Abstract
Ring, trophozoite, and schizont stages of Plasmodium knowlesi were cooled in dimethyl sulfoxide either by direct immersion in liquid nitrogen or by a two-step method in which the cells were held at temperatures slightly below 0°C for different lengths of time before they were cooled to -196°C. After the direct plunge treatment, thawed trophozoites and schizonts were found to be extensively damaged. Their survival was markedly increased by holding them at -31°C for 30 min before plunging them into liquid nitrogen. Freeze-substitution showed that cells cooled by the two-step procedure were grossly shrunken and had relatively few intracellular ice cavities. Large amounts of ice formed in trophozoites and schizonts preserved by direct immersion in liquid nitrogen. The two-step protocols investigated did not improve the survival of ring-stage parasites, 25-50% of which survived rapid cooling to -196°C. Infected cell agglutination tests were carried out with frozen and thawed schizonts. Variant specificity was demonstrated with cells that had been plunged directly to -196°C, but cells cooled by the two-step method tended to agglutinate spontaneously.
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