A rapid and accurate protocol for quantifying living Acanthamoeba castellanii cysts

The accurate quantification of living Acanthamoeba castellanii (AC) cysts after various interventions is essential for evaluating the effectiveness of AC cyst disinfection and their environmental stability. The current protocol for measuring living AC cysts requires 7 days to complete, posing a substantial challenge. In this study, we aimed to develop a rapid protocol that markedly reduces the time required to accurately quantify living AC cysts. Clinical and standard strain AC cysts were inoculated into 96-well plates and incubated in peptone-yeast extract-glucose (PYG) medium with varying concentrations of fetal bovine serum (FBS) and CO₂ levels. Excystation was monitored under an inverted microscope, and the living AC cyst counts (LACC) were calculated. We found that the excystation of AC cysts was enhanced by increasing the FBS concentration in the PYG medium and increasing the CO₂ concentration during culture, which collectively significantly reduced the duration required for quantification. Specifically, by supplementing PYG medium with 5% or more FBS and incubating at 2.5% CO₂ or higher, measurements could be completed within only 3 days for standard and clinical AC strain cysts. Notably, this rapid protocol maintained the same accuracy as the conventional 7 day protocol and provided high accuracy in the LACC assay for samples exposed to disinfectants. In summary, the rapid protocol we developed can reduce the time required for measurement by more than half compared to current protocols and will contribute to substantially expediting the evaluation of the effectiveness of disinfectants and other drugs against AC cysts.IMPORTANCEAcanthamoeba castellanii (AC) is a pathogenic microbe that causes refractory Acanthamoeba keratitis (AK). AC cysts are highly durable and resistant to various disinfectants, making effective disinfection, which is essential for mitigating AK, difficult. Currently, it takes 7 days to determine disinfection effectiveness against AC cysts because it involves measuring viable AC cyst counts after disinfection. However, the novel, accurate method for quantifying living AC cysts developed in this study can be completed within 3 days, reducing the time required for measurement by more than half compared to conventional methods, and will markedly streamline the evaluation of disinfectant effectiveness against AC cysts. Moreover, this quantitative method can potentially be applied to shorten the time required for quantifying living cysts of other amoebas.