Monitoring the Wet-Heat Inactivation Dynamics of Single Spores of Bacillus Species by Using Raman Tweezers, Differential Interference Contrast Microscopy, and Nucleic Acid Dye Fluorescence Microscopy

Abstract

Dynamic processes during wet-heat treatment of individual spores of Bacillus cereus, Bacillus megaterium, andBacillus subtilis at 80 to 90°C were investigated using dual-trap Raman spectroscopy, differential interference contrast (DIC) microscopy, and nucleic acid stain (SYTO 16) fluorescence microscopy. During spore wet-heat treatment, while the spores' 1:1 chelate of Ca 2؉ with dipicolinic acid (CaDPA) was released rapidly at a highly variable time T lag , the levels of spore nucleic acids remained nearly unchanged, and the T lag times for individual spores from the same preparation were increased somewhat as spore levels of CaDPA increased. The brightness of the spores' DIC image decreased by ϳ50% in parallel with CaDPA release, and there was no spore cortex hydrolysis observed. The lateral diameters of the spores' DIC image and SYTO 16 fluorescence image also decreased in parallel with CaDPA release. The SYTO 16 fluorescence intensity began to increase during wet-heat treatment at a time before T lag and reached maximum at a time slightly later than T release . However, the fluorescence intensities of wet-heat-inactivated spores were ϳ15-fold lower than those of nutrient-germinated spores, and this low SYTO 16 fluorescence intensity may be due in part to the low permeability of the dormant spores' inner membranes to SYTO 16 and in part to nucleic acid denaturation during the wet-heat treatment.Spores of Bacillus species are formed under starvation conditions and are metabolically dormant and extremely resistant to a variety of physical and chemical agents, including heat, radiation, and many chemical disinfectants (35). Since spores of many of these species can cause food spoilage and foodborne diseases (37), efforts to eliminate spores from foods are routinely made. High-temperature treatment of hydrated spores is very effective in spore killing and is the most common method used by the food industry for spore inactivation, even though hydrated dormant spores invariably resist much higher temperatures than do the growing cells of the same strain (40). Factors that contribute to spore wet-heat resistance include (i) the spore core's low water content (11,20,34), (ii) the core's high level of pyridine-2,6-dicarboxylic acid (dipicolinic acid [DPA]) plus its chelated divalent cations, predominantly Ca 2ϩ

Cold Atmospheric Air Plasma Sterilization against Spores and Other Microorganisms of Clinical Interest

“…The development of tools that can identify and quantify relevant plasma species is required, in combination with biochemical studies, for the investigation of the inactivation mechanism for microorganisms evoked by CAP. Therefore, physiological studies that combine different methods for the provision of multimodal information, e.g., on a single bacterial spore in situ, are supportive (22), as described recently for wet-heat inactivation dynamics (50). CAP studies of mutants that lack specific proteins that confer resistance are especially necessary for better understanding.…”

Inactivation of bacterial spores subjected to sub-second thermal stress

“…A second mechanism of inactivation is through permeabilization of the spore membrane, cortex and coat (encoded by genes like cotE), as supported by several lines of evidence [3,[39][40][41][42][43][44]. Damage to spore compartments after heating has been shown with compartment-specific staining techniques [45,46]. While the spore coat was identified as the primary barrier to oxidizing agents (hydrogen peroxide, hypochlorite, ozone, etc.)…”

Variability in DPA and Calcium Content in the Spores of Clostridium Species

“…It is presumed that the spores with the longest lag phase are the most resistant to wet heat. However, molecular, physiological, or environmental factors underpinning this variability in spore populations are poorly understood (Coleman et al, 2007; Zhang et al, 2010, 2011). …”