Tissue culture bottles

D-mannose agarose beads (Sigma-Aldrich Chemie GmbH, Munich, Germany) and sepharose beads (CL-4B, ø 40 μm to 165 μm, Sigma-Aldrich Chemie GmbH, Munich, Germany) were each diluted in a ratio of 1:10 in PBS and subsequently centrifuged at 1000 rpm for 5 min. The supernatant was removed and the washing procedure was repeated twice. Finally, the cleaned beads were diluted 1:10 in PBS. 3 × 10⁵A. castellanii or A. comandoni in 5 ml of PYG medium were seeded into tissue culture bottles (25 ml, Sarstedt AG & Co., Nümbrecht, Germany) and 0.5 ml of one of the bead solutions were added. The samples were observed with an inverted phase contrast microscope (Olympus CKX41) equipped with a camera (C9300, Hamamatsu, Hamamatsu, Japan) and 50 images of each bottle were taken right after the addition, after 2 h and after 4 h incubation time at room temperature. The number of acanthamoebae adhering to the beads and the total number of acanthamoebae in the resulting images were counted.

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.

A. castellanii (ATTC 30234) were cultured in PYG medium at room temperature in 75 ml tissue culture bottles (Sarstedt, Germany) as described previously58 (link)59 (link). Prior to experiments, about 10⁵ freshly harvested acanthamoebae (A. castellanii) per ml medium were seeded on a flat surface (tissue culture bottle or six-well plate, Sarstedt AG & Co., Nümbrecht, Germany) and incubated at room temperature for 30 min to 1 h to ensure their attachment to the tissue culture surface.