The effect of different environmental conditions on the encystation of Acanthamoeba castellanii belonging to the T4 genotype

Abstract

In this study, Acanthamoeba castellanii was cultivated under different stress conditions to induce possible encystation. The morphological and histological properties were analysed by light and electron microscopy as well as cyst-specific staining. The findings revealed that cysts prepared through liquid medium using higher osmolarity as a trigger (10% glucose with 50mM magnesium chloride for 72 h) are similar to cysts prepared using non-nutrient agar (nutrient deprivation as a trigger in plating assays for 14 days), as determined by SDS-resistance, cyst-specific Calcofluor white staining and transmission electron microscopy. Using liquid medium assay, A. castellanii encystation was studied by exposing trophozoites to media lacking growth ingredients (phosphate buffered saline or distilled water), inappropriate temperatures (4-45°C), pH (3-9), artificial light-dark cycles, 5% CO2, and microaerophilic conditions. Optimal encystation was observed when cells were incubated in PBS with 50mM MgCl2 and 10% glucose at 24-30°C at pH 7. Increasing temperature over 37°C or pH 9 adversely affected encystation, while light-dark cycles, 5% CO2 and microaerophilic conditions had no effect on encystation of A. castellanii. None of the aforementioned conditions had any effect on the viability of A. castellanii, as determined by Trypan blue exclusion assay. A complete knowledge of encystation in A. castellanii is crucial to our understanding of the biology of these ecologically and medically important organisms.

Keywords: Acanthamoeba; Encystation; Non-nutrient agar; Osmolarity; Physiological conditions; Transmission electron microscopy.