Breaking the ice − simplified freeze-fracture of parasitic protists: a cost-effective approach without highly specialised equipment

BACKGROUND We present a simplified freeze-fracture method to reveal the internal structure of protozoan parasites and their host cells, without the need for costly transmission electron microscopy (TEM) equipment. In a traditional procedure, the four key steps in making a freeze-fracture replica are (1) freezing, (2) fracturing, (3) replication, and (4) replica cleaning. In conventional protocols, visualisation is performed using a TEM. OBJECTIVES This study aimed to adapt the traditional freeze-fracture protocol to a more accessible method, eliminating the need for vacuum equipment and TEM, while preserving the capacity to visualise cellular structures. METHODS In our adapted method, vacuum equipment was not required for sample fracturing, shadowing, or replica preparation. Cells were fixed with glutaraldehyde, embedded in a gelatin-chitosan matrix, cryoprotected with dimethyl sulfoxide (DMSO), and subsequently frozen in Freon, followed by immersion in liquid nitrogen. We manually break the cells with a previously frozen razor blade and macerate them with osmium tetroxide (OsO4). The samples are visualised using scanning electron microscopy (SEM). FINDINGS We present various structures of Toxoplasma gondii, Trypanosoma cruzi, Trichomonas vaginalis, Giardia intestinalis, and cultured cells observed using our adapted freeze-fracture method. MAIN CONCLUSIONS This is a rapid and low-cost technique that reveals cell structures comparable to those observed using traditional freeze-fracture methods, although with reduced resolution.