## 3 Results and Discussion
### Tb-PAW Inactivation of E. coli after Different Storage Times and Temperatures  
In a third study, Tb-PAW was stored at three different temperatures (7 \({}^{\circ}\)C, 21 \({}^{\circ}\)C and 30 \({}^{\circ}\)C) and monitored at four time points (4 h, 8 h, 12 h and 24 h). The bactericidal efficacy of those Tb-PAWs against _E. coli_ was tested (Table 1). The comparisons between untreated TRIS-buffer control samples and the corresponding Tb-PAW samples showed significant reductions of the CFU in Tb-PAW at all temperatures and storage times. These ranged from 5.08 \(\pm\) 0.52 log steps (30 \({}^{\circ}\)C, 8 h) to 0.73 \(\pm\) 0.23 log steps (30 \({}^{\circ}\)C, 24 h). The residual microbial content of the Tb-PAW sample at 30 \({}^{\circ}\)C and 24 h was significantly higher compared to the 7 \({}^{\circ}\)C and 21 \({}^{\circ}\)C samples, whereas the other time points revealed no differences between the temperature groups. In addition, we found significantly higher bacterial counts in  
Figure 4: Comparison of the inactivation capacity of Tb-PAW on _E. coli_, _F. necrohorum_ and _P. levii_ under the influence of BSA. The treatment time for each bacterium was 1 min. Results represent the mean \(\pm\) standard deviation. * Significant differences between the TRIS-buffer (control) and Tb-PAW samples are defined as _p_\(\leq\) 0.05. Not significant differences between Tb-PAW and Tb-PAW with 5% BSA are defined as ns. ns = not significant; BSA = bovine serum albumin; Tb-PAW = TRIS-buffered plasma-activated water.  
the 30 \({}^{\circ}\)C treatment group at 24 h compared to the other three time points. We therefore verified that Tb-PAWs stored at 7 \({}^{\circ}\)C and 21 \({}^{\circ}\)C can be used for a period of 24 h and for at least 12 h when stored at 30 \({}^{\circ}\)C.  
These results are consistent with findings of the study by [PERSON] et al. [44], who also reported prolonged preservation of bactericidal activity when PAW was stored at low temperatures. In this study, the bactericidal properties of a PAW used against _S. aureus_ also increased with decreasing temperatures (25 \({}^{\circ}\)C \(<\) 4 \({}^{\circ}\)C \(<\) -20 \({}^{\circ}\)C \(<\) -80 \({}^{\circ}\)C). After one day of storage, the reduction rates dropped from 5.0 log CFU/mL (room temperature) to approx. 3.7 log CFU/mL (-80 \({}^{\circ}\)C) and 1.8 log CFU/mL (25 \({}^{\circ}\)C), respectively. The PAW at \(-\)80 \({}^{\circ}\)C retained a significantly higher reduction rate and revealed a better way of storage than 25 \({}^{\circ}\)C.  
[PERSON] et al. [17] investigated the bactericidal activity of PAW and plasma-activated PBS (PAPBS). They found that storage of PAW resulted in a decreasing reduction effect with time. The PALs were stored for seven days after preparation. Each day, _E. coli_ was incubated with the PALs for 15 min as well as for 3 h. Over two days, the 3 h exposure resulted in a 5.0 log reduction before it began to decline, while the 15 min exposure dropped to 2.4 log on the first day (30 min after production) and remained at about 1.0 log until the end of the second day. In contrast to the PAW, the PAPBS did not reach a high reduction at any of the time points and remained below 1.0 log.  
The effect of the PALs in these studies appears to decrease with time and at elevated temperature, which is in good agreement with our own findings. However, the decrease varies greatly depending on the study. In the researches described, much longer exposure times (4 min to 3 h) were used compared to our study. In addition, we were able to achieve high reductions rates with Tb-PAW compared to the PAPBS of [PERSON] et al. [17] using only 1 min exposure time resulting in reduction rates of up to 2.61 logs (7 \({}^{\circ}\)C) and 3.18 logs (21 \({}^{\circ}\)C) after 24 h.