Continuous-flow Cell Cycle Fractionation of Eukaryotic Micro-organisms

Previous methods of cell cycle fractionation have been based on sedimentation of organisms through density gradients in tubes or zonal centrifuge rotors (Warmsley & Pasternak, 1970). Both rate-sedimentation and equilibrium density methods have been used, for example for Saccharomyces cerevisiae and for Schizosaccharomyces pombe (Sebastian, Carter & Halvorson, 1971; Poole & Lloyd, 1973; Poole, Lloyd & Chance, 1974; Edwards & Lloyd, 1977). However, these methods suffer from a major drawback. Organisms must be harvested from their growth media, resuspended, loaded on to the gradient, then separated and recovered. Such procedures may take up to I h during which time the organisms are exposed to conditions of nutrient and oxygen deprivation, suboptimal growth temperatures and, in some cases, unfavourable osmotic environments. Continuous-flow cell cycle fractionation in a Sharples Supercentrifuge is completed in less than 15 min (for a 10 1 culture), and thus obviates the unfavourable conditions inherent in gradient methods ; size selection is just as efficient.