Hydrogen Sulfide (H2S) Production Test


What is the purpose of the test? 


This test determines whether the microbe reduces sulfur-containing compounds to sulfides during the process of metabolism.



How is the hydrogen sulfide production determined?


If sulfide is produced, it combines with iron compounds to produce FeS, a black precipitate.



What medium is used? 


Several media containing iron compounds allow detection of hydrogen sulfide production. One medium used is Sulfide-Indole-Motility (SIM) medium. This is a nutrient medium allowing the detection of three different traits in bacteria: (1) it contains sulfates to serve as the substrate for detecting sulfide production; (2) it has abundant tryptophan as a substrate for indole production; and (3) its content of 0.5% agar is sufficient to allow bacterial motility, thereby allowing detection of motility. SIM is not supported by VirtualUnknownTM Microbiology.


A second medium is triple sugar iron agar (TSIA), which is also used to detect the ability of the microbe to ferment up to three sugars. In either case, the chemistry of the test and its interpretation are the same.

VirtualUnknownTM Microbiology uses TSIA for detecting H2S production.



How is the test performed?  


An inoculum from a pure culture is transferred aseptically to a sterile triple sugar iron agar (TSIA) slant. The inoculated tube is incubated at 35-37 C for 24 hours and the results are determined. Present in TSIA is an iron compound. The iron ions (Fe2+) have a high affinity (strong attraction) for sulfide ions. The result is that H2S combines with the iron to make FeS, a black compound. In tubes of TSIA containing bacteria producing hydrogen sulfide, the agar turns black from the FeS.


Please note that this medium is also useful for monitoring fermentation properties of the unknown microbe. Three sugars are found in TSIA: glucose, sucrose, and lactose. If any of the sugars can be used, the microbe will accumulate acidic byproducts. In a positive test, the pH indicator in the medium changes color from its normal red to yellow, indicating acid production. For more information on how to interpret these changes, refer to the discussion of TSIA agar slants.



What reagents are required?





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


Inoculation of Medium


1. Select the TSIA slant medium.  

2. Start your Bunsen burner.  

3. Select the inoculating wire tool.  

4. Flame your inoculating wire 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. For TSIA, the proper technique is to streak the slant of the agar first, then stab through it to the butt of the tube. Be careful to disturb the medium as little as possible. To streak and stab this medium does not require two trips to the inoculum culture.  

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 regions of the agar have been turned black, the organism is producing hydrogen sulfide. Other, unrelated results can also be seen. Cracks in the agar indicate gas production from the fermentation of sugars. If the red agar has been turned yellow, acids are being produced from at lease one of the sugars contained within.  

17. Record test result.  

18. Dispose of the culture.