FISH and chips: Marine bacterial communities analyzed by flow cytometry based on microfluidics

Abstract

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 α-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.