed and two end products is formed. The reaction is as follows– sodium hippurate is broken into benzoic acid and glycine(amino acid).

The hydrolysis is detected by indicator methods. In classical method ferric chloride is added. If benzoic acid is present a persistent brown flocculent precipitate is formed and the test is positive. In rapid method ninhydrin reagent is used. If glycine is present deep purple or blue-violet color is produced and the result is positive.

It is used in clinical laboratories for presumptive identification of pathogens. It is mainly used to differentiate Streptococcus agalactiae(Group B Streptococcus) from other beta-hemolytic streptococci. It is also used for distinguishing Campylobacter jejuni from other Campylobacter species. It is helpful in identification of Listeria monocytogenes and Gardnerella vaginalis.

Purpose of Hippurate Hydrolysis Test

• A number of bacteria can be tentatively identified through the production of the enzyme hippuricase.

Principle of Hippurate Hydrolysis Test

It is based on detection of hippuricase(hippurate hydrolase) enzyme in bacteria. This enzyme is constitutive and it can hydrolyze the substrate sodium hippurate. If the organism possess the enzyme then hydrolysis is done and end products is released.

The reaction occurs when sodium hippurate is cleaved at amide linkage. Two products is formed benzoic acid and glycine(amino acid). The presence of these products is detected by indicator system.

In rapid method ninhydrin reagent is used. The glycine released is deaminated by ninhydrin and the reagent is reduced. A deep blue or purple colored complex is formed(Ruhemann’s purple) and it indicates positive test.

In classical method acidified ferric chloride is used. The ferric ions reacts with benzoate ions and a persistent brown flocculent precipitate is formed. This precipitate is ferric benzoate and it confirms positive result.

It is important that rapid test medium contains only sodium hippurate as protein source. Ninhydrin reacts with free amino acids also and it can give false positive reaction.

Media and Material Required for Hippurate Hydrolysis Test

Reagents and media

• Sodium hippurate substrate– sodium hippurate broth(heart infusion powder peptone sodium chloride sodium hippurate) or 1% aqueous sodium hippurate solution or rapid hippurate disks/tablets.
• Ninhydrin reagent– it is used in rapid method for detecting glycine. It is prepared by dissolving ninhydrin in acetone and butanol mixture.
• Ferric chloride reagent– it is used in classical method for detecting benzoic acid. It is prepared with ferric chloride concentrated hydrochloric acid and distilled/deionized water.
• Sterile diluent– sterile distilled water or saline(neutral pH) for preparing bacterial suspension.
• Growth media– Brain Heart Infusion Broth(BHI) is used in some disk inoculation methods.

Equipment and laboratory supplies

• Reaction containers– small optically clear test tubes(glass/plastic) or microtitre plates.
• Inoculation tools– sterile inoculating loop sterile cotton swab or sterile wooden stick.
• Incubation equipment– incubator or water bath maintained at 33°C to 37°C.
• Separation equipment– centrifuge is required in classical broth method for separating cells and supernatant.
• Liquid transfer– pipettes or droppers for adding reagent.
• Handling tools– clean/heated forceps for placing hippurate disk into tube.
• Sterilization equipment– loop sterilizer or incinerator.

Composition and Preparation of Hippurate Hydrolysis Broth

Composition of Hippurate Hydrolysis Broth(per litre)

• Heart infusion powder– 10.0 g.
• Peptone(or peptic digest of animal tissue)– 10.0 g.
• Sodium hippurate– 10.0 g.
• Sodium chloride– 5.0 g.
• Final pH– 7.4 ± 0.2 at 25°C.
• Total dehydrated powder– 35.0 g.

Preparation of Hippurate Hydrolysis Broth

1. Suspend– 35.0 g of dehydrated medium is added to 1000 ml distilled/purified water.
2. Dissolve– it is heated if required and the powder is dissolved completely.
3. Dispense– the medium is poured in test tubes in 5 ml amount.
4. Sterilize– it is autoclaved at 121°C(15 lbs pressure) for 15 minutes.
5. Storage– the prepared medium is stored in refrigerator(2–8°C) in tightly closed container.

Procedure of Hippurate Hydrolysis Test

Rapid ninhydrin method(detects glycine)

1. Inoculum preparation– a heavy bacterial suspension is prepared(at least No. 3 McFarland). It is made in small test tube using 0.1 ml to 0.4 ml sterile distilled water/saline(neutral pH 6.8–7.2). The organism is taken from pure 18–24 hour culture on non selective agar and agar is not picked.
2. Substrate addition– hippurate substrate is added by placing rapid hippurate disk aseptically into the tube or 0.4 ml of 1% aqueous sodium hippurate solution is added.
3. Initial incubation– the tube is capped and incubated at 35°C to 37°C for exactly 2 hours. Water bath is preferred for uniform heating.
4. Indicator addition– after 2 hours 2–4 drops(about 0.2 ml) of ninhydrin reagent is added.
5. Re-incubation– the tube is incubated again at 35°C to 37°C for 15 to 30 minutes.
6. Observation– the tube is checked at 10 minutes interval. Deep blue or purple colour within 30 minutes indicates positive test. The incubation is not done more than 30 minutes because false positive can occur.

Classical ferric chloride broth method(detects benzoic acid)

1. Inoculation– hippurate broth is inoculated with 1–2 drops of overnight broth culture or 1–2 isolated colonies of test organism. Positive and negative control tubes are also kept.
2. Incubation– tubes are incubated aerobically with loosened caps at 33°C to 37°C for 24 to 48 hours.
3. Supernatant separation– the broth is centrifuged after incubation and the cells is packed at bottom.
4. Transfer– the clear supernatant(about 0.8 ml) is transferred aseptically into new small test tube.
5. Reagent addition– acidified ferric chloride reagent is added to supernatant(usually 0.2 ml). The volume is decided by titrating uninoculated negative control broth and minimum reagent is selected which keeps solution clear.
6. Observation– the tube is shaken immediately and kept for 10 to 15 minutes. Persistent brown flocculent insoluble precipitate indicates positive test. If precipitate dissolves on shaking the test is negative.

Interpretations and results of Hippurate Hydrolysis Test

Ninhydrin method(rapid test)

• Positive result– deep blue or purple colour(similar to crystal violet) is developed within 30 minutes. It indicates hippurate is hydrolyzed and glycine is released.
• Negative result– no colour change is seen and solution remains colourless. Sometimes faint yellow-pink or faint blue-gray/purple colour is formed and it indicates no hydrolysis.

Ferric chloride method(classical test)

• Positive result– brown flocculent insoluble precipitate is formed. It persists after vigorous shaking and after standing for 10 to 15 minutes.
• Negative result– no precipitate is formed or the precipitate dissolves completely on shaking. The solution remains clear or slightly cloudy.

Organism interpretation

• Hippurate positive organism– Streptococcus agalactiae(Group B Streptococcus) Campylobacter jejuni Listeria monocytogenes Gardnerella vaginalis.
• Hippurate negative organism– Streptococcus pyogenes(Group A Streptococcus) Campylobacter coli Campylobacter laridis and most strains of Enterococcus faecalis.

Control Organisms of Hippurate Hydrolysis Test

Positive control organism

• Streptococcus agalactiae(ATCC 12386 or ATCC 4768)– it is the main positive control used for checking reagent sensitivity for Group B streptococci identification.
• Listeria monocytogenes(ATCC 7644)– it gives purple colour change and it is used as positive control.
• Klebsiella aerogenes– it can be used as positive control but the reaction is variable.

Negative control organism

• Streptococcus pyogenes(ATCC 19615)– it is the main negative control for checking reagent specificity and for differentiation from Group A streptococci.
• Enterococcus faecalis(ATCC 29212)– it is used as negative control to ensure no cross reaction with Group D organism.
• Enterobacter cloacae– it can be used as negative control.
• Streptococcus hominis– it can be used as negative control.

Limitations of Hippurate Hydrolysis Test

• Prolonged incubation can give false positive. If ninhydrin reagent incubation is done more than 30 minutes the colour can develop and it is read as positive.
• Amino acid interference is present. Ninhydrin reacts with free amino acids and proteins. If other protein source is present in medium or agar is picked during inoculation then false positive result can occur.
• Low inoculum can give false negative. Rapid method depends on pre formed enzyme and if suspension is not dense enough then reaction is not detected.
• Reagents are not stable. Sodium hippurate solution degrades fast and it lasts about 7 days at 4°C. Old solution can give false negative. Ninhydrin solution also deteriorates after about 6 months.
• Atypical strains can occur. Some Campylobacter jejuni isolates and some biotypes of Gardnerella vaginalis are hippurate negative so negative result cannot rule out these organism.
• Cross reaction is seen with other organism. Some viridans streptococci certain beta hemolytic enterococci and some Bacillus and Corynebacterium species can hydrolyze hippurate. Additional tests like PYR test is required for differentiation from Group B Streptococcus.
• Classical method needs strict steps. If tube is not shaken immediately after adding ferric chloride reagent then unreacted hippurate may not dissolve and false positive precipitate is formed. Iron concentration is also critical and titration is required.
• Culture restrictions is present. Fresh pure well isolated colonies is required and mixed culture cannot be used. Pigmented colonies from chromogenic agar should not be used because chromogen can interfere with colour reaction.
• It is only presumptive test. It cannot be used as single identification method and other biochemical immunological molecular or mass spectrometry tests are required for complete identification.

Uses of Hippurate Hydrolysis Test

• It is used for presumptive identification of Group B streptococci. It helps in identifying Streptococcus agalactiae and it is differentiated from other beta-hemolytic streptococci like Group A streptococci.
• It is used for speciation of Campylobacter. It is the definitive phenotypic test to distinguish Campylobacter jejuni(positive) from Campylobacter coli and Campylobacter laridis(negative).
• It is used for identification of Listeria species. It helps in identification of Listeria monocytogenes and it differentiates Listeria grayi(negative) from L. innocua L. ivanovii and L. monocytogenes(positive). It is also used for distinguishing Listeria from Erysipelothrix.
• It is used for detection of Gardnerella vaginalis. It helps in presumptive identification of Gardnerella vaginalis which is associated with bacterial vaginosis.
• It is used for differentiation of Legionella species. It separates Legionella pneumophila(positive) and L. feeleii(variable) from other Legionella species like L. bozemanii and L. micdadei(negative).
• It is used for differentiation of Actinobacillus species. It distinguishes Actinobacillus lignieresii(positive) from Actinobacillus equuli(negative).
• It is used for differentiation of Brevibacterium species. It distinguishes Brevibacterium iodinum(negative) from B. casei B. epidermidis and B. linens(positive).
• It is used for differentiation of Mobiluncus species. It separates Mobiluncus mulieris(negative) from M. curtisii subsp. curtisii and M. curtisii subsp. holmesii(positive).

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