Survival of monoxenically ( Crithidia sp.) and axenically grown Entamoeba histolytica cultures after storage in liquid nitrogen

Abstract

Monoxenically (in association with Crithidia sp.) and axenically grown E. histolytica cultures with the addition of dimethyl sulfoxide (5%, v/v concentration) were cooled slowly (0–5° C min ) to −45°C using a semiautomatic or automatic refrigerator and then transferred directly to liquid nitrogen container. The amoebae were stored in various types of plastic vessels and different organism concentrations. The viability of frozen material was tested by establishing cultures of the rapidly thawed amoebae suspension and also by staining them supravitally with eosin. In general all monoxenic cultures could be recovered successfully after storage in liquid nitrogen, independently of the different test conditions. E. histolytica amoebae grown axenically did not survive the freezing procedure in PVC tubing, but did so in all other plastic ampoules used. Staining supravitally with eosin does not indicate the potential of cryopreserved organisms to develop in culture. The longest period of storage that has been successfully tested so far is 5·1 years in respect of monoxenic cultures (strain F) and 1·9 years in respect of axenic cultures (strain HK 9).