Fig. 2.

Frozen and freshly-prepared inocula display similar growth and virulence characteristics, but only frozen stocks diluted directly (not washed) gave consistent survival results. a In vitro growth curves of subcultures started from fresh overnight cultures and frozen vials are similar. TSB cultures were seeded with equal numbers of fresh or frozen bacteria, and aliquots were removed at the time points indicated. Serial dilutions were plated to measure CFUs. Results are combined from six independent experiments. Median values are plotted with error bars denoting interquartile ranges. b Multiple preparations of bacterial inocula were prepared on different days for intravenous injection into mice, at the doses shown on the x-axis. Freshly-prepared inocula (“Fresh Culture”, squares) were quantitated based on O.D. readings on the day of preparation. Alternatively, frozen stocks were used to prepare infectious inocula, either by washing 3x to remove trace amounts of glycerol (“Washed”, triangles), or by directly diluting the frozen stock into PBS for injection (“Direct”, circles). Frozen stock inocula were based on the known concentrations of bacteria per vial following freezing. Each symbol represents a single preparation, injected into N = 5 mice per group on a different day. Survival percentages are shown on the y-axis