Citrate Utilization Test | Its Principle, Procedure and Applications

A citrate utilization test is an identification test for bacteria.

It is one of the IMVic series groups of tests where IMViC stands for Indole, Methyl red, VP, and citrate test.

A citrate utilization test helps detect the organism’s ability to produce citrase enzymes, i.e., citrate lyase.

The bacteria which rely on citrate as the sole source of carbon for their energy needs are determined.

Here, the bacteria utilize sodium citrate as its only source of carbon and inorganic ammonium dihydrogen phosphate (NH4H2PO4) as its only source of nitrogen.

This test helps differentiate the Gram-negative bacilli from the Enterobacteriaceae family.

It also helps to distinguish coliforms from fecal coliforms.

Coliforms:- These occur naturally in the soil and the aquatic environments.

An example includes Enterobacter aerogenes (Gram-positive)

Fecal coliforms: -Their presence is an indication of stool contamination.

Example:- Escherichia coli (Gram-negative)

Principle of citrate utilization test

When citrate, an organic acid, is used as the only source of carbon, two things are produced like

  1. Alkaline carbonates
  2. Bicarbonates

Besides, when the ammonium salts are used as the sole nitrogen source, ammonium hydroxide is also produced in the medium.

The organism that grows and develops on this medium produces an enzyme called citrate permease (citrate transport protein), which has the ability to break citrate to oxaloacetate and acetate by the enzyme citrate lyase.

After that, oxaloacetate is metabolized into pyruvate and CO2.

Then this will enter the metabolic cycle of the organism to produce energy.

If citrate is utilized, there would be profuse growth of the organism.

This will then become an intermediate metabolite in the Krebs cycle.

Production of Na2CO3 from the utilization of sodium citrate and NH3 from the utilization of ammonium salt results in alkaline pH.

This results in a change in the color of the medium from green to blue.

Citrate utilization test

After this step, Metabolic breakdown is dependent upon the pH of the medium.

  1. In alkaline conditions: – pyruvate is metabolized to acetate and formate.
  • pyruvate = acetate + formate
  1. At pH 7.0 and below:- lactate and acetoin are also produced.
  • pyruvate = acetate + lactate + CO2
  • pyruvate = acetoin + CO2

The carbon dioxide which is released reacts with water and the sodium ion present in the medium and produces sodium carbonate(alkaline compound) that will raise the pH.

As mentioned above, ammonium hydroxide is produced when the ammonium salts in the medium are used as the sole nitrogen source.

Due to this growth results in the bromothymol blue indicator, this will turn the media from green to blue.

At neutral pH, the bromothymol blue pH indicator is deep forest green in color.

With pH increase in medium up to above 7.6, bromothymol blue changes the color to blue.

Citrate Utilization Test Procedure

Media preparation for Simmon’s Citrate agar

In the citrate utilization test, the citrate medium is made by the standard formula of Simmons. Hence, it is known as Simmon’s citrate agar media.

The medium is poured into a tube to make a slant.

The composition of Simmons citrate agar

IngredientQuantity (g/L)
Dipotassium phosphate
Sodium chloride
Ammonium dihydrogen
Sodium chloride5g
Sodium citrate2g
Magnesium sulfate 0.20g
Agar 15g
Bromothymol blue 0.08g
Distilled water1000ml
pH of media6.9
  • Mix the salt content (Dipotassium phosphate sodium chloride and Ammonium dihydrogen phosphate)and dissolve in distilled water.
  • Now add both the agar and bromothymol blue.
  • Heat it till it mixes well until the agar is completely dissolved.
  • Put the mixture (about 5 ml) into a tube and autoclave at 121 degrees Celsius for 15 minutes under 15 psi pressure.
  • Allow to cool in a slanted position, so a slant is formed.
  • To extend the shelf life of the tube, it should be put in the refrigerator.
  • By this, it can extend its shelf life up to eight to nine weeks.

Note: The color of an uninoculated medium is deep forest green because of bromothymol blue’s pH level.


  1. Take a wire loop and sterilize the wire loop in the blue flame on the Bunsen burner and wait till the loop becomes red. The wire loop should be heated from the base towards the tip.
  1. Allow it to cool and remove the cap of the Simmon’s citrate agar slant tube and pass the neck of the test tube into the flame to avoid contamination.
  2. Take the loopful of microorganisms and inoculate on Simmon’s citrate agar lightly on the slant. Do this by touching the tip of a needle to a colony which is 18 to 24 hours old.
  3. Incubate at a temperature of 35°C to 37°C for 18 to 24 hours.
  • Note:- Some organisms may require up to 7 days of incubation due to their limited rate of growth on citrate medium.
  1. Observe the development of blue color after 24 hours.

Interpretation of citrate utilization test :

A) No bacteria. B) Citrobacter freundii, C) Has Escherichia coli. (Color change has occurred in ‘B’ because C. freundii can grow using citrate) {Credit: Swedish University of Agricultural Sciences}
  • Citrate positive: Prussian blue color growth will be observed on the slant surface and the medium.
    • Alkaline carbonates and bicarbonates are produced as by-products of citrate catabolism, this will raise the pH of the medium to above 7.6, this causes the bromothymol blue to change from the original green color to blue.
    • Examples:-
      • Klebsiella pneumonia
      • Enterobacter species (only some of the strains gives negative result)
      • Citrobacterfreundii
      • Salmonella other than Typhi and Paratyphi A
      • Serratia marcescens
      • Proteus mirabilis
  • Citrate negative: small amount(trace) or no growth will be visible.
    • e color change will not occur.
    • The medium will remain as it is I.e. the deep forest green color like the uninoculated agar.
    • Those bacteria which can utilize citrate as the sole carbon and energy source will be able to grow on the Simmons citrate medium.
    • So that citrate-negative test culture will be virtually differentiated from an uninoculated slant.
    • Examples :-
      • Escherichia coli
      • Shigella spp
      • Salmonella Typhi
      • Salmonella Paratyphi A
      • Morganella morganii
      • Yersinia enterocolitica
  • Some other strains that gives differentCitrate Test results:
    • Proteus vulgaris
    • Vibrio cholerae
    • Vibrio parahaemolyticus

Quality Control strains used in citrate utilization test :-

Positive control : Klebsiella pneumoniae

Negative control : Escherichia coli

Applications of Citrate Utilization Test

  • Citrate utilization test is used to identify
    • gram-negative pathogens of Enterobacteriaceae family.
    • And coliforms and non-fecal coliforms microorganisms.
  • Test kits are available such as the API-20E and Enterotube II that include citrate utilization medium as one of the diagnostic tests.

It helps to detect the organism’s ability to produce citrase enzyme

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