PROTECTIVE ACTION OF GLYCEROL IN THE FREEZING OF LEPTOSPIRAE

Scientists have attempted to keep leptospirae viable for long periods of time by freezing, by drying, by drying after freezing, and by storing sealed cultures at room temperature. Freezing methods have received the most attention, but viability after prolonged storage is uncertain due to the destruction of a high percentage of leptospirae (Weinman and McAllister, Am. J. Hyg., 45, 102-121, 1947). Glycerol protects cells of many kinds from the damage incurred in freezing and thawing, and rapid freezing is unnecessary with glycerol protected cells (Polge, Proc. Roy. Soc. (London), B, 147, 498-508, 1957). Slower rates of freezing thus may be used, avoiding the thermal shock suffered by many cells when frozen too rapidly. This study was undertaken to determine whether glycerol would reduce the proportion of leptospirae destroyed by the freezing and thawing process; long-term storage was not attempted. Leptospirae were grown at room temperature in Ringen's modification of Gardner's medium (Ringen and Gillespie, J. Bacteriol., 67, 252, 1954). To a sterile tube containing 0.3 ml of culture, 50 per cent glycerol (sterilized by autoclaving at 121 C for 15 min) in saline was added to the required concentration. Glycerol treated cultures (and controls) were allowed to equilibrate osmotically at room temperature, then frozen. Tubes of frozen cultures were thawed in tap water; motility examinations were carried out within 15 min. Subcultures were made in 3 ml of medium, and incubated at 33 C for 8 weeks or until dark-field examination revealed leptospiral growth. The proportion of motile organisms in thawed cultures was approximated by counting 100 organisms (200 if none of the first 100 was motile) and recording the number showing true motility. Glycerol was found to be toxic for many strains of leptospirae at room temperature, a 15 per cent concentration causing loss of motility in 30 to 60 per cent of the organisms within 30 hr. Five per cent glycerol was less toxic, but not without some inhibitory effect. When cultures were frozen rapidly (by immersion in a bath of Dry Ice and ethyl alcohol), 5 per cent glycerol had no significant protective effect, but there was a marked protective action on many strains when freezing was carried out at a slower rate (by placing the