title: Improvement of FISH-FCM enumeration performance in filamentous yeast species in activated sludge by snailase partial digestion.
abstract: This paper developed a novel strategy to improve the fluorescence in situ hybridization-flow cytometry (FISH-FCM) enumeration performance in filamentous yeast species in activated sludge by snailase partial digestion to fully disaggregate filamentous yeast chains into single cells. A 2 h 2% snailase partial digestion liberated more rod-shaped yeast single cells from intertwined filamentous yeast samples than did sonication disaggregation, based on an optical microscopic observation and the forward-light-scatter frequency histogram of FCM analysis. However, adding snailase resulted in a fluorescence-quenching phenomenon of the hybridized filamentous yeast cells, which was minimized by lowering the snailase concentration. An approximately 3 h 0.5% snailase partial digestion conducted between sonication and hybridization significantly improved the FISH-FCM enumeration performance for filamentous yeast species by 37%. The results presented here will facilitate the rapid detection, identification and exact enumeration of specific filamentous fungal species in environmental samples.
authors.affiliation: MOE Key Laboratory of Water and Sediment Sciences/State Key Laboratory of Water Environment Simulation, School of Environment, Beijing Normal University, People's Republic of China.
authors.forename: Cancan
authors.initials: C
authors.lastname: Cui
authors.forename: Yanyan
authors.initials: Y
authors.lastname: Zhang
authors.forename: Hui
authors.initials: H
authors.lastname: Han
authors.forename: Shaokui
authors.initials: S
authors.lastname: Zheng
mesh_terms: D015169:Colony Count, Microbial / Q000379:methods*; D057075:Enzyme Assays; D005434:Flow Cytometry / Q000379:methods*; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000737:chemistry; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D059748:Proteolysis; D012722:Sewage / Q000032:analysis / Q000382:microbiology*; D013010:Sonication; D013997:Time Factors; D014250:Trichosporon / Q000737:chemistry / Q000166:cytology / Q000302:isolation & purification*
chemical_list: D005456:Fluorescent Dyes; D012722:Sewage