Citric Acid (Citrate) Utilization Test –

Salmonella typhi and Escherichia aerogen bacteria obtain carbon from citrate. This ability of utilizing citrate as the carbon and energy source is used as a distinguishing test for some Grods.


Simmons citrate agar containing sodium citrate as the carbon source is used to detect whether or not citrate is utilized by the organism. This agar contains Bromothymol blue indicator which undergoes a colour change from green to blue when bacterial growth utilize citrate and turns the media alkaline. This colour change from green to blue indicates a positive test.

Materials Required

  1. Simmon’s citrate agar medium*,
  2. Cultures of E. coil and Enterobacter aerogenes,
  3. Inoculation needle,
  4. Culture tubes, and
  5. Incubator.
*Simmon’s Citrate Agar Medium
Sodium citrate 2.0gm
MgSO4 0.2gm
(NH4)H2PO4 1.0gm
K2H.PO4 1.0gm
NaCl 5.0%
Bromthymol blue 0.08gm
Agar 15.0gm
Distilled water 1 litre
pH 7.0


  1. A few slants of Simmon’s citrate agar medium are prepared.
  2. The slants are then inoculated by stabbing to the base of the slant.
  3. The medium surface is streaked.
  4. The tubes are incubated for 2 days at 37°C.
  5. The tubes are then examined.
  6. If bacterial growth occurs the culture turns blue due to pH change as it becomes alkaline after citrate utilization (as in case of E. aerogenes); while if there is no visible growth the culture undergoes no change in colour (and remains green) due to unutilized citrate (as in case of E. coil).


Formation of an intense blue colour on the slant renders the citrate utilization test positive (Klebsiella sp., Salmonella sp., Citrobacter sp. and Enterobacter sp. are citrate-positive bacteria); while no colour change renders the test negative (E. coil, Edwardsiella, and S. typhi are citrate-negative bacteria).

Read More Topics
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Scanning electron microscopy (SEM)
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