Marine microorganisms are a rich potential source of novel metabolites with unique biotherapeutic applications. Conventional isolation methods include plating on liquid selective enrichment media or specially formulated solid media. However, only about 5% of these microorganisms can be isolated because many fail to form colonies on solid media. Viable slow growing or non-growing organisms, which comprise up to 80% of some collections, are also quickly overgrown by fast growers using liquid culture media. To date, isolation based on metabolic activity, especially in the case of bacteria smaller than 5 mu m, has not been possible. This Phase I proposal aims to develop a rapid, high throughput, automated assay to analyze and isolate bacteria from environmental samples based on their biochemical properties, and metabolic and proliferative activities. Using phytoplankton as a model system, a gel microdrop (GMD) assay which permits flow cytometric sorting of different bacteria based on autofluorescence will be developed. To isolate bacteria based on proliferative activity, GMD encapsulation and fluorescence activated cell sorting (FACS) will be combined. To isolate bacteria based on metabolic activity, GMD encapsulation, 5-cyano-2,3-ditolyl tetrazolium chloride(CTC) which detects cellular respiratory activity, and FACS will be combined.
The proposed assays will facilitate drug discovery by increasing the number of isolated strains of marine microorganisms available for drug screening.
