This Article Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Sekar, R. Articles by Amann, R. Search for Related Content PubMed PubMed Citation Articles by Sekar, R. Articles by Amann, R. Agricola Articles by Sekar, R. Articles by Amann, R.

 Previous Article  |  Next Article 

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

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.