Analytical challenges and perspectives of assessing viability ofcysts andoocysts by live/dead simultaneous staining.

Abstract

andare pathogenic protozoa often present in the environment in their infective form(cysts and oocysts). These parasites are very resistant to disinfection, which makes them important target organisms in environmental quality monitoring and sanitation. Viability assessment provides an interpretation of cell inactivation, and it can be evaluated by membrane integrity as well as enzyme activity, using different staining methods. These are straightforward and adequate to laboratories that lack infrastructure for molecular-based technologies or animal infectivity tests. This study investigated simultaneous staining by a commercial live/dead kit, in order to assess viability ofoocysts andcysts, comparing it to propidium iodide (PI) incorporation, a common stain applied in viability estimation. Results suggested that, although the central hypothesis of one-panel visualization ( = 0.05) was met, simultaneous staining impaired (oo)cyst detection by immunofluorescence assay (IFA), which was found to be essential to enumeration, as the live/dead test led to poor (oo)cyst labelling or a 10-fold lower recovery when carried out concomitantly to IFA. As for the viability assessment itself, although red dye uptake occurred as expected by dead or weakened organisms, neither livecysts oroocysts present any green fluorescence by esterase metabolism. This may have been caused by low enzyme activity in the infective form and/or wall thickness of these parasites. The results do not exclude the possibility of simultaneous fluorescence staining for protozoa, but it is a starting point for a broader analysis, that may consider, for instance, different incubation conditions.