Encystation of Acanthamoeba castellanii: dye uptake for assessment by flow cytometry and confocal laser scanning microscopy.
OBJECTIVE
To develop rapid means of distinguishing between cysts and trophozoites of the opportunistic pathogen, Acanthamoeba castellanii, the causative agent of keratitis.


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.


CONCLUSIONS
These data provide a rapid alternative to traditional haemocytometer or plate counts for discrimination of trophozoites from cysts.


CONCLUSIONS
Rapid and precise determination of the growth cycle of a dangerous ocular pathogen.
Connell
C
C
Microbiology (BIOSI 1), Cardiff University, Wales, UK.
Rutter
A
A
Hill
B
B
Suller
M
M
Lloyd
D
D
D000048:Acanthamoeba / Q000302:isolation & purification* / Q000378:metabolism; D000818:Animals; D001463:Barbiturates / Q000378:metabolism; D001557:Benzenesulfonates / Q000378:metabolism; D003224:Congo Red / Q000378:metabolism; D005434:Flow Cytometry; D005456:Fluorescent Dyes / Q000378:metabolism*; D007555:Isoxazoles / Q000378:metabolism; D018613:Microscopy, Confocal
D001463:Barbiturates; D001557:Benzenesulfonates; D005456:Fluorescent Dyes; D007555:Isoxazoles; C018622:oxonol dyes (isoxazole); C052795:bis(1,3-dibutylbarbiturate)trimethine oxonol; D003224:Congo Red; C007061:C.I. Fluorescent Brightening Agent 28