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Applied and Environmental Microbiology, May 2003, p. 2928-2935, Vol. 69, No. 5
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.5.2928-2935.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

An Improved Protocol for Quantification of Freshwater Actinobacteria by Fluorescence In Situ Hybridization

Raju Sekar,1 Annelie Pernthaler,1 Jakob Pernthaler,1* Falk Warnecke,1 Thomas Posch,2 and Rudolf Amann1

Max Planck Institute for Marine Microbiology, Bremen, Germany,1 Institute of Zoology and Limnology, University of Innsbruck, Innsbruck, Austria2

Received 23 September 2002/ Accepted 19 February 2003

We tested a previously described protocol for fluorescence in situ hybridization of marine bacterioplankton with horseradish peroxidase-labeled rRNA-targeted oligonucleotide probes and catalyzed reporter deposition (CARD-FISH) in plankton samples from different lakes. The fraction of Bacteria detected by CARD-FISH was significantly lower than after FISH with fluorescently monolabeled probes. In particular, the abundances of aquatic Actinobacteria were significantly underestimated. We thus developed a combined fixation and permeabilization protocol for CARD-FISH of freshwater samples. Enzymatic pretreatment of fixed cells was optimized for the controlled digestion of gram-positive cell walls without causing overall cell loss. Incubations with high concentrations of lysozyme (10 mg ml-1) followed by achromopeptidase (60 U ml-1) successfully permeabilized cell walls of Actinobacteria for subsequent CARD-FISH both in enrichment cultures and environmental samples. Between 72 and >99% (mean, 86%) of all Bacteria could be visualized with the improved assay in surface waters of four lakes. For freshwater samples, our method is thus superior to the CARD-FISH protocol for marine Bacteria (mean, 55%) and to FISH with directly fluorochrome labeled probes (mean, 67%). Actinobacterial abundances in the studied systems, as detected by the optimized protocol, ranged from 32 to >55% (mean, 45%). Our findings confirm that members of this lineage are among the numerically most important Bacteria of freshwater picoplankton.

* 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, May 2003, p. 2928-2935, Vol. 69, No. 5
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.5.2928-2935.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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