Investigation of lotic microbial aggregates by a combined technique of fluorescent in situ hybridization and lectin-binding-analysis

Abstract

A technique combining fluorescent in situ hybridization and lectin-binding-analysis (FISH-LBA) was developed and applied for the simultaneous detection of cellular components and glycoconjugates in lotic microbial aggregates (river snow). River snow aggregates were directly collected from the bulk water phase into coverslip chambers, in which the complete procedure including fixation, fluorescent in situ hybridization, lectin-binding and optical analysis by confocal laser scanning microscopy was performed. Neither autofluorescence originating from phyotosynthetic organisms nor inorganic particles did negatively interfere with the FISH-LBA technique. In river snow samples obtained from the river Elbe, Germany, distinct compartments of the river snow structure could be visualized with FITC-labelled lectins from Triticum vulgaris, Limulus polyphemus, Arachis hypogaea, Phaseolus vulgaris and Pseudomonas aeruginosa, binding to frequently occurring saccharide residues in the river snow matrix. The analysis could be performed on different levels of complexity. The combined technique visualized bacteria of different phylogenetic groups in the entire river snow structure as well as glycoconjugate components linked with various microcolonies. Different lectins stained slime layers and cell-envelopes of individual eukaryotic and prokaryotic cells. Consequently, application of the FISH-LBA technique allows the linkage between cellular and glycoconjugate identity in complex microbial communities.