USE OF ATTENUATED TOTAL REFLECTANCE INFRARED MICROSPECTROSCOPY TO DISCRIMINATE BACILLUS SPORES S. DE LAMO-CASTELLVÍ 1,3 and L.E. RODRÍGUEZ-SAONA 2 1 Departament d’Enginyeria Quimica, Universitat Rovira i Virgili, Avinguda Païssos Catalans, 26 campus Sescelades, Tarragona 43007, Spain 2 Department of Food Science and Technology, The Ohio State University, Columbus, OH 3 Corresponding author. TEL: +34-977-559-673; FAX: +34-977-559-621; EMAIL: silvia.delamo@urv.cat Accepted for Publication February 17, 2011 doi:10.1111/j.1745-4565.2011.00313.x ABSTRACT In the present study, the potential of Fourier-transform infrared microspectroscopy to differentiate and discriminate Bacillus spp. spores that sporulated in two nonse- lective media were evaluated. Spore crops of Bacillus subtilis (ATCC 11779), Bacillus thuringiensis (ATCC 13368, ATCC 35866 and ATCC 55177), Bacillus amyloliquefa- cies (ATCC 496763) and Bacillus stearothermophilus (ATCC 7953) were obtained with Nutrient and Trypticase Soy Agar supplemented with 10 ppm of MnSO4.H2O. Spores were sonicated, washed with deionized water, and pellets (10 8 cfu/mL) were deposited onto the grids of hydrophobic membrane filters and dried to produce a uniform and thin film. Spectra were collected in the attenuated total reflectance mode in the mid-infrared region (4,000–700 cm -1 ). Our classification models, soft independent modeling of class analogy obtained from derivatized infrared spectra (1,800–900 cm -1 ), showed that the discrimination among spore crops was mainly attributed to infrared frequencies of spore coat proteins. PRACTICAL APPLICATIONS Despite regulatory efforts and emergence of new processing technologies, food- related illnesses remain a major concern for consumers and producers. Detection of bacterial spores using conventional methods is time consuming and challenging. Therefore, there is a need to develop rapid, simple and cost-effective methods to detect Bacillus spores. Fourier-transform infrared microspectroscopy is a viable technology to meet these demands. INTRODUCTION Bacillus and Clostridium spores are very resistant to variety of treatments including heat, desiccation, radiation, pressure and chemicals (Gerhardt and Marquis 1989; Gould 2006). Bacterial spores are challenging to detect and enumerate by conventional methods (Gould 2006). Several techniques such as pyrolysis/mass spectrometry (Ryzhov et al. 2000), enzyme- linked immunosorbent assays (Smith and Ulrich 1983), pyrolysis-gas chromatography-ion mobility spectrometry (Snyder et al. 1999) and molecular approaches (Fasanella et al. 2003; Guidi et al. 2010) have been tested, but these pro- cedures require special personnel training and may not be easily adapted to routine testing. Therefore, there is a need to develop rapid, simple and cost-effective methods to detect them. Fourier-transform infrared (FT-IR) spectroscopy is a simple technique that has been used to detect and identify vegetative cells and spores (Ngo-Thi et al. 2003). For instance, Helm and Naumann (1995) have studied the formation of endospores in Clostridia and Bacilli by FT-IR. Attenuated total reflectance infrared microspectroscopy (IRMS), com- bined with multivariate data analysis could be an interesting technique to detect and discriminate Bacillus spores due to its potential of detecting subtle compositional differences between microorganisms (Baldauf et al. 2006; Grasso et al. 2009a; De Lamo-Castellví et al. 2010). The aim of this research was to evaluate the potential of using attenuated total reflectance IRMS to discriminate among Bacillus spore crops sporulated on different media composition using hydropho- bic membranes as a support. Journal of Food Safety ISSN 1745-4565 401 Journal of Food Safety 31 (2011) 401–407 © 2011 Wiley Periodicals, Inc.