Rapid and simple method for the most-probable-number estimation of arsenic-reducing bacteria
Rapid and simple method for the most-probable-number estimation of arsenic-reducing bacteria
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DOI：
10.1128/aem.67.7.3168-3173.2001
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发表时间：
2001-07-01
影响因子：
4.4
通讯作者：
Polz, MF
中科院分区：
文献类型：
--
作者：
Kuai, L;Nair, AA;Polz, MF
提出了一种快速、简便的最大可能数计数法（MPN），用于异化砷还原菌（DARB）的计数。该方法是基于特定的检测亚砷酸盐，厌氧砷酸呼吸的最终产物，通过沉淀反应与硫化物。孵育1周后，将MPN方法的培养基酸化至pH 6，并加入硫化物至终浓度约为1 mM。亮黄色的三硫化二砷立即沉淀，并且在亚砷酸盐浓度低至0.05 mM时可容易地进行评分。加入硫化物后砷酸盐的非生物还原可能产生假阳性，显然产生可溶性As-S中间体，其直到加入硫化物后约1小时才沉淀。使用新的MPN方法，DARE纯培养物的群体估计与直接细胞计数相似。环境水和沉积物样品的MPN分别产生每毫升或每克（干重）10（1）和10（5）个细胞之间的DARB数量。中毒和灭菌对照表明，环境样品中潜在的非生物还原剂不干扰MPN估计。一个主要的优点是，该测定可以很容易地缩放到微量滴定板格式，使得能够通过使用多通道移液器分析大量样品。总的来说，MPN方法提供了一种快速和简单的方法来估计DARB的群体大小，DARB是一组不同的生物体，尚未开发出全面的分子标记物。
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 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 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 DARE were similar to direct cell counts. MPNs of environmental water and sediment samples yielded DARB numbers between 10(1) and 10(5) cells per mi 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.