Estimation of bacterial hydrogen sulfide production in vitro

Abstract

Oral bacterial hydrogen sulfide (H2S) production was estimated comparing two different colorimetric methods in microtiter plate format. High H2S production was seen for Fusobacterium spp., Treponema denticola, and Prevotella tannerae, associated with periodontal disease. The production differed between the methods indicating that H2S production may follow different pathways.

Keywords: Fusobacterium spp; bacterial metabolites; bismuth sulfide; cysteine; hydrogen sulfide; methylene blue; oral bacteria; periodontitis.

Fig. 1

(a) The BS method. The production of H₂S was estimated by black BS precipitation and recorded as no color production (−; score 0) to maximum black color production (+ + + + +; score 5). (b) The methylene blue (MB) method. The production of MB in the presence of H₂S was estimated from no color production (−; score 0) to maximum blue color production (+ + + + +; score 5).

Fig. 2

NaHS was tested with two colorimetric tests: the BS method (black) and the methylene blue (MB) method (blue). The color production for known concentrations of NaHS was a) determined (mean) with optical density analysis (λ=405 nm for the bismuth method and λ=668 nm for the MB method), b) scored on a visual scale by three individuals (median). Data shown are individual values for triplicates of the test.

Fig. 3

Bacterial H₂S production was determined by BS precipitation and optical density (OD₄₀₅) measurements up to 7 h at different time points. The bacteria were analyzed at a concentration of approximately 5×10⁸ cells/mL. Data shown are mean values for triplicate wells for three repetitions of the experiment.