Titration experiments made with relapsing fever spirochetes before and after freezing showed the following:

1. With each freezing and thawing there is a slight but regular decrease in virulence, which decrease bears no relation to the duration of storage at – 78°C. Ordinarily infectivity is destroyed by more than 4 refreezings.

2. There was not always close correlation between motility and infectivity.

3. Cooling spirochetes from 0°C. to –78°C. over a 2 to 6 hour period damages them only slightly more than does rapid cooling, but warming from – 78°C. to 0°C. over a 2 to 6 hour period kills most of the organisms. Rapid thawing, as in a water bath, damages the spirochetes less than thawing more slowly, as at room temperature.

4. At storage temperatures of –12°C. and –20°C. there is a gradual decrease in virulence over a period of days or weeks, and by the 6th week the infectivity of the material is markedly reduced.

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