The power of neutralization: the critical step for the accurate antimicrobial potential of plasma-activated water

Cold atmospheric plasma (CAP) has emerged as a promising alternative technology for water disinfection due to its strong antimicrobial activity mediated by plasma-activated water (PAW). In this study, CAP generated using a flow-through dielectric barrier discharge (DBD) reactor was evaluated for its antimicrobial efficacy against Escherichia coli and Staphylococcus aureus in model hard water, with particular emphasis on post-treatment reactivity and the need for neutralization in antimicrobial testing. CAP treatment for 3 min resulted in log reductions of 1.09 for E. coli and 3.27 for S. aureus, confirming effective microbial inactivation. Storage of PAW at 4°C for 24 h led to complete inactivation of both strains, demonstrating persistent antimicrobial activity driven by long-lived reactive oxygen and nitrogen species (RONS). Quantification of hydrogen peroxide, ozone, nitrite, and nitrate revealed significant depletion of ozone and hydrogen peroxide during storage, particularly in the presence of bacteria, indicating ongoing chemical-biological interactions. Nitrite and nitrate remained comparatively stable, suggesting a secondary or synergistic role in prolonged antimicrobial effects. The persistence of PAW activity highlighted the necessity of immediate neutralization to avoid overestimation of antimicrobial efficacy. Several chemical neutralizers recommended in standardized antimicrobial testing protocols were evaluated, with a combined "Mix" formulation (PBS, NaCl with tryptone, polysorbate 80, lecithin, and sodium thiosulphate) providing the most effective quenching of residual RONS while remaining non-toxic to bacteria. In parallel, the influence of solid culture media on bacterial recovery was assessed. We recommend combining the Mix neutralizer with non-selective or mildly selective media (NEA for E. coli and NMSA for S. aureus) to improve reproducibility and reliability in PAW antimicrobial testing. These findings contribute to methodological standardization and support the development of CAP-based water disinfection technologies.