Activity of Individual Aquatic Bacteria by Flow Cytometry; Instrumentation and Protocols

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This award will support the development of flow cytometry instrumentation and associated protocols that can analyze aquatic bacteria with emphasis on their growth and viability status. Flow cytometry has become an established research tool for study of blood cells and of a variety of cells and organisms that can be grown in culture. However, its use to analyze natural populations of bacteria such as those found in aquatic systems is complicated by the small cell size typical of such bacteria. Few commercial instruments are dedicated to the detection of these small targets. The main effort of this project is to design additional modifications to a commercial flow cytometer to enhance its ability to determine the growth and viability status of aquatic bacteria. Output signals will be organized for manipulation to reflect the rate of growth in mixed populations as judged by DNA content. Two additional probes for activity will be tested in the system, one for RNA content and the other for membrane potential, both important indicators of bacterial growth and viability. The equipment will be particularly important in study of the microbial generation of greenhouse gasses at high latitudes where both ice cover and bacterial activity is highly temperature sensitive.