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Rapid and Simple Method for the Most-Probable-Number Estimation of Arsenic-Reducing Bacteria

机译:快速简单的方法估算减少砷的细菌的最大数量

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摘要

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 anaerobic arsenate respiration, by a precipitation reaction with sulfide. After 4 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 yellow arsenic trisulfide precipitates immediately and can easily be scored at arsenite concentrations as low as 0.05 mM. Abiotic reduction of arsenate upon sulfide addition, which could yield false positives, apparently produces a soluble As-S intermediate, which does not precipitate until about 1 h after sulfide addition. Using the new MPN method, population estimates of pure cultures of DARB were similar to direct cell counts. MPNs of environmental water and sediment samples yielded DARB numbers between 101 and 105 cells per ml or gram (dry weight), respectively. Poisoned and sterilized controls 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 large numbers of samples by use of multichannel pipettors. Overall, the MPN method provides a rapid and simple means for estimating population sizes of DARB, a diverse group of organisms for which no comprehensive molecular markers have been developed yet.
机译:提出了一种快速,简单的最可能数(MPN)程序,用于枚举异化减少砷的细菌(DARB)。该方法基于通过与硫化物的沉淀反应,对厌氧性砷酸盐呼吸的最终产物砷的特定检测。孵育4周后,将用于MPN方法的培养基酸化至​​pH为6,并添加硫化物至终浓度约为1 mM。淡黄色的三硫化二砷立即沉淀出来,在低至0.05 mM的亚砷酸盐浓度下很容易被打分。加入硫化物后砷的非生物还原,这可能会产生假阳性结果,显然会产生可溶的As-S中间体,该中间体要等到加入硫化物约1小时后才会沉淀。使用新的MPN方法,DARB纯培养物的种群估计与直接细胞计数相似。环境水和沉积物样品的MPNs产生的DARB数分别为每毫升或克(干重)101和105个细胞。中毒和消毒后的对照表明,环境样品中潜在的非生物还原剂不会干扰MPN估算值。一个主要的优点是该测定法可以轻松地缩放到微量滴定板的形式,从而可以通过使用多通道移液器来分析大量样品。总体而言,MPN方法为估算DARB的种群规模提供了一种快速简便的方法,该DARB是目前尚未开发出全面分子标记的多种生物。

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