Packer, Elliot L. (University of California, Davis), John L. Ingraham, and Stanley Scher. Factors affecting the rate of killing of Escherichia coli by repeated freezing and thawing. J. Bacteriol. 89:718-724. 1965.-Repeated freezing and thawing of cultures of Escherichia coli grown in a minimal medium and frozen in the same medium without carbon source resulted in a linear decrease in the log of the number of surviving cells as a function of the number of freeze-thaw cycles. The slope of this curve, which can be determined accurately, is an index of susceptibility of a culture to death by freezing and thawing. The effect of the physiological state of the culture on the killing rate was determined. Contrary to previous reports, the phase of growth, the state of aerobiosis, and the density of the culture had no effect on the degree of susceptibility to death by freezing and thawing. However, presence of spent growth medium (a filtrate of a stationary culture) in the freezing medium protected cells against death by freezing and thawing. Protection by spent growth medium is effective at high dilutions (1:10(5)), and is lost if spent growth medium is heated in the presence of alkali. It is suggested that the protection afforded by spent growth medium accounts for differences between our results and those reported in the literature.