title: Development of mass cytometry methods for bacterial discrimination.
abstract: Fluorescent flow cytometry has become the method of choice for interrogation of bacterial populations at the single-cell level. However, limitations of this technique include issues of dynamic range, spectral overlap, photobleaching, and overall low signal intensity due to the small size of bacteria. The recent development of mass cytometry allows single-cell analysis with the resolution of inductively coupled plasma mass spectrometry, facilitating multiparametric analysis. Using a combination of a metal-based membrane stain and lectins conjugated to lanthanide-chelating polymers, we demonstrate that individual Escherichia coli cells can be differentiated based on their cell surface polysaccharides using mass cytometry. The model E. coli system involves evaluation of three different surface polysaccharides using element-tagged concanavalin A and wheat germ agglutinin lectins. Finally, this technique enabled experiments designed to follow the export of O-antigen substituted lipopolysaccharide in a conditional mutant. These studies revealed that the culture responds as a uniform population and that lipopolysaccharide export is approximately 10 times faster than the logarithmic bacterial doubling time.
authors.affiliation: Department of Chemistry, University of Toronto, Toronto, Ontario, Canada M5S 3H6.
authors.forename: Michael D
authors.initials: MD
authors.lastname: Leipold
authors.forename: Olga
authors.initials: O
authors.lastname: Ornatsky
authors.forename: Vladimir
authors.initials: V
authors.lastname: Baranov
authors.forename: Chris
authors.initials: C
authors.lastname: Whitfield
authors.forename: Mark
authors.initials: M
authors.lastname: Nitz
mesh_terms: D002462:Cell Membrane / Q000737:chemistry; D002469:Cell Separation; D002614:Chelating Agents / Q000737:chemistry; D003592:Cytophotometry / Q000295:instrumentation / Q000379:methods*; D004926:Escherichia coli / Q000145:classification / Q000302:isolation & purification*; D028581:Lanthanoid Series Elements / Q000737:chemistry; D037102:Lectins / Q000737:chemistry; D008070:Lipopolysaccharides / Q000737:chemistry; D013058:Mass Spectrometry; D009154:Mutation; D004369:Pentetic Acid / Q000737:chemistry; D059010:Single-Cell Analysis / Q000379:methods*; D013194:Staining and Labeling / Q000379:methods*
chemical_list: D002614:Chelating Agents; D028581:Lanthanoid Series Elements; D037102:Lectins; D008070:Lipopolysaccharides; D004369:Pentetic Acid