Hydrogen sulphide (H2S) production test is used for the detection of hydrogen sulphide (H2S) gas produced by an organism. It is used mainly to assist in the identification of members of family Enterobacteriaceae and occasionally to differentiate other bacteria such as Bacteroides sps and Brucella sps. H2S is produced when sulphur – containing amino acids are decomposed. This test can be performed in many ways using different biochemicals.


Use of Kligler iron agar (KIA) to detect H2S

This medium is suitable for detecting H2S production by enterobacteria. H2S is detected by the ferric citrate contained in the medium. Inoculate the test organism into KIA and incubate it at appropriate temperature over night. Observe blackening of the medium

Use of sulphite indole motility (SIM) medium to detect H2S

This medium contains ferrous ammonium sulfate and sodium thiosulfate, which together serve as indicators for the production of hydrogen sulfide. Hydrogen sulfide production is detected when ferrous sulfide, a black precipitate, is produced as a result of ferrous ammonium sulfate reacting with H2S gas.

Lead acetate paper test to detect H2S

When a sensitive technique for detecting H2S production is required, the lead acetate paper test is recommended. For this, you have to:

Inoculate a tube or bottle of sterile peptone water or nutrient broth with the test organism.

Insert a lead acetate paper strip in the neck of the bottle or tube above the medium, and stopper well.

Incubate the inoculated medium at 35-37oC, and examine daily for a blackening of the lower part of the strip. 

Expected Results

Blackening —————————– Positive test result-  H2S produced

No blackening ————————- Negative test result -no H2S produced.


Positive control: Proteus vulgaris

Negative control: Shigella sps

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