Simmons’ citrate agar is differential growth media recommended for gram-negative bacteria or members Enterobacteriaceae on the basis of citrate utilization.That is,useful for organisms that uses citrate as a carbon and source of energy. It is also defined as a selective and differential medium that tests for an organism’s ability to use citrate as a sole carbon source and ammonium ions as the sole nitrogen source.
Simmons’ citrate agar contains citrate, ammonium ions, and other inorganic ions needed for bacteria growth and bromothymol blue as pH indicator. Bromothymol blue is green at pH below 6.9, and then turns blue at a pH of 7.6 or greater.
Principle of Simmons’ citrate agar
- The presence of ammonium dihydrogen phosphate and sodium citrate serve as the sole nitrogen and carbon source respectively.
- Some microorganisms also use inorganic ammonium salts as their sole nitrogen source. Metabolism of these salts causes a change pH of the medium to become alkaline.This is indicated by a change in colour of the pH indicator from green to blue.
- Bromothymol blue is the pH indicator. The medium should be freshly prepared because in dry conditions, changes in colour may appear even before inoculation, especially at the bottom of the slant.
Composition of Simmons’ citrate agar
|Ammonium dihydrogen phosphate||1.000|
|Final pH at temperature 25°C||6.8±0.2|
How to Prepare Simmons’ citrate agar
- Suspend 24.28 grams of the Cream to yellow homogeneous free flowing powder in 1000 ml distilled water.
- Heat, to boiling, to dissolve the medium completely
- Followed by Mixing well and distribute in tubes or flasks.
- Sterilize by autoclaving at 15 lbs pressure (121°C) for a time of 15 minutes
Note: Before using water, ensure pH of water is 6.5 to 7.0.Initial colour of the medium may deviate from expected colour, if the above note is ignored.
Appearance of Simmons’ citrate agar
It has a forest green coloured slightly opalescent gel forms in tubes as slants
Type of Inoculums
Use pure isolate from clinical and non clinical samples when using this media
Cultural characteristics after Incubation at 35-37°C for 18-24 hours.
|Klebsiella aerogenes ATCC 13048||good-luxuriant||positive reaction, blue colour|
|Escherichia coli ATCC 25922||inhibited||No reaction|
|Salmonella Typhi ATCC 6539||fair-good||negative reaction, green colour|
|Salmonella Typhimurium ATCC 14028||good-luxuriant||positive reaction, blue colour|
|Shigella dysenteriae ATCC||inhibited||No reaction|
|Salmonella Choleraesuis ATCC||good-luxuriant||positive reaction, blue colour|
|Salmonella Enteritidis ATCC 13076||good-luxuriant||positive reaction, blue colour|
How to store or conserve Simmons’ citrate agar
- Store below 30°C in a tightly closed container and the prepared medium at temperature 2 – 8°C.
- Make sure to properly stored dry, after tightly capping the bottle in order to prevent lump formation due to the hygroscopic nature of the product.
- Also Improper storage of the this medium may lead to lump formation. Store in dry ventilated area protected from extremes of temperature and sources of ignition
- Seal the container tightly after use
Some Limitations with Simmons’ citrate agar
- Note that before using water, ensure that the pH of the water is 6.5 to 7.0.
This is to avoid a deviation in Initial colour of the medium from expected colour because the pH affects the performance of the medium and must be correctly monitored.
- Koser, 1923, J. Bact., 8:493.
- Simmons, 1926, J. Infect. Dis., 39:209.
- MacFaddin J., 1985, Media for Isolation-Cultivation-Identification-Maintenance of Medical Bacteria, Vol. 1, Williams and Wilkins, Baltimore.
- Eaton A. D., Clesceri L. S., Rice E. W., and Greenberg A W., (Eds.), 2005, Standard Methods for the Examination of Water and Wastewater, 21st Ed., APHA, Washington, D.C. Revision : 03 / 2017
- American Public Health Association, Standard Methods for the Examination of Dairy Products, 1978, 14th Ed., Washington D.C.