Hydrogen Sulfide (H₂S) Production Test

Last updated on March 25th, 2020

Certain bacterial species liberate sulfur from sulfur-containing amino acids or other compounds in the form of H₂S. This ability of these bacteria can be used as an important characteristic of their identification.

Hydrogen sulfide-positive organisms

  1. Citrobacter freundii
  2. Salmonella species
  3. Proteus mirabilis
  4. Proteus vulgaris
  5. Edwardsiella tarda


Bacterial species capable of producing H₂S release sulfide from cysteine or thiosulfate present in the medium by their enzymatic action. Bacteria that produce cysteine desulfhydrase are able to remove the sulfhydryl and amino groups from cysteine, yielding hydrogen sulfide, ammonia, and pyruvic acid. Hydrogen sulfide is also produced by the reduction of thiosulfate in anaerobic respiration by the enzyme thiosulfate reductase. 

Thus formed H₂S gas, which is colorless, combines with H₂S indicators (iron, bismuth or lead) present in the medium producing insoluble, heavy metal sulfides that appear as a black precipitate.

Media for the detection of Hydrogen Sulfide (H₂S)

Commonly used media for the detection of hydrogen sulfide production and the sources for sulfur and the sulfide indicators are as follows:

Media Sulfur source H₂S indicator
Bismuth sulfite Peptones plus sulfite Ferrous sulfate
Citrate sulfide agar Sodium thiosulfate Ferric ammonium citrate
Deoxycholate citrate agar (DCA) Peptones Ferric citrate
Lysine iron agar (LIA) Sodium thiosulfate Ferric ammonium citrate
Kligler iron agar (KIA) Sodium thiosulfate Ferrous sulfate
Triple sugar iron (TSI) agar Sodium thiosulfate Ferrous sulfate
Lead acetate agar Sodium thiosulfate Lead acetate
Salmonella-Shigella (SS) agar Sodium thiosulfate Ferric citrate
Sulfide-indole-motility (SIM) Medium Sodium thiosulfate Peptonized iron
Xylose-lysine-deoxycholate (XLD) agar medium Sodium thiosulfate Ferric ammonium citrate
Hektoen enteric agar Sodium thiosulfate Ferric ammonium citrate

As various types of media are available for the detection of H₂S production with varying degrees of sensitivity, microbiologists can choose a specific detection system based on their needs and characteristics of the test isolate. For example, lead acetate, the most sensitive indicator, should be used whenever bacteria that produce only trace amounts of H₂S are tested.

Note: When incorporated in culture media, lead acetate may inhibit the growth of many fastidious bacteria so while testing, instead of incorporating it into the media, a lead acetate impregnated filter paper should be draped under the cap of a culture tube.

Hydrogen sulfide production test

As H₂S detected in one medium may not be detected in another, it is necessary to know the test system used when interpreting identification charts. In diagnostic microbiology, SIM, KIA or TSI tubes are commonly used for the detection of H₂S production. Among these three biochemical test media, TSI is least sensitive. It is believed that sucrose present in this test medium suppresses the production of hydrogen sulfide. SIM is more sensitive than TSI and KIA. Lack of carbohydrates to suppress H₂S formation, and the use of peptonized iron as the indicator makes SIM a better test medium for the detection of H₂S production.

With all H₂S detection systems, the endpoint is an insoluble, heavy metal sulfide, which produces a black precipitate in the medium or on the filter paper strip. Because hydrogen ions must be available for H₂S formation, the blackening is first seen in test media in which acid formation is maximal, that is, along the inoculating line, within the deeps of slanted agar media, or in the centers of colonies growing on agar surfaces.

References and further reading

  1. Koneman’s Color Atlas and Textbook of Diagnostic Microbiology, Seventh Edition.
About Acharya Tankeshwar 467 Articles
Hello, thank you for visiting my blog. I am Tankeshwar Acharya. Blogging is my passion. I am working as an Asst. Professor and Microbiologist at Department of Microbiology and Immunology, Patan Academy of Health Sciences, Nepal. If you want me to write about any posts that you found confusing/difficult, please mention in the comments below.