# Biological Decontamination by Microplasma
## 3 Results  
Three bacterial strains were used to assess the antimicrobial efficacy of microplasma under two experimental designs (Figure 5).  
Figure 4: Ozone concentration increases with discharge voltage.  
The bacteria were inoculated on agar plates and the microplasma was applied at \(-1.5\) kV and \(-1.7\) kV for 5 min. At \(-1.5\) kV, only two out of the three bacterial strains were inhibited. The experiments were carried in duplicate, and both replicates only had a clear inhibition zone for _S. aureus_. At \(-1.7\) kV, the development of bacterial colonies was inhibited for all three strains, with clear inhibition zones that could be measured and compared (Figure 6). The microplasma had the best effect against _S. aureus_ in this case as well, followed by _E. coli_, and _P. aeruginosa_, which was the least susceptible from the tested strains. Where possible, the colony-forming units (CFU) were counted (Table 1). For both _E. coli_ and _S. aureus_, a decrease in CFU was observed; however, the results were not statistically significant compared to the untreated controls.  
The selection of experimental parameters such as discharge voltage, exposure time, type of gas and gas-flow rate was based on our previous research presented in [1; 2; 16], theoretical considerations, and also preliminary optimizations.  
The microplasma discharge occurs within some values of discharge voltage. These values are given by the electrode characteristics and the gas pressure; thus, in air at atmospheric pressure, microplasma discharge occurred above \(-1.2\) kV. We carried out preliminary experiments at \(-1.3\) kV and \(-1.4\) kV, but the sterilization effect was not visible; thus, we increased the voltage at \(-1.5\) kV and \(-1.7\) kV. Above \(-1.7\) kV, we have considered that the values of generated ozone will be too high and will affect the human health. Also, above some voltages, the dielectric layer between the electrodes will break due to the intensity of the electric field. The sterilization effect at lower voltages is also desirable, since microplasma is a technology that has an advantage over other nonthermal plasma technologies due to its energy efficiency and safety.  
The preliminary optimizations experiments that were carried out at exposure times below 5 min showed less sterilization effects, especially at lower discharge voltages. Considering the choice of discharge voltage values and exposure time, the results are reliable and there are no potential flaws in the methodology.  
Figure 5: Representative images of the bacterial strains treated with microplasma; experimental design 1: bacteria inoculated on agar plates and incubated for 24 h after microplasma treatment; experimental design 2: 24 h bacteria inoculated on agar plates, treated with microplasma at \(-1.7\) kV for 5 min, and re-incubated for an additional 24 h.  
Two replicates were used for each bacterial strain. In case of _P. aeruginosa_, all three plates had dense bacterial populations and the CFU could not be calculated. We considered selecting a lower bacterial concentration to begin with; however, the results would not have been comparable with the other two bacterial samples, so we did not further investigate this scenario. In future research, we will specifically investigate the effect of microplasma on _P. aeruginosa_; thus, we will be able to adjust the initial concentration to a value that will allow us to count the CFU.