A simple method for the cryopreservation of free-living amoebae belonging to the genera Naegleria and Acanthamoeba

Abstract

The cryopreservation of free-living amoebae of the genera Naegleria and Acanthamoeba from axenic or monoxenic culture with Escherichia coli was achieved using a two-step cooling process in laboratory freezers and storage in liquid nitrogen. Trophozoites were suspended in axenic culture medium and dimethylsulphoxide (DMSO) added to a final concentration of 5%. Ampoules were placed directly at -20°C for 60 min, followed by a further 60 min at -70°C and then stored in liquid nitrogen. On rapid thawing of the ampoules at 37°C, recovery rates with standard errors from three separate experiments performed in triplicate were: N. fowleri 8% ± 3%, N. lovaniensis 12% ± 3%, N. gruberi 25% ± 6% and A. polyphaga 34% ± 5.5% from axenic cultures, and21% ± 7%, 31% ± 10%, 44% ± 11%, 49% ± 9%, respectively from monoxenic cultures. Although not optimal, the method is proposed as a simple and reproducible means of cryopreserving Naegleria and Acanthamoeba strains.