Classification of endospores of Bacillus and Clostridium species by FT-IR reflectance microspectroscopy and autoclaving

Fourier transform infrared reflectance microspectroscopy was applied to the classification of bacterial endospores, both untreated and those pretreated with autoclaving. Mid-infrared vibrational spectra were collected following minimal sample preparation from endospores of five species of bacteria: Bacillus cereus, Bacillus globigii, Bacillus megaterium, Bacillus subtilis, and Clostridium perfringens. The amide I and amide II regions were found to provide the best classification of the native spores. For autoclaved bacterial spores, the N-H and C-H stretching regions (2700-3500 cm^-1) were found to be useful for classification. Autoclaving endospores also augmented the classification of untreated endospores in the same region. Speciation of these microorganisms was accomplished by application of principal component analysis (PCA), hierarchical cluster analysis (HCA), and soft independent method of class analogy (SIMCA). The methods described can potentially be applied by homeland security personnel to screen for pathogenic microorganisms.