Ultrastructural appearance of freeze-substituted schistosomula of Schistosoma mansoni frozen by a two-step cooling schedule

Schistosomula of the parasitic helminth Schistosoma mansoni were frozen by two-step cooling, then examined for ultrastructural changes by the freeze-substitution method. Samples were cooled at 1 °C min^?1 to ?20, ?25, ?28, and ?38 °C before being cooled at 10,000 °C min^?1 to ?196 °C. The results showed that progressive partial dehydration of the parasites occurred during slow cooling. Numerous cavities, indicating the presence of intracellular ice crystals, were observed in organisms which did not become shrunken. The sizes of the ice cavities varied between organisms and also within the same cell type in individual organisms indicating that intracellular ice nucleation may occur at any time during the slow cooling step. Some organisms cooled first to ?28 or ?38 °C contained no evidence of ice crystal formation. When correlated with previously reported infectivity studies, the results indicated that successful cryopreservation of schistosomula requires slow cooling to approximately ?30 °C to induce cryodehydration, followed by rapid cooling to ?196 °C to prevent ice nucleation or crystal growth.