Responses of Escherichia coli, Listeria monocytogenes, and Staphylococcus aureus to Simulated Food Processing Treatments, Determined Using Fluorescence-Activated Cell Sorting and Plate Counting

Abstract

Three common food pathogenic microorganisms were exposed to treatments simulating those used in food processing. Treated cell suspensions were then analyzed for reduction in growth by plate counting. Flow cytometry (FCM) and fluorescence-activated cell sorting (FACS) were carried out on treated cells stained for membrane integrity (Syto 9/propidium iodide) or the presence of membrane potential [DiOC 2 (3)]. For each microbial species, representative cells from various subpopulations detected by FCM were sorted onto selective and nonselective agar and evaluated for growth and recovery rates. In general, treatments giving rise to the highest reductions in counts also had the greatest effects on cell membrane integrity and membrane potential. Overall, treatments that impacted cell membrane permeability did not necessarily have a comparable effect on membrane potential. In addition, some bacterial species with extensively damaged membranes, as detected by FCM, appeared to be able to replicate and grow after sorting. Growth of sorted cells from various subpopulations was not always reflected in plate counts, and in some cases the staining protocol may have rendered cells unculturable. Optimized FCM protocols generated a greater insight into the extent of the heterogeneous bacterial population responses to food control measures than did plate counts. This study underlined the requirement to use FACS to relate various cytometric profiles generated by various staining protocols with the ability of cells to grow on microbial agar plates. Such information is a prerequisite for more-widespread adoption of FCM as a routine microbiological analytical technique.

Flow cytometry as a potential method of measuring bacterial viability in probiotic products: A review

“…This data indicates the complexity of the relationship between cell membrane damage, individual strain and resultant cultivability. Another factor in the above relationship is the influence of media type, while Khan et al (2010) plated cells onto standard nutrient agar, Kennedy et al (2011), noted differences in the percentage recovery of stressed cells following cell sorting onto either nutrient or selective agars. In terms of method development, Khan et al (2010) established protocols for staining, detection and analysis of viable and VBNC populations using FCM and recommended a cell concentration of 10 4 /ml as being optimal for this analysis.…”

“…Cell sorters are cytometers with the ability to physically sample cells originating from a sub-population of interest (Nebevon-Caron, Stephens, Hewitt, Powell, & Bradley, 2000). A cell sorter has the ability to encapsulate a single cell within a single droplet which is then given either a positive or negative charge and deflected through a high voltage electrical field (∼5000 V) to be deposited into various receptacles e.g agar plate, test tube, or a microtitre plate well (Kennedy et al, 2011;Muller & Nebe-von-Caron, 2010). Hence, stained or stained/antibody-labelled cells belonging to a sub-population e.g live, dead, damaged can be examined to verify and correlate their cytometric profile to various criteria including viability, possible VBNC status, and resuscitation potential on various agar media.…”

“…The robustness of this particular methodology was attributed to the stringent sample preparation protocol used, which appeared to minimise any matrix particle interference on subsequent cytometric analysis. Kennedy et al (2011) examined the responses of the food pathogens E. coli, Listeria monocytogenes and Staphylococcus aureus when subjected to the effects of various stressors typically encountered by pathogens during food processing. The strains were analysed by FCM for viability using SYTO9/PI or for the presence/ absence of a functioning membrane potential by staining with DiOC 2 (3).…”

“…Bacterial viability currently refers to ability of a cell to grow and reproduce itself under a set of defined environmental conditions (Kennedy, Cronin, & Wilkinson, 2011). The characteristics of viable cells include the presence and functioning of a range of structural, metabolic, physiological and genetic properties.…”

Fluorescence-based in situ assay to probe the viability and growth kinetics of surface-adhering and suspended recombinant bacteria

“…Membrane integrity can be tested optically by using a combination of membrane permeable and impermeable fluorescent dyes that selectively enter live and dead bacteria ( Figure 1a) [8,[14][15][16][17]. While being broadly employed as endpoint staining assays to determine the viability of single bacteria and bacterial colonies directly on the test surface, these assays are not optimized for realtime in situ bacterial viability monitoring.…”