Hydrogen Sulfide Test: Principle, Procedure, Uses, & Interpretation

Hydrogen Sulfide Test Definition

The capacity of some microbes to break down molecules containing Sulphur (Sulphur) into hydrogen sulphide (H2S) during metabolism is frequently used as a laboratory test for microbiological confirmation. Methods for detecting H2S generation by bacteria are affected by the supply of Sulphur and the metal salts employed to indicate H2S creation.

Due to its semi-solid form, absence of interfering carbohydrates and utilization of peptonized iron as an indication, Sulfide-indole-motility (SIM) is more sensitive than Triple sugar iron (TSI) or Kligler iron agar (KIA) in detecting H2S. But compared to other media, lead acetate paper is ten times more sensitive.

Hydrogen Sulfide Test Objectives

If the bacterium converts substances containing Sulphur to sulphide in order to create hydrogen sulphide gas.

Principle of Hydrogen Sulfide Test

The test medium comprises both an iron compound and a Sulphur compound to determine if hydrogen sulphide gas is produced. If the bacterial strain reduces the Sulphur component, hydrogen sulphide is created. In this way, the test evaluates whether the bacterium converts substances containing Sulphur to sulphide as part of its metabolism.

Certain bacteria produce H2S when they reduce sulfur-containing amino acids like cystine and methionine, as well as when they reduce inorganic Sulphur compounds like thiosulfates, sulphates, or sulfites during the breakdown of proteins or when anaerobic respiration causes electrons to be transferred to Sulphur rather than to oxygen.

In either scenario, hydrogen sulphide gas, or H2S, is created. This gas reacts with the iron component to form the black precipitate ferric sulphide. The black coloration identifies the existence of hydrogen sulphide. The primary objective of recognizing H2S gas produced by an organism is to assist in identifying that organism.

Hydrogen Sulfide Test Media

Numerous media can be used for this test, including lead acetate paper, TSI, KIA, and SIM medium.

Sulphite indole motility (SIM) medium to Detect H2S

This medium comprises sodium thiosulfate and ferrous ammonium sulphate, which serve as indicators for the production of hydrogen sulphide. Ferrous sulphide, a dark precipitate, is produced by the interaction of ferrous ammonium sulphate with H2S gas. Hydrogen sulphide production can be identified.

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3.0 g beef extract, 30.0 g peptone, 0.2 g ferrous ammonium sulphate, and 0.025 g sodium thiosulfat; 1000 ml of distilled water

Iron Agars to Detect H2S

This medium may be used to identify enterobacteria that produce H2S. The ferric citrate in the medium allows for the detection of H2S.

H2S Detection Test on Paper With Lead Acetate

Lead acetate paper testing is advised when a sensitive method of identifying H2S generation is needed.

Hydrogen Sulfide Test Procedure

1. In Sulphite indole motility (SIM) medium

  • Carefully inoculate the organism into the tube with the label on it.
  • The infected tubes should be kept at 37 °C for 24–48 hours.
  • Keep an eye out for the development of a black precipitate on the medium.

2. In Kligler iron agar (KIA) and Triple Sugar Iron Agar (TSIA)

  • Inject the test organism into KIA, then incubate it overnight at the proper temperature.
  • Keep an eye out for the medium getting darker.

3. Lead-acetate paper test

  • Introduce the test organism to a tube or bottle of sterile peptone water or nutritional broth.
  • Place a lead acetate paper strip above the medium and stopper in the bottle’s or tube’s neck.
  • Incubate the infected medium between 35 and 37 degrees Celsius, and check the strip’s lower portion every day for signs of blackening.

Hydrogen Sulfide Test Results

  • Positive result: blackening of the medium
  • Negative result: no blackening on the medium.

Hydrogen Sulfide Test Uses

  • It is mostly employed to help distinguish between members of the Enterobacteriaceae family and other bacteria, such as Bacteroides spp. and Brucella spp.
  • The test assists in identifying and distinguishing Enterobacteriaceae (enteric) from other gram-positive bacilli.
  • It is particularly useful for identifying the species of Francisella, Proteus, and Salmonella.

Hydrogen Sulfide Test Limitations

  • H2S production may be prevented in organisms that use sucrose and block the enzyme process that results in H2S generation.
  • The growth of certain bacteria may be inhibited by lead acetate, which is poisonous to bacteria.
  • Do not let the strip’s media touch it.
  • For comprehensive identification, it is advised to run biochemical, immunological, molecular, or mass spectrometry tests on colonies from pure cultures.


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