A modification of the methylene blue method to measure bacterial sulfide production in feces

Human feces may contain large numbers of sulfate reducing bacteria. These bacteria oxidize H₂ and other compounds during the reduction of sulfate to sulfide, and this metabolism could play a role in colonic disease and influence the numbers and activity of other colonic bacteria. While fecal sulfide concentrations have been determined by a methylene blue method used to measure sulfide in water, we found the accuracy of this assay to be limited by turbidity and interference by fecal material with the colorimetric reaction. Centrifugation after color development largely eliminated turbidity; however, an acidified sample blank was necessary to correct for residual turbidity. Interference with the colorimetric reaction was markedly reduced by analysis of a 1:200 dilution of the fecal sample, and analysis of a sample spiked with sulfide allowed for correction for residual interference. Analysis of feces from eight healthy volunteers using this modified technique showed an average fecal sulfide concentration of 1.6 ± 0.85 μmol/g feces, a value roughly ten times higher than previously reported with the unmodified technique.