False-positive H2S production on TSI medium (left).
Hydrogen Sulfide Production
DESCRIPTION
Some bacteria have the enzymatic capability to degrade amino acids  (cysteine, cystine etc.) that contain sulfhydryl group (-SH)
producing hydrogen sulfide. Hydrogen sulfide reacts with heavy metals such as lead or iron forming a black precipitate. You can use
TSI medium (contains iron) or prepare a nutritive agar with lead acetate (1g Pb acetate to 100 ml nutritive agar).
MEDIUM PREPARATION
TSI composition:
Peptic peptone 10g, Casein peptone 10g, Meat extract 3g, Yeast extract 3g, Lactose
10g, Sacharose 10g, Dextrose 1g, NaCl 5g, Fe2SO4 0.2g, Na-thyosulphate 0.3g,
Phenol red 0.024g, Agar 12g, H2O ad 1000 ml.

PROCEDURE
Harvest a well isolated colony and inoculate a TSI tube by stabbing the medium.
Incubate at 37 °C, 24 hours. Reaction is positive if a black color appears.

EXPLANATION
Bacteria growing in TSI degrade aminoacids  forming ferrous sulfide which blackens
the medium.
REFERENCES:
1. H. Raducanescu, V.Bica-Popii,1986. Bacteriologie veterinara, Ed. Ceres, Bucuresti.
2. Margaret Barnett, 1992. Microbiology Laboratory Exercises. Wm. C. Brown
Publishers.
TSI Medium blackening due to H2S production
(left tube) -
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NOTES
Do not incubate more than 24 hours. False-positive or false-negative results may
appear.
Klebsiella oxytoca strains can produce a dark brown pigment when growth on media
containing gluconate and ferric citrate. The TSI slant is darkened by pigment, not by
hydrogen sulfide production.