## 3 Results
### Heat Stability of Plasma Activated Water  
High stability of the bactericidal activity of PAW at supra-ambient temperatures was recorded. Both SD15 and GD15 PAW heated to between 50 and 100 \({}^{\circ}\)C for 5 min retained the ability to reduce bacterial concentrations below the limit of detection (data not shown). Similar peroxide concentrations were recorded between the heated and nonheated PAW. There were no adverse effects of heating SD15 and GD15 PAW at 100 \({}^{\circ}\)C for different time periods (5, 10, or 15 min) on the retention of antimicrobial efficacy (Figure 5A). Hydrogen peroxide concentration of SD PAW was observed to be relatively stable, for the different heating times (Figure 5B). Peroxide concentration was similar to that of hydrogen peroxide for SD PAW, indicating that the peroxides present are almost exclusively hydrogen peroxide (Figure 5B). Regarding GD PAW, hydrogen peroxide was not detected, but the concentration of peroxides indicated similar amounts as measured in SD PAW, which decreased slightly as the heating times increased. However, in nonbuffered conditions, an increase in peroxides was found (Figure 5B). Figure 3: Bactericidal effects of PAW after 5, 10, or 15 min plasma activation, made by spark and glow discharges, on _Escherichia coli_ (_E. coli_) and _Staphylococcus aureus_ (_S. aureus_) with contact times of 15, 30, 60 min and 24 h on the day of PAW generation. (**A**) _E. coli_—15 min; (**B**) _S. aureus_—15 min; (**C**) _E. coli_—30 min; (**D**) _S. aureus_—30 min; (**E**) _E. coli_—60 min; (**F**) _S. aureus_—60 min; (**G**) _E. coli_—24 h; (**H**) _S. aureus_ 24 h (data shown as mean \(\pm\) SD, average of three experiments). Limit of detection: 2 log\({}_{10}\) CFU/mL. Figure 4: Bactericidal stability of PAW after 5, 10, or 15 min plasma activation, made by spark and glow discharges on _E. coli_ and _S. aureus_ with 30 and 60 min contact time a week after PAW generation. (**A**) _E. coli_—30 min; (**B**) _S. aureus_—30 min; (**C**) _E. coli_—60 min; (**D**) _S. aureus_—60 min (data shown as mean \(\pm\) SD, average of three experiments). Limit of detection: 2 log\({}_{10}\) CFU/mL. Figure 5: Effect of heating at 100 \({}^{\circ}\)C and heating time duration on retention of antimicrobial efficacy of PAW (SD15, GD15). (**A**) Bactericidal effects after 60 min contact time. (**B**) Chemical composition of hydrogen peroxide measured by TiOSO\({}_{4}\), peroxidoides measured by buffered KI and nonbuffered KI. Data shown as mean \(\pm\) SD; nd = no colonies detected. Limit of detection: 2 log\({}_{10}\) CFU/mL. Controls (CTL) consisted of bacterial suspension exposed to untreated deionised water under the same conditions. Based on the high temperature stability observed at 100 \({}^{\circ}\)C, PAW samples were further heat treated by autoclaving at 121 \({}^{\circ}\)C, 15 psi for 20 min and their chemical composition and antibacterial effects analysed. The liquids showed remarkable stability to high temperature and pressure in terms of their bactericidal activity. Autoclaved samples of SD15 showed comparable or even enhanced antibacterial activity to the original PAW for all contact times (Figure 6). Autoclaved PAW GD15 on the other hand retained a similar ability to reduce _E. coli_ and _S. aureus_ in some cases, but demonstrated reduced antibacterial efficacy in others. Analysis of hydrogen peroxide and other peroxides in these samples indicated a strong reduction in the concentrations of these species but the extent of reduction varied significantly between the 3 sets of PAW analysed (Table 1). The stark differences in concentrations of hydrogen peroxides and other peroxides between all three sets after autoclaving (despite similar initial concentrations in sets 1 and 2) cannot be explained at present. However, while the autoclave run itself was fixed at 20 min, the heating and cooling phase were not precisely controlled in these experiments and may have varied depending on the initial temperature of the autoclave and the overall load.