溴脱氧尿嘧啶核苷（BrdU）标记和文化独立的鉴定溶解有机随后的荧光激活细胞分选碳有辱人格的浮游细菌
Summary
环境浮游细菌培养与溶解有机碳（DOC）的化合物和一个DNA标记试剂，溴脱氧尿嘧啶核苷（BrdU）的一种模式。随后，商务部降解细胞分离升高BrdU使用荧光激活细胞分选（FACS）纳入散装社会。随后的分子分析这些细胞，然后确定。
Abstract
微生物介导许多溶解的有机碳（DOC）基板在水生环境退化的主要代理商。然而，鉴定，改造大自然的DOC的具体池的细菌类群构成一个技术上的挑战。
在这里，我们描述了一个方法，夫妇溴脱氧尿嘧啶核苷（BrdU）掺入，荧光激活细胞分选（FACS），和16S rRNA基因的分子分析功能，使文化有辱人格的特定DOC的化合物在水生环境的浮游细菌能够独立识别。设置为接收BrdU和模型DOC化合物（DOC修订），或只BrdU（不加控制）一式三份浮游细菌的缩影。 BrdU替代品在新合成的细菌的DNA和BrdU标记的DNA的胸苷的位置可以随时immunodetected 1,2。通过一个24小时孵化，浮游细菌，可以使用添加的DOC复合预计将有选择地激活，因此有较高水平的BrdU掺入比在DOC的修订和细胞非反应细胞（您好细胞）在无除了控制（低BrdU掺入细胞，李细胞）。荧光免疫检测后，您好细胞区分和身体从李细胞分离，荧光激活细胞分选（FACS）3 。排序DOC – 反应的细胞（您好细胞）的DNA提取和分类学上通过随后的16S rRNA基因为基础的分析，包括PCR，克隆库的建设和测序鉴定。
Protocol
Discussion
我们的做法夫妇BrdU掺入，流式细胞仪和16S rDNA序列分析，让物种一级的浮游细菌的代谢水环境中的个人的DOC组件的识别。 BrdU掺入检测标签的细菌细胞的代谢活动，只允许对活性细菌的分析，因此不包括休眠细胞。在我们的方法， 在原位 immunodetected和细菌有不同层次的BrdU掺入BrdU掺入随后可视化，使用流式细胞仪进行分组和排序。的BrdU纳入细胞的分析也有报道使用的BrdU标记的DNA磁珠immu…
Divulgations
The authors have nothing to disclose.
Acknowledgements
这个项目的经费是由国家科学基金会资助OCE1029607（XM）和MCB0702125（MAM），戈登和贝蒂摩尔基金会（MAM）的。
Materials
| Name | Type | Company | Catalog number | Comments |
| BrdU | Reagent | Sigma | B5002-5G | |
| Lysozyme | Reagent | Sigma | L6876-5G | |
| Proteinase K | Reagent | Sigma | P2308-25MG | |
| In Situ Cell Proliferation Kit, FLUOS | Kit | Roche | 11810740001 | Consume more of Anti-BrdU-FLUOS and Incubation buffer per reaction than suggested by the manufacturer. |
| Frame-Seal Incubation Chambers | Material | Bio-Rad | SLF-1201 | |
| Polycarbonate Membrane Filters (142-mm-diameter, 1.0 μm-pore-size) | Material | Millipore | FALP14250 | |
| Polycarbonate Membrane Filters (25-mm-diameter, 0.2 μm-pore-size) | Material | Millipore | FGLP02500 | |
| illustra PuReTaq Ready-To-Go PCR Beads | Kit | GE Healthcare | 27-9559-01 | |
| QIAquick gel extraction kit | Kit | QIAGEN | 28704 | |
| FailSafe PCR System | Kit | EPICENTRE | FS99060 |
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