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Hydrogen sulphide test is used to identify bacteria that produce the gas, hydrogen sulphide (H2S). Hydrogen sulphide (H2S) production test is used for the detection of hydrogen sulphide (H2S) gas produced by an organism. H2S is produced by bacteria during the anaerobic metabolism of sulphur containing amino acids (e.g. cysteine). This gas (H2S) is also produced from the reduction of inorganic sulphur compounds such as sulphides and sulphites with ferrous, ferric and lead salts. Hydrogen sulphide test is used mainly to assist in the identification of members of the family Enterobacteriaceae and occasionally to differentiate other bacteria such as Bacteroides species and Brucella species. H2S gas is produced when sulphur – containing amino acid compounds are decomposed. This test can be performed in many ways using different biochemical reagents including indicator paper, sulphite indole motility (SIM) medium, Kligler iron agar (KIA), triple sugar iron agar (TSIA) and Lead acetate paper.


  1. Prepare indicator paper by cutting Whitman filter paper No. 1 into long strips.
  2. Soak the filter paper in saturated lead acetate solution.
  3. Prepare the basal medium (e.g. nutrient broth or sterile peptone water) by adding 0.01 % cysteine to peptone H2O or nutrient broth.
  4. Distribute the prepared basal medium into 10 ml portions in test tubes.
  5. Inoculate each test tube with a loopful of the test bacteria while leaving one of the tubes uninoculated. This uninoculated tube will serve as the negative control tube.
  6. Insert a strip of lead acetate indicator paper between plug and glass in such a way that the indicator paper stays just above the medium.
  7. Incubate the medium at 37oC for 18-24 hrs.
  8. Observe the tubes containing the indicator paper for H2S production after incubation. H2S production normally develops during the incubation period of the agar medium. The blackening of the lead acetate indicator paper indicates H2S production and this shows a positive test result. Absence of the blackening of the lead acetate indicator paper shows a negative test result (Figure 1). Proteus vulgaris is H2S positive while Shigella species is H2S negative.
Figure 1. Hydrogen sulphide test. The tube on the left is positive while the tube on the right is negative. Photo courtesy: https://www.microbiologyclass.com

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