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.

Aims: To validate an in vitro model for the analysis of physiological and ecological responses to sugar challenge in bacterial populations, and subsequent changes in enamel mineralization.Methods and Results: A seven-organism bacterial consortium was grown in a biofilm mode on enamel and hydroxyapatite (HA) surfaces in a continuous culture system and exposed to repeated sucrose challenges. This produced 'pH-cycling' conditions within the system. Populations on HA surfaces were enumerated. Changes in relative proportions of the different populations, and in the total viable count, were observed, between different treatments. Microradiography of the enamel sections showed increasing demineralization with increasing sucrose concentration. The lesions formed were similar to 'white-spot' lesions found in vivo. Differences in the quality of biofilms formed were also observed using Confocal Laser Scanning Microscopy.Conclusions: An in vitro model has been validated for the analysis of both physiological and ecological responses to sucrose challenges in bacterial populations, and subsequent changes in enamel mineralization.Significance and Impact of the Study: This model should facilitate the study of changes in bacterial populations in response to application of putative anticaries agents and concomitant changes in enamel mineralization.

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.