LS-7-P-1910 Improved preservation of Toxoplasma gondii by High Pressure Freezing and Freeze-Substitution

Toxoplasma gondii, the agent of Toxoplasmosis is an obligatory intracellular parasite of warm-blooded animals, including humans [1]. The ultrastructure of T. gondii was described mainly based in scanning (SEM) and transmission (TEM) electron microscopy of chemically fixed specimens. Figure 1 displays a ultrathin section of a typical tachyzoite form fixed with 2.5% glutaraldehyde in 0.1M cacodylate buffer followed by post-fixation in 1%OsO₄ in the same buffer. Chemical fixatives typically have a slow rate of penetration and usually cells suffer hypo or hyperosmotic shock during fixation. High pressure freezing (HPF) followed by freeze substitution can avoid these artifacts, bringing a more realistic view of the ultrastructure. Tachyzoites from culture cells were inserted by
capillarity in cellulose capillaries and quickly frozen in a Balzers HPM010. Freeze substitution was carried out in a Leica AM EFS2. Four protocols were tested. Best results were obtained with 0.1% tannic acid in absolute acetone at -70^oC for 24h, rinsed in acetone at ^-90oC, followed by 0.1%uranyl acetate, 1% OsO₄,and 0.5% H₂O. Temperature was raised at 1^oC per hour up to 4^oC; samples rinsed in absolute acetone and embedded in Epoxy resin. General organization of the cell was confirmed, except for ribosomes that were not randomly scattered in the cytoplasm, but rather formed clusters of
polysomes. Rhoptries did not have the spongy and biphasic appearance, but were homogeneously stained. These two features probably are more faithful to the real ultrastructure of T. gondii because of the speed of immobilization resulting from HPF. However, impregnation of the fixatives in internal membranes under low temperatures did not result in contrast, so the inner membranes were clear and the cytoplasm was very electron dense (Figure2). The inner membrane complex internal space was also electron lucent. Addition of tannic acid enhanced preservation of the microtubules.
Although, new information was brought by this fixation protocol, it also had some artifacts, as the tendency of the outer membrane to detach from the surface. In conclusion, routine chemical fixation, reliably preserves the ultrastructure of Toxoplasma gondii, but high pressure freezing and freeze substitution techniques have revealed some novel aspects of the ultrastructural organization of this parasite.