The object of this proposal is to develop and optimize a novel experimental approach, based on scanning microfluorescence, to characterize cellular physiology as a function of both time and position within immobilized-cell biocatalysts. The approach will then be used to investigate plasmid stability within immobilized Escherichia coli cells. The method employs a biocatalyst configuration which allows visual observation of cells entrapped at various distances from the nutrient supply, so that well-nourished, as well as starving cells may be viewed. A recently developed scanning microfluorescence instrument is also used. This instrument has advanced image- analysis capabilities, and its excitation laser beam can be focused to a diameter less than one micron. These features allow fluorescence of individual bacterial cells to be measured. Plasmid stability will be monitored using fluorescent marker genes attached to target plasmids. This new analytical approach is both powerful and versatile. A wide variety of immobilized-cell phenomena can be studied by changing fluorescent stains. Results of the study will enhance the fundamental understanding of immobilized-cell physiology and allow refinements in mathematical models for industrially important immobilized-cell processes.
