Development of rapid molecular methods for the detection of food-borne pathogens based on immunostaining and physiological profiling using  FACS technology

The use of flow cytometry (FCM) and fluorescence activated cell sorting (FACS) for food applications has received little research attention. In this thesis, novel FCM and FACS methods were developed for the detection and determination of physiological status of pathogenic microorganisms, chiefly Staphylococcus aureus. Initially, FCM and FACS methods were used to correlate the physiology of bacteria with viability on traditional agars. Two commercially available species specific monoclonal antibodies were identified as capable of tagging S. aureus and amplification of the primary antibody-antigen reaction was enabled using a phyocerythrin (PE) labelled secondary antibody. This formed the prototype immuno-FCM assay and was further evaluated in a number of studies. The effects of food processing stressors on antibody binding were assessed using physiological indicators such as SYTO 9/ propidium iodide, DiOC2(3) and calcein acetoxymethylester. Different degrees of physiological heterogeneity were revealed using these various probes and chemical stressors were also shown to interfere with antibody binding in broth systems. In food systems, immuno-FCM assays compared favourably with existing microbiological plate count methods but were dependant on the populations of the bacterium in foods. FACS was shown to be essential for validation of this novel methodology by allowing confirmation of species detection and physiological status of microorganisms by sorting directly onto agar plates. This novel immuno-FCM method for S. aureus developed may prove to be a potential commercial advantage for the Irish food industry, especially the chilled meals sector which requires such rapid assays.