Gerdts G, Luedke G
Alfred Wegener Institute Foundation for Polar and Marine Research, Marine Station Helgoland, 27498 Helgoland, Germany.
J Microbiol Methods. 2006 Feb;64(2):232-40. doi: 10.1016/j.mimet.2005.05.001. Epub 2005 Jun 23.
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 2100 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.
为揭示天然海洋浮游细菌群落的结构，过去已成功应用基于聚合酶链反应（PCR）的技术以及荧光原位杂交（FISH）技术。使用荧光显微镜或共聚焦激光扫描显微镜（CLSM）分析FISH实验，能够区分细菌群落，但迄今为止，大多数将流式细胞术与FISH结合用于此目的的尝试均告失败。在此，我们展示了使用基于微流体的流式细胞仪（安捷伦2100生物分析仪）对天然海洋浮游细菌群落进行FISH实验的成功分析。海水样本在聚碳酸酯滤膜上富集，并与不同系统发育深度的Cy5标记基因探针杂交。细菌从滤膜上分离下来，随后在安捷伦2100生物分析仪的细胞芯片中进行分析。样本用SYTOX复染。在所有样本中，EUB338阳性信号可与NON探针的信号清晰区分。此外，还观察到α-变形菌占优势（如探针ALF968和GrB所示）。基于微流体的流式细胞术是分析海洋环境中天然细菌群落的一种有前景的技术。
