Encystation of Acanthamoeba castellanii: dye uptake for assessment by flow cytometry and confocal laser scanning microscopy.

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.