Characterization of cold atmospheric plasma inactivation of individual bacterial spores using Raman spectroscopy and phase contrast microscopy

Shiwei Wang, Christopher J. Doona, Peter Setlow and Yong-qing Li

Raman spectroscopy and phase-contrast microscopy were used to examine calcium dipicolinate (CaDPA) levels and rates of nutrient and non-nutrient germination of multiple individual Bacillus subtilis spores treated with cold atmospheric plasma (CAP). Major results for this work are: 1) >5 logs of spores deposited on glass surfaces were inactivated by CAP treatment for 3 min, while deposited spores placed inside an impermeable plastic bag were inactivated only ∼2 logs in 30 min. 2) >80% of the spores treated for 1-3 min with CAP were non-culturable, and retained CaDPA in their core, while >95% of spores treated with CAP for 5-10 min lost all CaDPA. 3) Raman measurements of individual CAP-treated spores without CaDPA showed differences from spores that germinated with L-valine in terms of nucleic acids, lipids, and proteins. 4) 1-2 min CAP treatment killed 99% of spores, but these spores still germinated with nutrients or exogenous CaDPA, albeit more slowerly and to a lesser extent than untreated spores, while spores CAP-treated for >3 min that retained CaDPA did not germinate via nutrients or CaDPA. However, even after 1-3 min of CAP-treatment, spores germinated normally with dodecylamine. These results suggest that exposure to the present CAP configuration severely damages spore's inner membrane and key germination proteins, such that the treated spores either lose CaDPA or can neither initiate nor complete germination with nutrients or CaDPA. Analysis of the various CAP components indicated that UV photons contributed minimally to spore inactivation, while charged particles and reactive oxygen species contributed significantly.

DOI

Characterization of Wet-Heat Inactivation of Single Spores of Bacillus Species by Dual-Trap Raman Spectroscopy and Elastic Light Scattering

Pengfei Zhang, Lingbo Kong, Peter Setlow, and Yong-qing Li

Dual-trap laser tweezers Raman spectroscopy (LTRS) and elastic light scattering (ELS) were used to investigate dynamic processes during high-temperature treatment of individual spores of Bacillus cereus, Bacillus megaterium, and Bacillus subtilis in water.Major conclusions from these studies included the following. (i) After spores of all three species were added to water at 80 to 90°C, the level of the 1:1 complex of Ca²⁺ and dipicolinic acid (CaDPA; 25% of the dry weight of the spore core) in individual spores remained relatively constant during a highly variable lag time (T_lag), and then CaDPA was released within 1 to 2 min. (ii) The T_lag values prior to rapid CaDPA release and thus the times for wet-heat killing of individual spores of all threespecies were very heterogeneous. (iii) The heterogeneity in kinetics of wet-heat killing of individual spores was not due to differences in the microscopic physical environments during heat treatment. (iv) During the wet-heat treatment of spores of all three species, spore protein denaturation largely but not completely accompanied rapid CaDPA release, as some changes in protein structure preceded rapid CaDPA release. (v) Changes in the ELS from individual spores of all three species were strongly correlated with the release of CaDPA. The ELS intensities of B. cereus and B. megaterium spores decreased gradually and reached minima at T₁ when 80% of spore CaDPA was released, then increased rapidly until T₂ when full CaDPA release was complete, and then remained nearly constant. The ELS intensity of B. subtilis spores showed similar features, although the intensity changed minimally, if at all, prior to T₁. (vi) Carotenoids in B. megaterium spores' inner membranes exhibited two changes during heat treatment. First, the carotenoid's two Raman bands at 1,155 and 1,516 cm^–1 decreased rapidly to a low value and to zero, respectively,well before T_lag, and then the residual 1,155-cm^–1 band disappeared, in parallel with the rapid CaDPA release beginningat T_lag.

DOI