The viability and virulence of an 8-day-old culture of a hamster-virulent strain of serotype canicola in Stuart's medium were successfully preserved by the use of liquid nitrogen for freezing and storage. Glycerol was added to the culture to give a final 10% concentration. The culture was rapidly cooled at a rate of 65 C degrees per min from 0 C to -130 C and then stored in the vapor portion of the liquid-nitrogen refrigerator. Prior to freezing, the viability count and LD0 titer for hamsters were 1.2 X 10 per ml and 10 respectively. One day after freezing, the viability and virulence values were reduced 10- to 100-fold but remained essentially unchanged thereafter over a 5-year observation period. The serological properties of the strain did not appear to be altered after prolonged storage. All except 9 of 103 additional cultures of diverse serotypes grown in either Stuart's medium or in a Tween-80 albumin medium were successfully preserved by liquid-nitrogen refrigeration over a 7- to 38-month observation period with glycerol as a cryoprotective agent and a controlled rate of freezing of 35 or 60 C drop per min. Failures to recover leptospiras could be attributed to either the presence of contaminating microorganisms or the sparse concentration of organisms in the original preparation. The viability of thawed cultures persisted for 7 days when held at room temperature. The feasibility of storage of stock leptospiral cultures by use of liquid-nitrogen refrigeration was demonstrated.


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