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Applied and Environmental Microbiology, November 1998, p. 4115-4127, Vol. 64, No. 11
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Automated Confocal Laser Scanning Microscopy and Semiautomated Image Processing for Analysis of Biofilms

Martin Kuehn,1 Martina Hausner,1 Hans-Joachim Bungartz,2 Michael Wagner,3 Peter A. Wilderer,1 and Stefan Wuertz1,*

Institute of Water Quality Control and Waste Management, Technical University of Munich, D-85748 Garching,1 and Department of Computer Science2 and Department of Microbiology,3 Technical University of Munich, D-80290 Munich, Germany

Received 16 March 1998/Accepted 23 July 1998

The purpose of this study was to develop and apply a quantitative optical method suitable for routine measurements of biofilm structures under in situ conditions. A computer program was designed to perform automated investigations of biofilms by using image acquisition and image analysis techniques. To obtain a representative profile of a growing biofilm, a nondestructive procedure was created to study and quantify undisturbed microbial populations within the physical environment of a glass flow cell. Key components of the computer-controlled processing described in this paper are the on-line collection of confocal two-dimensional (2D) cross-sectional images from a preset 3D domain of interest followed by the off-line analysis of these 2D images. With the quantitative extraction of information contained in each image, a three-dimensional reconstruction of the principal biological events can be achieved. The program is convenient to handle and was generated to determine biovolumes and thus facilitate the examination of dynamic processes within biofilms. In the present study, Pseudomonas fluorescens or a green fluorescent protein-expressing Escherichia coli strain, EC12, was inoculated into glass flow cells and the respective monoculture biofilms were analyzed in three dimensions. In this paper we describe a method for the routine measurements of biofilms by using automated image acquisition and semiautomated image analysis.

* Corresponding author. Mailing address: Institute of Water Quality Control and Waste Management, Technical University of Munich, Am Coulombwall, D-85748 Garching, Germany. Phone: 49 (89) 2891 3708. Fax: 49 (89) 2891 3718. E-mail: stefan.wuertz{at}wga.bauwesen.tu-muenchen.de.

Applied and Environmental Microbiology, November 1998, p. 4115-4127, Vol. 64, No. 11
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.


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