Acanthamoeba castellanii Castellani was obtained from the American Type Culture Collection (ATCC 30011) (Rockville, Maryland, USA). A. castellanii trophozoites were axenically cultured in PYG medium (0.75% proteose peptone, 0.75% yeast extract, and 1.5% glucose) at 25°C incubator. A. castellanii encystation was induced in an encystation media (0.1 M KCl, 0.008 M MgSO₄, 0.0004 M CaCl₂, and 0.02 M 2-amino-2-methyl-1,3-propanediol pH 9.0) for 3 days [13]. Mature cysts were counted under a light microscope after treating them with 0.5% SDS, and encystation ratios were calculated [14].

All tested MPDS made in Korea and their respective contents are listed in Table 1. All MPDS were taken from their original wrappings and were used before their stated expiry date. Amoebicidal activity of MPDS was determined using a most probable number (MPN) technique for amoebic enumeration [15]. Trophozoites or cysts were exposed to 15 MPDS and a control solution (0.9% PAS). Aliquots (0.1 ml) of trophozoite or cyst culture were added to 9.9 ml of each MPDS and control in sterile glass bottles to give initial concentrations of 104 trophozoites or cysts/ml. All bottles were incubated in a 25°C incubator at predefined intervals (0, 4, 8, and 24 hr), and 1 ml aliquots from each MPDS and the control solution were placed in 9 ml of Dey Engley (DE) broth for disinfectant neutralization. These were sealed and incubated at 25°C for 15 min. Seventeen 1-ml aliquots of the one in 10 dilution prepared in DE broth were inoculated onto agar plates covered with heat-killed bacteria. The 6-well tissue culture plates were used for the 1-ml aliquots, and the wells of 12-well plates were used for the 0.1- and 0.01-ml aliquots. All plates were sealed and incubated at 25°C. The plates were examined for the presence of viable trophozoites after 3 and 7 days of incubation. Each MPDS was tested in triplicate against trophozoites and cysts.

Human corneal epithelial (HCE) cells were cultured in endothelial cell growth media kits (KGMTM BulletKitTM) (Lonza, Portsmouth, New Hampshire, USA) in 5% CO₂ incubator. Monolayered HCE cells were placed in 96-well plates and incubated with 3 kinds of MPDS or control media in 5% CO₂. Cytopathic effects (CPE) were assessed visually after Giemsa staining and measurement of optical density at 590 nm, with 0.1 ml of cells solubilized in 5% SDS. Percent CPE was calculated according to the following formula: % CPE=100 − [(OD of experimental well − OD of HCE alone)/OD of control cells] ×100. The CPE assay was performed in triplicate.

Data are expressed as mean±SD in 3 independent experiments. Statistical significance was analyzed using an unpaired Student’s t-test. A P-value of <0.05 was interpreted as statistically significant.

To determine the amoebicidal activity of Korean MPDS, the most probable number (MPN) technique was used. All tested Korean MPDS and their respective contents were listed in table 1. Fifteen kinds of MPDS and control solution were tested for amoebicidal activity. The average and standard deviation from MPN at 0, 4, 8, and 24 hr of trophozoites and cysts after incubation with MPDS were shown in Table 2. The trophozoites and cysts MPN average and SD were presented respectively in Fig. 1. No significant difference was detected between trophozoites and cysts. After an exposure time of 4 hr, solution N showed high amoebicidal activity to trophozoites and cysts. After 8 hr of exposure, solution N and M showed moderate amoebicidal activity to trophozoites (Fig. 1A). After 24 hr of exposure, solution N and M killed almost all trophozoites and cysts (Fig. 1A, B). Solutions A, B, G, H, L, and O showed positive amoebicidal effects with 24 hr exposure (Table 2; Fig. 1). Especially, solutions G and H showed moderate amoebicidal activity to trophozoites. However, cysts were more resistant to solutions G and H than solutions N and M. Among 15 MPDS made in Korea, solutions N and M showed higher amoebicidal effects to trophozoites and cysts than the others but not enough to eradicate the amoeba.

To investigate the cytopathic effects of MPDS which possessed amoebicidal effects, solutions A, N, and M were incubated with human corneal epithelial cells for 30 min, 1 hr, 2 hr, and 4 hr. The concentrations of MPDS were designed so that a linear range of 0% to 100% (Fig. 2). A few amoebicidals effective MPDS-A was not cytotoxic at any of the concentrations tested (Fig. 2A). Solution A was cytotoxic for higher concentrations (70–100%) after 4 hr exposure; however, the solution is dilute to appropriate concentrations by eye drops.

Moderate amoebicidal solution M showed 58% cytopathic effects at 50% concentration and 4 hr exposure (Fig. 2B). Even at 1 hr exposure to 50% diluted solution M, 43% cytopathic effects were shown. The best amoebicidal solution N showed 62% cytopathic effects at 50% concentration and 4 hr exposure, and 32% cytopathic effects at 50% concentration and 1 hr exposure (Fig. 2C). Both of these solutions M and N showed a time-dependent cytotoxicity towards HCE cells. These results showed that amoebicidal solutions also have various ranges of cytopathic effects against human cells.