Citrate utilization test: Principle, Procedure, expected results and positive organisms

Principle: Citrate utilization test is commonly employed as part of a group of tests, the IMViC (Indole, Methyl Red, VP and Citrate)  tests, that distinguish between members of the Enterobacteriaceae family based on their metabolic by-products.  Citrate utlilization test is used to determine the ability of bacteria to utilize sodium citrate as its only carbon source and inorganic(NH4H2PO4) is the sole fixed nitrogen source.

When an organic acid such as citrate (Remember Krebs cycle) is used as a carbon and energy source, alkaline carbonates and bicarbonates are produced ultimately.  In addition, ammonium hydroxide is produced when the ammonium salts in the medium are used as the sole nitrogen source.

The color change of the indicator is due to alkali production by the test organism as it grows on the medium.

Growth usually results in the bromothymol blue indicator, turning from green to blue. The bromothymol blue pH indicator is a deep forest green at neutral pH.  With an increase in medium pH to above 7.6, bromothymol blue changes to blue

Citrate utilization test A: Negative B: Positive

Citrate utilization test (Image source: Microbelibrary)
A: Negative
B: Positive

Procedure of citrate utilization test:

  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.
  2. Incubate at 35oC to 37oC for  18 to 24 hours. Some organisms may require up to 7 days of incubation due to their limited rate of growth on citrate medium.
  3. Observe the development of blue color; denoting alkalinization.

Expected results in citrate utilization test:

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

Quality Control strains used in citrate utilization test

  • Positive: Klebsiella pneumoniae
  • Negative: Escherichia coli

 List of Bacteria which gives positive citrate utilization test

  1. Citrobacter spp
  2. Klebsiella pneumoniae
  3. Enterobacter aerogenes

Members of enterobacteriaceae such as Escherichia coli, Proteus mirabilis etc gives negative citrate utilization test.