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

Abstract

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 microb~al cells within the aggregates was shown with general nucleic acid specific stains (SYTO 9, SYTO 64). The polymeric matnx 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 unaging 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 lotlc 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.

The utiquity and many roles of exopolymers (EPS) in aquatic systems

“…As would be expected, these flocs are also found in lakes and rivers and are referred to as "lake snow" and "river snow" respectively (Grossart et al, 1997;Neu, 2000). Flocs are moved by currents and thus collect other materials and organisms by impaction.…”

Microbial ecology of organic aggregates in aquatic ecosystems

“…2A; Suzuki & Kato 1953). They have been studied extensively in marine systems since then and more recently, to a lesser extent, also in lakes, rivers and estuaries (Fowler & Knauer 1986, Alldredge & Silver 1988, Alldredge 1992, Herndl 1992, Grossart & Simon 1993,b, Berger et al 1996, Lampitt 1996, Grossart et al 1997, Zimmermann 1997, Neu 2000. The term 'lake snow' was adopted from marine snow for limnetic macroaggregates (Grossart & Simon 1993) and most recently 'river snow' for riverine aggregates (Neu 2000).…”

“…The recent and ongoing introduction of highly intense fluorescent dyes such as 5-(4, 6-dichlorotriazynil)aminofluorescein (DTAF) and fluorescent lectins to detect specific poly- (Schumann & Rentsch 1998, Grossart 1999, Neu 2000. Scanning (SEM) or transmission electron microscopy also has been used to examine the composition of micro-and macroaggregates (e.g.…”

Fluorescent lectin assay to quantify particulate marine polysaccharides on 96‐well filtration plates

“…Data were fourth root-transformed to achieve homogeneity of variances. visualize colloidal polysaccharide distribution in river snow (Neu 2000). Diatoms exude polysaccharides and, because of their abundance in many aquatic systems, act as a major source for exopolymer substances (Hama and Handa 1983, Staats et al 1999, Passow 2002a.…”

“…Lectins are ubiquitous in nature (Lis and Sharon 1998) and play an important role in infection, host defense, fertilization, cancer, and protein transport (Gabius and Gabius 1997). The proteinaceous nature of lectins makes them amenable to labeling, and they have become important tools in medicine, histology, and biology (Van Damme et al 1998), as exemplified by cell-labeling experiments (Farina and Wilkins 1980) and microscopic examination of sugar distributions in river snow, biofilms, and diatoms (Waite et al 1995, Neu 2000, Neu et al 2001). Thus, lectins may provide new insights into polysaccharide composition, distribution, and quantity.…”