Sensitive Detection of Escherichia coli O157:H7 in Food and Water by Immunomagnetic Separation and Solid-Phase Laser Cytometry

Abstract

Rapid, direct methods are needed to assess active bacterial populations in water and foods. Our objective was to determine the efficiency of bacterial detection by immunomagnetic separation (IMS) and the compatibility of IMS with cyanoditolyl tetrazolium chloride (CTC) incubation to determine respiratory activity, using the pathogenEscherichia coli O157:H7. Counterstaining with a specific fluorescein-conjugated anti-O157 antibody (FAb) following CTC incubation was used to allow confirmation and visualization of bacteria by epifluorescence microscopy. Broth-grown E. coli O157:H7 was used to inoculate fresh ground beef (<17% fat), sterile 0.1% peptone, or water. Inoculated meat was diluted and homogenized in a stomacher and then incubated with paramagnetic beads coated with anti-O157 specific antibody. After IMS, cells with magnetic beads attached were stained with CTC and then an anti-O157 antibody-fluorescein isothiocyanate conjugate and filtered for microscopic enumeration or solid-phase laser cytometry. Enumeration by laser scanning permitted detection of ca. 10 CFU/g of ground beef or <10 CFU/ml of liquid sample. With inoculated meat, the regression results for log-transformed respiring FAb-positive counts of cells recovered on beads versus sorbitol-negative plate counts in the inoculum were as follows: intercept = 1.06, slope = 0.89, andr 2 = 0.95 (n = 13). The corresponding results for inoculated peptone were as follows: intercept = 0.67, slope = 0.88, andr 2 = 0.98 (n = 24). Recovery of target bacteria on beads by the IMS-CTC-FAb method, compared with recovery by sorbitol MacConkey agar plating, yielded greater numbers (beef, 6.0 times; peptone, 3.0 times; water, 2.4 times). Thus, within 5 to 7 h, the IMS-CTC-FAb method detected greater numbers of E. coli O157 cells than were detected by plating. The results show that the IMS-CTC-FAb technique with enumeration by either fluorescence microscopy or solid-phase laser scanning cytometry gave results that compared favorably with plating following IMS.

Polystyrene/Fe3O4 composite latex via miniemulsion polymerization‐nucleation mechanism and morphology

“…5,6 For example, the magnetic property of Fe 3 O 4 and catalytic activity property of TiO 2 were widely well known and useful, and the inorganic particles could be coated on the polymer surface or be encapsulated in the interior of polymer matrix. Among variety of composite particles, magnetic polymer particles have been investigated enormously because of their diversified applications in cell separation, 7 food analysis, 8 and targeting drug delivery. 9,10 For magnetic polymer particles, a variety of encapsulation techniques have been developed including conventional emulsion polymerization, [11][12][13][14] precipitation polymerization, 15 suspension polymerization, 16 seeded polymerization, 17,18 soapless emulsion polymerization, 19,20 miniemulsion polymerization, [21][22][23] and so on.…”

Rapid detection of Escherichia coli in waters using fluorescent in situ hybridization, direct viable counting and solid phase cytometry

“…). The enumeration of stained fluorescent cells with SPC such as the Scan RDI instrument offers an ultra-sensitive quantification of targeted cells for the detection of rare cells in the water volume analysed (generally 100 ml), such as E. coli or more generally, as pathogens (Pyle et al 1999;Pougnard et al 2002;Cools et al 2005;West et al 2006;Aurell et al 2004). Our results showed that DVC-FISH performed with either single or two monolabelled probes is not efficient to allow cell detection by SPC.…”

Thermally responsive complex polymer networks containing Fe3O4 nanoparticles: Composition/morphology/property relationship

“…11,[15][16][17] Hence, these gels can be expected to act as intelligent materials used in many fields, such as controlled or targeted drug release, 16,18,19 extraction, 20,21 and enzyme activity control. 22 Besides the intelligent polymeric networks, more and more attention has been drawn to the preparation of different kinds of magnetic polymeric composites in the past decades because of the applications of magnetism to various fields, such as cell separation, 23 protein purification, 24 targeted drug delivery, 25 environmental and food analyses, 26 organic and biochemical syntheses, 27 and industrial water treatment. 28 Endowed with unique magnetic properties that turn on upon the application of a strong magnetic field and turn off upon the removal of the field, superparamagnetic nanoparticles attract great interest in the form of polymeric composites.…”