Fluctuations in buoyant density during the cell cycle of Escherichia coli K12: significance for the preparation of synchronous cultures by age selection

Abstract

The buoyant densities of Escherichia coli K12 were investigated by isopycnic centrifugation in gradients of colloidal silica (Ludox) and polyvinylpyrrolidone. Bacteria from an exponential culture in a defined medium supplemented with hydrolysed casein banded at densities between 1-060 and 1-115 g ml-1; the mean density was 1-081 g ml-1. At the higher densities, two populations of cells were present: smaller cells were approximately twice as numerous as, and half the modal volume of, the population of larger cells. A homogeneous population of cells of intermediate volume equilibrated in the least dense region of the density band. Synchronous cultures were established by inoculating cells selected from the most or least dense regions of the band into spent growth medium. The results are consistent with a fluctuation between maximal density at cell birth and division, and minimal density near the middle of the cell cycle. In synchronous cultures prepared by continuous-flow age selection, the first division occurred after a period that was significantly shorter than the length of subsequent cell cycles. Cells selected by this procedure were of similar mean density to those in the exponential culture but were more homogeneous with respect to size. The possibility that the smallest (and densest) cells in an exponential culture are retained in the rotor, and are thus excluded from the synchronous culture, is discussed.