Development of a Fluorescence-based Microplate Assay to Screen Strains of Listeria Monocytogenes for Surface Attachment
Abstract
Our objectives were to develop a fluorescence-based method to screen for strains of L. monocytogenes attached to microplate surfaces and to establish optimum conditions for its evaluation. Four strains of L. monocytogenes were tested at various incubation temperatures (25o, 30o, and 37oC) to determine a good screening regimen. A Biotec Elx405 Magna plate washer was used to remove planktonic cells and wash attached cells. Retained cells were incubated at various time/temperatures with 5,6-carboxyfluorescein diacetate (5,6-CFDA) to determine optimal detection of attached cells using four laboratory strains of L. monocytogenes. Fluorescence from the internalized fluorescing substrate byproduct was determined with a Tecan GENios fluorescent plate reader. Attachment of additional strains was examined using this procedure and identified strong and weakly adhering strains showing > 50-fold difference in fluorescence measurements that was further confirmed by microscopic analysis and enzymatic detachment that allowed enumeration of the retained cells on microbiological media.
Collections
- OSU Theses [15752]