Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo, Japan.
Appl Microbiol Biotechnol. 2010 Jan;85(4):1211-7. doi: 10.1007/s00253-009-2330-z. Epub 2009 Nov 20.
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.
我们开发了一种从环境样本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直接活菌计数、5-羧基荧光素二乙酸乙酰氧基甲酯活细胞染色以及微操作和随后在培养基中的培养的组合。为了评估该方法，我们将其应用于研究稻田中的反硝化群落。通过传统的最大可能数分析和我们的 FSC 分离方法获得了相似的反硝化计数。使用 FSC 分离方法，分离出 37 株反硝化细菌，其中一些含有含铜亚硝酸盐还原酶基因（nirK）。16S rRNA 基因分析表明，属于固氮螺菌属和氧化硫杆菌属的成员可能是稻田中的主要反硝化菌。这些结果表明，FSC 分离方法是从环境样本中获得功能活性单细胞的有用工具。
