title: Comparison of antibody-direct epifluorescent filter technique with the most probable number procedure for rapid enumeration of Listeria in fresh vegetables.
abstract: The antibody-direct epifluorescent filter (Ab-DEFT) technique was evaluated as a rapid alternative to the most probable number (MPN) method for enumeration of artificially inoculated Listeria monocytogenes in ready-to-eat packaged salads and other fresh vegetables. Ab-DEFT was performed by homogenization of food in mesh-lined Stomacher bags, followed by prefiltration of homogenate through a 5 microns pore nylon filter, and passage of filtrate through a 0.4 micron pore black polycarbonate filter to collect and concentrate Listeria cells. After cells were stained with a fluorochrome-labeled polyclonal antibody to Listeria, the filter surface was examined by epifluorescence microscopy, and fluorescent cells were counted. A 3-tube MPN procedure was performed by successive enrichments of homogenized foods in Listeria enrichment and Fraser broths, followed by selective plating. Ab-DEFT provided quantitative determinations of Listeria cells that correlated with plate counts and MPN estimates in a linear response over a range of cell concentrations from 10 to 10(7) colony forming units (CFU)/mL. Microbial backgrounds as high as 10(8) CFU/mL did not affect performance of Ab-DEFT. In contrast to the MPN method, which required 5 days to perform, quantitation by Ab-DEFT could be completed in less than 1 h. Despite cross-reactivities demonstrated by the polyclonal fluorescent antibody, the potential of Ab-DEFT as a rapid alternative to MPN for microbial cell enumeration was evident.
authors.affiliation: U.S. Food and Drug Administration, National Center for Food Safety and Technology, Summit-Argo, IL 60501, USA.
authors.forename: M L
authors.initials: ML
authors.lastname: Tortorello
authors.forename: K F
authors.initials: KF
authors.lastname: Reineke
authors.forename: D S
authors.initials: DS
authors.lastname: Stewart
mesh_terms: D000918:Antibody Specificity; D001431:Bacteriological Techniques; D015169:Colony Count, Microbial / Q000379:methods; D005374:Filtration / Q000295:instrumentation; D005455:Fluorescent Antibody Technique; D008089:Listeria monocytogenes / Q000302:isolation & purification*; D011336:Probability; D013997:Time Factors; D014675:Vegetables / Q000382:microbiology*