1.5 ml reaction tubes

The 1BU trophozoites or cysts were suspended at 2 × 10⁴/100 µL of PYG or anti-amoebic agent-containing medium (see Methods) in 1.5 mL reaction tubes (Sarstedt, Nümbrecht, Germany) and incubated in a Thermomixer (Eppendorf AG, Hamburg, Germany) with a shaking rate of 650 rpm at 30°C for 2 hours. All reaction tubes were then centrifuged at 200 × g for 5 minutes, and 80 µL supernatant was removed from each tube. Next, 20 µL of a 0.4% trypan blue solution (Sigma-Aldrich) was added to each tube and the trophozoites or cysts were incubated at room temperature for 5 minutes. The 1BU trophozoites and cysts without trypan blue staining served as negative controls. Next, 10 µL of the trypan blue-treated cells were pipetted onto a hemocytometer (C-Chip, NanoEnTek, Waltham, MA) and bright field images were taken with a Leica DMI4000 B microscope (Leica Microsystems, Wetzlar, Germany) at 10-fold magnification, using Leica Application Software (LAS) version 3.7. These experiments were repeated three times on different days.
Because natamycin formed a suspension due to its high molecular weight, trophozoites or cysts could not be distinguished from natamycin itself; therefore, this assay was not performed on natamycin-treated samples.

Cells grown as monolayers were washed with PBS and detached from cell culture wells with 100 µL trypsin/EDTA (PAN-Biotech GmbH, Aidenbach, Germany) for 3 min. The reaction was stopped by addition of 500 µL FCS-containing cell culture medium. All cell containing fluids were pooled in 15 mL tubes (Sarstedt, Nümbrecht, Germany) and centrifuged at 500 rcf for 5 min (Eppendorf centrifuge 5810 R, Eppendorf AG, Hamburg, Germany). The supernatants were discarded, and the cell pellet was resuspended in 120 µL PBS.
Multicellular spheroids were harvested from the 96-well plates using a cut pipette tip (1250 µL). 3–4 spheroids were pooled into 1.5 mL reaction tubes (Sarstedt, Nümbrecht, Germany), washed with PBS and single cell suspensions were prepared by incubation with trypsin for 5 min. Reaction was stopped by addition of 500 µL FCS-containing cell culture medium. Cells were centrifuged at 300 rcf for 5 min. The supernatant was discarded and cells were resuspended in 200 µL PBS.