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Applied and Environmental Microbiology, October 2004, p. 6210-6219, Vol. 70, No. 10
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.10.6210-6219.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Flow Sorting of Marine Bacterioplankton after Fluorescence In Situ Hybridization

Raju Sekar, Bernhard M. Fuchs, Rudolf Amann, and Jakob Pernthaler*

Max Planck Institute for Marine Microbiology, Bremen, Germany

Received 8 March 2004/ Accepted 31 May 2004

We describe an approach to sort cells from coastal North Sea bacterioplankton by flow cytometry after in situ hybridization with rRNA-targeted horseradish peroxidase-labeled oligonucleotide probes and catalyzed fluorescent reporter deposition (CARD-FISH). In a sample from spring 2003 >90% of the cells were detected by CARD-FISH with a bacterial probe (EUB338). Approximately 30% of the microbial assemblage was affiliated with the Cytophaga-Flavobacterium lineage of the Bacteroidetes (CFB group) (probe CF319a), and almost 10% was targeted by a probe for the ß-proteobacteria (probe BET42a). A protocol was optimized to detach cells hybridized with EUB338, BET42a, and CF319a from membrane filters (recovery rate, 70%) and to sort the cells by flow cytometry. The purity of sorted cells was >95%. 16S rRNA gene clone libraries were constructed from hybridized and sorted cells (S-EUB, S-BET, and S-CF libraries) and from unhybridized and unsorted cells (UNHYB library). Sequences related to the CFB group were significantly more frequent in the S-CF library (66%) than in the UNHYB library (13%). No enrichment of ß-proteobacterial sequence types was found in the S-BET library, but novel sequences related to Nitrosospira were found exclusively in this library. These bacteria, together with members of marine clade OM43, represented >90% of the ß-proteobacteria in the water sample, as determined by CARD-FISH with specific probes. This illustrates that a combination of CARD-FISH and flow sorting might be a powerful approach to study the diversity and potentially the activity and the genomes of different bacterial populations in aquatic habitats.

* Corresponding author. Mailing address: Max-Planck-Institut für Marine Mikrobiologie, Celsiusstraße 1, D-28359 Bremen, Germany. Phone: 49 421 2028940. Fax: 49 421 2028580. E-mail: jperntha{at}mpi-bremen.de.

Applied and Environmental Microbiology, October 2004, p. 6210-6219, Vol. 70, No. 10
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.10.6210-6219.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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