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Generation time statistics of Escherichia coli using synchronous culture techniques

The distribution of generation times and the correlation coefficient between the generation times of mother and daughter cells are determined for a B strain of Escherichia coli under various conditions of growth. Synchronously dividing cultures of E. coli are produced by the Mitchison-Vincent density gradient centrifugation technique. Very precise data are yielded by cell number counts at frequent intervals with a Coulter electronic particle counter. Culture growth is followed for three and sometimes four generations. Doubling times between 30 and 80 minutes are obtained at 37°C with mineral salts medium supplemented with suitable carbon sources, namely, glucose + methionine + histidine, glucose, sucrose, glycerol, or succinate. On glucose, doubling times between 40 and 90 minutes are obtained by varying the temperature between 37° and 26°C. A mathematical description of synchronous growth is taken from the literature. The rate of increase of cell number concentration is expressed as the sum of a series of functions with properties related to the generation time distribution of the cells and the correlations between the generation times of related cells. Smoothing of the cell number data by digital filtering and subsequent numerical differentiation yields a series of peaks having little apparent skewness for all growth rates. This is confirmed by fitting a sum of Gaussian functions to the peaks by a nonlinear least squares procedure. The parameters of the generation time distribution and the correlation coefficients between the generation times of parent and progeny cells are then extracted directly from the means and variances of the Gaussian functions. The uncertainty associated with each data point is estimated to be within 1½% and the errors in the extracted parameters are determined by repeated simulation of the data analysis procedure using computer generated noisy data. Under all growth conditions the generation time distribution is of Gaussian form with a coefficient of variation of 0.22 ± 0.02. The mother-daughter generation time correlation coefficient was significantly negative at doubling times between 40 and 64 minutes; a constant value of -0.47 ± 0.06 was consistent with the observations. At doubling times of 30 and 80-90 minutes this coefficient tended to be closer to zero but with a higher uncertainty. In succinate medium at 37°C (doubling time 80 minutes) synchrony was noticeably weaker after the first generation compared with the results at higher growth rates. The growth data for this medium indicate a correlation coefficient very close to zero. The implications of the results are discussed in terms of the predictions of a number of models for the control of cell division that have appeared in the literature.
Type of thesis
The University of Waikato
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