A fast and fully automated image analysis technique for the enumeration of fluorescence-labeled bacteria in oral sampleswas developed. This paper describes the system configuration, application strategy, automated operation, and initial validation experiments using fluorescent microspheres, bacterial cultures, in vitro grown biofilms and human dental plaque. Following a series of brief operator-controlled calibration steps, the technique automatically performs all necessary microscope operations (stage translation, focus, sampling and analysis) on slides with up to 48 wells for as many different samples. It quantifies bacteria from differential interference contrast images, images showing cells that had been labeled by immunofluorescence with monoclonal antibodies, or images with cells labeled by a fluorescent DNA stain. With all evaluated samples, close agreement between the automated system and the assessor's visual counts was observed. This novel automated image grabbing and analysis procedure is applicable to the enumeration of specific taxa in clinical samples by both immunofluorescence and fluorescent in situ hybridization.

Assessment of the role of biofilm microstructure in biofilm-specific activities requires non-destructive measurement techniques for parameterization of structural characteristics in parallel with relevant biochemical and physiological data. This paper briefly reviews some current methods for biofilm structural analysis, with emphasis on new developments in optical imaging and mathematical modeling methods. Fluorescence imaging studies of bacterial colonization events occurring on exposed model tooth surfaces indicated that bacterial adhesion to sessile organisms was of central importance to the early colonization process and that this occurred in a non-random manner. Structural studies of mature biofilms by confocal microscopy demonstrated the spatial distribution of individual species using fluorescent antibodies. Biofilms grown under different physiological conditions exhibited differences in structure, and methods were developed for parameterizing the spatial orientations of the bacteria. Diffusive processes within biofilm microstructures were studied using a random walk model in both 2-D and 3-D. Modeling of convective flow within biofilm microstructures was achieved by application of lattice Boltzmann methodology.