We describe a method for obtaining synchronously dividing cells of bacteria (Escherichia coli B and K-12 and Bacillus subtilis 168) and fission yeasts (Schizosaccharomycespombe) by the use of Percoll density gradients.Several synchronization techniques have been developed for the study of sequential changes during the division cycle of bacteria. The methods consist of induction of synchrony by specific environmental shifts (such as temperature [3] or nutrition [11]) or by separation of newly divided cells by filtration (4) or by centrifugation (6). One of the best methods for selection of newly divided cells is the membrane elution technique developed by Helmstetter (2). The method consists of selectively eluting newly divided cells from a randomly growing culture adsorbed to an inverted Millipore membrane filter. The advantage of this technique is that the physiological perturbations are minimal. The major disadvantages are the low yield of cells, which renders conventional biochemical methods inapplicable, and the fact that satisfactory synchrony is obtained only with strains B/r (2) and B (10) of Escherchia coli. Since many cell division mutants were isolated in E. coli K-12, which is not easily synchronized by the membrane elution technique (13), we looked for another method for obtaining newly divided cells which will be more general and also applicable for E. coli K-12. Our results with sucrose gradients (1, 6) were unsatisfactory due to an inhibitory effect on the cells and to poor synchrony, and Ludox gradients, which have also been used (9), are toxic and very viscous.We describe here separation of newly divided cells by the use of Percoll gradients. Percoll has been previously used in mammalian tissue cultures for separation of cells and cell organelles (7) and for enrichment of viable cells. Percoll is colloidal silica which is coated with polyvinylpyrrolidone to protect the cells from toxic effects. One of the major advantages of Percoll is that there is no free polymer in the solution, and therefore the viscosity is relatively low, even at high densities, and the osmotic properties are good. It was found by Pertoft et al. (8) that mammalian cells are less inhibited by Percoll than by other density gradients such as Ficoll, metdizamide, and sodium metrizoate.We studied the effect of Percoll on several microorganisms and its possible use for obtaining synchronously dividing cultures. The results presented in this communication indicate that synchronized cultures can be obtained in bacteria (E. coli B and K-12 and Bacillus subtilis 168) and in yeasts (Schizosaccharomyces pombe) by selecting newly divided cells from an exponentially growing culture.MATERIALS AND METHODS Strains and growth conditions. E. coli K-12 DG-76 (F-thyA47 leu-6 dra-3 str-153; kindly supplied by B. Bachmann) and E. coli B (thyA ilvA; obtained in our laboratory by mutagenesis of E. coli B) were grown in a minimal salts medium (2) containing 0.2% glucose and supplemented, when required, with 20 ,ug of thymine, 50 ,ug of the required ...
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