Fluctuations in Buoyant Density during the Cell Cycle of Escherichia coli K12: Signiacance for the Preparation of Synchronous Cultures by Age Selection

Abstract

K I~ 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 1.1 15 g ml-l; the mean density was 1-081 g ml-l. 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.

The Influence of Growth Substrate and Capacity for Oxidative Phosphorylation on Respiratory Oscillations in Synchronous Cultures of Escherichia coli K12

“…2). This is probably a result of the fluctuations in the buoyant density of cells that occur during the cell cycle of this organism (Poole, 1977). Furthermore, when the technique is applied to cultures grown with glycerol in the absence of Casamino acids, cells collected in the rotor effluent divided asynchronously with a doubling time considerably greater than that of the unfractionated culture (unpublished observations).…”

“…Strain A 1 0 0 2 (KC2 Y me1 ilv-lad-metE-) is the parental strain from which the ATPase-deficient mutant, A I 03c, was derived (Schairer & Haddock, 1972). Both were grown at 37 "C in mineral salts medium (Poole, 1977) containing either 0.5 %'(w/v) glucose or 0.5 % (w/v) glycerol as carbon source, in the absence or presence of 0.1 % (wlv) vitamin-free Casamino acids (Difco). Strain ~1 0 3~…”

“…Preparation of synchronous cultures by continuous-flow centrifugation. This was as described previously (Poole, 1977). The total centrifugal force (g-min) was calculated for a radius of 2 .…”

“…Zero time for the synchronous culture was taken as the time when half the effluent volume subsequently used for analysis had been collected. and Channelyzer C I 000 (Coul ter Electronics, Harpenden, Hertfordshire) as described by Poole (1977).…”

Which Cells are Selected from Exponential Cultures by Continuous-flow Centrifugation? The Selection of Small Cells from Cultures of Escherichia coli and Schizosaccharomyces pombe that Exhibit Minimal Density Fluctuations during their Cell Cycles

“…x 5 min, 20 "C) and washed once under the same conditions using 50 m-KH2P04/K2HP04 buffer (pH 7.5). The separation on gradients of colloidal silica and polyvinylpyrrolidone was similar to that described previously (Poole, 1977~ About 1 x lo9 cells from an exponentially growing culture of S. pombe were harvested by centrifugation (7000 g at rave x 5 min, 20 "C), washed once with water and finally resuspended in 1 ml water. Clumped cells were disaggregated by 10 s sonication at 20 "C and a peak-to-peak amplitude of 5 pm using the titanium vibrator micro-probe (end diam.…”