title: Experimental improvements in combining CARD-FISH and flow cytometry for bacterial cell quantification.
abstract: Flow cytometry and Fluorescence In Situ Hybridization are common methods of identifying and quantifying bacterial cells. The combination of cytometric rapidity and multi-parametric accuracy with the phylogenetic specificity of oligonucleotide FISH probes has been regarded as a powerful and emerging tool in aquatic microbiology. In the present work, tests were carried out on E. coli pure culture and marine bacteria using an in-solution hybridization protocol revealing high efficiency hybridization signal for the first one and a lower for the second one. Other experiments were conducted on natural samples following the established CARD-FISH protocol on filter performed in a closed system, with the aim of improving cell detachment and detection. The hybridized cells were then subsequently re-suspended from the membrane filters by means of an optimized detachment procedure. The cytometric enumeration of hybridized marine bacteria reached 85.7%±18.1% of total events. The quality of the cytograms suggests that the procedures described may be applicable to the cytometric quantification of phylogenetic groups within natural microbial communities.
authors.affiliation: Department of Earth, Life and Environmental Sciences, University of Urbino Carlo Bo, Urbino, Italy. anita.manti@uniurb.it
authors.forename: Anita
authors.initials: A
authors.lastname: Manti
authors.forename: Paola
authors.initials: P
authors.lastname: Boi
authors.forename: Stefano
authors.initials: S
authors.lastname: Amalfitano
authors.forename: Alberto
authors.initials: A
authors.lastname: Puddu
authors.forename: Stefano
authors.initials: S
authors.lastname: Papa
mesh_terms: D058491:Bacterial Load / Q000379:methods*; D004926:Escherichia coli / Q000302:isolation & purification*; D005434:Flow Cytometry / Q000379:methods*; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D014871:Water Microbiology