Rapid and simple method for the most-probable-number estimation of arsenic-reducing bacteria

A rapid and simple most-probable-number (MPN) procedure for the enumeration of dissimilatory arsenic-reducing bacteria (DARB) is presented. The method is based on the specific detection of arsenite, the end product of anaerob ic arsenate respiration, by a precipitation reaction with sulfide. After 1 weeks of incubation, the medium for the MPN method is acidified to pH 6 and sulfide is added to a final concentration of about 1 mM. The brightly yell ow arsenic trisulfide precipitates immediately and can easily be scored at arsenite concentrations as low as 0.05 mM. Abiotic reduction of arsenate up on sulfide addition, which could yield false positives, apparently produces a soluble As-S intermediate, which does not precipitate until about 1 h af ter sulfide addition. Using the new MPN method, population estimates of pur e cultures of DARE were similar to direct cell counts. MPNs of environmenta l water and sediment samples yielded DARB numbers between 10(1) and 10(5) c ells per mi or gram (dry weight), respectively, Poisoned and sterilized con trols showed that potential abiotic reductants in environmental samples did not interfere with the MPN estimates. A major advantage is that the assay can be easily scaled to a microtiter plate format, enabling analysis of lar ge numbers of samples by use of multichannel pipettors, Overall, the MPN me thod provides a rapid and simple means for estimating population sizes of D ARB a diverse group of organisms for which no comprehensive molecular marke rs have been developed yet.