Hydrogen Sulfide Gas Production Test

In this hydrogen sulfide gas production test post we have briefly explained hydrogen sulfide test principle, objectives, requirements, H2S production test procedure, uses and limitations of H2S production test.

H2S production test

H2S production test is based on the fact that enteric bacteria convert sulphur to hydrogen Sulfide, which has a distinct odour and generates a black iron Sulfide precipitate when ferrous iron is present.

A recent comprehensive review of the H2S production test described numerous evaluations of its performance, and concluded that until more comprehensive and rigorous studies are performed, the H2S production test could not be widely recommended for testing faecal contamination of drinking water.

H2S production test was recommended with caution in specific situations: where other water quality tests are not available and for educational or motivational purposes.


Bacterial species that produce H2S use their enzymatic activities to liberate Sulfide from cysteine or thiosulfate in the medium.

Cysteine desulfhydrase is a bacterial enzyme that removes the sulfhydryl and amino groups from cysteine, resulting in hydrogen Sulfide, ammonia, and pyruvic acid. The reduction of thiosulfate in anaerobic respiration by the enzyme thiosulfate reductase also produces hydrogen Sulfide.

The resulting colourless H2S gas reacts with H2S indicators (iron, bismuth, or lead) in the medium to generate insoluble heavy metal Sulfides, which appear as a black precipitate.

Media Composition

Several media containing iron compounds allow detection of hydrogen sulfide production. One medium used is Sulfide-Indole-Motility (SIM) medium. This is a nutrient medium allowing the detection of three different traits in bacteria. 

It contains sulfates to serve as the substrate for detecting sulfide production. It has abundant tryptophan as a substrate for indole production; and its content of 0.5% agar is sufficient to allow bacterial motility, thereby allowing detection of motility.

A second medium is triple sugar iron agar (TSIA), which is also used to detect the ability of the microbe to ferment up to three sugars. In either case, the chemistry of the test and its interpretation are the same.

TSI Agar

Ingredients Gms / Litre: Beef extract – 3.000, Peptone – 20.000, Yeast extract – 3.000, Lactose – 10.000, Sucrose – 10.000, Dextrose monohydrate – 1.000, Ferrous sulphate – 0.200, Sodium chloride – 5.000, Sodium thiosulphate – 0.300,  Phenol red – 0.024, Agar -12.000.

Test Procedure

Tube Method

1. Touch the core of a well-isolated colony with a sterile inoculating needle. Stab the tube to within 3 to 5 mm of the bottom. Streak the whole surface of the agar slant with TSI agar. Put a strip of lead acetate paper to the top of the tube and secure it with the tube cap so that it extends 1 inch inside the tube.

2. Place a loose cap on the tube. To allow for the escape of gas in the tube, do not tighten the cap. Incubate aerobically for 18 to 24 hours at 35 to 37°C. Look for a dark precipitate, which indicates the presence of hydrogen Sulfide.

3. If desired, extend incubation only to detect H2S production. Campylobacters may take 3 days for production of H2S.

Plate Method

1. Touch the core of a well-isolated colony with a sterile inoculating needle. Streak plate so as to obtain isolated colonies.

2. Incubate aerobically for 18 to 24 hours at 35 to 37°C. Look for a dark precipitate, which indicates the presence of hydrogen Sulfide.

Test Result

Hydrogen Sulfide Test

Positive reactions

Black colour throughout the medium, a black ring at the junction of the butt and slant, or any black precipitate in the butt is all signs of H2S generation in tube media. Blackening normally starts near the inoculation line.

In plate medium, H2S production appears as black colonies surrounded by a brownish-black zone or a metallic gleam.

Negative reactions

Production of H2S in tube media: no tube blackening. In plate media, no blackening or metallic-sheen colonies were seen.


1. H2S production test is mostly used to identify members of the Enterobacteriaceae family and, on rare occasions, to distinguish other bacteria such as Bacteroides sps and Brucella sps.

2. H2S production test facilitates in the identification and distinction of Enterobacteriaceae (enterics) bacteria from other Gram-bacilli bacteria.

3. H2S production test notably useful for Salmonella, Escherichia coli  Francisella, Citrobacter freundii, Salmonella species, Proteus mirabilis, Proteus vulgaris, Edwardsiella tarda, and Proteus species identification.


1. Bacteria are poisonous to lead acetate, and it may impede the growth of some bacteria. Allow no media to come into contact with the strip.

2. For complete identification, biochemical, immunological, molecular, or mass spectrometry testing on colonies from pure culture is indicated.

Further Readings