A hydrogen sulfide test is a procedure used to identify a specific organism in a laboratory setting. There are microorganisms having the ability to compounds containing sulfur into hydrogen sulfide during metabolism.

Two test tubes Hydrogen Sulfide Test

Image 1: Two test tubes – the one on the left tests negative as no changes in color is observed. The test tube on the right tests positive as a black precipitate is formed.
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The principle of the hydrogen sulfide test is to check for the production of sulfide gas. To do so, an iron and sulfur compound is added to the test medium. Hydrogen sulfide is formed if a specific strain of bacteria reduces the sulfur compound.

Hydrogen sulfide is produced by a particular type of bacteria by reducing the sulphur-containing amino acids such as methionine, cysteine, or by reducing inorganic sulfur compounds like sulfates, thiosulfates, or sulfites during degradation of protein or when anaerobic respiration turns the electrons to sulfur instead of turning it to oxygen.

The hydrogen sulfide gas is produced and reacts with the iron compound causing the black precipitation of ferric sulfide. You will be able to notice the presence of hydrogen sulfide because of the color (black), which serves as an indicator. (1, 2, and 3)

Media Used

Various media can be used to perform hydrogen sulfide test such as:

  • SIM (Sulphite Indole Motility) medium
  • TSI (Triple Sugar Iron)
  • Lead acetate paper
  • Kligler’s iron agar (4)
Hydrogen sulfide test using SIM medium

Image 2: Hydrogen sulfide test using SIM medium.
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SIM medium to check for hydrogen sulfide gas

SIM medium has ferrous ammonium sulfate and sodium thiosulfate. They act as indicators when hydrogen sulfide is produced. The presence of black precipitate is an indicator that hydrogen sulfide is formed. It is a reaction process – ferrous ammonium sulfate reacts with hydrogen sulfide gas. (4, 5)

The test tubes above are tested using iron agar medium

Image 3: The test tubes above are tested using iron agar medium.

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Iron agar to detect hydrogen sulfide gas

It is the perfect medium to use when checking the presence of hydrogen sulfide gas as caused by enterobacteria. A positive hydrogen sulfide gas is when ferric citrated is contained in the medium.

Hydrogen sulfide test using lead acetate paper

Image 4: Hydrogen sulfide test using lead acetate paper.

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Lead acetate paper test to check for hydrogen sulfide gas

A lead acetate paper test is used when a delicate means for checking hydrogen sulfide gas is needed. (6)


  • Beef extracts
  • Peptone
  • Ferrous ammonium sulphate
  • Sodium thiosulphate
  • Final pH
  • Distilled water (6, 7)

Hydrogen Sulfide Test Procedures

When using SIM (sulphite indole motility) as a medium:

  • The organism to be tested is inoculated into a labeled test tube using the stab inoculation technique.
  • The inoculated tube should be incubated at a temperature of 37 degrees Celsius for at least one to two days.
  • Watch out for the formation of a black precipitate. (2, 3)

When using iron agars such as triple sugar iron agar and Kligler iron agar as a medium:

  • The organism to be tested should be inoculated into Kligler iron agar and incubated at the right temperature overnight.
  • Check for any changes in the medium, specifically if the medium turns to black.

When using a lead acetate paper test as a medium:

  • A bottle or a tube containing sterile peptone water or nutrient broth containing the organism to be tested should be inoculated.
  • A lead acetate paper strip is inserted in the beck of the tube or bottle; just above the medium.
  • The inoculated medium should be incubated at a temperature of 35 to 37 degrees Celsius. Check for any signs of blackening on the lower part of the strip. (4, 5, and 6)

Interpreting results

The result of the test is positive if you notice a change of color on the medium, specifically, blackening of the medium. On the other hand, the result is negative if the medium does not turn to black.


  • Make sure that when you use the lead acetate paper strip the media does not touch the strip. Lead acetate is a toxic substance for bacteria and it may inhibit the growth of some types of bacteria.
  • The production of hydrogen sulfide gas may be inhibited on TSI for microorganisms that use sucrose. It can suppress the enzyme mechanism leading to the production of hydrogen sulfide gas.
  • For complete identification of organisms, other testing methods should be used such as molecular, mass spectrometry, immunological, and biochemical testing. (5, 6, and 7)

Why is the hydrogen sulfide test ordered and what are the uses?

  • The test is performed to help identify and differentiate members of Enterobacteriaceae from other Gram negative bacilli.
  • It aids in identifying microorganisms like Francisella, Salmonella, and Proteus species.
  • It is used to help identify the members of Enterobacteriaceae family and to differentiate microorganisms like Bacteroidessps and Brucella species. (1, 3, and 5)


  1. https://microbiologyinfo.com/hydrogen-sulfide-test/
  2. https://www.vumicro.com/vumie/help/VUMICRO/Hydrogen_Sulfide_Production_Test.htm
  3. https://water-research.net/index.php/sulfur
  4. https://microbenotes.com/hydrogen-sulfide-h2s-production-test/
  5. https://sciencing.com/test-hydrogen-sulfide-6757052.html
  6. http://microbesinfo.com/2015/02/hydrogen-sulphide-h2s-production-test/
  7. https://www.intertek.com/petroleum/h2s-test/

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