In situ cell and glycoconjugate distribution in river snow studied by confocal laser scanning microscopy

Aggregates from the river Elbe (Germany) were collected and examined by confocal laser scanning microscopy. The river snow was directly transferred into coverslip chambers and observed without any embedding and fixation procedure. River snow samples were examined in the reflection and fluorescence mode to record mineral content and autofluorescence signals. Spatial distribution of microbial cells within the aggregates was shown with general nucleic acid specific stains (SYTO 9, SYTO 64). The polymeric matrix of the river snow was demonstrated by using a panel of fluorescently labelled lectins. Staining with FITC and TRITC labelled. lectins and simultaneous dual channel imaging showed the distribution of different types of glycoconjugates. The binding pattern observed included bacterial cell surface labelling and cloud-type labelling of extracellular polymeric substances (EPS). In addition, dual staining combined with triple-channel recording was employed to clearly separate the signal in the red channel originating from TRITC-lectin or SYTO 64 nucleic acid stain from the far red autofluorescence originating from chlorophyll-containing organisms. With this approach the fully hydrated, living, 3-dimensional architecture of aquatic aggregates was investigated. Application of lectins demonstrated the heterogeneity of the EPS matrix in between the cellular constituents of the river snow community. The chemical heterogeneity of river snow may be significant for sorption and transport of nutrients and contaminants in lotic aquatic environments. This in situ technique may be also useful to examine interfacial microbial consortia in other habitats. Finally, it is suggested to employ in situ lectin staining to probe for the glycoconjugate distribution at the polymer level similar to in situ hybridisation with rRNA targeted oligonucleotides at the cellular level.