Assessment of lectin-binding analysis for in situ detection of glycoconjugates in biofilm systems

An assessment of lectin-binding analysis for the characterization of extrac ellular glycoconjugates as part of the extracellular polymeric substances i n environmental microbial communities was performed using fully hydrated ri ver biofilms, The applicability of the method was evaluated for single, dua l and triple staining with a panel of fluor-conjugated lectins, It was show n that lectin-binding analysis was able to stain glycoconjugates within bio film communities. Lectin staining also demonstrated spatial heterogeneity w ithin the biofilm matrix. Furthermore, the application of two or even three lectins was possible if suitable combinations were selected, The lectin-bi nding analysis can be combined with general nucleic acid stains to collect both nucleic acid and glycoconjugate signals. The effects of incubation tim e, lectin concentration, fluor labelling, carbohydrate inhibition, order of addition and lectin interactions were studied. An incubation time of 20 mi n was found to be sufficient for completion of lectin binding. It was not p ossible to ascertain saturating concentration for individual lectins, there fore a standard concentration was used for the assay, Carbohydrate inhibiti on tests indicated that fluorescein isothiocyanate (FITC)-conjugated lectin s had more specific binding characteristics than tetramethyl rhodamine isot hiocyanate (TRITC)- or cyanine dye (CY5)-labelled lectins. The order of add ition and the nature of the fluor conjugate were also found to influence th e binding pattern of the lectins, Therefore the selection of a panel of lec tins for investigating the EPS matrix must be based on a full evaluation of their behaviour in the biofilm system to be studied. Despite this necessit y, lectin-binding analysis represents a valuable tool to examine the glycoc onjugate distribution in fully hydrated biofilms, Thereby, chemical heterog eneities within extracellular biofilm locations can be identified in order to examine the role (e.g, sorption properties, microenvironments. cell-extr acellular polymeric substance interactions) of the extracellular polymeric substances in environmental biofilm systems.