Survival of bacteria upon repeated freezing and thawing.

The following bacteria were employed as test organisms: Lactobacillus fermenti strain 69L-3, Escherichia coli strain 69L-15, and Serratia marcescens strain ATCC 274. They were cultivated as outlined previously (Major et al., 1955). Broth cultures of the organisms were centrifuged, the spent broth pipetted off, the cells resuspended in a minimal amount of fresh broth, and then the suspension diluted with additional broth to the desired cell concentration. The broth used as the suspending medium had the following percentage composition: yeast extract (B.B.L., dehydrated), 1.0; K2HP04, 0.2; MgSO4. 7H20, 0.01; and "tween 80" (Atlas Powder Co.), 0.1. In some experiments with L. fermenti the suspending medium contained 1 per cent trypticase (B.B.L., dehydrated) and 2 per cent glucose as additional ingredients. In experiments where water was used as the suspending medium the cells from the centrifuged culture were resuspended in an equal volume of distilled water and again centrifuged, the supernatant pipetted off, the cells resuspended in a minimal amount of distilled water, and then the suspension diluted with additional water to the desired cell concentration. The suspension was dispensed in 5-ml aliquots into test tubes (15 mm by 150 mm) fitted loosely with aluminum caps. Freezing was accomplished by immersion of the tubes in an alcohol bath at -22 C. After the contents of the tubes had