Citrate Utilization Test: Objective, Principle, Procedure and Results

By Prof Jeremiah Seni


  • To detect the ability of organisms to produce citrase enzyme.The citrate test detects the ability of an organism to use citrate as the sole source of carbon and energy.


Bacteria are inoculated on a medium containing sodium citrate and a pH indicator such as bromothymol blue. The medium also contains inorganic ammonium salts, which are utilized as sole source of nitrogen. Use of citrate involves the enzyme citrase, which breaks down citrate to oxaloacetate and acetate. Oxaloacetate is further broken down to pyruvate and carbon dioxide (CO2). Production of sodium bicarbonate (NaHCO3) as well as ammonia (NH3) from the use of sodium citrate and ammonium salts results in alkaline pH. This results in a change of the medium’s color from green to blue.

Bacterial colonies are picked up from a straight wire and inoculated into slope of Simmons citrate agar and incubated overnight at 37 °C. If the organism has the ability to use citrate, the medium changes its color from green to blue.


Reagents And Materials Required

  • Test tubes
  • Conical flask
  • Cotton plugs
  • Inoculating needle
  • Autoclave
  • Bunsen burner
  • Laminar flow chamber
  • Dispose jar, incubator
  • Simmons citrate agar
  • Isolated colonies or pure cultures of bacteria


  1. The ingredients of Simmons citrate agar medium (containing sodium citrate, NH4H,P04 and bromothymol blue as the main ingredients) or its ready-made powder required for 100 ml of the medium is weighed and dissolved in 100 ml of distilled water in a 250 ml conical flask by shaking and swirling.
  2. Its pH is determined using a pH paper or pH meter and adjusted to 6.9 using 0.1N HC1 if it is more or using 0.1N NaOH if it is less.
  3. The flask is heated to dissolve the agar in the medium completely.
  4. Before it solidifies, the medium in warm molten condition is distributed into 5 test tubes (approximately 20 ml each).
  5. The test tubes are cotton-plugged, covered with craft paper and tied with thread or rubber band.
  6. They are sterilised at 121°C (15 psi pressure) for 15 minutes in an autoclave.
  7. After sterilisation, they are removed from the autoclave and kept in a slanting position to cool and solidify the medium, so as to get citrate agar slants.
  8. The test bacteria is inoculated aseptically, preferably in a laminar flow chamber, into the slants by stabbing into the butt and streaking on the surface of the slants with the help of a flame- sterilised needle. The needle is sterilised after each inoculation.
  9. The inoculated slants are incubated at 37°C for 24 to 48 hours in an incubator.

Observations And Results Interpretation

  • Citrate Positive:  growth will be visible on the slant surface and the medium will be an intense Prussian 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 Negative:  trace or no growth will be visible. No color change will occur; the medium will remain the deep forest 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, thus a citrate-negative test culture will be virtually indistinguishable from an uninoculated slant.

Bacteria which gives positive citrate utilization test

  • Klebsiella pneumoniae
  • Enterobacter species (minority of strains gives negative result)
  • Citrobacter freundii
  • Salmonella other than Typhi and Paratyphi A
  • Serratia marcescens
  • Proteus mirabilis (minority of strains gives negative result)
  • Providencia

Bacteria which gives negative citrate utilization test

  • Escherichia coli
  • Shigella spp
  • Salmonella Typhi
  • Salmonella Paratyphi A
  • Morganella morganii
  • Yersinia enterocolitica

Bacteria that give variable results on citrate utilization test

  • Proteus vulgaris
  • Vibrio cholerae
  • Vibrio parahaemolyticus