The formation of methylene blue after the reaction of hydrogen sulfide with dimethyl-p-phenylenediamine and iron(III) at pH 0.4 – 0.7 is used to determine by photometric measurements sulfide concentration in the range 0.020 to 1.50 mg/L (20 ppb to 1.5 ppm). The test is very sensitive and the blue coloration developing upon contact of the reagents with dissolved HS is stable for 60 min. Ready-to-use kits such as the Spectroquant sulfide test facilitate routine analyses. The methylene blue sulfide test is a convenient method often used in soil microbiology to quickly detect in water the metabolic activity of sulfate reducing bacteria (SRB). It must be noted that in this colorimetric test, methylene blue is a product formed by the reaction and not a reagent added to the system.
The addition of a strong reducing agent, such as ascorbic acid, to a sulfide-containing solution is sometimes used to prevent sulfide oxidation from atmospheric oxygen. Although it is certainly a sound precaution for the determination of sulfide with an ion selective electrode, it might however hamper the development of the blue color if the freshly formed methylene blue is also reduced, as described here above in the paragraph on redox indicator.