Citrate Utilization Test- Principle, Procedure, and Result

The citrate utilization test is used to detect the organism’s ability to utilize citrate as a sole source of energy. The medium containing sodium citrate as the only carbon source and inorganic ammonium salt is used as a sole source of nitrogen.

The citrate utilization test is a part of a group of tests, which is the IMViC (Indole, Methyl red, Voges Proskauer, and Citrate utilization) test. That is used to distinguish between members of the Enterobacteriaceae family based on their metabolic activity.

What is the Principle of Citrate utilizing test?

The basic principle of the citrate utilization test is to detect the organism’s ability to utilize citrate as a sole source of energy. The medium containing sodium citrate is the only carbon source and inorganic ammonium salt is used as a sole source of nitrogen.

citrate utilization Test

Mechanism of Citrate utilization

As we know bacteria mainly produce an intracellular enzyme so that if the cell needs to utilize any substrate it must be transported into the cell. So, how much citrate is utilized is directly depends on how much citrate is transported into the cell.

Here, the main two enzymes play a crucial role in the citrate utilization. The first enzyme, that induced citrate metabolism is citrate permease. It involves bringing citrate into the cell. The enzyme citrate permease function at pH below 6.0 with an optimum pH of around 5.0.

Once the citrate transport inside the cell. The enzyme citrate lyase breakdown the citrate into oxaloacetate and acetate. Citrate lyase is also known as “citritase”. The optimal pH for the citritase activity is 7.4 to 7.6.

In addition to citritase, the cell also contains oxaloacetate dehydrogenase that breakdown oxaloacetate into pyruvate and CO2. The end product obtained from the citrate metabolism depends on the resulting pH of the medium. As shown as the above pathway If the medium is alkaline, mostly Acetic acid and Formic acid are produced.

Pyruvic acid  = Formic acid + Acetic acid + CO2

If the medium is acidic, acetoin and lactic acid are produced:

Pyruvic acid = acetoin + lactic acid + CO2

As we can see there is 2 molecules of CO2 are produced. First is when oxaloacetate converted into pyruvate and second is pyruvate is converted into either acetic acid or lactic acid. That CO2 molecule combines with the sodium present in the medium to form sodium carbonate.

But the reaction is not that simple. Where the sodium ion come from? As we know we are using sodium citrate as a primary carbon source. When the citrate is breakdown the free sodium ion is released. And when the ammonium salt is used as a nitrogen source free molecule of ammonia is released. That increases the pH of the medium. In the medium, we also add the pH indicator dye which is Bromothymol blue. At the neutral pH it appears as a green color when pH sifts into alkaline ( above pH 7.6)  it turned into blue color.

Media Composition

Ammonium hydrogen phosphate (NH4H2PO4)1 gm/l
Dipotassium phosphate (HK2O4P)1 gm/l
Sodium chloride (NaCl)5 gm/l
Sodium citrate (C6H5Na3O7)2 gm/l
Magnesium sulfate (MgSO4)0.2 gm/l
Bromothymol blue0.08 gm/l
Agar15 gm/l
Distilled water1000 ml
Final pH6.9 +/- 0.02

How to Prepare Simmon’s Citrate Medium

  1. Dissolve above salts in deionized water.
  2. Adjust pH to 6.9.
  3. Add agar and Bromothymol blue.
  4. Gently heat, with mixing, to boiling until agar is dissolved.
  5. Dispense 4.0 to 5.0 ml into 16-mm tubes.
  6. Autoclave at 121 °C under 15 psi pressure for 15 minutes.
  7. Cool in slanted position (long slant, shallow butt).
  8. Tubes should be stored in a refrigerator to ensure a shelf life of 6 to 8 weeks.
  9. The uninoculated medium will be a deep forest green due to the pH of the sample and the bromothymol blue.


  1. Inoculate Simmons citrate agar lightly on the slant by touching the tip of a needle to a colony that is 18 to 24 hours old. Do not inoculate from a broth culture, because the inoculum will be too heavy.
  2. Incubate at 35°-37°C for up to 7 days.
  3. Observe for growth and the development of blue color, denoting alkalinization.

Result Interpretation

Positive: Growth on the medium, with or without a change in the color of the indicator. Growth typically results in the bromthymol blue indicator turning from green to blue.
Negative: Absence of growth.

citrate Utilization test


Some organisms are capable of growth on citrate and do not produce a color change. Growth is considered a positive citrate utilization test, even in the absence of a color change.

Quality Control

Positive: Enterobacter aerogenes (ATCC13048)—growth, blue color
Negative: Escherichia coli (ATCC25922)—little to no growth, no color change