Optical Detection of Microbial Contamination in Food Matrices

Approach:
A collection of category B bacterial pathogens are maintained at the USDA-ARS, Richard B. Russell Research Center, Poultry Microbiological Research Unit. Bacterial cultures were isolated from poultry samples consisting of either whole carcass rinses or fecal/cecal specimens from conventionally-reared broiler chickens or processing plants. The collection of category A bacterial pathogens are maintained at the USDA-ARS, Richard B. Russell Research Center, FSIS, Federal Response Emergency Network (FERN) Laboratory. We are collaborating with FERN and they maintain ownership of the pathogens. All work will be conducted at the Bio-Safety Level 2 and a Standard Operation Procedure (SOP) has been written and reviewed by Ms. Frankie J. Beacorn, Biosecurity Officer, USDA, FSIS, OPHS, FERN Division, Athens, GA and Joseph P. Kozlovac, ARS Biosafety Officer, Beltsville , MD. Stock cultures of non-virulent strains of four pathogen (two category B) and bio-threat agents (two category A) will be grown in solution and enriched to at least three concentrations. Additionally, common background microflora will also be grown and enriched. The pathogens, bio-threat agents, and background microflora will all be spot plated on appropriate growth media. Hyperspectral imaging systems (400-900 nm, 1000-2500 nm, and hyperspectral microscope) will be used to collect spectral libraries of the organisms and growth media. Multivariate models will then be developed to classify the various organisms. Additionally, the enriched solutions of the organisms will also be measured with a Fourier-transform Infrared (FTIR) spectrometer and a Raman spectrometer and multivariate models once again developed. Further modeling will then be used to determine the minimum detection level for all pathogens and bio-threat agents. Once reasonable results are obtained for pure cultures, important food matrices will be spiked with pathogens and bio-threat agents at varying concentration levels and hyperspectral images will be collected on both the total contaminated food matrices and on a rinsate from the infected food matrices. Multivariate calibration models, based on the spectral libraries of the bio-threat agents and food matrices, will then be developed and the lower limit of detection for each pathogen and bio-threat agent will be determined.