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The Simmons citrate test procedure, commonly referred to as the citrate utilization test, is used to identify bacteria that can break down citrate.
S.A. Koser devised the citrate utilization test in the early 1920s while he studied the bacterial catabolism of organic acids using Koser’s medium. The test proved useful in distinguishing Escherichia coli which led to fecal contamination from Enterobacter aerogenes which occur naturally in water and soil.
Koser’s medium, however, had a downside, heavy inoculums resulted in false positive results. Simmons later built on the invention and devised Simmons medium, which contained a bromothymol blue pH indicator and agar.
The test forms part of the IMVIC tests, Indole test, Methyl Red test, Voges-Proskauer test, and Citrate test.
Simmons citrate test principle
Bacteria break down Citrate into products which include ammonia. The ammonia raises the pH of the medium and the bromothymol blue in the medium, at a pH above 7.6, turns color from green to blue.
The Simmons citrate agar contains Citrate as the only degradable carbon. The agar is prepared in a slant in a tube, a flat surface makes it difficult to quantify bacterial growth. The slant also increases the surface area for Oxygen utilization by bacteria.
Bacteria that utilize citrate produce the enzyme citrate-permease that converts citrate to pyruvate which provides the bacteria with energy.
Test guidelines and limitations
Check the Simmon citrate agar for signs of contamination such as drying and cloudiness. Contamination of the agar with other nutrients results in false positive results.
Some bacteria grow in the agar but do not elicit a color change, you should report those results as Positive for citrate utilization.
Ensure that the test cultures are pure, grown on a solid medium, and not broth suspensions.
Requirements for the Simmons citrate test include inoculating loop, test colony, Simmons citrate agar prepared in a slant, and incubator.
Simmons citrate test procedure
- Innoculate the test colony on the Simmons citrate agar.
- Incubate the preparation aerobically at 37oC for 4 – 7 days.
- Check for bacteria growth and color change.
- Report Citrate utilization positive, if there is bacterial growth, or Citrate utilization negative if there is none.
- Also, note the color change, some bacteria utilize citrate, and their by-products do not raise the pH of the medium.
Citrate-positive bacteria
Examples of bacteria that break down citrate into pyruvate include Salmonella of subgenus II, III, and IV, Salmonella enteritidis, Serratia proteamaculans, Klebsiella, Providencia, Citrobacter, Yokenella regensburgei, Proteus rettgeri, Acidovorax delafieldii, and Leminorella grimontii.
Citrate-negative bacteria
Examples of bacteria that cannot utilize citrate include Salmonella paratyphi A, Salmonella gallinarum, Salmonella typhi, Salmonella pullorum, Edwardsiella spp, Escherichia coli, Morganella morganii biogroups 1 and 2, Shigella spp, Yersinia pseudotuberculosis, Yersinia enterocolitica, Hafnia alvei, Leminorella richardii, Acidovorax facilis, and Acidovorax temperans.
How to prepare Simmons Citrate agar
| Ingredient | Quantity | Function |
|---|---|---|
| Deionized water | 1 l | solvent |
| Agar | 5.0 g | solidifying agent |
| Bromothymol blue | 0.08 g | pH indicator |
| Magnesium Sulfate (heptahydrate) | 0.2 g | metabolic co-factor |
| Dipotassium Phosphate | 1 g | buffer |
| Ammonium Dihydrogen Phosphate | 1 g | Nitrogen source |
| Sodium Citrate (dehydrate) | 2 g | Citrate carbon source |
| Sodium Chloride | 5 g | maintain osmotic pressure |
| (pH) | 6.9 +-0.2 | optimum pH |
- Dissolve 5 mg of sodium chloride, 2g sodium citrate, 1g ammonium dihydrogen phosphate, 1g dipotassium phosphate, and 0.2g Magnesium Sulfate (heptahydrate) dissolve in 100ml deionized water.
- Add 0.08g bromothymol blue and 15g agar into the solution. Ensure the final pH is at 6.9.
- Heat the preparation while mixing until all the agar dissolves.
- Pour out the mixture into a petri dish or test tubes.
- Autoclave for 15 minutes at 121oC at 15psi.
- If you used a test tube, the medium should solidify while the tube is in a slant position – shallow butt, long slant.
- hold it in a slant position dispense the medium then allow it to solidify in a slant position – shallow butt, long slant.
- Finally, the color of the medium should be deep forest green. For extended use, refrigerate the media.
- above salts in deionized water.
References
Koser, S. A. 1923. Utilization of the salts of organic acids by the colon-aerogenes group. J. Bacteriol. 8:493–520. 6.
Bailey & Scott’s Diagnostic Microbiology 13th Edition: Citrate Utilization
Clinical Microbiology Procedures Handbook 2nd Edition: Citrate Utilization Test.
