Quantitative analysis and isolation of Escherichia coli O157:H7 in a food matrix using flow cytometry and cell sorting

Abstract

Flow cytometry is a potentially valuable analytical method in microbiology providing the ability to analyze rapidly large numbers of individual microorganisms by several parameters. With a flow cytometer with enhanced light scatter sensitivity and a conventionally configured sorting cytometer, a series of comparative studies to determine the ability of the two flow systems and the antibody-direct epifluorescent filter technique (Ab-DEFT) to detect and enumerate Escherichia coli O157:H7 were made. Initial experiments used culture-derived mixtures of non-pathogenic E. coli and serial dilutions of E. coli O157:H7. Subsequent studies involved analysis of enrichment cultures from ground beef inoculated with E. coli O157:H7. Comparison of flow cytometry with microscopy and plate counts produced similar results at higher concentrations in both culture mixtures and beef enrichments. At the lowest concentrations Ab-DEFT was more sensitive, however, the time required for analysis was much less with flow cytometry. With a cytometer with enhanced light scatter sensitivity designed for bacterial analysis, O157:H7 could be distinguished from E. coli strain HB101 on the basis of light scatter. This instrument also provided direct count data for selected populations. In experiments using cell sorting to isolate target organisms, the purity of fluorescent-labeled E. coli O157:H7 sorted from beef enrichment cultures and plated was not affected by the level of background organisms, as is often the case in conventional plating procedures.

The growth, physiology and toxigenic potential of Bacillus cereus in cooked rice during storage temperature abuse

“…FCM has previously been employed to study the physiology of microbes in a variety of foodstuffs including milk (Gunasekera, Veal, & Attfield, 2003;Holm, Mathiasen, & Jespersen, 2004), drinking water (Pianetti et al, 2005), cheese (Sheehan, O'Loughlin, O'Cuinn, FitzGerald, & Wilkinson, 2005) and ground beef (Tortorello, Stewart, & Raybourne, 1998). Previously, the authors reported the use of FCM to demonstrate a heterogenous response by B. cereus endospores to simulated cooking regimes and refrigerated storage (Cronin & Wilkinson, 2008a data and provided a deeper insight into the physiological status of populations of B. cereus stored at various temperatures over the course of the 6 days incubation.…”

Effect of Bicozamycin on the Eradication of Shiga Toxin‐Producing Escherichia coli in Calves

“…For these purposes, screening and quantitative assay systems for detecting STEC were required. To date, most techniques employed have been focused on the selection of serotype 0157 (8,19,27,29,32). However, the bacteriological, immunological and molecular biological techniques adopted may influence the estimates of prevalence when detecting STEC other than serotype 0157.…”

Updates on the applications of flow cytometry in microbiology

“…FCM is rapid, can be done quantitatively, can be automated and produces real time as well as archival data, all of which offer important information for rapid detection systems as well as critical control point monitoring. In addition, FCM equipped with cell sorting capability can be used to isolate cells or bacteria of interest for further analysis or confirmatory culture [17].…”

“…The detection of specific pathogenic bacteria within meats (and eggs), namely Salmonella spp., E. coli O157:H7 and Campylobacter spp. [62], is an important focus in the development of hazard analysis critical control point (HACCP) procedures for meat processing [17]. Food safety regulators, food producers, distributors, and retailers need effective microbiological methods with improved sensitivity, specificity, and speed for quality control purposes to eliminate or reduce product contamination by foodborne pathogens (American Type Culture Collection (ATCC), 2012).…”