Effects of pH on a fluorogenic vital dyes assay (4′,6′-diamidino-2-phenyl-indole and propidium iodide) for Cryptosporidium sp. oocysts

The fluorogenic vital dyes assay utilizing 4′,6′-diamidino-2-phenyl-indole (DAPI) and propidium iodide (PI) can be used for determining the viability of individual Cryptosporidium parvum oocysts isolated from environmental samples; however this assay can be affected by the pH of the oocyst suspending medium, resulting in formation of fluorescent yellow DAPI crystals at neutral or alkaline pH. Interference from DAPI crystals can lead to oocyst occlusion, making their viability assessment difficult or subjective. When the oocyst suspending medium is rinsed with HBSS adjusted to pH 4, DAPI crystallization is reduced or prevented without affecting oocyst staining characteristics with these two dyes or with fluorescein isothiocyanate conjugated anti-Cryptosporidium monoclonal antibody.