Citrate Utilization Test: Introduction Principle, Procedure, Result and Interpretation, Citrate Utilization Test Positive and Negative Bacteria List and keynotes


A citrate utilization test is the most commonly used routine biochemical test for Gram-negative rods in bacteriology for the identification of bacteria on the basis of citrate utilization. Klebsiella pneumoniae is the citrate utilization-positive organism while Escherichia coli is negative.

Principle of Citrate Utilization Test

A citrate utilization test is applied to determine the ability of a bacterium to utilize sodium citrate as its only carbon source and inorganic ammonium salts as its only nitrogen source. When the organisms metabolize citrate, the ammonium salt is disassociated with ammonia, which increases alkalinity turning the bromthymol blue indicator from green to blue.

Citrate Utilization Test: Introduction Principle, Procedure, Result and Interpretation, Citrate Utilization Test Positive and Negative Bacteria List and keynotes
Citrate Utilization Test-Negative (left) and Positive (right)

Requirements for test

  1. Simmons citrate agar slant
  2. Cotton plug
  3. Sterile Inoculating wire/ sticks
  4. Test organism
  5. Bunsen burner
  6. Incubator
  7. Test tube rack
  8. Control strains ( Positive and negative controls for validation of the test)

Procedure of Citrate Test

  1. Streak the slant back and forth with a light inoculum picked from the midpart of a well-isolated colony.
  2. Put a cap or cotton plug loosely on the tube.
  3. Incubate the tube aerobically at 35°C to 37°C for 18 to 24 hours.
  4. Observe a color change from green to blue along the slant.
  5. Follow the above steps to control stains too.

Quality control

Quality Control strains used in citrate utilization test

Positive Control (PC)-Klebsiella pneumoniae ATCC 13883

Negative Control (NC)-Escherichia coli ATCC 25922


  • Positive: Growth on the medium with color change from green to intense blue.
  • Negative: Absence of growth and color change

Result and interpretation in citrate test

  • Citrate utilization test positive:  The growth will be visible on the slant surface and the medium will be an intense blue. The alkaline carbonates and bicarbonates produced as by-products of citrate catabolism raise the pH of the medium to above 7.6, causing the bromothymol blue to change from the original green color to blue.
  • Citrate utilization test negative:  Trace or no growth will be visible.  No color change will occur; the medium will remain the green color of the uninoculated agar.  Only bacteria that can utilize citrate as the sole carbon and energy source will be able to grow on the Simmons citrate medium, therefore a citrate utilization negative test culture will be virtually indistinguishable from an uninoculated slant.
  • Uninoculated (UN): No growth and no color change; slant remains green
  • Negative control: No growth and no color change; slant remains green
  • Positive control: Growth with color change from green to intense blue along the slant
  • Test organism/bacteria: Positive i.e. growth with color change from green to intense blue along the slant as shown in the footage.

Citrate Utilization Test Positive and Negative Bacteria List

Citrate Positive BacteriaCitrate Negative Bacteria
Klebsiella pneumoniaeEscherichia coli
Citrobacter freundiiShigella species
Enterobacter cloacaeSalmonellaTyphi
Salmonella other than Typhi and Paratyphi ASalmonella Paratyphi A
Serratia marcescensMorganella morganii
Proteus mirabilisYersinia enterocolitica
Providencia alcalifaeciensEdwardsiella tarda
Vibrio vulnificusVibrio holisae
Euringella Americana
Achromobacter oxylosoxidans
Citrate positive and negative bacteria

Citrate Test: variable (different strains give different results)

 Proteus vulgaris
Vibrio cholerae
Vibrio parahaemolyticus
Citrate variable bacteria

Keynotes on Citrate utilization test

  1. Generally, incubate aerobically at 35°C to 37°C for 18 to 24 hours but if negative some organisms may require up to 4 days of incubation due to their limited rate of growth on citrate medium.
  2. Other methods of citrate utilization test available are-IMViC the test kit method and the API test kit method
  3.  As we know, Escherichia coli is citrate utilization test negative; although uncommon, natural E. coli variants that are citrate positive have been isolated.  Citrate-negative strains of E. aerogenes have also been found.
  4. Other media used for the citrate utilization test are Koser’s citrate and Christen’s citrate.

Koser’s citrate medium

  • It is a liquid medium lacking agar.
  • It is a medium without any indicator.
  • A positive test is indicated by the presence of turbidity in the medium.

Christen’s citrate medium

  • Same as the Simmons citrate medium in which the bromothymol blue indicator is replaced by phenol red.

Limitations of Citrate Test 

The limitations of this test are as follows:

  1. Luxuriant growth on the slant without an accompanying color change may indicate a positive test. However, if the agar does not turn blue on further incubation, the test should be repeated with less inoculum.
  2. Do not stab the slant, since the test requires an aerobic environment.
  3. Do not inoculate from the broth culture, due to carryover of media.
  4. Tests with equivocal results should be repeated.
  5. To avoid false-positive reactions, use a light inoculum to prevent the carryover of t substances from the previous medium.
  6. The reaction of this medium alone is not sufficient for identification at the species level.


  1. Jean F. Mac Faddin Biochemical tests for Identification of Medical Bacteria
  2. Mackie and McCartney Practical Medical Microbiology-14th Edition
  3. Bailey’s and Scott’s Diagnostic Microbiology-13th Edition
  4. ASM Clinical Microbiology Procedures Handbook-2nd Edition
  5. Monica Cheesbrough Distinct Laboratory Practice in Tropical Countries…2nd Edition
  6.  Textbook of practical Microbiology – Subhash Chandra Parija

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