Impact of Microwave Plasma Torch on the Yeast Candida glabrata
Round 1
Reviewer 1 Report
An interesting use of a novel technique for controlling pathogenic yeast.
A couple of minor things, all species names are lower case - cerevisiae rather than Cerevisiae and glabrata rather than Glabrata etc.
The choice of English is occasionally incorrect so it should be grown rather than grown up. Candnida in line 80.
Line 81 - better known rather than more known. I think the paper could do with a degree of proof-reading.
Methods - Its unclear why those settings were chosen for the microwave in terms of W and time of use.
For figs 3-5 and 10, it would help the reader if the different experiments had different symbols. Also its unclear why the 12W experiments didn't go all the way to 200s. Why is there no data for 24 hrs in fig 5?
The data could do with a statistical analysis to determine significance of the different conditions.
Author Response
Dear referee
Many thanks for your valuable notes and comments. We have tried to improve the whole text according to your recommendations. Our detailed response is given point to point below.
- A couple of minor things, all species names are lower case - cerevisiae rather than Cerevisiae and glabrata rather than Glabrata etc.
Terminology corrected.
- The choice of English is occasionally incorrect so it should be grown rather than grown up. Candnida in line 80. Line 81 - better known rather than more known. I think the paper could do with a degree of proof-reading.
English was carefully corrected in the whole manuscript.
- Methods - Its unclear why those settings were chosen for the microwave in terms of W and time of use.
The use of argon is based on its good properties in case of the surface wave sustained discharge and lower price than helium, so it is better for the real application out of the academic research. The selected mass flow, powers and treatment times are based on our previous experience with treatment of fruits and artificial wounds on model mice. The goal is to find good combination of applied power, gas flow and inactivation efficiency without surface heating that can lead to the treated surface damage (like living skin).
- For figs 3-5 and 10, it would help the reader if the different experiments had different symbols. Also its unclear why the 12W experiments didn't go all the way to 200s. Why is there no data for 24 hrs in fig 5?
Symbols were changed according to the recommendation. We tried to get some system in the used symbols across the figures.
The experiment at applied power of 12 W was finished at 120 s of the treatment because we supposed higher inactivation efficiency (based on preliminary experiments) and thus it was not necessary to continue. The general aim is to go to shorter treatment times because of future patients comfort.
Inhibition zones at the 100-fold diluted culture after 24 h from plasma exposure are not presented because the yeast concentration was too low and no visible inhibition zone was formed within 24 hours. Information was added in Fig. 5 caption and in the text related to Figs. 10 and 11.
- The data could do with a statistical analysis to determine significance of the different conditions.
The statistical analysis was added into the manuscript in a form of Table 1. It was concluded that differences are significant at all treatment conditions.
Reviewer 2 Report
This article describes the influence of microwave (MW) torch plasma on the yeast Candida Glabrata. The article is interesting, especially concerning the antifugal effect of the MW plasma. Additionally, this paper seems to be interested of readers for this journal. However, the article contains some inconsistencies.
Detailed comments:
1. Please specify in "Introduction" section, the advantage of applied plasma system in comparison with other cold atmospheric plasma source (e.g. barrier discharge, corona discharge, atmospheric pressure glow discharge ).
2. What is temperature of agar plates during the treatment of Candida Glabrata yeast by MW plasma? How about the temperature of MW plasma in the core zone?
3. Why did you use the argon gas, instead helium?
4. Please provide the information about the optimization of plasma system
5. The discussion should be improved in the term of production of reactive nitrogen and oxygen species, radicals and so on.
6. Please specified the interaction zone of microwave plasma torch. What is diameter ?
7. Please add information about the reflected power
Author Response
Dear referee
Many thanks for your valuable notes and comments. We have tried to improve the whole text according to your recommendations. Our detailed response is given point to point below.
Please specify in "Introduction" section, the advantage of applied plasma system in comparison with other cold atmospheric plasma source (e.g. barrier discharge, corona discharge, atmospheric pressure glow discharge ).
The information was added into Introduction:
„Standardly, we use argon as a carrier gas for the plasma generation and the yeast treatment because it is cheaper than helium and colder than common atmospheric pressure discharges in air such as DBDs, corona or glow discharges [34].“
What is temperature of agar plates during the treatment of Candida Glabrata yeast by MW plasma? How about the temperature of MW plasma in the core zone?
Information was added into Material and Methods section:
„Based on our previous results obtained by the thermal camera, temperature of the treated object does not exceed 40 °C even after 30 s of the treatment [34]. The applied powers of 9 W and 12 W were selected based on our experience presented in the same study. The rotational temperature (indicating the real temperature of the plasma core) was obtained from the emission spectrum of the OH radical and it reached (650±50) K at the end of the quartz capillary and decreased linearly to (600±50) K at the treated surface [34].“
Why did you use the argon gas, instead helium?
Argon is about 1 order cheaper than helium and the surface wave sustained discharge is well operating in both gasses. So, argon is preferred with respect to the future application. In our following research, we plan to investigate mixtures of argon and helium to optimize conditions and simultaneously keep operational costs as low as possible. Information about the use of argon is added into Introduction.
Please provide the information about the optimization of plasma system
The plasma system parameters selected for this particular study are based on broader research carried out during the last two years. The general aim is to get the system operating at sufficiently low power at applicable operational costs that will effectively inactivate different pathogens. The prepared research will be focused on the application of argon-helium mixtures with the aim to further decrease the applied energy and to keep the biologic activity of plasma.
Information about this is added into Introduction (current settings) and Conclusions (future plans).
- The discussion should be improved in the term of production of reactive nitrogen and oxygen species, radicals and so on.
Production of various active species was observed in our previous studies. The supplementary information was added in Results and Discussion section:
„Based on our previous experimental results obtained by optical emission spectroscopy [34], we suppose the action of reactive oxygen and nitrogen species (RONS) produced by our torch that can play a significant role in the decontamination process. Even though we use pure argon as the carrier gas, molecules contained in the surrounding air (oxygen, nitrogen, water vapor, etc.) are swept into the plasma flow where they are excited or ionized. Therefore, the presence of RONS such as nitrogen monoxide radical (NO), hydroxyl radical (OH), atomic oxygen (O), peroxynitrite molecule (ONOOH) or ozone (O3) was determined in our plasma torch [34]. The NO radical is very reactive and, in a reaction with water, forms nitrous acid, which decreases the substrate pH. This effect might contribute to the deactivation of some kinds of microorganisms. Also the OH radical is well known as one of the strongest non-selective oxidative species. Further, UV radiation of the NO radical belongs to the UVC region, which is well known for its bactericide effects [14].“
Please specified the interaction zone of microwave plasma torch. What is diameter ?
This is a very good point but correct answer is really complicated. The active discharge part is not in the direct contact with the treated surface (distance is 5 mm at 9 W and 3.5 mm at 12 W), so interaction with the surface is with the post-discharge part, only. In this region, active particles are presented in sufficient amount (see reference [33]) up to couple of centimetres in case of the free torch. But when plasma interacts with the surface, vortexes are formed that completely change plasma-surrounding air interactions. We have observed this effect using the Schlieren imaging. The flow affects couple of cm in diameter but the question is about active particles concentration in such case. We have tried a preliminary experiment to verify the interaction zone by indigo dye immobilized in the agar plate. The decoloured area was similar as the inhibition zones presented here. We plan the exact observation of the relation between inhibition zones size and decoloured zones using selected dyes at different discharge conditions just after holidays because we know that this is one of critical points with respect to the application. Brief information is added into Conclusions.
Please add information about the reflected power
Information about reflected power was added into Materials and Methods section. The reflected power (as well as outgoing power) was indicated by the power supply panel with 1W resolution, so not precise enough. Reflected power of 0 W was indicated during all experiments, so we suppose that the reflected power was below 0.5 W.
