Forschungs- & Entwicklungsinformationsdienst der Gemeinschaft - CORDIS

An improved CARD-FISH protocol to identify marine bacteria with oligonucleotide rRNA probes

A protocol is presented for the phylogenetic identification of microorganisms in environmental samples (water and sediments) by means of fluorescence in situ hybridization with rRNA-targeted oligonucleotide probes (FISH) and signal amplification (catalysed reporter deposition, CARD). The FISH probes are labeled with the enzyme horseradish peroxidase. A subsequent deposition of fluorescently labeled tyramides results in substantially higher signal intensities of target cells than after FISH with probes directly labeled with fluorochromes. Sample preparation and cell permeabilization strategies for various microbial cell wall types are discussed. The custom labeling of tyramides with different fluorochromes is described. A sequential multi-color CARD-FISH protocol is outlined for the simultaneous detection of different phylogenetic groups of bacteria.

This method serves as a tool for rapid identification of marine bacteria newly developed during the BASICS project. The protocol provides a standardized approach that allows scientists from different laboratories to produce comparable information on the abundances of different bacterial species in marine waters. It thus serves as a methodological base for a comparative monitoring of microbes in different European Seas. It is the central tool for a dissemination of this method for bacterial single cell identification in specific workshops on FISH planned within the framework of the project and thereafter.

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Max Planck Institute for Marine Microbiology
Celsiustrasse 1
D-28359 Bremen
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