Discrimination between viable and dead Encephalitozoon cuniculi (microsporidian) spores by dual staining with Sytox Green and Calcofluor White M2R

Microsporidia are obligate intracellular parasites, recognized as causing c hronic diarrhea and systemic disease in AIDS patients, organ transplant rec ipients, travelers, and malnourished children. Species of microsporidia tha t infect humans have been detected in drinking-water sources, and methods a re needed to ascertain if these microsporidia are viable and capable of cau sing infections. In this study, Calcofluor White M2R and Sytox Green stains were used in combination to differentiate between live (freshly harvested) and dead (boiled) Encephalitozoon cuniculi spores. Calcofluor White M2R bi nds to chitin in the microsporidian spore wall. Dual-stained live spores ap peared as turquoise-blue ovals, while dead spores appeared as white-yellow ovals at an excitation wavelength of 395 to 415 nm used for viewing the Cal cofluor stain. Sytox Green, a nuclear stain, is excluded by live spores but penetrates compromised spore membranes. Dual-stained dead spores fluoresce d bright yellow-green when viewed at an excitation wavelength of 470 to 490 nm, whereas live spores failed to stain with Sytox Green. After live and d ead spores mere mixed at various ratios, the number of viably stained spore s detected in the dual-staining procedure correlated (P = 0.0025) with the expected numbers of viable spores. Spore mixtures were also assayed for inf ectivity in a focus-forming assay, and a correlation (P = 0.0002) was measu red between the percentage of focus-forming microsporidia and the percentag e of expected infectious spores in each mixture, By analysis of variance, n o statistically significant differences were measured between the percentag e of viably stained microsporidia and the percentage of infectious microspo ridia (P = 0.964) in each mixture. These results suggest that Calcofluor Wh ite M2R and Sytox Green stains, when used together, may facilitate studies to identify viable microsporidia.