Cryopreservation of pathogenic free-living amebae

A variety of conditions of cryopreservation were evaluated in order to define a single procedure for freezing the amebae of pathogenic Naegleria and Acanthamoeba. The average best conditions for freezing the three species studied were: 1 x 10(6) exponentially growing amebae/ml of freezing medium consisting of 12% dimethylsulfoxide, 20% heat-inactivated bovine calf serum, 4% glucose, in Mix ameba medium; 30 min equilibration at 23 degrees C (room temperature), followed by 60 min at -20 degrees C, with storage at -70 degrees C. Under these conditions viability after 1 month of freezing was 53% for Acanthamoeba castellanii, 64% for Naegleria fowleri, and 66% for Naegleria australiensis. After 12 months of freezing, viability was 39% for A. castellanii, 47% for N. fowleri, and 53% for N. australiensis.