Flow-through imaging cytometry for characterization of Salmonella subpopulations in alfalfa sprouts, a complex food...

We recently developed an approach combining fluorescence in situ hybridization (FISH) and flow cytometry for detecting low levels of Salmonella spp. (approximately 10(3) cells/mL sprout wash) against high levels of naturally occurring sprout flora (approximately 10(7)-10(8) CFU/g sprouts). Although this "FISH and flow" approach provided rapid presence/absence testing for Salmonella in this complex food system, it was not capable of more nuanced tasks, such as probing the phenotypic complexity of the microbes present in sprouts or determining the physical interactions of Salmonella with these microbes, or with sprout debris. In the present study, we have combined rapid FISH-based labeling of Salmonella spp. in sprout washes with flow-through imaging cytometry (FT-IC), using the ImageStream 100, a commercial FT-IC instrument. This approach enables image-based characterization of various subpopulations of interest occurring within these samples. Here, we demonstrate the ability of FT-IC to unambiguously identify cells, cell aggregates and other events within these subpopulations based on both cell morphology and hybridization status after reaction with a Salmonella-targeted probe cocktail. Our ability to directly explore the nature of these events expands the layers of information possible from cytometric analyses of these complex samples and clearly demonstrates that "a picture is worth a thousand dots".