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17 Cards in this Set

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Simmons' Citrate Agar is a defined, enrichment medium that tests for an organism's ability to use citrate as a sole carbon source and ammonium ions as the sole nitrogen source.

Developed by


Used for

This media is used for the differentiation between Enterobacteriaceae and the members of aerogenes group on the basisof citrate utilization as sole carbon source
Ingredients Gms / Litre

Magnesium sulphate -0.200

Ammonium dihydrogen phosphate - 1.000 Dipotassium phosphate -1.000

Sodium citrate - -2.000 Sodium chloride -5.000 Bromothymol blue -0.080 Agar -15.000

The purpose of sodium citrate in the agar
It is the sole carbon source. Bacteria that possess citrate-permease can transport the citrate molecules into the cell and produce pyruvate
- Ammonium dihydrogen phosphate and sodium citrate serve as the sole nitrogen and carbon source respectively.

-Metabolism of these salts causes the mediumto become alkaline, indicated by a change in colour of the pH indicator from green to blue.

-Bromothymol blue is the pHindicator.

Bromothymol blue
Bromothymol blue is green at pH below 6.9, and then turns blue at a pH of 7.6 or greater.
enzyme involved in the Citrate test

Produce pyruvate from the carbon source citrate.

pH of the media


Incubation conditions
Incubate at 35oC (+/- 2oC) for 18 to 48 hours. Some organisms may require up to 7 days of incubation due to their limited rate of growth on citrate medium.
Why is the test performed on slants?
O2 is necessary for citrate utilization
Interpretation of Result
If the medium turns blue, the organism is citrate positive. If there is no color change, the organism is citrate negative.

Some citrate negative organisms may grow weakly on the surface of the slant, but they will not produce a color change.

Why does the media turn blue?
Bacteria that can survive the medium and utilize the citrate also convert the ammonium phosphate to ammonia and ammonium hydroxide both of which tend to alkalinize the agar. At pH 7.6 it is blue.



The citrate test is often part of a battery of tests used to identify gram-negative pathogens and environmental isolates

For instance, test kits such as the API-20E (bioMerieux) and Enterotube II (BD Diagnostics) include citrate utilization medium as one of the diagnostic tests.

Use a light inoculum to streak the slant; a heavy inoculum may result in false-positive results.

When inoculating multiple biochemicals from the same culture, inoculate this medium first, or flame inoculating needle prior to streaking this medium.

Carryover of glucose or other nutrients onto this medium may result in false-positive results

For more information .....