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bAtmospheric cold plasma (ACP) is a promising nonthermal technology effective against a wide range of pathogenic microorganisms. Reactive oxygen species (ROS) play a crucial inactivation role when air or other oxygen-containing gases are used. With strong oxidative stress, cells can be damaged by lipid peroxidation, enzyme inactivation, and DNA cleavage. Identification of ROS and an understanding of their role are important for advancing ACP applications for a range of complex microbiological issues. In this study, the inactivation efficacy of in-package high-voltage (80 kV [root mean square]) ACP (HVACP) and the role of intracellular ROS were investigated. Two mechanisms of inactivation were observed in which reactive species were found to either react primarily with the cell envelope or damage intracellular components. Escherichia coli was inactivated mainly by cell leakage and low-level DNA damage. Conversely, Staphylococcus aureus was mainly inactivated by intracellular damage, with significantly higher levels of intracellular ROS observed and little envelope damage. However, for both bacteria studied, increasing treatment time had a positive effect on the intracellular ROS levels generated.A tmospheric cold plasma (ACP) refers to nonequilibrated plasma generated at near-ambient temperatures and pressure. ACP is composed of particles, including free electrons, radicals, and positive and negative ions, but it is low in collision frequency of gas discharge compared to that with equilibrated plasma (1, 2). ACP technologies have widely been applied for many surface treatments and environmental processes. Recently, they have been studied for food sterilization and plasma medicine (2-5).ACP provides inactivation effects against a wide range of microbes, mainly by the generation of cell-lethal reactive species (6-8). By discharging in air, groups of reactive species are generated, such as reactive oxygen species (ROS), reactive nitrogen species (RNS), UV radiation, energetic ions, and charged particles (5). However, the inactivation efficacy can be varied by changing the working gases, which results in different types or amounts of reactive species generated (9-11). ROS are often identified as the principal effecting species, with a relatively long half-life and strong antimicrobial effects, which are generated in oxygen-containing gases (12).ROS generated during plasma discharge in air or oxygen-containing mixtures are assemblies of ozone, hydrogen peroxide, and singlet and atomic oxygen, while ozone is considered the most microbicidal species (13). With strong oxidative stress, cells are damaged by lipid peroxidation, enzyme inactivation, and DNA cleavage. The generation of plasma in air or a nitrogen-containing gas mixture can also generate NO x species. However, higher inactivation efficacy has been reported with the combined application of NO and H 2 O 2 on Escherichia coli than that with a treatment with NO or H 2 O 2 alone (14). Reactive nitrogen species are highly toxic and can lead to cell death by increas...