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Citrate test is used to identify Enterobacteriaceae that utilize citrate or citric acid as their sole carbon and energy source. Simmon’s citrate agar or citrate tablet is used in performing this test. The Simmons citrate agar tests the ability of organisms to utilize citrate as a sole carbon source. Simmons citrate agar contains sodium citrate as the sole source of carbon; and ammonium dihydrogen phosphate as the sole source of nitrogen, and other nutrients required for microbial growth. The pH indicator in the Simmons citrate agar is called bromothymol blue.

The test organism produces the enzyme, citritase which breaks down citrate to oxaloacetate and acetate. Citrate positive enterobacteria grow on citrate agar slants or medium. Enterobacteria that do not require citrate as carbon or energy source (i.e. citrate negative bacteria) do not grow on citrate agar medium. Simmon’s citrate agar medium contains a pH indicator called bromothymol blue which changes colour to blue when bacteria  that utilize citrate starts growing on it upon inoculation. The medium changes from green to blue, and this indicates a positive result. Klebsiella pneumoniae and Proteus mirabilis are typical citrate positive organisms.


  1. Prepare Simmon’s citrate agar slant according to manufacturer’s instruction. In the preparation, test tubes containing the citrate medium must be kept in slanting position in order to make a slope. A well prepared Simmon’s citrate medium looks greenish.
  2. Inoculate the slant by streaking the slope with a speck or loopful of the test isolate.
  3. Incubate inoculated tube(s) overnight at 37oC.
  4. Observe the tube(s) for a change in colour after incubation. A change in colour from green to blue indicates an alkaline reaction arising from citrate utilization and growth of citrate positive bacteria (Figure 1). The bromothymol-blue greenish colour remains unchanged when the citrate is not utilized, and this is a negative test result because the inoculated organism did not grow in the medium or produce the enzyme, citrase.
Figure 1. Citrate test. The tube on the left is positive as indicated by the blue colour; the tube at the center is uninoculated while the tube on the right is negative.

Further reading

Brooks G.F., Butel J.S and Morse S.A (2004). Medical Microbiology, 23rd edition. McGraw Hill Publishers. USA.

Goldman E and Green L.H (2008). Practical Handbook of Microbiology, Second Edition. CRC Press, Taylor and Francis Group, USA.

Madigan M.T., Martinko J.M., Dunlap P.V and Clark D.P (2009). Brock Biology of Microorganisms, 12th edition. Pearson Benjamin Cummings Inc, USA.

Mahon C. R, Lehman D.C and Manuselis G (2011). Textbook of Diagnostic Microbiology. Fourth edition. Saunders Publishers, USA.

Patrick R. Murray, Ellen Jo Baron, James H. Jorgensen, Marie Louise Landry, Michael A. Pfaller (2007). Manual of Clinical Microbiology, 9th ed.: American Society for Microbiology.

Wilson B. A, Salyers A.A, Whitt D.D and Winkler M.E (2011). Bacterial Pathogenesis: A molecular Approach. Third edition. American Society of Microbiology Press, USA.

Woods GL and Washington JA (1995). The Clinician and the Microbiology Laboratory. Mandell GL, Bennett JE, Dolin R (eds): Principles and Practice of Infectious Diseases. 4th ed. Churchill Livingstone, New York.

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