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<OL> <LI> To generate highly specific vibrational spectroscopic signature profiles of pathogenic and spore-forming bacteria commonly encountered in contaminated foods by using infrared spectroscopy. <LI> To develop multivariate classification models for the reliable and reproducible identification and classification of food-borne pathogens.
NON-TECHNICAL SUMMARY: Rapid and cost-effective techniques to detect foodborne pathogens are required to assure a safe product while preserving food quality and profitability The long-term research goal is to develop rapid, simple, sensitive and specific microbial detection strategies to identify microbe-contaminated food products. FT-IR spectroscopy provides information about the unique chemical composition of bacteria that will allow fast discrimination among species at the individual strain level present in contaminated foods.
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APPROACH: Procedure: Vegetative cells: Different pathogenic strains will be grown in Plate Count agar or selective media. The bacterial suspension will be either applied to an IR transparent polyethylene membrane or to a ZnSe window for transmittance measurements. The heterogeneity of the chemical composition of the microbial colonies will be tested by collecting the IR spectra of small microcolonies obtained after 6 to 8 h of culturing and mature colonies obtained after 18-20 h of culturing. Different methods for microbial isolation such as hydrophobic grids and immuno-magnetic beads will be evaluated. The detection limits of the IR method will be tested by preparing overnight cultures, preparing dilutions and enumerating the suspension by the standard plate count method. Bacillus spores: Spores from Bacillus species that are significant agents of food spoilage and food-borne disease will be evaluated. Spores will be harvested as described by Sala et al. (1995). The spore suspension will be subjected to High Pressure or Heat treatments to inactivate the spores. The IR spectra will be determined by applying the spore suspension to a ZnSe window for transmittance measurements. The IR spectra will be used to develop classification models to discriminate among different Bacillus spp. spores and to develop rapid predictive models for evaluation of the level of spore inactivation based on chemical information. FT-IR spectrometry FT-IR spectra will be recorded on a FTS Excalibur 3500GX Mid/Near-IR spectrometer (Digilab, Randolph, MA). The spectral resolution to be used is 4 cm-1 and to improve the signal-to-noise ratio, 128 spectra will be co-added and averaged. Multivariate Analyses Soft Independent Modeling Class Analogy (SIMCA) and KNN analysis will be carried out by Pirouette pattern recognition software (Version 3.02 for Windows NT, Infometrix, Inc., Woodinville, WA). The data will be comprised of repeated observations on culture cells and spores and also from cultures grown on subsequent days (replicates). Also, data from the different stages of the life cycle of the spore-forming Bacillus spp. will be analyzed.