H2S Test- Objective, Principle, Procedure, Results, Limitations

Objective of Hydrogen Sulfide (H2S) Test

To determine the ability of the organism to produce hydrogen sulfide.

Principle of Hydrogen Sulfide (H2S) Test

Hydrogen sulfide (H2S) production test is used for the detection of hydrogen sulfide (H2S) gas produced by an organism. It is used mainly to assist in the identification of members of the family Enterobacteriaceae. H2S is produced by certain bacteria through the reduction of sulfur-containing amino acids like cysteine, methionine or through the reduction of inorganic sulfur compounds such as thiosulfates, sulfates, or sulfites. The hydrogen sulfide production can be detected by incorporating a heavy metal salt containing iron or lead as an H2S indicator to a nutrient culture medium containing cysteine and sodium thiosulfates as the sulfur substrates. Hydrogen sulfide, a colorless gas, if produced reacts with the metal salt-forming visible insoluble black precipitate of ferrous sulfide.

Media Used

SIM Medium

Composition per liter:

Pancreatic digest of casein………………………….. 20.0 gm

Peptic digest of animal tissue……………………….. 6.1 gm

Agar………………………………………………………….. 3.5 gm

Fe(NH4)2(SO4)2·6H2O…………………………………. 0.2 gm

Na2S2O3·5H2O……………………………………………. 0.2 gm

pH 7.3 ± 0.2 at 25°C

Procedure of Hydrogen Sulfide (H2S) Test

1. Inoculate the organism into labeled tube by means of stab inoculation in SIM medium.
2. Incubate the inoculated tubes at 37°C for 24-48 hours.
3. Observe for the formation of black precipitate on the medium.

Result Interpretation of Hydrogen Sulfide (H2S) Test

Positive result: blackening on the medium

Negative result: no blackening on the medium

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Limitations

1. H2S production may be inhibited on TSI for organisms that utilize sucrose and
   suppress the enzyme mechanism that results in production of H2S.
2. SIM is more sensitive in the detection of H2S than either TSI or KIA, because
   of its semisolid nature, its lack of interfering carbohydrates, and the use of
   peptonized iron as an indicator.
3. Lead acetate paper is 10 times more sensitive than other media.
4. Lead acetate is toxic to bacteria and may inhibit the growth of some bacteria. Do not allow the media to touch the strip.

Quality Control

Positive control: Proteus vulgaris

Negative control: Escherichia coli