Hippurate Broth Principles and Hippurate Hydrolysis Test

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Hippurate Test

Hippurate Broth is a liquid medium which is recommended for use in qualitative determination of the ability of an organism producing the enzyme Hippuricase to hydrolyze sodium hippurate mainly Streptococcal species such as  Streptococcus agalactiae

Principle of Hippurate Broth

  • Peptones and heart infusion supply nitrogenous compounds and amino acids necessary for the growth of streptococci.
  • Also sodium chloride is a source of essential electrolytes that  maintains osmotic equilibrium.
  • Dextrose sugar is the energy source which also stimulates the production of streptococcal antigenic hemolysin.
  • Disodium phosphate and sodium carbonate acts as buffers which counteract the effects of acid produced during the fermentation of dextrose and prevent inactivation of the hemolysin.
  • Hippuricase hydrolysis sodium hippurate in the medium to form benzoic acid and glycine. The addition of ferric chloride results in ferric ion combining with benzoic acid to form an insoluble precipitate know as  ferric benzoate.

Composition of Hippurate Broth

Casein Peptone10.0 g
Meat Peptone10.0 g
Sodium Hippurate10.0 g
Heart Infusion3.1 g
Sodium Carbonate2.5 g
Dextrose2.0 g
Sodium Chloride2.0 g
Disodium Phosphate .0.4 g
Demineralized Water1000.0 ml
Final pH at temperature  25°C7.8 ± 0.2

How to Prepare Hippurate Broth

  1. Start by suspending 35 grams in 1000 ml distilled water.
  2. Heat if necessary to dissolve the medium completely.
  3. Dispense 5 ml amounts in tubes.
  4. Sterilize by autoclaving at 15 lbs pressure (121°C) for 15 minutes.

Appearance of  Hippurate Broth After Preparation

It has a yellow coloured, clear solution without any precipitate

Type of Inoculums

  • Wound Exudates
  • Blood
  • Nose or throat swab

Hippurate Hydrolysis Test (Using Ferric Chloride)

Hippurate Hydrolysis Test Reagents

Ferric Chloride 12.0 g
Concentrated Hydrochloric Acid5.4 ml
Demineralized Water94.6 ml
Also read  How to prepare Blood and Chocolate agar using Columbia Agar base

Note that these reagent is corrosive  and may cause skin irritation or burns ,eyes or respiratory tract so avoid breathing vapor and eye or skin contact contact by wearing Personal protective equipments

Procedure of Hippurate Hydrolysis Test (Using Ferric Chloride)

  1. Start by adding approximately 75 ml of distilled water to a 100 ml volumetric flask.
  2. With a transfer pipette, add 5.4 ml of HCl to the flask, running down the acid along the sides of the flask.
  3. Add 12 gram of ferric chloride.
  4. Dissolve by warming the flask gently, swirling the contents to mix well. Bring the volume up to 100 ml with distilled water. The solution appears orange in colour.
  5. Inoculate tubes with 1 to 2 drops of 18 to 24 hours old pure broth culture of a confirmed beta-haemolytic Streptococcus or with one to two isolated colonies from an original isolation plate.
  6. Include an uninoculated tube as a negative control and a positive control ( Streptococcus agalactiae ).
  7. Incubate tubes with loosened caps for 48 hours at 35 ± 2°C in an aerobic atmosphere.
  8. After incubation, centrifuge all cloudy cultures and use the supernatant fluid in the test.
  9. Aseptically transfer a specific aliquot of culture (or its supernatant) to a small test tube.
  10. Add ferric chloride solution (0.2 ml of 12% FeCl3 solution).
  11. Shake the tube immediately. Stand for 10-15 minutes before interpretation.
  12. If the test is positive, brown flocculant insoluble precipitate persists on shaking after 10 minutes i.e. hippurate is hydrolyzed.
  13. If the precipitate gets dissolved on shaking, hippurate is not hydrolyzed and the test is negative.
Also read  Papanicolaou stains : Principle ,Procedure and Interpretation

Cultural response after Incubation

Incubation is done at temperature 35-37°C for 24-48 hours

OrganismHippurate Hydrolysis
Enterococcus faecalis negative reaction,precipitate if any, dissolves on shaking
Streptococcus agalactiaepositive reaction, brown flocculent precipitate persisting on shaking after 10 minutes
Streptococcus pyogenesnegative reaction, precipitate if any, dissolves on shaking

Hippurate Hydrolysis Test (Using Ninhydrin Method)


  • The bacterial enzyme hippuricase hydrolysis sodium hippurate present in the medium to form glycine and benzoic acid.
  • Glycine is deaminated by the oxidizing agent ninhydrin which gets reduced and becomes purple.

Procedure of Hippurate Hydrolysis Test (Using Ninhydrin Method)

  1. Into hippurate broth, add 0.2ml (3-4 drops) of distilled water at a pH of 6.8-7.2.
  2. Using a heavy inoculum from an 18-24 hour culture, make a heavy suspension of the organism in the Hippurate Reagent with a standard inoculating loop (The tube should be cloudy looking after inoculation, turbid.).
  3. Incubate the tube for two hours at 35-37°C.
  4. During the incubation period, reconstitute the Ninhydrin indicator solution in the dropper bottle by adding 2 ml of distilled water at a pH of 6.8-7.2. Replace the cap tip and cap, and vigorously shake for one minute. Let stand at room temperature for 30 minutes or until all the substrate has dissolved.
  5. After the two hour incubation period, add two drops of the Ninhydrin indicator solution to the hippurate reagent and organism mixture.
  6. Re-incubate at 35-37°C for 30 minutes. Observe the tubes at 10 minute intervals for the appearance of a deep blue color, which is a positive test. The color change will usually appear in 10 to 15 minutes after the Ninhydrin indicator solution has been added.
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Observation and Interpretation

Positive testDeep blue colour.
Negative test resultPale blue colour.

Quality Control

Streptococcus agalactiaeModerate to heavy growth and Hydrolysis of Hippurate
Streptococcus pyogenesModerate to heavy growth; Hippurate not hydrolyzed.

Hippurate test positive Organism

  • Gardnerella vaginalis,
  • Campylobacter jejuni,
  • Listeria monocytogenes and
  • Group B streptococci (Streptococcus agalactiae)

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About the Author: Arthur Westmann

DEFFE ARTHUR (AMOEBAMANN) is the founder and author of MLTGEEKS and MLTEXPO.He’s from Cameroon and is currently a Final year State Medical Laboratory Technician (MLT MA). Beyond lab works, he’s a passionate internet user with a keen interest in web design and blogging. Furthermore He likes traveling, hanging around with friends and social networking to do in his spare time.

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