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.

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