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The microorganisms Escherichia coli DH5 alpha and Bacillus thuringiensis HD-1 show an increased tolerance to freeze-drying when dried in the presence of the disaccharides trehalose and sucrose. When the bacteria were dried with 100 mM trehalose, 70% of the E. coli and 57% of the B. thuringiensis organisms survived, compared with 56 and 44%, respectively, when they were dried with sucrose. Only 8% of the E. coli and 14% of the B. thuringiensis organisms survived drying without the sugars. Fourier transform infrared spectroscopy was used to investigate the role of membrane phase transitions in the survival of the organisms during drying and rehydration. Both E. coli and B. thuringiensis showed an increase of 30 to 40 degrees C in the temperature of their phospholipid phase transition when dried without the sugars, while phase transition temperatures of those dried with the sugars remained near those of the hydrated cells. A Fourier transform infrared spectroscopy microscope made it possible to investigate the effects of drying on the protein structure in the intact cells. The amide II peak shifts from 1,543 cm-1 in the hydrated cells to about 1,533 cm-1 in the cells dried without sugar. There is no shift in the amide II peak when the cells are dried with trehalose or sucrose. We attribute the increased survival to the sugars' ability to lower the membrane phase transition temperature and to protect protein structure in the dry state.(ABSTRACT TRUNCATED AT 250 WORDS)

Freeze drying and exposure to oxygen of E. coli causes damage to the bacterial cytoplasmic membrane. Freeze-drying itself produces an injury to the transport system for ONPG and potassium, so as to make the membrane leaky to these compounds. This damage is partially repaired upon incubation of the reconstituted bacteria in nutrient medium. When, however, freeze dried bacteria are not held in vacuo before reconstitution, but exposed to oxygen, this damage to the bacterial membrane becomes more extensive and irreversible.

The damage occurring in freeze-dried bacteria exposed to oxygen is mainly in the bacterial membrane and involves the DNA-initiation complex. This injury occurs in two stages: The primary damage is due to the freeze-drying itself, and is repaired upon reconstitution of bacteria and their subsequent incubation in nutrient broth. The repair process requires protein synthesis. In the next step, the exposure of freezedried bacteria to oxygen, the injury becomes irreversible and the bacteria “die.” The lethal effects of oxygen can be counteracted by either engaging the initiation sites on the membrane (e.g., by colicin E 1 ), or by arresting the activity of the initiation complex. Such arrest occurs in ts-mutants at nonpermissive temperature, or when appropriate mutants are starved of amino acids or thymine. After freeze drying and storage in vacuo , the reconstituted bacteria grow in filaments and synthetize DNA multifocally; they do not form septa and do not divide. After exposure to oxygen of the freeze-dried bacteria the ability to initiate DNA synthesis is lost. It is assumed therefore that oxygen specifically acts on the initiation complex in the bacterial membrane of the freeze-dried bacteria. When an external DNA synthesis control is provided, e.g., by infection with phage the productivity of the freeze-dried bacteria exposed to oxygen is the same as of the lyophilized controls held in vacuo .

Summary: Escherichia coli strain BT-, freeze dried from distilled water, loses viability by exposure to air in the dry state, and decay curves of such bacteria were determined. The amounts of tritiated thymidine incorporated in the DNA of such ‘dead’, rehydrated bacteria are compatible with an assumption that the synthesis of DNA proceeds till the completion of its replication cycle, but not beyond. Induction of β-galactosidase served as an indicator of RNA and protein synthesis in the dead bacteria. Such induction occurred after a short lag after which it proceeded at a rate similar to that observed in lyophilized, untreated bacteria. The loss of viability of these bacteria cannot be accounted for by defects in the production of DNA or RNA and protein synthesis; it is most probably due to damage to the DNA initiation point.