# Multimodal Comparison of Cold Atmospheric Plasma Sources for Disinfection
## 2 Setup  
The setup (Figure 1) is designed to unify all diagnostics in a single treatment protocol. Above a grounded metallic table, a 35 mm diameter glass Petri dish is positioned. A set of spacers is fixed on the table such that the dish is always placed back in the exact same position. A metallic flange is fixed in place above the table with a set of custom supports, and used to sustain the different plasma sources. Custom 3D-printed adapters are designed for each source, such that each source tip is positioned at the same distance from the bottom of the Petri dish. The treated sample consists of 6 mL of ultrapure water (VWR Purnity TU 3 UV/UF+); the choice of ultrapure water reflects the aim of simplifying as much as possible the chemistry involved, and avoid complex reactions and species which can be generated in standard culture media when interacting with the plasma; such a large sample volume, with respect to the microlitre scales usually reported in the literature [8], was chosen to prevent liquid overheating and evaporation while better simulating real-world applications. The distance between the source nozzle and the water surface is fixed at 6 mm. The treatment duration was fixed at 10 min for all samples. The bacteria abatement capabilities of the different sources have been evaluated using _Escherichia coli_ (K-12, MG1655) as a model organism. Samples of bacteria, selected in their exponential phase after overnight incubation and reactivation, and centrifuged to remove the growth medium (Luria-Bertani broth), are resuspended in ultrapure water immediately before the treatment. The initial concentration is estimated to be around \(10^{6}\)-\(10^{7}\) CFU/mL. The treated solutions are then sampled just after the treatment, serially diluted, and inoculated onto agar plates. After overnight incubation, the colonies are counted and the colony-forming units (CFU) estimated. The results are reported in terms of logarithmic abatement:  
\[\text{logAbat}:=\log_{10}\frac{\text{CFU}_{\text{control}}}{\text{CFU}_{\text{ treated}}}\]  
Multiple verification tests have been carried out to confirm that the various steps of the protocol do not affect cell viability. No significant CFU variations have been observed even when performing the entire protocol, including exposure to the sources and gas flows, if the plasma is not switched on. All measurements reported in this work are the results of at least three independent experiments, and the uncertainties in the final values are estimated by statistically combining the standard deviation of the replicates with the nominal uncertainty of each method. Figure 1: Schematic of the setup. The plasma source treats the liquid present in the Petri dish. The source can be replaced with the other ones, keeping the surrounding setup fixed. These results will be labelled in the following as _direct_ treatments. Another set of measurements, labelled as _indirect_ treatments, has been carried out following the same protocol previously described, but inoculating the bacteria in the water just after, and not just before, the plasma treatment. Mimicking the _direct_ protocol, ultrapure water has been treated for 10 min. Immediately after, the bacteria sample is injected in the water and left in the setup for 10 min (without plasma). The protocol is then resumed with serial dilutions and inoculation in Agar dishes. By exploring the causal chain which connects the plasma to the bacteria, the treated water has been characterised from a physiochemical point of view. A fast measurement of the activation of the treated water is performed by testing the oxidation-reduction potential (ORP) immediately after the treatment with a dedicated probe (Metrohm, Herisau, Switzerland). The measurement of pH proves to be more challenging because, due to the low conductivity of the solutions (below 100 uS/cm), the use of standard probes results in unreliable data [25]. A rough estimation of the pH, however, is provided in Section 4, based on indicator strips and observations on the chemistry.