David H. Hollander and E. Ellen Nell

Preservation of bacteria at the temperature of dry ice (-78.5 C) is a particularly valuable method for the maintenance of such organisms as Treponema pallidum which otherwise require continuous animal-to-animal passage. Turner (1938), Turner and Fleming (1939), and Turner and Brayton (1939) demonstrated that at the temperature of dry ice T. pallidum and relapsing fever spirochetes retain their virulence for long periods. However, in practice it has become apparent that the freezing method of preservation, especially in the case of spirochetes, is not completely reliable, and that much activity may be lost in the process, so that on occasion it has been difficult or impossible to reactivate frozen strains of trepoinemes (Turner, 1953). The influence of low temperatures on living cells has been reviewed by Belehra'dek (1935) and by Luyet and Gehenio (1940). Prudden in 1887 distinguished two types of damage which may result from the freezing of bacteria. Although subsequent investigators have not always separated these two effects, all the available evidence tends to support this distinction. This is clearly demonstrated in the studies of Weiser and Osterud (1945) who state in summary: "Death by freezing involves a rapidly acting or "immediate" death, caused by the freezing and thawing per se, and a "storage death" which is a direct function of time and temperature." The present study is primarily concerned with the damage which occurs during the freezing and thawing. Prudden (1887) was the first of many observers to report that repeated freezing and thawing was more injurious than a single freezing. Weiser and Lief (1940) in unpublished experiments, determined that bacteria which are repeatedly frozen and thawed are damaged at a rate which is the same for each cycle of freezing and thawing so that the loss is exponential. Different species of bacteria vary in their susceptib)ility to damage by freezing and thawing. Haines 1 This study was supported by a grant from the National Institutes of Health, U. S. Public Health Service. (1938) found that a single freezing and thawing killed 89 per cent of Saccharomyces cerevisiae, but only 5 per cent of Staphylococcus aureus. The susceptibility of several other bacteria was intermediate. Keith (1913) showed that if the einvironmental coniditions are changed, the resistance of the organism may be greatly altered. When certain substances, such as sugar, milk, or glycerol, were added to the suspending medium, the bacteria were partially protected from the damage of freezing at -20 C. Recently, analogous observations have been made of the effect of glycerol when other types of cells are frozen. Polge, Smith and Parkes (1949) and Smith and Polge (1950) have reported that in the presence of dilute glycerol solutions spermatozoa of various species can regain their motility and their physiological activity (Polge, 1951) after being frozen at the temperature of dry ice; Smith (1950) has reported that red blood cells in glycerol are not hemolyzed by freezing; and Hartmann and Conley (1952) have found that blood platelets in the presence of glycerol are protected from the destructive action of freezing. The amount of damage from freezing and thawing may be influenced by the rate and by the temperature of the freezing and of the thawing. Bacteria ordinarily show the least amount of damage when rapidly frozen and rapidly thawed (Turner and Brayton, 1939; Weiser and Osterud, 1945). According to Luyet and Gehenio (1940), who believed that the damage is caused by crystal formation, quick freezing and thawing is essential in order to pass rapidly over the temperature range in which crystallization occurs. Luyet (1951) found 28 per cent of a red blood cell suspension was hemolyzed after quick freezing and thawing, whereas 96 per cent was hemolyzed during slow freezing. More recently Luyet and Keane (1952) have reported that the speed of freezing was not important in preserving chicken embryo cells with ethylene glycol, and Smith and Polge (1950) have found that very 164