Preservation of intraerythrocytic forms of malarial parasites by one-step and two-step cooling procedures

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 degrees C for different lengths of time before they were cooled to -196 degrees 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 degrees 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 degrees 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 degrees C, but cells cooled by the two-step method tended to agglutinate spontaneously.