2011 Annual Report
1a.Objectives (from AD-416)
To improve analytical methods to enhance and validate detection of a wide spectrum of biological and chemical threats against food supply systems.
1b.Approach (from AD-416)
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.
This project is related to in-house Objective 1A: To improve analytical methods to enhance and validate detection of a wide spectrum of biological and chemical threats against food supply systems.
A visible and near-infrared (VNIR) hyperspectral imaging technique was developed to detect and differentiate Campylobacter in Petri dishes from five primary contaminants commonly found in commercial poultry carcass rinses. A protocol for imaging the spots of bacteria growing in agar media and processing their hyperspectral images was developed such that spectral libraries of the pure bacteria were collected for development of classification and validation models. From the research based on spot-plate imaging, the research was extended to image spread plates using the known mixture formula of pure bacteria. Currently, the protocol for imaging spread plates of pure pathogenic bacteria has been established and the performance of detection is being validated with results obtained by phase contrast microscopic tests.
A short-wavelength infrared (SWIR) hyperspectral imaging system was developed to image Petri dishes in the wavelength range from 1,000 to 2,500 nanometers (nm). The developed SWIR hyperspectral imaging system was designed to extract spectral fingerprints beyond the wavelength range most commonly used for hyperspectral imaging (400 to 1000 nm). The SWIR imaging system contains a Mercury Cadmium Telluride (MCT) camera with a spectrograph, a moving stage with precision motion and location control, a sample holder, and custom application software for image acquisition via motion and camera controls. The system is also capable of imaging a Petri dish either in the reflectance or transmittance mode. Next, the potential of the system for detection of pathogens in agar media was investigated by conducting a study with a spectrometer and the SWIR imaging system. The study found the spectral region from 1,000 to 2,500 nm was not viable for detecting pathogen colonies on agar plates because the water in the agar media dominated the spectral responses.