Self-Pressurised Rapid Freezing (SPRF): an easy-to-use and low-cost alternative cryo-fixation method for nematodes

Abstract

Self-Pressurised Rapid Freezing (SPRF), an easy-to-use and low-cost alternative cryo-fixation method, was evaluated based on a comparative analysis of the ultrastructure of spermatozoa of the nematodes Acrobeles complexus and Caenorhabditis elegans. Sealed copper tubes, packed with active nematodes in water, were plunged into nitrogen slush, a semi-solid form of nitrogen. The water inside the capillary copper tube expands upon cooling due to the formation of hexagonal ice, thereby generating high pressure intrinsically for cryo-fixation of the sample. For sperm cells cryo-fixed by SPRF, the preservation of the ultrastructure was comparable to that achieved with high pressure freezing. This was evidenced by the clear details in mitochondria, membranous organelles and cytoskeleton in the pseudopod. It was demonstrated that SPRF fixation did not destroy antigenicity, based on the results of the immunolocalisation of the major sperm protein in both species. In conclusion, SPRF is a low-cost alternative cryo-fixation method for nematodes.