Citrate Utilization Test (Simmons)


What is the purpose of the test? 


The purpose is to see if the microbe can use the compound citrate as its sole source of carbon and energy for growth.



How is citrate use determined? 


If a microbe can use citrate for carbon and energy, it will grow on Simmons citrate agar. The use of citrate leads to a rise in pH of the medium, and a pH indicator changes color.



What medium is used?  


The medium used is Simmons citrate agar slant. It contains mineral salts, sodium citrate for carbon, and ammonium phosphate for its nitrogen source. The pH indicator is brom thymol blue, which is green at neutral pH, yellow at acidic pH <6.0 and turns blue at alkaline (basic) pH >7.6.



How is the test performed?  


An inoculum from a pure culture is transferred aseptically to a sterile tube of Simmons citrate agar. The inoculated tube is incubated at 35-37 C for 24 hours and the results are determined. Abundant growth on the slant and a change from green to blue in the medium indicates a positive test for growth using citrate.


What reagents are added?





To perform this test in VirtualUnknown™ Microbiology, complete the following steps:


Inoculation of Medium


1. Select the Simmons’ citrate agar slant medium.  

2. Start your Bunsen burner.  

3. Select the inoculating loop tool.  

4. Flame your inoculating loop to sterilize it.  

5. Remove the caps from your test tubes.  

6. Flame the mouths of your test tubes.  

7. Use the sterile inoculating tool to pick up an inoculum from the culture tube of the unknown bacterium.  

8. Immediately transfer the inoculum into the fresh, sterile medium.  

9. Flame the mouths of your tubes once again.  

10. Replace the caps on the test tubes.  

11. Re-flame the inoculating tool.  


Incubation of the Inoculated Medium


12. Place the inoculated tube into the 35-37 C incubator.  

13. Press the New Day button to move forward 24 hours.  


Determination of Test Results


14. Incubate for the appropriate length of time. For this test, 24 hours is sufficient for determining the result.  

15. Retrieve desired incubated culture from the incubator.  

16. Observe test result. If the test was followed as described above, the sample will have changed to blue in the presence of alkali/bases (positive result). In negative tests, there is no growth.  

17. Record test result.  

18. Dispose of the culture.