# Anti-Bacterial Action of Plasma Multi-Jets in the Context of Chronic Wound Healing
## 2 Materials and Methods
### Biological Samples  
#### 2.2.1 Bacterial Lawn Preparation and Plasma Treatment  
As shown in Figure 3, a membrane filtration system composed of a funnel and a vacuum valve was used to prepare samples with different bacterial strains. The bacterial inoculum solution was set in the funnel, the base of which was equipped with a membrane 47 mm in diameter and having a porosity of 0.45 um. This membrane, set on a filter stone, acted as filter for the inoculum when the vacuum pump was switched on. In this work, an inoculum (10\({}^{3}\) CFU/mL) volume of 100 mL was filtered across the membrane. The membrane was then set on an agar disk (50 mm in diameter) prepared in a petri dish. Following a 24-h incubation period at 37 \({}^{\circ}\)C, a 10 cm\({}^{2}\) bacterial lawn was produced as shown in Figure 3d.  
As mentioned in the results section, the plasma treatments were performed right after the inoculated membrane was set on the agar substrate, i.e., before the 24-h incubation period, meaning before the growth of the bacterial lawn.  
The plasma treatments consisted of 1, 5 or 10-min exposure with a gap distance between multi-jet bottom base and the membrane of 5 mm, a gas flow rate of 4 L/min, peak voltage amplitude of 14 kV and pulse repetition rate of 2 kHz (both multi-spot and diffuse mode applicators were used). All experiments have been performed in triplicate. Control samples consisted in gas flow only exposure of the membrane on agar substrates for the same durations (1 to 10 min) but with no plasma ignition.  
A larger surface bacterial lawn of 12 \(\times\) 12 cm\({}^{2}\) inoculated with patients' collected resistant bacteria have also been prepared and exposed to plasma. A 100 uL volume of bacterial solution having 10\({}^{4}\) colony forming units per milliliter (CFU/mL) was spread over the surface of the agar plate. These large surface inoculated agar samples were then incubated at 37 \({}^{\circ}\)C for 24 h with or without preliminary plasma treatment. The plasma treatment with the multi-spot applicator was performed in a scanning protocol with the  
\begin{table}
\begin{tabular}{c c c c} \hline \hline  & **1 st Option** & \multicolumn{2}{c}{**2 nd Option**} & **3 rd Option** \\  & **Single Jet** & **Multi-Jets from One Primary Jet** & **Bunch of Individual Jets** \\ \hline \multirow{4}{*}{Plasma impact} & One two mm spot & 4 + 12 + 12 + 24, & Same as multi & 4/9/13 two mm \\  & One two mm spot & one mm spots & spot with & spots distributed \\  & moving over a & distributed over & additional diffuse & over a 2/3/3 in \\  & 2 cm\({}^{2}\) disk & 8/18/28/38 mm in & & volume between \\  & & diameter rings & the multi jets & diameter disk \\ \hline \hline \end{tabular}
\end{table}
Table 1: \(Cont.\)  
Figure 3: **(a)** Funnel and valve connected to a vacuum pump (not shown) used to initiate the bacteria inoculum preparation, **(b)** Top view of funnel, whose base is covered with a membrane used to filter the bacterial inoculum, **(c)** zoom on the membrane set on agar prepared in a petri dish right at the end of the filtering process, **(d)** view of the grown bacterial lawn following a 24-h incubation at 37 \({}^{\circ}\)C.  
help of a programmable displacement board allowing various scanning patterns and speeds with the fixed plasma applicator and the moving agar sample as shown in Figure 4.  
Two scanning patterns have been experienced. First was a translation pattern with back and forth linear displacements, the back and forth duration being 12 s and having an extension of 100 mm. This translation pattern was repeated respectively 5, 25 or 50 times, thus corresponding to a whole plasma treatment duration of 1, 5 and 10 min. The second pattern, zigzag-like, is shown in Figure 4c, and combined translation along the x and y axis of 100 and 2 mm, respectively. The time set to process the full pattern was one-minute long and this was repeated for 5 or 10 times, corresponding to 5 or 10-min plasma exposure durations.