Hydrogen Sulfide (H₂S) Production Test

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.

Principle

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:

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.

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 the 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.

Procedure

1. Tube Media
   1. Warm medium to room temperature and examine for cracks. Do not use if cracks appear.
   2. Using a sterile inoculating needle, touch the center of a well-isolated colony.
   3. Stab to within 3 to 5 mm from the bottom of the tube.
   4. Withdraw the needle.
   5. For KIA or TSI, streak the entire surface of the agar slant.
   6. Optional for fastidious organisms: add a strip of lead acetate paper to top of tube and hold in place with the cap of the tube so that it extends 1 in. into the tube.
   7. Place cap loosely on tube. Do not tighten the cap to allow for release of gas in the tube.
   8. Incubate aerobically at 35 to 37°C for 18 to 24 h.
   9. Observe for black precipitate indicating hydrogen sulfide production.
   10. If desired, extend incubation only to detect H2S production. Campylobacters may take 3 days for production of H2S.
2. Plate Media
   1. Streak plate so as to obtain isolated colonies.
   2. Incubate aerobically at 35 to 37°C
   3. Observe for blackened colonies

Result and Interpretation

• Positive reactions
  • H2S production in tube media: black color throughout the medium, a black ring at the junction of the butt and slant, or any black precipitate in the butt. Blackening usually begins at the line of inoculation.
  • H2S production in plate media: black colonies surrounded by a brownish-black zone or metallic sheen
  • Lead acetate paper: brownish-black coloration of the paper strip
• Negative reactions
  • H2S production in tube media: no blackening in tube
  • H2S production in plate media: no blackening and no metallic-sheen colonies
  • Lead acetate paper: no change in color of the strip

Hydrogen sulfide-positive organisms

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

Limitations

1. H2S production may be inhibited on TSI for organisms that utilize sucrose and suppress the enzyme mechanism that results in the production of H2S.
2. Lead acetate is toxic to bacteria and may inhibit the growth of some bacteria.

References and further reading

1. Koneman’s Color Atlas and Textbook of Diagnostic Microbiology, Seventh Edition.
2. Clinical Microbiology Procedures Handbook, Fourth Edition. (2016). American Society of Microbiology.