2.4. In vitro effect against the cyst stage of A. castellanii Neff (ATCC300010)

The cysticidal activity was determined by the alamarBlue™ assay at 168 h and confirmed visually by inverted microscopy. A. castellanii Neff cysts were prepared as described by Lorenzo-Morales et al. (2008). Briefly, trophozoite were transferred from PYG medium based cultures (trophozoite medium) to Neff's encystment medium (NEM; 0.1 M KCl, 8 mM MgSO4·7H2O, 0.4 mM CaCl2·2H2O, 1 mM NaHCO3, 20 mM ammediol [2-amino-2-methyl-1,3-propanediol; Sigma Aldrich Chemistry Ltd., Madrid, Spain], pH 8.8, at 25 °C) and were cultured in this medium with gently shaking for a week in order to obtain mature cysts. After that, mature cysts were harvested and washed twice using PYG medium.

A serial dilution of the most active eye drop was prepared in PYG medium in sterile 96-well microtiter plates (NUNC, Thermo Scientific™). After this step, 5 × 10⁴ Acanthamoeba spp. mature cysts ml−1 were added, obtained a final volume of 100 μL in each well. Plates were then incubated for 168 h at 26 °C with slight agitation as it has been described before (Sifaoui et al., 2018). After this initial incubation period, the 100 μL of each well were removed and added 100 μL of fresh PYG medium, in order to evaluate the excystation capacity of the treated cysts. Finally, 10 μL of the alamarBlue™ Reagent (Life Technologies, Madrid, Spain) was placed into each well, and the plates were then incubated for another 168 h at 26 °C with slight agitation. Subsequently, the plates were analysed, with an Enspire® microplate reader (PerkinElmer, Massachusetts, USA) using the emitted fluorescence (570/585 nm). Percentages of growth inhibition, 50% inhibitory concentration (IC₅₀) was calculated by linear regression analysis with 95% confidence limits. All experiments were performed three times each in duplicate, and the mean values were calculated.