## 2 Materials and Methods
### Plasma Source and Specimens' Treatment
#### 2.2.1 Plasma-Processed Air (PPA) Generation and Plasma-Treated Water (PTW) Functionalization
Furthermore, small amounts of hydrogen peroxide (H\({}_{2}\)O\({}_{2}\)), a reactive oxygen species (ROS), were detected [39]. Both RNA and ROS are known as antimicrobial agents. PPA was subsequently used to functionalize distilled, sterile water to produce PTW (Figure 1B). The volume of water used was 10 mL in a 1-L glass bottle. A volume of 1.3 mL was used for PTW treatment of the inoculated PE strips. The most important antimicrobial components of PTW are nitrite (NO\({}_{2}\)\({}^{-}\)), nitrate (NO\({}_{3}\)\({}^{-}\)) and H\({}_{2}\)O\({}_{2}\)[39]. A plasma-on time of 5, 15 or 50 s was selected for the single treatment of the inoculated PE strips with antimicrobial functionalized PPA (pre-treatment time). In these time windows, the compressed air was functionalized to PPA. Subsequently, the samples were treated with the functionalized PPA for 1, 3 or 5 min (post-treatment time). The post-treatment time was stopped by means of pure compressed air introduced into the sample-loaded reaction chamber. Thus, the PPA was displaced. This step was repeated twice in a row. The treatment chamber had a volume of 4.51 (270 \(\times\) 185 \(\times\) 150 mm\({}^{3}\)), and the samples were fixed freestanding in the chamber to keep the contact areas as small as possible. The inactivation of the yeast cells thus depended on the amount of short- and long-lived reactive chemical species (RONS) produced during the pre-treatment time and which could act as a fungicide during the post-treatment time with PPA. For reference, the post-treatment time was given as 0 min, which means that these samples were exposed to an airflow instead of a PPA flow. PTW Treatment  
With the help of PPA, distilled, sterile water was functionalized to PTW. For this purpose, the produced PPA was introduced into a 1-L glass bottle with 10 mL water. Here too, the plasma-on time was 5, 15 or 50 s (pre-treatment time), and the subsequent post-treatment time was again limited to 1, 3 or 5 min. During this post-treatment time, the antimicrobial functionalized PTW could act on the inoculated PE strips. Therefore, a volume of 1.3 mL PTW was added to the prepared PE strips in the screw cap tubes to start the inactivation process. Stopping the post-treatment time was done by decanting the PTW. Finally, the PTW-treated PE strips were transferred to 10 mL nutrient broth (tryptic soy broth). The nutrient broth has a very high buffer capacity, which prevented any chemical reaction. The surviving/proliferating yeast cells were then detected as described in Chapter 2.3. The observed inactivation of yeast cells depended on incubation with long-lived reactive species (RONS) in PTW with low pH during the post-treatment time. Combined Treatment  
To demonstrate possible additive and synergistic effects of a combined PTW/PPA treatment, the individual steps described above for the PTW and PPA single treatments were coordinated. In a first step, PTW was added to the inoculated PE strips to be decontaminated, and in a second step, these treated samples were dried and decontaminated with PPA. All investigated different pre- and post-treatment times of the single treatments with PPA and PTW were also tested for the combination of PTW and PPA.