The agar Simmons citrate it is a solid medium used as biochemical identification test microorganisms, especially gram - negative bacilli. The original medium was created by Koser in 1923.
Koser Citrate medium consisted of a broth containing sodium phosphate, ammonium phosphate, monopotassium phosphate, magnesium sulfate, and sodium citrate.
A. Dehydrated Simmons Citrate Agar commercial medium B. Prepared Simmons Citrate Agar. Source: A. Photo taken by MSc. Marielsa Gil B. Roto2esdios, from Wikimedia Commons
As can be seen, the only source of carbon in the medium is citrate, and of nitrogen is ammonium phosphate, omitting proteins and carbohydrates as a source of these elements, they are commonly present in other media.
Therefore, the bacteria inoculated in this medium can only reproduce if it is capable of taking up the carbon from citrate. The test was positive if there was turbidity in the medium, however it had the disadvantage that non-specific turbidity could occur.
This problem was solved by Simmons by adding bromothymol blue and agar to Koser's original formula. Although the principle is the same, it is interpreted differently.
Basis
Some bacteria have the ability to survive in the absence of fermentation or lactic acid production, needing to obtain energy through the use of other substrates. In this test the only carbon source offered is citrate.
Bacteria that are able to survive under these conditions rapidly metabolize citrate in an alternative to the traditional route, using the tricarboxylic acid cycle or the citrate fermentation cycle.
The catabolism of citrate by bacteria involves an enzymatic mechanism without the intervention of coenzyme A. This enzyme is known by the name of citricase (citrate oxaloacetate-lyase) or citrate desmolase. The reaction requires the presence of a divalent cation, which in that case is supplied by magnesium.
The reaction generates oxaloacetate and pyruvate, which then give rise to organic acids in the middle of an alkaline pH formed by the use of the nitrogen source. These organic acids are used as a carbon source generating carbonates and bicarbonates, further alkalizing the environment.
Sowing mode
Simmons citrate medium should be lightly inoculated into fishtail using a straight loop or needle, and incubated for 24 hours at 35-37 ° C. After the time, the results are observed.
Seeding is done only on the surface of the agar. Do not puncture.
Interpretation
If the medium remains the original color (green) and there is no visible growth, the test is negative, but if the medium turns blue, it indicates the presence of alkaline products, which is detected by the pH indicator. In this case the test is positive.
Citrate (+) and (-). Source: MSc. Marielsa gil
This happens because if the bacterium uses the carbon from the citrate, it is also capable of taking up the nitrogen from the ammonium phosphate with which it releases ammonia, alkalizing the medium.
On the other hand, if growth of the bacteria is observed in the medium, but there is no color change, the test must also be considered positive, since if there is growth, it means that the bacteria was able to use citrate as a carbon source, although there is no change in pH at the moment (sometimes it may take time).
If there is any doubt in the interpretation of the final color, it can be compared with an uninoculated citrate tube.
Preparation
Weigh 24.2 g of the dehydrated medium for one liter of water. Mix and let it rest for approximately 5 minutes. Finish dissolving the medium by heating for 1 or two minutes, shaking frequently.
Pour 4 ml into test tubes and autoclave at 121 ° C for 15 minutes. When leaving the autoclave, incline with the help of a support in such a way that the agar solidifies in the shape of a flute beak with little block or bottom and more bevel.
The final pH of the citrate medium is 6.9 (green color). This medium is very sensitive to changes in pH.
At pH 6 or below, the medium turns yellow. This color is not observed in the bacteria test.
And at pH 7.6 or above, the medium turns deep Prussian blue.
Use
Simmons Citrate Agar is used for the identification of certain microorganisms, especially bacilli belonging to the Enterobacteriaceae family and other non-glucose fermenting bacilli.
Source: Koneman E, Allen S, Janda W, Schreckenberger P, Winn W. (2004). Microbiological Diagnosis. 5th ed. Editorial Panamericana SA Argentina.
Final thoughts
Simmons citrate medium is a very delicate test, as false positives can be obtained if certain mistakes are made.
The care that must be taken are the following:
Inoculum
A very thick or loaded bacterial inoculum should not be made, as it can cause a coppery yellow color to develop in the sowing place, without affecting the rest of the medium, but it can lead to believe that there is growth. It does not mean positivity of the test.
Also, a thick inoculum can generate a false positive, because organic compounds preformed within the cell walls of dying bacteria can release enough carbon and nitrogen to turn the pH indicator.
Therefore, the ideal is to sow using the needle instead of the platinum handle, to avoid taking excess material.
Sown
On the other hand, when the battery of biochemical tests for the identification of the microorganism in question is being seeded, it is important that the citrate test is the first to be inoculated, to avoid the carry-over of proteins or carbohydrates from another medium.
Under this circumstance it is possible to obtain a false positive, because any of these substances that are introduced by mistake will be metabolized and will cause a change in pH.
Another way to avoid the carry-over of substances is to burn the loop well and take a new inoculum between one test and another.
Care must also be taken when touching the colony to perform the inoculum, since it should be avoided to drag part of the agar from the culture from which the bacteria come, due to the above explained.
In this sense, Matsen, Sherris and Branson recommend diluting the inoculum in physiological solution before inoculating the citrate test to avoid the transfer of other carbon sources.
Color intensity
It should be taken into account that the intensity of the color produced when the test is positive may vary according to the commercial house.
In addition, there are microorganisms that test positive at 24 hours, but there are other strains that require 48 hours or more to produce a change in pH.
References
- Mac Faddin J. (2003). Biochemical tests for the identification of bacteria of clinical importance. 3rd ed. Editorial Panamericana. Buenos Aires. Argentina.
- Forbes B, Sahm D, Weissfeld A. (2009). Bailey & Scott Microbiological Diagnosis. 12 ed. Editorial Panamericana SA Argentina.
- Koneman E, Allen S, Janda W, Schreckenberger P, Winn W. (2004). Microbiological Diagnosis. 5th ed. Editorial Panamericana SA Argentina.
- BD Laboratories. BBL Simmons Citrate Agar Slants. 2015.Available at: bd.com
- Britannia Laboratories. Simmons Citrate Agar. 2015.Available at: britanialab.com
- Valtek Diagnostic Laboratories. Simmons Citrate Agar. 2016.Available at: andinamedica.com.