Wilson R J, Farrant J, Walter C A
Bull World Health Organ. 1977;55(2-3):309-15.
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.
诺氏疟原虫的环状体、滋养体和裂殖体阶段在二甲基亚砜中冷却，冷却方式要么是直接浸入液氮，要么是两步法，即细胞在冷却至-196℃之前，先在略低于0℃的温度下保持不同时长。直接投入处理后，发现解冻后的滋养体和裂殖体受到广泛损伤。在将它们投入液氮之前，先在-31℃保持30分钟，其存活率显著提高。冷冻置换显示，通过两步法冷却的细胞严重皱缩，细胞内冰腔相对较少。直接浸入液氮保存的滋养体和裂殖体中形成了大量冰块。所研究的两步方案并未提高环状体阶段寄生虫的存活率，其中25%-50%的寄生虫在快速冷却至-196℃后存活。对冷冻和解冻后的裂殖体进行了感染细胞凝集试验。直接投入-196℃的细胞表现出变异特异性，但通过两步法冷却的细胞往往会自发凝集。
