Flow cytometry for determination of the efficacy of contact lens disinfecting solutions against Acanthamoeba spp

Abstract

Flow cytometric analyses of cellular staining with fluorescent viability dyes and direct microscopic observations of methylene blue exclusion were compared for evaluation of the effects of a chlorhexidine gluconate-based contact lens disinfectant solution and a polyhexamethylene biguanide solution against cysts and trophozoites of Acanthamoeba castellanii and Acanthamoeba polyphaga. The flow cytometric procedure with propidium iodide (used to stain dead cells) indicated that more than 90% of trophozoites of both species (inocula of 10(5) to 10(6)/ml) at 22 degrees C lost their viability after 4 h of exposure to chlorhexidine. When propidium iodide was used in combination with fluorescein diacetate (for live cells), the apparent number of propidium iodide-stained cells was reduced, but the relative efficacies of the two biguanide solutions appeared unchanged from those evident with the single dyes; the chlorhexidine solution was more effective than the polyhexamethylene biguanide solution. Similar data were obtained with the more cumbersome methylene blue exclusion procedure. Flow cytometric analyses provided a statistically reproducible and rapid procedure for determining the relative antiamoebal efficacies of the disinfecting solutions.

FIG. 1

Effect of chlorhexidine solution (50 μg/ml) on trophozoites (A) and cysts (B) on A. polyphaga (2 × 10⁶ cells/ml) stained with PI. FL2-H versus FSC-H dot plots were used to discriminate viable (PI excluded; lower quadrant) from dead (PI stained; upper quadrant) cells. The dot plots were gated by forward scatter to eliminate analysis of noise and smaller particles (<3 μm in diameter). Analysis gates were set with fluorescence from control live (FL2-H, ≤540) and heat-killed (FL2-H, >540) amoebae.

FIG. 2

Effect of PHMB solution (0.5 μg/ml) on viability of trophozoites (A) and cysts (B) of A. castellanii; cells were stained with PI. Analysis gates were set as described previously.

FIG. 3

Effect of staining with PI alone (A) and PI and FDA simultaneously (B) after exposure of A. polyphaga cysts to PHMB for 4 h. Of 10,000 treated cells, 50% appeared viable after staining with PI alone (A [lower quadrant]) and 80% appeared viable after staining with PI and FDA (B [upper right quadrant]). The analysis gates (from amoebae stained with PI and FDA) were set with fluorescence from control live (FL1-H, ≥400; FL2-H, ≥520) and heat-killed (FL1-H, <400; FL2-H, ≥520) cells. Analysis gates for cells stained with PI were set as previously described.