A Sensitive Visual Method for the Detection of Hydrogen Sulfide Producing Bacteria

Hydrogen sulfide-producing bacteria can utilize sulfur-containing amino acids and proteins to produce hydrogen sulfide. Here, we established a sensitive and simple method to detect the hydrogen sulfide in bacteria. This method is a modified version of bismuth sulfide precipitation that used 96-well transparent microtiter plates.

Bacteria culture was combined with bismuth solution containing L-cysteine and cultured for 20 minutes, at the end of which a black precipitate was observed. Demonstrating the procedures will be Wei Zhu, a research assistant from our laboratory. Begin by transferring the bacterial colonies from a Luria-Bertani or LB agar plate to 100 milliliters of LB medium, and culture at 37 degrees Celsius for 12 to 16 hours until the bacterial concentration is about 1 billion cells per milliliter.

Transfer one bacterial colony of Fusobacterium nucleatum and Proteus vulgaris from Trypticase soy broth or TSB agar plates to 100 milliliters of TSB medium, and culture at 37 degrees Celsius anaerobically for 24 hours until the bacterial concentration is about 1 billion cells per milliliter. Mix 100 microliters of bacterial culture with 100 microliters of newly prepared bismuth solution in the 96-well microtiter plates, and culture for 20 minutes at 37 degrees Celsius. For each bacterial strain, perform the analysis in triplicate.

After 20 minutes, check for color change. If the color of the solution changes from light yellow to black, this indicates that the bacteria is able to produce hydrogen sulfide or H2S. Repeat this measurement three times.

Determine the sensitivity of the method using different concentrations of sodium hydrosulfide mixed with bismuth sulfide solution. Finally, determine the presence of bisulfide and sulfide ions by observing the formation of a black bismuth sulfide precipitate. Score the color of the wells using a visual scale from no color production to the darkest black color production.

The performance of the hydrogen sulfide test was investigated using pure cultures of selected bacterial strains. The results indicated that Salmonella paratyphi B, Fusobacterium nucleatum, Enterococcus faecalis, Pseudomonas aeruginosa, and Proteus vulgaris can produce hydrogen sulfide with black bismuth sulfide precipitate. While Salmonella paratyphi A, Staphylococcus aureus, Aeromonas hydrophila, and Klebsiella pneumoniae failed to show any black precipitate.

The key advantage of this method is that it is easy to replicate and does not require specialized equipment.