Differentiation between Viable and Dead Cryptosporidium Oocysts Using Fluorochrome Staining

• Published in: Kobe journal of the medical sciences Vol. 61; no. 5; pp. E138 - E143
• Main Authors: Tomonaga, Tatsuya, Rai, Shiba Kumar, Uga, Shoji
• Format: Journal Article
• Language: English
• Published: Japan 2015
• Subjects: Animals; Cryptosporidiosis - parasitology; Cryptosporidiosis - prevention & control; Cryptosporidiosis - transmission; Cryptosporidium - cytology; Cryptosporidium - pathogenicity; Drinking Water - parasitology; Fluorescent Dyes; Humans; Oocysts - cytology; Staining and Labeling - methods; Viable; SYTO-17; Nucleic acid staining; Differentiation; Cryptosporidium oocyst
• ISSN: 1883-0498

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.