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Applied and Environmental Microbiology, March 1999, p. 1259-1267, Vol. 65, No. 3
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

In Situ Identification of Cyanobacteria with Horseradish Peroxidase-Labeled, rRNA-Targeted Oligonucleotide Probes

Wilhelm Schönhuber,1,2 Boris Zarda,2 Stella Eix,1 Rosmarie Rippka,3 Michael Herdman,3 Wolfgang Ludwig,2 and Rudolf Amann1,2,*

Max-Planck-Institut für Marine Mikrobiologie, Bremen,1 and Lehrstuhl für Mikrobiologie, Technische Universität München, Munich,2 Germany, and Unité de Physiologie Microbienne, Institut Pasteur, Paris, France3

Received 9 October 1998/Accepted 16 December 1998

Individual cyanobacterial cells are normally identified in environmental samples only on the basis of their pigmentation and morphology. However, these criteria are often insufficient for the differentiation of species. Here, a whole-cell hybridization technique is presented that uses horseradish peroxidase (HRP)-labeled, rRNA-targeted oligonucleotides for in situ identification of cyanobacteria. This indirect method, in which the probe-conferred enzyme has to be visualized in an additional step, was necessary since fluorescently monolabeled oligonucleotides were insufficient to overstain the autofluorescence of the target cells. Initially, a nonfluorescent detection assay was developed and successfully applied to cyanobacterial mats. Later, it was demonstrated that tyramide signal amplification (TSA) resulted in fluorescent signals far above the level of autofluorescence. Furthermore, TSA-based detection of HRP was more sensitive than that based on nonfluorescent substrates. Critical points of the assay, such as cell fixation and permeabilization, specificity, and sensitivity, were systematically investigated by using four oligonucleotides newly designed to target groups of cyanobacteria.

* Corresponding author. Mailing address: Max-Planck-Institut für Marine Mikrobiologie, Celsiusstraße 1, D-28359 Bremen, Germany. Phone: 49 421 2028 930. Fax: 49 421 2028 580. E-mail: ramann{at}mpi-bremen.de.

Applied and Environmental Microbiology, March 1999, p. 1259-1267, Vol. 65, No. 3
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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