title: Differentiation between Viable and Dead Cryptosporidium Oocysts Using Fluorochrome Staining.
abstract: The use of nucleic acid staining with a fluorochrome dye to differentiate viable and dead (heat-killed) Cryptosporidium oocysts was assessed. The specificities (percentage of unstained viable oocysts) and sensitivities (percentage of stained dead oocysts) of the seven tested dyes (SYTO-17® and SYTO-59® to 64®) ranged from 65 to 76% (average 71%) and 83 to 95% (average 91%), respectively. SYTO-59 and SYTO-17 imparted greater color (4+) intensity than the other dyes (2+ or less). Of these two dyes, SYTO-17 exhibited more brightness and slower discoloration and was selected for use in further experiments. The optimum staining time for SYTO-17 at 37℃ was one hour or more (sensitivity of 96%). Dye concentrations of 20 and 30 µM resulted in maximal color intensity, and no further improvement was observed with further increases in dye concentration. Staining a mixture of viable and dead oocysts (1:1 ratio) with 20 µM dye at 37℃ for one hour yielded the expected results (approximately 50%), but no remarkable increase in the percent staining with time (up to 8 hours) was observed. In this study, no ghost oocysts were observed. The present study indicated that the fluorogenic nucleic acid dye SYTO-17 could be used to discriminate between live and dead Cryptosporidium oocysts.
authors.affiliation: Department of Microbiology and Infectious Disease, Kobe University Graduate School of Medicine, Kobe 654-0142, Japan.
authors.forename: Tatsuya
authors.initials: T
authors.lastname: Tomonaga
authors.affiliation: Department of Pathology, Institute of Medicine (IOM), Tribhuvan University Teaching Hospital (TUTH), Kathmandu, Nepal.
authors.forename: Shiba Kumar
authors.initials: SK
authors.lastname: Rai
authors.affiliation: Department of Parasitology, Faculty of Nursing, Kobe-Women's University, Kobe 650-0046, Japan.
authors.forename: Shoji
authors.initials: S
authors.lastname: Uga
mesh_terms: D000818:Animals; D003457:Cryptosporidiosis / Q000469:parasitology / Q000517:prevention & control / Q000635:transmission; D003458:Cryptosporidium / Q000166:cytology* / Q000472:pathogenicity; D060766:Drinking Water / Q000469:parasitology; D005456:Fluorescent Dyes; D006801:Humans; D033661:Oocysts / Q000166:cytology*; D013194:Staining and Labeling / Q000379:methods*
chemical_list: D060766:Drinking Water; D005456:Fluorescent Dyes
keywords: Cryptosporidium oocyst; Differentiation; Nucleic acid staining; SYTO-17; Viable