A microporous polyethylene disposable optical film (DOF) that is mostly transparent to infrared light was used to characterize bacterial strains by Fourier transform infrared (FT-IR) spectroscopy for subsequent classification by principal components analysis (PCA) and hierarchical cluster analysis (HCA). Developing a methodology with a disposable infrared substrate is desirable for the FTIR measurement of pathogenic bacteria. The bacteria studied were <i>Escherichia coli</i> HB101, <i>E. coli</i> ATCC 43888, a wild-type <i>E. coli</i>, and one strain each of <i>Pseudomonas aeruginosa</i>, <i>Bacillus cereus</i>, and <i>Listeria innocua</i>. The cultures were harvested by centrifugation and the wet cells (5 mg) were placed onto DOF and dried under vacuum at room temperature for one hour. For each strain, four replicates were prepared and measured on each of six different days in order to investigate the possible sources of variance in the sampling procedure. PCA obtained for second derivative, mean-centered spectra for selected frequency ranges (1312-1339, 1026-1061, and 903-930 cm<sup>-1</sup>) explained over 98.1% of the total variability using the first three principal components. The scores plots exhibited six tight clusters of 24 points each (4 replicates × 6 days). Out of a total of 144 spectra, one outlier was found. The dendrograms resulting from HCA (centroid linkage method) correctly clustered all 143 remaining samples. The analysis provided excellent discrimination between the different species and, more significantly, between the different <i>E. coli</i> strains. This work demonstrates the potential of this procedure for the rapid, repeatable, and precise classification of bacteria, with minimum sample preparation, in food safety laboratories.

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