Aims: To develop rapid means of distinguishing between cysts and trophozoites of the opportunistic pathogen, Acanthamoeba castellanii, the causative agent of keratitis. Methods and Results: Fluorescence of Congo Red, Calcoflor White was specific for the endocyst wall; trophozoites did not become fluorescent. The anionic oxonol dye, DiBAC(4)(3), did not penetrate the cytoplasmic membrane after short-term (<5 min) exposure, whereas cysts are permeable and become fluorescent. Confocal scanning laser microscopy confirmed these properties and large populations of organisms were analysed by flow cytometry. Conclusions: These data provide a rapid alternative to traditional haemocytometer or plate counts for discrimination of trophozoites from cysts. Significance and Impact of the Study: Rapid and precise determination of the growth cycle of a dangerous ocular pathogen.