title: Direct counting of Cryptosporidium parvum oocysts using fluorescence in situ hybridization on a membrane filter.
abstract: This report describes the development of a direct and rapid detection method for the pathogenic protozoan, Cryptosporidium parvum, from environmental water samples using fluorescence in situ hybridization (FISH) on a membrane filter. The hydrophilic polytetrafluoroethylene (PTFE) membrane filter with FISH-stained oocysts yielded the highest signal to noise (S/N) ratio of the different membrane filters tested. PTFE membranes retained 98.8+/-0.4% of the concentrated oocysts after washing, simultaneous permeabilization and fixation with a hot ethanol solution, and hybridization with a fluorescently labeled oligonucleotide probe. This procedure eliminates subsequent time-consuming recovery steps that often result in a loss of the actual oocysts in a given environmental water sample. Furthermore, C. parvum was successfully distinguished from Cryptosporidium muris and other species in environmental water samples with the addition of formamide into the hybridization solution. In tap water samples, the S/N ratio was heightened by washing the membrane filter prior to FISH with a 1 M HCl solution in order to reduce the large amounts of impurities and background fluorescence from the non-specific adsorption of the fluorescently labeled oligonucleotide probe.
authors.affiliation: Department of Biotechnology, Tokyo University of Agriculture and Technology, 2-24-16 Naka-cho, Koganei, Tokyo 184-8588, Japan.
authors.forename: Tomoyuki
authors.initials: T
authors.lastname: Taguchi
authors.forename: Youhei
authors.initials: Y
authors.lastname: Shinozaki
authors.forename: Haruko
authors.initials: H
authors.lastname: Takeyama
authors.forename: Satoshi
authors.initials: S
authors.lastname: Haraguchi
authors.forename: Masato
authors.initials: M
authors.lastname: Yoshino
authors.forename: Masao
authors.initials: M
authors.lastname: Kaneko
authors.forename: Yoshio
authors.initials: Y
authors.lastname: Ishimori
authors.forename: Tadashi
authors.initials: T
authors.lastname: Matsunaga
mesh_terms: D000818:Animals; D002232:Carbocyanines / Q000737:chemistry; D016785:Cryptosporidium parvum / Q000235:genetics / Q000302:isolation & purification*; D015342:DNA Probes / Q000235:genetics; D005456:Fluorescent Dyes / Q000737:chemistry; D006801:Humans; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D008567:Membranes, Artificial; D008856:Microscopy, Fluorescence; D008857:Microscopy, Interference; D033661:Oocysts; D014867:Water / Q000469:parasitology*
chemical_list: D002232:Carbocyanines; D015342:DNA Probes; D005456:Fluorescent Dyes; D008567:Membranes, Artificial; C089187:cyanine dye 3; D014867:Water