Inducing Cysts in Pathogenic Amoebae

Cysts or precysts (did not fully showed the 2-distinct cyst walls) of N. fowleri, A. castellanii, and A. polyphaga were induced by cultivating on 3 kinds of encystment media (buffer 1: 120 mM NaCl, 0.03 mM MgCl₂, 1 mM NaHPO₄, 1 mM KH₂ PO₄, 0.03 mM CaCl₂, 0.02 mM FeCl₂, H 6.8; buffer 2 [11 (link)]: 95 mM NaCl, 5 mM KCl, 8 mM MgSO₄, 0.4 mM CaCl₂, 1 mM NaHCO₃, 20 mM Tris-HCl, pH 9.0; buffer 3 [6 ]: 100 mM KCl, 8 mM MgSO₄, 0.4 mM CaCl₂, 20 mM 2-amino-2-methyl-1,3-propanediol, pH 7.6) (Table 1). Amoebic trophozoties (approximately 2×10⁶ cells) were washed with PBS (pH 7.4) twice, and incubated in 24-well plates with 5 ml of each medium, at 30°C or 37°C. Using an optical microscope (Olympus, Shinjuku, Tokyo, Japan), the morphological changes were observed after encystation, and final cysts were re-cultured with fresh Nelson or PYG media, in order to observe the recovered trophozoites.