Studies on the factors which determine the survival of Entamoeba histolytica in cryopreservation

Abstract

Maintenance of E. histolytica in vitro requires frequent subcultures which are time-consuming and may lead to changes in the biological characteristics of stocks. Cryopreservation avoids these problems, since amoebae can be stored at low temperature soon after isolation with their biological characteristics relatively unaltered. To investigate those factors which may influence survival after cryopreservation, a standard, reproducible method of cultivation was developed using Robinson's medium, which was found to be the best culture medium for growth of amoebae from low inocula. It was possible, using this medium, to prepare genetically homogenous populations of E. histolytica by cloning. A sensitive viability assay, based on titration of infectivity to cultures, was developed to measure the success of cryopreservation. The effect of various cryoprotectants on the viability of E. histolytica stocks before and after freezing was studied. It was found that polyvinylpyrrolidone (PVP), dimethyl-sulfoxide (DMSO) and glycerol, though cryoprotective, are toxic to amoebae. Although sorbitol, methanol and ethanol at the concentrations used were not in themselves toxic, they conferred no cryoprotection. Up to 12.5% of the amoebic population survived when the amoebae were equilibrated for 15 minutes at 37 C with 7.5% (v/v) DMSO and then cooled at 1° C/min to -196° C followed by thawing at 37° C and inoculation into medium in less than two minutes. However, there was a partial loss of viability over a storage period of six months in liquid nitrogen. Conditions which influenced survival were: the protectant, its concentration, the temperature and period of equilibration, freezing rate, suspending medium, culture medium and thawing temperature. A period of 'structural reconstitution' after thawing led to a decrease in viability. It was possible to show, by characterization of isoenzyme type, observation of concanavalin A-induced agglutination, reaction with antibody in the indirect fluorescent antibody test and studies on leucocytotoxicity, that the biological characteristics of E. histolytica had been retained during cryopreservation. The generation-time of some amoebal stocks was found to change after cryopreservation. It was not possible to demonstrate selection of a population with reduced sensitivity to freezing damage.