# Low Temperature Plasma Strategies for
## 3 Results
### XfDD Growth Conditions and Plasma Treatments  
Treatment of Agar Surface-Grown Bacteria  
Preliminary investigations showed that the plasma pre-treatment of the agar plates did not alter the growth of XfDD cells (data not shown). When bacteria (10\({}^{7}\) CFU mL\({}^{-1}\)) were plated out on Buffered Charcoal Yeast Extract (BCYE) agar and exposed to SDBD for 200 s, complete inhibition of cell growth was observed, whereas after 100 s and 10 s of exposure (Figure 6) there was less pronounced but time-dependent inhibition. Since it is difficult to quantify the effect of plasma treatment by counting colonies at a concentration of 10\({}^{7}\) CFU mL\({}^{-1}\) (Figure 7), decimal dilutions from 10\({}^{5}\) to 10\({}^{3}\) CFU mL\({}^{-1}\) of XfDD inoculum were applied to the BCYE agar plates in subsequent experiments (trials). Figure 5: Emission spectra of SPS. Figure 6: Time-dependent inhibition of XfDD growth by plasma exposure of cells discharged 1 day after plating. Photos were taken after 15 days post-exposure. The figure reports typical results for cells untreated (**A**) and treated for 10 (**B**), 100 (**C**), and 200 (**D**) s.  
Final SDBD plasma treatment conditions were therefore performed on cells grown 1-5 days after seeding, with a maximum exposure time of 200 s.  
Table 1 shows the results for three different dilutions (\(10^{3}\), \(10^{4}\) and \(10^{5}\)) for untreated and 200 s treated plates pre-cultured for one or five days before LTP exposure. The treatment was effective and completely removed all bacteria by plasma, at both 1 and 5 days. When plasma interacts with a surface, its energy (in the form of ions, RONS and UV radiation) is deposited in the exposed matrix. Multiple exposures result in a cumulative effect of absorbed doses leading to progressive destruction of the microorganism. To evaluate the LTP dose-effect, we performed a repetitive plasma treatment of 200 s on plates pre-cultured for seven days before LTP exposure with two different frequencies: 1 treatment per day for a period of 10 days (Trial a, treatment performed in days from 0 to 9) and once per week for a period of three weeks (Trial b, treatment performed in days 0, 7, 15). The results of these experiments are given in Table 2.  
If we compare the log growth of NT samples to treated samples, we can observe that in Trial (a) a strong effect of cumulative dose was observed, with complete removal of bacteria at the lowest initial concentration (\(10^{3}\) CFU mL\({}^{-1}\)) and almost a LOG 3 lower growth for higher concentration. In Trial (b), cells were counted just after the last treatment, on day 15, and on day 20 after the first LTP treatment. Treatment resulted on day 20 in lower growth of almost log 4 for the \(10^{3}\) concentrations and log3 for the \(10^{4}\), while at \(10^{5}\) \begin{table}
\begin{tabular}{c c c c c} \hline \hline  & **Time** & **N of Replicas** & **11 Days CFU mL\({}^{-1}\)** & **13 Days CFU mL\({}^{-1}\)** \\ \hline \(\mathbf{10^{5}}\) & & 3 & \(3.81\times 10^{8}\pm 3.7\times 10^{6}\) & \(9.79\times 10^{8}\pm 8.7\times 10^{6}\) \\ \(\mathbf{10^{4}}\) & untreated & 3 & \(4.17\times 10^{5}\pm 2.7\times 10^{5}\) & \(2.17\times 10^{7}\pm 3.8\times 10^{5}\) \\ \(\mathbf{10^{3}}\) & & 3 & 0.00 & \(1.92\times 10^{5}\pm 3.8\times 10^{4}\) \\ \hline \(\mathbf{10^{5}}\) & & 3 & 0.00 & 0.00 \\ \(\mathbf{10^{4}}\) & T0 & 3 & 0.00 & 0.00 \\ \(\mathbf{10^{3}}\) & & 3 & 0.00 & 0.00 \\ \hline \(\mathbf{10^{5}}\) & & 3 & 0.00 & 0.00 \\ \(\mathbf{10^{4}}\) & T5 & 3 & 0.00 & 0.00 \\ \(\mathbf{10^{3}}\) & & 3 & 0.00 & 0.00 \\ \hline \hline \end{tabular}
\end{table} 
Table 1: Results of the experiment performed on agar surface grown XfDD. Treatment time was fixed at 200 s following the finding of the previous experiment. We performed experiments in triplicate for a total of 9 dishes each experiment. The experiments were conducted on plates just at T0 = 0 and T5 = 5 days after XfDD seeding.