[5] In Situ analysis of microbial biofilms by rRNA-targeted oligonucleotide probing

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Publisher Summary

In situ hybridization allows the detection of specific nucleic acid sequences in eukaryotic and prokaryotic cells by binding oligonucleotide probes to their complementary target sequences. Fluorescent in situ hybridization (FISH) in combination with advanced microscopic techniques, such as confocal laser scanning microscopy (CLSM) and digital image analysis, became an important part of a polyphasic approach in microbial ecology for the identification and localization of microorganisms within complex environments. Defined bacterial populations in biofilms were investigated by FISH in anaerobic multispecies biofilms, in drinking water and associated biofilms, in marine photosynthetic biofilms, and in activated sludge that can be regarded as immobilized biofilm. This chapter provides a brief description of the design, evaluation, and fluorescent labeling of oligonucleotides, the preparation of biofilms for hybridization, and the use of FISH for the identification of individual bacterial cells in complex biofilms.

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