Microbiology (2001), 147, 299–313 Printed in Great Britain Assessment of lectin-binding analysis for in situ detection of glycoconjugates in biofilm systems Thomas R. Neu, 1 George D. W. Swerhone 2 and John R. Lawrence 2 Author for correspondence : Thomas R. Neu. Tel : 49 391 8109 800. Fax: 49 391 8109 150. e-mail : neugm.ufz.de 1 Department of Inland Water Research Magdeburg, UFZ Centre for Environmental Research, Leipzig-Halle, Brueckstrasse 3A, 39114 Magdeburg, Germany 2 National Water Research Institute, Saskatoon, Saskatchewan, Canada An assessment of lectin-binding analysis for the characterization of extracellular glycoconjugates as part of the extracellular polymeric substances in environmental microbial communities was performed using fully hydrated river biofilms. The applicability of the method was evaluated for single, dual and triple staining with a panel of fluor-conjugated lectins. It was shown that lectin-binding analysis was able to stain glycoconjugates within biofilm communities. Lectin staining also demonstrated spatial heterogeneity within the biofilm matrix. Furthermore, the application of two or even three lectins was possible if suitable combinations were selected. The lectin-binding analysis can be combined with general nucleic acid stains to collect both nucleic acid and glycoconjugate signals. The effects of incubation time, lectin concentration, fluor labelling, carbohydrate inhibition, order of addition and lectin interactions were studied. An incubation time of 20 min was found to be sufficient for completion of lectin binding. It was not possible to ascertain saturating concentration for individual lectins, therefore a standard concentration was used for the assay. Carbohydrate inhibition tests indicated that fluorescein isothiocyanate (FITC)-conjugated lectins had more specific binding characteristics than tetramethyl rhodamine isothiocyanate (TRITC)- or cyanine dye (CY5)-labelled lectins. The order of addition and the nature of the fluor conjugate were also found to influence the binding pattern of the lectins. Therefore the selection of a panel of lectins for investigating the EPS matrix must be based on a full evaluation of their behaviour in the biofilm system to be studied. Despite this necessity, lectin-binding analysis represents a valuable tool to examine the glycoconjugate distribution in fully hydrated biofilms. Thereby, chemical heterogeneities within extracellular biofilm locations can be identified in order to examine the role (e.g. sorption properties, microenvironments, cell–extracellular polymeric subtance interactions) of the extracellular polymeric substances in environmental biofilm systems. Keywords : biofilms, lectins, extracellular polymeric substances, glycoconjugates, confocal laser scanning microscopy INTRODUCTION Extracellular polymeric substances (EPS) represent, in terms of weight and volume, a major structural com- ................................................................................................................................................. Abbreviations : CLSM, confocal laser scanning microscopy ; CY5, cyanine dye ; EPS, extracellular polymeric substances ; FITC, fluorescein isothio- cyanate ; ICBA, intensity-corrected binding area ; LSD, least significant difference ; TRITC, tetramethyl rhodamine isothiocyanate ; UEA-I, Ulex euroaeus lectin I. ponent of interfacial microbial communities (Cooksey, 1992). EPS are responsible for the interaction of micro- organisms with each other as well as with interfaces (Neu & Marshall, 1990) and EPS act as a sorptive biofilm constituent (Flemming et al., 1996). Generally, EPS are understood to be extracellular polymers mainly composed of microbial polysaccharides. However, by definition, other extracellular polymeric substances may also be associated with biofilm systems, for example proteins, nucleic acids and polymeric lipophilic com- 0002-4267  2001 SGM 299