Ultrastructural changes on various Trypanosoma spp. after a 30-year storage period in liquid nitrogen

Four Trypanosoma species were examined for damage following prolonged storage in liquid nitrogen (-196 degrees C). The stabilates were successfully recovered after a cryopreservation period of approximately 30 years. The structure of specimens was studied by means of light microscopy and scanning (SEM) and transmission (TEM) electron microscopy. All of the species tested--T. evansi, T. equinum, T. brucei, and T. congolense--proved to be infective to mice. However, as compared with controls, the trypomastigote bloodstream forms, which had been frozen and later recovered, showed clear differences. Formerly deep-frozen organisms usually appeared to have shrunk as a result of solution effects, which occur during freezing and thawing. Ultrastructural changes such as separation of the cytoplasm from the pellicle, the occurrence of large vacuoles in the cytoplasm and karyoplasm, a loss of cytoplasmatic ribosomes, membrane injuries, enlargement of the flagellar pocket, and denaturation of chromatin became obvious. The extent of the ultrastructural alterations appeared to be much greater after a cryopreservation period of approximately 30 years than those previously reported after a 13-year storage period. These changes, however, did not result in a complete loss of infectivity to mice.