EFFECTS OF FREEZING, FREEZE-DRYING, AND STORAGE IN THE FREEZE-DRIED AND FROZEN STATE ON VIABILITY OF ESCHERICHIA COLI CELLS

Abstract

Escherichia coli cells in concentrations of 2 × 10⁹cells per ml were resistant to freezing at −78 °C and to low temperatures encountered during freeze-drying when suspended in distilled water, 7.5% glucose, 4.5% glycerol, skim milk, or serum but not when in saline. Survival immediately after freeze-drying varied with the drying interval and composition of the suspending medium and was highest (70–100%) in media containing 7.5% added glucose. Survival during storage in freeze-dried suspensions in serum containing 7.5% added glucose was inversely related to storage time and temperature; after 1 year at 32 °C, 21 °C, and 4 °C, survival was 0, 25%, and 80% respectively. When suspensions in water, glucose, glycerol, and serum were frozen directly and stored in the frozen state at −18 °C, −23 °C, and −40 °C, the cells showed a diminishing death rate and eventually attained a stable state. The final survival level varied with the composition of the suspending medium and the storage temperature. Glycerol provided most consistent protection (minimum survival 65%). Survival in water and glucose was inversely proportional to the storage temperature. Viability in water was higher than in glucose and was equal to glycerol in storage at −40 °C (80% after 2 years).