Flow cytometric assessment of Bacillus spore response to high pressure and heat A. Mathys a, ⁎ , B. Chapman b,1 , M. Bull b,1 , V. Heinz c,2 , D. Knorr a,3 a Berlin University of Technology, Department of Food Biotechnology and Food Process Engineering, Koenigin-Luise-Str. 22, D-14195 Berlin, Germany b Commonwealth Scientific and Industrial Research Organisation, Food Futures National Research Flagship, P.O. Box 93, North Ryde 1670, NSW, Australia c German Institute of Food Technology, P.O. Box: 1165, D-49601, Quackenbrück, Germany Received 7 December 2006; accepted 18 June 2007 Abstract The physiological response of Bacillus licheniformis spores to high pressure and thermal inactivation in sodium citrate buffer and nutrient broth was investigated using multiparameter flow cytometry. Spores were treated by heat-only at 121 °C, by high pressure at 150 MPa (37 °C), or by a combined high pressure and heat treatment at 600 MPa and 77 °C, and then dual stained with the fluorescent dyes SYTO 16 and propidium iodide (PI). For pressure treated spores, but not heat-only treated spores, four distinct sub-populations were detected by flow cytometry, and for these we suggest a three step model of inactivation involving a germination step following hydrolysis of the spore cortex, an unknown step, and finally an inactivation step with physical compromise of the spore's inner membrane. © 2007 Elsevier Ltd. All rights reserved. Keywords: Flow cytometry; High pressure; Spore germination; Spore inactivation Industrial relevance: This preliminary study offers a simple and fast flow cytometric method for the rapid assessment of the physiological state of bacterial spores following high pressure and thermal processing. An improved understanding of the mechanisms of spore inactivation will aid in the food safety assessment of pressure assisted thermal sterilisation in particular, and also assist in the commercialisation of these processes facilitating adoption by industry. 1. Introduction Many years before the flow cytometer became a practical instrument for microbiological research, Gucker, O'Konski, Pickard and Pitts (1947) developed an instrument for the analysis of dust particles, primarily for determining the efficiency of gas mask filters. In collaboration with the United States Army this device, often quoted as the first flow cytometer (Shapiro, 1988), was also applied for the detection of airborne bacterial spores during World War II. Interestingly, the application of flow cytometry for the detection and identification of bacterial spores has again come into favour, particularly with reference to the potential biological weapon Bacillus anthracis (Laflamme, Lavigne, Ho, & Duchaine, 2004; Stopa, 2000). However, flow cytometry has still only rarely been applied to the analysis of the physiological state of bacterial spores, with the first detailed flow cytometric studies of Bacillus spores published in 1995 (Ueckert et al., 1995) and 2000 (Stopa, 2000). Even more recently, the first use of the functional dyes 3,3′-dihexyloxacarbocyanine iodide [DiOC 6 (3)] and bis-(1,3- dibutylbarbituric acid) trimethine oxonol [DiBAC 4 (3)] was reported for the flow cytometric assessment of membrane potential activation in the inner membrane of spores germinated by nutrients (Laflamme, et al., 2005). de Vries (2006) used fluorescent esterase markers (5,[and 6]-Carboxyfluoresceindia- cetate [cFDA]) and DNA-binding dyes (SYTO BC) for the quantification of individual germination events in Bacillus spores. Available online at www.sciencedirect.com Innovative Food Science and Emerging Technologies 8 (2007) 519 – 527 www.elsevier.com/locate/ifset ⁎ Corresponding author. Tel.: +49 30 314 71250; fax: +49 30 832 76 63. E-mail address: alexander.mathys@tu-berlin.de (A. Mathys). 1 Tel.: +61 2 9490 8333; fax: +61 2 9490 8499. 2 Tel.: +49 5431 183 232; fax: +49 5431 183114. 3 Tel.: +49 30 314 71250; fax: +49 30 832 76 63. 1466-8564/$ - see front matter © 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.ifset.2007.06.010