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 sa mples, however this assay can be affected by the pH of the oocyst suspendin g medium, resulting in formation of fluorescent yellow DAPI crystals at neu tral or alkaline pH. Interference from DAPI crystals can lead to oocyst occ lusion, making their viability assessment difficult ol subjective. When the oocyst suspending medium is rinsed with HBSS adjusted to pH 4, DAPI crysta llization is reduced or prevented without affecting oocyst staining charact eristics with these two dyes or with fluorescein isothiocyanate conjugated anti-Cryptosporidium monoclonal antibody. (C) 1999 Elsevier Science Ltd. Al l rights reserved.