Harvey L. Bank

Visualization of freezing damage. II. Structural alterations during warming

Abstract There is a growing amount of indirect evidence which suggests that the loss in viability of rapidly cooled cells is due to recrystallization of intracellular ice. This possibility was tested by an evaluation of the formation of morphological artifacts in rapidly cooled cells to determine whether this process can account for the loss in viability. Samples of the common yeast Saccharomyces cerevisiae were frozen at 1.8 or 1500 °C/min, and the structure of the frozen cells was examined by the use of freeze-fracturing techniques. Other cells cooled at the same rate were warmed to temperatures ranging from −20 ° to −50 °C and then rapidly cooled to −196 °C, a procedure that should cause small ice crystals to coalesce by the process of migratory recrystallization. Cells cooled at 1500 °C/min and then warmed to temperatures above −40 °C formed large intracellular ice crystals within 30 min, and appreciable recrystallization occurred at temperatures as low as −45 °C. Cells cooled at 1.8 °C/min and warmed to temperatures as high as −20 °C underwent little structural alteration. These results demonstrate that intracellular ice can cause morphological artifacts. The correlation between the temperature at which rapid recrystallization begins and the temperature at which the cells are inactivated indicates that recrystallization is responsible for the death of rapidly cooled cells.

Confidence Intervals for the Relative Frequency of Responding Cells in Limiting Dilution Assays

SUMMARY Confidence intervals for the relative frequency of responding cells in limiting dilution assays (LDAs) have not been examined closely. Generally, confidence intervals are calculated by using the normal approximation, sometimes preceded by a log transform. We evaluate and compare with analytical approaches: the normal approximation, the log transform, and two binomial methods-the quadratic approximation and a modification of the Clopper-Pearson exact method. We evaluate these confidence interval methods for use with the maximum likelihood and jackknife estimators of the relative frequency. Our results show that confidence interval construction with maximum likelihood point estimates is more accurate than construction with jackknife estimates. When using maximum likelihood estimates, the normal approximation produces acceptable two-sided confidence intervals, with the smallest length, at a = .05. At a = .01, the normal approximation is anticonservative. In all cases, the normal approximation is unable to produce adequate one-sided confidence intervals. The log transform and both binomial confidence interval methods are shown to be generally superior to