Adhesion of Acanthamoeba to SIRC cells after pretreatment with different water conditions. (A) Acanthamoeba trophozoites were incubated with PYG medium, PAS, domestic tap water, CaCl₂, or DDW, followed by staining with calcein AM (green) for one hour. The stained amoebae were then co-cultured with SIRC cells. After incubation, fluorescence microscopy was performed to assess adhesion. The upper panel shows bright-field images, while the lower panel displays dark-field fluorescence images, where green fluorescence indicates Acanthamoeba. (B) The adhesion percentage of Acanthamoeba were examined after pretreatment with PYG medium, PAS, domestic tap water, CaCl₂, or DDW subsequent incubation with SIRC cells. Bar graphs depict the mean ± SD (*p ≤ 0.05).