Journal of General Microbiology (1g77), gg, 223-227 Printed in Great Britain 223 Continuous-flow Cell Cycle Fractionation of Eukaryotic Micro-organisms By D, LLOYD, LYNDA JOHN, MERRYL HAMILL, CAROL PHILLIPS, J. KADER AND S. W. E D W A R D S Department of Microbiology, University College, Newport Road, Card18 CF2 I TA (Received 12 August 1976;revised 25 September 1976) INTRODUCTION Previous methods of cell cycle fractionation have been based on sedimentation of or- ganisms through density gradients in tubes or zonal centrifuge rotors (Warmsley & Paster- nak, 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 separa- ted 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 tem- peratures 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. METHODS Organisms. The methods used for the maintenance and growth of organisms have been described for Schizosaccharomyces pombe 972h- (Poole, Lloyd & Kemp, 1973)~ Candida utilis NCYCI93 (Light & Garland, 1971), Saccharomyces cerevisiae D22a (Deutsch et al., 1974),Acanthamoeba castellanii (Griffiths & Chagla, 1972)~ Crithidia fasciculata (Edwards & Lloyd, 1973) and Tetrahymena pyriformis strain ST (Lloyd et al., 1971). Carbon sources in the yeast cultures were glucose, acetate and glycerol for Schiz. pombe, C. utilis and S. cerevisiae respectively. Coulter counter analysis. The frequency distribution of particle volumes was determined using a model & Coulter counter (Coulter Electronics, Dunstable, Bedfordshire) together with a Coulter Channelyzer and XY recorder 11. Samples were diluted in Isoton (Coulter Electronics), except, for A . castellanii and T. pyriformis, filtered growth medium was used. Particle counts were made immediately using a 70 ,um orifice (except, for T. pyriformis, the orifice diameter was I 40 pm). Frequency distributions of particle volumes were accumulated in the Channelyzer over arbitrary periods of sample analysis until 2048 particles had been counted in the peak channel; they were then plotted out on the recorder. Total particle counts were made on 0.5 ml samples; dilutions were such that the number of particles did not exceed 4 x 104 in this sample volume. Settings were I x A = 1.0 (for Schiz. pombe, S. cerevisiae and Cr. fasciculata), I x A = 0.5 (for C. utilis), I x A = 16 (for A. castellanii) and I x A = 16 (for T. pyriformis); edit switch, on; upper threshold, off; lower threshold, 10; gain switch, 5; matching resistance, 20 kQ. 15 MIC 99