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Abstract：
We developed a novel method to isolate functionally active single cells from environmental samples and named it the functional single-cell (FSC) isolation method. This method is based on a combination of substrate-responsive direct viable counts, live-cell staining with 5-carboxyfluorescein diacetate acetoxymethyl ester, and micromanipulation followed by cultivation in a medium. To evaluate this method, we applied it to study a denitrifying community in rice paddy soil. Similar denitrifier counts were obtained by the conventional most probable number analysis and our FSC isolation method. Using the FSC isolation method, 37 denitrifying bacteria were isolated, some of which harbored copper-containing nitrite reductase gene (nirK). The 16S rRNA gene analysis showed that members belonging to the genera Azospirillum and Ochrobactrum may be the major denitrifiers in the rice paddy soil. These results indicate that the FSC isolation method is a useful tool to obtain functionally active single cells from environmental samples.
摘要：
我们开发了一种从环境样品中分离功能活性单细胞的新方法，并将其命名为功能单细胞（FSC）分离方法。该方法基于底物响应性直接活菌计数的组合，用5-羧基荧光素二乙酸乙酰氧基甲酯进行活细胞染色，和显微操作，然后在培养基中培养。要评估此方法，我们将其应用于研究稻田土壤中的反硝化群落。通过常规的最可能数量分析和我们的FSC分离方法获得了相似的反硝化细菌计数。使用FSC隔离方法，分离出37株反硝化细菌，其中一些含有含铜的亚硝酸还原酶基因（nirK）。16SrRNA基因分析表明，在稻田土壤中，主要的反硝化因子可能是拟氮螺旋菌属和苍白杆菌属的成员。这些结果表明，FSC分离方法是从环境样品中获得功能活性单细胞的有用工具。
