## 2 Materials and Methods  
### Experimental Design  
In order to unravel the CAP inactivation mechanism for mature biofilms, it was examined how the generated CAP species and their (lethal) effect on the biofilms were influenced by three plasma characteristics (i.e., the electrode configuration, the oxygen level of the gas flow, and the power of the discharge). Regarding the effect on the biofilm-associated cells and the biofilm matrix, it was examined whether (i) the lethal effect of CAP was due to an altered membrane integrity and/or damage to the cellular DNA, (ii) the specifically generated CAP species were able to penetrate into the deeper layers of the biofilm, and (iii) the structure of the biofilm was altered. For each of the tested plasma characteristics, two different levels were examined, i.e., two electrode configurations (dielectric barrier discharge (DBD) and surface barrier discharge (SBD) electrode), two oxygen levels (helium + 0.0 and 1.0% (\(v\)/\(v\)) oxygen), and two different discharge power values. For the latter influencing factor, it should be mentioned that this was altered by changing the input voltage, i.e., 13.88 and 21.88 V were applied. The selected levels were similar to those investigated in the research of [PERSON] et al. [17], although only the outer values of previously examined ranges were considered within the presented study. Moreover, in order to comment on the influence of the Gram type and/or the biofilm forming species on CAP's inactivation mechanism, two different model biofilms were treated. These biofilms were developed by a Gram-positive species (i.e., _Listeria monocytogenes_) and a Gram-negative species (i.e., _Salmonella_ Typhimurium).  
To investigate the generated CAP species, three different experiments were performed. First of all, the generation of UV light was detected by means of UV strips (5-60 mJ/cm\({}^{2}\), 250-420 nm). Secondly, a small volume of demineralized water (500 uL) was CAP treated to simulate the chemical reactions that can occur between the generated CAP species and the intracellular fluid and/or the water molecules present within the biofilm matrix. After CAP treatment of the water, the concentration of three (relatively stable) CAP species was quantified (i.e., hydrogen peroxide, nitrite, and nitrate) and the pH was measured. Finally, to determine the generation of intracellular ROS, fluorescence measurements were applied in combination with a selective fluorescent component.  
To examine the (lethal) effect of the generated CAP species on the biofilm-associated cells and the biofilm matrix, four different experiments were performed. First of all, the contribution of UV light to the CAP inactivation efficacy was examined by covering the biofilm samples with a quartz disc, preventing all other reactive species to interact with them. Viable plate counts were applied to determine the cell density of the CAP treated model biofilms. Secondly, the membrane integrity of the CAP treated biofilm-associated cells, expressed based on the amount of DNA leakage, was assessed by means of absorbance measurements at 260 nm. Thirdly, cellular DNA damage was investigated by means of fluorescence measurements in combination with SYBR Green I, a selective detector of double-strand DNA (dsDNA). Finally, to examine the depth of penetration of the generated CAP species and the structure of the (CAP treated) model biofilms, confocal laser scanning microscopy (CLSM) was used in combination with live-dead staining. Moreover, the CLSM images can be used as well to comment on the integrity of the cell membranes since the ability of the selective dyes to penetrate into the cells depends on this cell attribute.