Citrate Utilization Test: Principle, Procedure, and Results

What is Citrate Utilization Test?

By measuring an organism’s ability to use citrate as its sole form of energy, the IMViC test (Indole, Methyl Red, Vogues-Proskauer, and Citrate Test) can discriminate between different types of organisms. One part of this examination is the Citrate Utilization Test.

  • Along with the other IMViC tests, the citrate test is used to identify distinct Enterobacteriaceae family Gram-negative bacilli.
  • It is a crucial test that identifies Enterobacteriaceae family members at the species level.
  • With the exception of a few species, Escherichia, Shigella, Morganella, and Yersinia often cause a negative reaction, while Salmonella, Edwardsiella, Citrobacter, Klebsiella, Enterobacter, Serratia, and Providencia typically cause a positive reaction. A citrate variable is protos.
  • Simmon’s citrate agar, which includes citrate as the primary energy source, is used in the test, which is also known by the name Simmon’s citrate test.
  • The only form of nitrogen in the medium is ammonium hydrogen phosphate (NH4H2PO4).
  • Positive test results indicate that the organisms can ferment citrate when the enzyme citrase (Citrate lyase) is present.

Principle of Citrate Utilization Test

  • Citrate agar is used to assess an organism’s ability to use citrate as an energy generator.
  • The primary sources of nitrogen and carbon in the agar medium are citrate and inorganic ammonium salts, respectively.
  • Citrate is a Krebs cycle intermediate metabolite, and the organism’s growth indicates that it is being used. Oxaloacetate and acetate are produced when citrate is broken down by the enzyme citrase, while pyruvate and carbon dioxide are produced when oxaloacetate is further broken down.
  • The creation of ammonia or sodium carbonate, all of which increase the medium’s alkalinity, is a result of the metabolism of ammonium salts, which is triggered by carbon dioxide emission.
  • Above pH 7.6, the medium’s bromthymol blue indicator changes from green to blue due to the pH change.

Citrate → Oxaloacetic acid → Pyruvic acid + CO2

  • Na2CO3 is created when sodium citrate, CO2, and water are combined.
  • A positive citrate test results from the organism growing on the medium, followed by a change in color brought on by citrate metabolism.

Microorganisms Tested

  • Members of the family Enterobacteriaceae are part of the species-level identification process.

Media, Reagents, and Supplies 


  • The test medium for determining whether an organism can use citrate as its only source of energy is Simmon’s Citrate Agar.
  • Different retailers provide commercially available dehydrated powder versions of Simmon’s Citrate agar. However, if the appropriate materials are on hand, it may also be made in a lab.
  • The ingredients that makeup Simmon’s Citrate Agar are as follows:
S/N Ingredients Gram/liter
1 Ammonium dihydrogen phosphate 1
2 Magnesium sulfate 0.2
3 Dipotassium phosphate 1
4 Sodium citrate 2
5 Sodium chloride 5
6 Bromo thymol blue 0.08
7 Bacteriological Agar 15
Final pH at 25ºC: 6.8±0.2


  • Vaccination needles or loops
  • 35-37°C in an incubator

Procedure of Citrate Utilization Test

1. Preparation of the media

  • One thousand milliliters of pure distilled or deionized water is combined with 24.28 grams of the dehydrated powder or lab-prepared media in a beaker.
  • The medium is then thoroughly dissolved by heating the solution until it comes to a boil.
  • After being dissolved, the medium is poured into tubes and sterilized for 15 minutes at 121°C under 15 lbs of pressure in an autoclave.
  • The tubes are withdrawn once the autoclaving process is complete and cooled to a temperature of around 40–45 °C when kept in a tilted position. Maintaining good posture is necessary to get butts 1.5 to 2.0 cm deep.

2. Utilization test

  • A sterile inoculating needle is used to remove a well-isolated colony from an 18–24-hour culture.
  • In order to inoculate the citrate agar tubes, the surface of the slant is streaked. The loop or inoculating stick should be used to streak the slant back and forth.
  • To guarantee appropriate aeration, the test tube caps should be left off.
  • After that, the tubes are incubated aerobically for up to 4 days at 35–37 °C.
  • Four days of daily checks on the test tubes are required before the findings are deemed untrustworthy.
  • If present, the color shift is seen.

3. Quality Control

  • Aseptic Simmon’s Citrate Agar ought to resemble gel slants that are forest green in color and slightly opalescent.
  • Before using the agar, it should be checked for signs of freezing, contamination, cracks, dehydration, and bubbles. Any tubes that could seem blue before being used must be thrown away.
  • The test’s positive and negative controls are as follows:
  • Citrate Positive- Positive Control for K. pneumonia
  • Citrate negative; negative control for Escherichia coli

Result Interpretation of the Citrate Utilization Test

  • Growth with a color change from green to bright blue along the slant serves as evidence of a positive test.
  • No growth or color change, and the slant’s color remaining green, are indicators of a negative test.
  • The results of key medical microorganisms using citrate are shown in the following table:

Citrate Utilization Test Result

S/N Organism Growth Citrate Test
1 Escherichia coli Inhibited Negative; the color of the medium remains green.
2 Enterobacter aerogenes Good-Luxuriant Positive reaction; change in the color of the medium from green to blue.
3 Salmonella Enteritidis Good-Luxuriant Positive reaction; change in the color of the medium from green to blue.
4 Salmonella Typhimurium Good-Luxuriant Positive reaction; change in the color of the medium from green to blue.
5 Shigella dysentriae Inhibited Negative; the color of the medium remains green.

Uses of the Citrate Utilization Test

  • An organism’s capacity to utilize citrate as its only source of energy is assessed using the citrate test.
  • The IMViC test uses a citrate test to distinguish between Enterobacteriaceae species.
  • Based on citrate usage, the test is often utilized to differentiate between fecal E. coli and the aerogenes family of bacteria.

You may also like to read: Hydrogen Sulfide Test

Limitations of Citrate Utilization Test

  • A positive test could be indicated by luxuriant growth on the slant without an accompanying color change. If the agar does not turn blue after additional incubation, the test must be redone with a smaller inoculum.
  • Tests that yield ambiguous findings ought to be repeated.
  • The test requires an aerobic setting, so the slant shouldn’t be punctured.
  • Due to the possibility of media carryover, broth culture inoculations should be avoided.
  • A mild inoculum should be used to prevent the carryover of chemicals from prior media to avoid false-positive responses.
  • Further tests should be carried out for confirmation because the reactivity of this medium alone is insufficient for species-level identification.


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