FISH and chips: Marine bacterial communities analyzed by flow cytometry based on microfluidics
FISH and chips: Marine bacterial communities analyzed by flow cytometry based on microfluidics
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DOI：
10.1016/j.mimet.2005.05.001
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发表时间：
2006-02-01
影响因子：
2.2
通讯作者：
Luedke, G
中科院分区：
文献类型：
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作者：
Gerdts, G;Luedke, G
为了揭示天然海洋中上层细菌群落的结构，过去成功地进行了基于聚合酶链式反应的技术以及荧光原位杂交(FISH)。使用荧光显微镜或激光共聚焦扫描显微镜(CLSM)分析FISH实验，可以区分细菌群落，但大多数将流式细胞术和FISH相结合的尝试到目前为止都失败了。在这里，我们提出了一个成功的鱼类实验分析天然海洋浮游细菌群落使用基于微流控的流式细胞仪(安捷伦2 100生物分析仪)。海水样品经聚碳酸酯滤膜浓缩后，与不同系统发育深度的Cy5标记基因探针杂交。细菌从过滤器中分离出来，随后在Agilent 2100生物分析仪的细胞芯片中进行分析。所有标本用SYTOX复染，EUB338阳性信号与非探针信号明显区别。此外，还可以观察到α-蛋白细菌的优势(如探针ALF968和G-RB所示)。基于微流控的流式细胞术是分析海洋环境中自然细菌群落的一种很有前途的技术。(C)2005 Elsevier B.V.保留所有权利。
To unveil the structure of natural marine pelagic bacterial communities, PCR-based techniques as well as fluorescence in situ hybridizations (FISH) were successfully performed in the past. Using fluorescence microscopes or confocal laser scanning microscopes (CLSM) for the analysis of FISH experiments, it was possible to differentiate bacterial communities, but most attempts to combine flow cytometry and FISH for this purpose have failed till now. Here we present a successful analysis of FISH experiments of natural marine pelagic bacterial communities using a flow cytometer based on microfluidics (Agilent 2 100 bioanalyzer). Marine water samples were enriched on polycarbonate filters and hybridized with Cy5 labeled gene probes of different phylogenetic depth. Bacteria were detached from the filters and subsequently analyzed in the Cell Chip of the Agilent 2100 Bioanalyzer. Samples were counter-stained using SYTOX In all samples the EUB338 positive signals could be clearly differentiated from those of the NON probe. Furthermore a dominance of alpha-protebacteria (as indicated by the probes ALF968 and G rB) could be observed.Microfluidics based flow cytometry is a promising technique for the analysis of natural bacterial communities from the marine environment. (c) 2005 Elsevier B.V. All rights reserved.