The effects of freeze–thaw events on the inactivation of Cryptosporidium parvum oocysts in soil were examined. Oocysts were inoculated into distilled water in microcentrifuge tubes or into chambers containing soil the water content of which was maintained at 3%, 43%, or 78% of the container capacity. The chambers and tubes were then embedded in 3 soil samples from different aspects of a hillside landscape (Experiments 1 and 2) and in 3 distinct soil types (Experiment 3) and frozen at −10 C. Containers were thawed every 3 days for a period of 24 hr in 1–9 freeze–thaw cycles over 27 days (Experiments 1 and 2) and 2–5 freeze–thaw cycles over 15 days (Experiment 3). Oocyst viability was measured using the fluorescent dyes 4′6-diaminidino-2-phenylindole and propidium iodide. Inactivation rates were greater in soils than in water and greater in dry soil than in moist and wet soils. Soil type showed no effect on inactivation. Oocysts subjected to freeze–thaw cycles had inactivation rates not significantly different from those of oocysts subjected to −10 C under static conditions. The results indicated that 99% of oocysts exposed to soils that are frozen at −10 C will become inactivated within 50 days whether or not freeze–thaw cycles occur.
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