About this 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 VirtualUnknown(C) 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. VirtualUnknown 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?
Performing this test in the VUMIE Online lab
Inoculation of Medium
1. Select the TSIA slant medium.
2. Using an inoculating wire, complete the process of a tube-to-tube aseptic transfer to inoculate the medium by streaking the slant and stabbing the butt of the medium.
Incubation of the Inoculated Medium
3. Place the inoculated tube into the 35-37 C incubator.
4. Press the New Day button to move forward 24 hours.
Determination of Test Results
5. Incubate this test for only 24 hours.
6. Retrieve the incubated culture from the incubator.
7. 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.
8. Record test result.