Biochemical tests


Written by MicroDok

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 but only the Simmon’s citrate agar method is expanded in this book. 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 37o
  4. Observe the tube(s) for a change in colour. A change in colour from green to blue indicates an alkaline reaction arising from citrate utilization and growth of citrate positive bacteria. 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.

Illustration of citrate test

Tube on left side is negative, tube in the middle or center is weak positive while tube on right side is strong positive as indicated by the clear blue colour.


Cheesbrough M (2006). District Laboratory Practice in Tropical Countries. 2nd Cambridge University Press, UK. Pp. 178-187.

Willey J.M, Sherwood L.M and Woolverton C.J (2008). Harley and Klein’s Microbiology. 7th ed. McGraw-Hill Higher Education, 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.

World Health Organization (1993). Laboratory Biosafety Manual, 2nd edn. Geneva: WHO.

World Health Organization (2003). Guidelines for the Safe Transport of Infectious Substances and Diagnostic Specimens.  WHO/EMC/97.3. Geneva: WHO.

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