2 m glucose

A. castellanii (ATTC^® 30234, derived from ATCC^® 30011) and A. comandoni (Pb30/40, group 1, genotype T7 [7 ]) were cultured at room temperature in tissue culture bottles (75 ml, Sarstedt AG & Co., Nümbrecht, Germany). A Peptone Yeast Glucose (PYG) Medium 712 consisting of 20 g proteose peptone (BD, Sparks, USA), 1 g yeast extract (BD, Sparks, USA), 950 ml distilled water, 8 ml 0.05 M CaCl₂ (AppliChem GmbH, Darmstadt, Germany), 10 ml 0.4 M MgSO₄ × 7H₂O (AppliChem GmbH, Darmstadt, Germany), 10 ml 0.25 M Na₂HPO₄ × 7H₂O (Carl Roth GmbH & Co. KG, Karlsruhe, Germany), 10 ml 0.25 M KH₂PO₄ (Carl Roth GmbH & Co. KG, Karlsruhe, Germany), 34 ml 0.1 M Na citrate × 2H₂O (Merck KGaA, Darmstadt, Germany), 10 ml 0.005 M Fe(NH₄)₂(SO₄)₂ × 6H₂O (AppliChem GmbH, Darmstadt, Germany), and 50 ml 2 M glucose (Sigma-Aldrich Chemie GmbH, Munich, Germany)) was used. The medium was renewed once a week by shaking the bottles, removing the old medium with swimming acanthamoebae, and adding fresh medium to the remaining cells.

Trophozoites of A. castellanii (ATTC 30234) were cultured at room temperature in Peptone Yeast Glucose (PYG) 712 medium (20.0 g proteose peptone (BD, Sparks, USA), 1.00 g yeast extract (BD, Sparks, USA), 950 mL distilled water, 10.0 mL 0.4 M MgSO₄·7H₂O (AppliChem, Darmstadt, Germany), 8.00 mL 0.05 M CaCl₂ (AppliChem, Darmstadt, Germany), 34.0 mL 0.1 M sodium citrate dihydrate (Merck, Darmstadt, Germany), 10.0 mL 0.005 M (NH₄)₂Fe(SO₄)₂·6H₂O (AppliChem, Darmstadt, Germany), 10.0 mL 0.25 M Na₂HPO₄·7H₂O (Roth, Karlsruhe, Germany), 10.0 mL 0.25 M KH₂PO₄ (Roth, Karlsruhe, Germany), 50.0 mL 2 M glucose (Sigma–Aldrich Chemie GmbH, Steinheim, Germany)). In this axenic culture, the PYG 712 medium was regularly exchanged in the cell culture flasks in order to avoid an encystment of A. castellanii trophozoites.