Hydrogen Sulfide (H2S) Production Test

Hydrogen Sulfide (H2S) Production Test

This test can be performed with the use of several media including Triple Sugar Iron (TSI), Kligler's Iron Agar (KIA), and SIM medium. 

An iron compound and a sulfur compound are included to test for the production of hydrogen sulfide gas.  Hydrogen sulfide is produced if the sulfur compound is reduced by the bacterial strain.  This happens when the strain either degrades the amino acid cysteine during protein degradation, or when anaerobic respiration shuttles the electrons to sulfur instead of to oxygen.  In either case H2S is produced (hydrogen sulfide gas) which reacts with the iron compound to form the black precipitate of ferric sulfide.  The black color acts as an indicator for the presence of hydrogen sulfide.  (If the tube becomes largely black, it may be difficult to read the tube for other tests.)

Purpose:

This test aids in the identification and differentiation of members of Enterobacteriaceae (enterics) from other Gram- bacilli.  It is especially helpful in identifying Salmonella, Francisella, and Proteus species.

Procedure:

1. Refer to the medium that you are using for the procedure as they differ by medium.
2. Observe the medium in the tube and record its color and appearance in your notebook.

Results:

1. Look for the occurrence of H2S production (black coloration), comparing your tube to an uninoculated control if needed.
2. The black coloration may mask the color of the the tube, especially the lower end.
3. Make your interpretations and record your results and interpretations.

Instructor Notes:
TSI is less sensitive in detecting the production of hydrogen sulfide gas than other similar tests (SIM media, Kligler's Iron Agar, etc.).  So, it is possible to observe the production of hydrogen sulfide in another test and not observe it in TSI.  The sucrose in TSI may suppress the formation of hydrogen sulfide.  Both TSI and Kligler's Iron Agar also use ferrous sulfate to detect the production of H2S, and this detector is less sensitive than other hydrogen sulfide detectors.  TSI has an added sulfur source (sodium thiosulfate), but sulfur may also be obtained from certain amino acids present in the protein in the media.

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