Hydrogen sulphide test is used to identify bacteria that produce the gas, hydrogen sulphide (H2S). 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.


  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 by adding 0.01 % cysteine or cystine to peptone H2O or nutrient broth.
  4. Distribute the prepared basal medium (e.g. peptone broth) 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 tube will serve as the 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. 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.

Illustration of hydrogen sulphide production in H2S test. Tube on left side shows positive test result (due to production of black colouration or medium turning black) while tube on right side shows negative test result (due to absence of dark/black colouration)


Cheesbrough M (2006). District Laboratory Practice in Tropical Countries. 2nd Cambridge University Press, UK. Pp. 178-187.

Willey J.M, Sherwood L.M and Woolverton C.J (2008). Harley and Klein’s Microbiology. 7th ed. McGraw-Hill Higher Education, USA.

Woods GL and Washington JA (1995). The Clinician and the Microbiology Laboratory. Mandell GL, Bennett JE, Dolin R (eds): Principles and Practice of Infectious Diseases. 4th ed. Churchill Livingstone, New York.

World Health Organization (1993). Laboratory Biosafety Manual, 2nd edn. Geneva: WHO.

World Health Organization (2003). Guidelines for the Safe Transport of Infectious Substances and Diagnostic Specimens.  WHO/EMC/97.3. Geneva: WHO.

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