Encystation of Acanthamoeba castellanii: dye uptake for assessment by flow cytometry and confocal laser scanning microscopy |
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Authors: | Connell C Rutter A Hill B Suller M Lloyd D |
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Institution: | Microbiology (BIOSI 1), Cardiff University, Wales, UK. |
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Abstract: | 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, DiBAC4(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. CONCLUSION: 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. |
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