Citrate Utilization Test

Citrate utilization test distinguishes different genera of enteric bacteria based on citrate utilization by an enzyme catalyst (Citrase) produced by the test organism. During citrate utilization, many by-products form. By the complete hydrolysis of citrate, an alkaline product forms, namely sodium carbonate that cause alkalinity in the medium and change the colour of media from green to blue by the change in pH of bromothymol indicator.

This test is based on the production of an enzyme named Citrase that provides energy to the microorganisms by the break down of carbon-containing compound Citrate. The organisms also gets energy by the degradation of a nitrogen-containing ammonium salt present in the media.

Content: Citrate Utilization Test

  1. Definition
  2. Citrate Test Media
  3. Principle
  4. Procedure
  5. Result Interpretation


It can define as a part of the IMViC test, where ‘C’ is an acronym for the process refers as Citrate utilization test. Citrate test is a standard method for the identification of enteric bacteria based on the property of exploiting energy in the form of carbon and nitrogen from the sources like sodium citrate and inorganic ammonium salts, respectively. It also distinguishes the organism based on the production of enzyme citrase that catalyses the conversion of sodium citrate into pyruvate.

Citrate Test Media

For citrate utilization test, Simmons citrate agar media is used that includes the following components:

Ammonium dihydrogen phosphate: 1g
Dipotassium phosphate: 1g
Sodium chloride: 5g
Sodium citrate: 2g
Magnesium sulphate: 0.20g
Agar: 15g
Bromothymol blue: 0.08g
Distilled water: 1L
Final pH: 6.9

Principle of Citrate Utilization Test

Citrate is an organic acid, which hydrolysis will ultimately result in the production of alkaline carbonates or bicarbonates. Simmons citrate agar is a media used to detect whether the test organism can exploit citrate or not. The citrate metabolism undergoes subsequent chemical reactions.
Principle of Citrate Test
First, sodium citrate hydrolyses by the aid of enzyme “Citrase lyase” into oxaloacetate and acetate. Then, oxaloacetate further decarboxylates by the enzyme “oxaloacetate decarboxylase” that converts it into pyruvate and carbon dioxide.

In acidic pH pyruvate further breaks down into acetoin, lactate and CO2. In alkaline pH pyruvate breaks down into acetate, formate and CO2. The released CO2 binds with the sodium ions of the media used to form sodium carbonate. The test organism makes the use of nitrogen from the ammonium salts of the media. The metabolism of ammonium salts by the microorganisms yields ammonia, which later combines with H2O to form ammonium hydroxide.

Compounds like sodium carbonate and ammonium hydroxide cause alkalinity of the media, which can be visualized by a colour change in the pH indicator bromothymol from green to blue colour due to change of pH from 6.9 to more than 7.5. In neutral pH, the colour of bromothymol indicator appears deep green coloured, but as the pH rises, it seems deep Prussian blue in colour.


A protocol for the citrate test includes the following steps:

  1. Weigh the exact quantity of Simmons citrate agar in a weighing machine and add distilled water as per the need.
  2. Autoclave the media to get rid of any contamination at 121 degrees C temperature for 15-20minutes.
  3. Under sterilized conditions, pour 3-4ml of media to the test tubes.
  4. After pouring, keep the test tubes in a slightly inclined position to prepare a slant.
  5. After solidification, take the pure culture of an organism to be tested via a sterilized inoculating loop.
  6. Streak inoculum over the inclined surface of slant and incubate it up to 24-48 hours at a temperature of 35 degrees Celsius.
  7. Observe the test tubes for any colour change in the media.

Result Interpretation

Positive result: Confirms by the bacterial growth, and the colour change of media from green to deep Prussian blue colour.
Examples: Salmonella sp, Klebsiella sp, Citrobacter sp, Proteus sp etc.

result interpretation

Negative result: There will be no bacterial growth and no colour change in the media.
Examples: Escherichia sp, Yersinia sp, Shigella sp etc.

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