Volumetric measurements of bacterial cells and extracellular polymeric substance glycoconjugates in biofilms

Abstract

In this study an enrichment culture developed from activated sludge was used to investigate the architecture of fully hydrated multispecies biofilms. The assessment of biofilm structure and volume was carried out using confocal laser scanning microscopy (CLSM). Bacterial cell distribution was determined with the nucleic acid-specific stain SYTO 60, whereas glycoconjugates of extracellular polymeric substances (EPS) were stained with the Alexa-488-labeled lectin of Aleuria aurantia. Digital image analysis was employed for visualization and quantification of three-dimensional CLSM data sets. The specific volumes of the polymeric and cellular biofilm constituents were quantified. In addition, gravimetric measurements were done to determine dry mass and thickness of the biofilms. The data recorded by the CLSM technique and the gravimetric data were then compared. It was shown that the biofilm thicknesses determined with both methods agree well for slow-growing heterotrophic and chemoautotrophic biofilms. In addition, for slow-growing biofilms, the volumes and masses calculated from CLSM and the biomass calculated from gravimetric measurements were also comparable. For fast-growing heterotrophic biofilms cultivated with high glucose concentrations the data sets fit to a lesser degree, but still showed the same common trend. Compared with traditional gravimetric measurements, CLSM allowed differential recording of multiple biofilm parameters with subsequent three-dimensional visualization and quantification. The quantitative three-dimensional results recorded by CLSM are an important basis for understanding, controlling, exploiting, and modeling of biofilms.

Confocal Laser Microscopic Analysis of Biofilm on Newer Feldspar Ceramic

“…[22][23] CLSM presents non-invasive and non-destructive characteristics and is actually considered the tool of choice for study of in situ biofilm. [27][28] Fluorescent dyes, such as the LIVE\DEAD kit (Bac light), have been used together with CLSM to describe the architecture of biofilm, 29 quantify the viability of microorganisms 19,21,25 and evaluate the mean thickness 23,26,[29][30] and biovolume of biofilm. 26,[30][31] Quantitative data calculated using the COMSTAT program represents a way to obtain numeric information from biofilm, allowing for comparisons.…”

Evaluation of Different Methods for Extracting Extracellular DNA from the Biofilm Matrix

“…EPSs are composed mainly of high-molecular-weight compounds, including polysaccharides, proteins, and amphiphilic polymers (19,20), that are secreted by microorganisms into their environment (32). The majority of proteins in the EPSs are bridged by divalent ions, including Ca 2ϩ and Mg 2ϩ , and a small fraction of carbohydrates and nucleic acids are linked to these divalent ions.…”

Structure and Composition of Aggregates in Two Large European Rivers, Based on Confocal Laser Scanning Microscopy and Image and Statistical Analyses

“…Quantification of CLSM data sets of the whole aggregate (optically sectioned material) and cryo-sections was done with the freely available software ImageJ (http://rsb.info.nih.gov/ij/), developed in Java (45). To analyze cryo-sections with the software ImageJ, special plug-ins were developed.…”