ORIGINAL RESEARCH article
Front. Microbiol.
Sec. Antimicrobials, Resistance and Chemotherapy
This article is part of the Research TopicExpanding Multidisciplinary Potential of Cold Atmospheric Plasma Beyond Antimicrobial ResistanceView all 3 articles
The Power of Neutralization: The critical step for the Accurate Antimicrobial Potential of Plasma-Activated Water
Provisionally accepted
- 1Univerza v Ljubljani zdravstvena fakulteta, Ljubljana, Slovenia
- 2Univerza v Ljubljani Biotehniska fakulteta, Ljubljana, Slovenia
- 3Laboratory of Oxidative Stress Research, Faculty of Health Sciences, University of Ljubljana, Ljubljana, Slovenia
Select one of your emails
You have multiple emails registered with Frontiers:
Notify me on publication
Please enter your email address:
If you already have an account, please login
You don't have a Frontiers account ? You can register here
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.
Keywords: Antibacterial, cold atmospheric plasma, Disinfection, neutralization, Plasma activated water
Received: 24 Dec 2025; Accepted: 09 Feb 2026.
Copyright: © 2026 Lunder, Dahle, Poljšak and Fink. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
* Correspondence: Rok Fink
Disclaimer: All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher.