# Anti-Bacterial Action of Plasma Multi-Jets in the Context of Chronic Wound Healing
## 3 Results
### Inactivation of Staphylococcus aureus
Two main reasons may be invoked to explain this observation: first, as discussed in Figure 5, the multi jets on the center are more intense; and, secondly, for a constant rotation speed of the sample, the plasma exposure duration per surface unit is obviously shorter for the larger diameter circles. The key role of the treatment time on the inhibition zone size is confirmed in Figure 7f, where a one-minute multi-jet exposure was processed in a static mode, i.e., without a sample rotation. This short exposure is enough to inactivate the bacteria, but the inhibition spots are much smaller and correlate with the 5 min and rotating sample observations in Figure 6e along the two larger diameter inhibition circles. The use of the multi-spot plasma exposure protocol including the rotation of the sample, demonstrates that the increase of the number of secondary jets for larger diameters is convenient to expand the plasma applicator action on large surface and that scanning of the sample at moderate speed is efficient as will be confirmed in Section 3.4. The optimal protocol results from the balance between the goal to set a high scanning speed to allow for multi pass over the sample during a short plasma treatment duration and the limitation imposed by the necessity to prevent too strong helium gas flow disturbance at the outlets of the channels of the applicator. This latter point depends on the helium gas flowrate and the design (length and diameters) of the channels. Another strategy assessed in this work, was to generate multi-jets in a more helium-rich volume to try to achieve a more diffuse and larger volume plasma discharge at the surface of the sample. The Figure 8a presents the diffuse mode plasma treatment of _S. aureus_ inoculated membrane set on agar substrates, in various experimental conditions. Figure 8b-d present the bacteria inactivation for 1, 5 and 10-min exposures in static mode with the diffuse mode applicator. Three main observations can be done. First, the inhibition diameter is time exposure-dependent, indicating that the inactivation is not only the result of the direct impact of the plasma on the sample but that the plasma-induced effect includes either some diffusion mechanism or bystander processes previously speculated for plasma cancer treatment experiment analysis [51; 52]. In this work, the results discussed from Figure 6 showing that the reactive species delivery in agar  
Figure 8: Inactivation of _S. aureus_ with the diffuse mode plasma applicator. **(a)** Diffuse mode plasma treatment of inoculated membrane on agar substrate, H24 images of the sample exposed at H0 to 1 (**b**), 5 (**c**) and 10 (**d**) minute plasma treatment, **(e)** H24+10 min image of the sample exposed at H24 to 10 min plasma treatment. H0 and H24 stand hour zero as the time origin and 24 h later, respectively. sample was plasma exposure duration dependent tend to support that the inhibition zone diameter increase is likely due to the diffusion of the reactive species. Nevertheless, as during plasma jet exposure, the bacteria are also submitted to intense transient electric field during each voltage pulse, it may be suspected that the accumulation of these electric field stimulation in combination with the delivery of reactive species may play a role in the gradual spread on the inhibition zone. Indeed, the electric field driven by the space charge at the tip of the ionization wave front, such as developing for kHz plasma jets, combines a longitudinal and a radial component, the latter exhibiting a decaying intensity versus the distance from the plasma jet axis [45; 53; 54; 55; 56; 57]. With our experimental set-up, and with a slightly higher peak voltage amplitude applied to the plasma reactor 16 kV instead of 14 kV in this work, electric field with amplitudes ranging from 3 to 8 kV/cm were reported along the plasma jet inside the capillary [45], while amplitude as high as 45 kV/cm were measured at the tip of the plasma plume [55].