Susceptibility of Staphylococcus epidermidis to Argon Cold Plasma Jet by Oxygen Admixture

Round 1

Reviewer 1 Report

In this study, the author mainly used cold atmosphere plasma jet to sterilize medical device.  However, the author did not explain the difference between non-thermal atmospheric plasma and cold atmospheric plasma.

On the other hand, the author only discussed that several techniques have been used to overcome the restrictions to generate non-thermal plasma at atmospheric pressure such as using Dielectric-barrier discharges (DBD) [4], pulsed power source [5], segmented cathode [6] and the micro-hollow cathode system [7].  Which method of atmosphere plasma generation is best？

In addition, APPJs can be used with heat-sensitive materials and with biological samples as it is a non-thermal plasma. Therefore, what are the differences between Atmospheric pressure plasma jets (APPJs) and Cold atmospheric pressure plasma jet (CAPPJ)?  Can you add an explanation？

The gas temperature of the CAPPJ was measured by evaluating the rotational (0,0) band of the second positive N2 system.  The recorded spectrum was observed side-on, from the nitrogen second positive system N2, and compared to a simulated spectrum.  The result show that the measurements of the gas temperature and vibrational temperature result in an estimated 320 K gas temperature (rotational temperature) and 1200 K vibrational temperature.  Is the temperature of CAPPJ significantly lower than that of APPJs?

The antibacterial test is based on which literature is designed and what is the mechanism of low temperature plasma sterilization？  Can you add an explanation？

Author Response

Reply to the comments and Suggestions of reviewers:

Thank you and appreciate for critical and accrete revisions

• In this study, the author mainly used cold atmosphere plasma jet to sterilize medical devices.  However, the author did not explain the difference between non-thermal atmospheric plasma and cold atmospheric plasma.

 Plasma can be classified mainly as thermal plasma in which the electrons temperature is equal to all other plasma species temperatures and non-thermal plasma in which electrons temperature is much higher than other plasma species temperature. Non-thermal plasma can be called cold plasma especially when its gas temperature approaches room temperature.  

• On the other hand, the author only discussed that several techniques have been used to overcome the restrictions to generate non-thermal plasma at atmospheric pressure such as using Dielectric-barrier discharges (DBD) [4], pulsed power source [5], segmented cathode [6] and the micro-hollow cathode system [7].  Which method of atmosphere plasma generation is best？

The method by which the non-thermal atmospheric pressure plasma is generated will depend on the plasma properties required such as volume of the generated plasma, gas temperature, electron density and temperature, and homogeneity of the plasma. For large-scale surface plasma DBD techniques can be used but cannot be used for direct current plasma as well as the electron density of the generated plasma is limited especially when a low-frequency power source is used. The pulsed power source can be used to generated room temperature homogeneous plasma when the pulse duration is in the nano second range below the time required for the glow to arc transition, but the coast for the power source is very expensive. The micro-hollow cathode system is used to generate high electron density plasma, but the size of the generated plasma is very limited.   We believe that a combination between the different techniques can be used to fulfill the requirement for generating nonthermal plasma with high rate of reactive species generation and large-scale plasma.

•    In addition, APPJs can be used with heat-sensitive materials and with biological samples as it is a non-thermal plasma. Therefore, what are the differences between Atmospheric pressure plasma jets (APPJs) and Cold atmospheric pressure plasma jet (CAPPJ)?  Can you add an explanation？

Atmospheric pressure plasma jets (APPJs) can be thermal or non-thermal plasma jets while cold atmospheric pressure plasma jet (CAPPJ) is non-thermal plasma jet.

• The gas temperature of the CAPPJ was measured by evaluating the rotational (0,0) band of the second positive N2 system.  The recorded spectrum was observed side-on, from the nitrogen second positive system N2, and compared to a simulated spectrum.  The result shows that the measurements of the gas temperature and vibrational temperature result in an estimated 320 K gas temperature (rotational temperature) and 1200 K vibrational temperature.  Is the temperature of CAPPJ significantly lower than that of APPJs?

The statement is true if the APPJs are non-thermal plasma.

• The antibacterial test is based on which literature is designed and what is the mechanism of low temperature plasma sterilization？  Can you add an explanation？

The mechanism of low temperature or non-thermal plasma was explained in details in the manuscript

Reviewer 2 Report

Review of the article "Susceptibility of Staphylococcus epidermidis to argon cold plasma jet by 0.2% oxygen admixture". The work is devoted to the use of cold atmospheric pressure plasma (CAP), namely, plasma jet (APPJ), for sterilization of surfaces of biological and non-biological objects. The article investigates the dependence of the survival rate of Staphylococcus epidermidis (cultivated in vitro) on the concentration of oxygen O2 admitted as an admixture to the working gas (argon) APPJ. The methods used are beyond doubt, the results are relevant to the methods. The concentration of O and OH radicals arising in APPJ, which play a key role in the ability of CAP to sterilize surfaces, was estimated from the plasma emission spectra. A relationship was established between the flow rate of the additional gas O2 and the production of O and OH radicals in the gas-plasma phase, as well as the survival rate of S. epidermidis. The optimal flow rate of the supplemental O2 gas for the APPJ source used by the authors in this work has been determined. This result is the novelty of the work, it is positioned as the main one in the submitted manuscript, and is placed in the title. However, this optimal operating mode can vary significantly when using other APPJ sources, including certified and commercially available sources. Replication of research results in other laboratory conditions seems to be difficult. Thus, the scientific and practical significance of the main result of the work is low. The discussion of the main results of the work needs to be improved. I advise the authors to compare the results obtained in the article with other known results. Nevertheless, the study of the effect of the component composition of the APPJ working gas on various biological objects is urgent. I believe that the manuscript can be published in the status of a brief scientific report after the English language editing and proof-reading. 

Author Response

Reviewer 2:

• The article "Susceptibility of Staphylococcus epidermidis to argon cold plasma jet by 0.2% oxygen admixture". The work is devoted to the use of cold atmospheric pressure plasma (CAP), namely, plasma jet (APPJ), for sterilization of surfaces of biological and non-biological objects. The article investigates the dependence of the survival rate of Staphylococcus epidermidis (cultivated in vitro) on the concentration of oxygen O2 admitted as an admixture to the working gas (argon) APPJ. The methods used are beyond doubt, the results are relevant to the methods. The concentration of O and OH radicals arising in APPJ, which play a key role in the ability of CAP to sterilize surfaces, was estimated from the plasma emission spectra. A relationship was established between the flow rate of the additional gas O2 and the production of O and OH radicals in the gas-plasma phase, as well as the survival rate of S. epidermidis. The optimal flow rate of the supplemental O2 gas for the APPJ source used by the authors in this work has been determined. This result is the novelty of the work, it is positioned as the main one in the submitted manuscript, and is placed in the title. However, this optimal operating mode can vary significantly when using other APPJ sources, including certified and commercially available sources. Replication of research results in other laboratory conditions seems to be difficult. Thus, the scientific and practical significance of the main result of the work is low. The discussion of the main results of the work needs to be improved. I advise the authors to compare the results obtained in the article with other known results. Nevertheless, the study of the effect of the component composition of the APPJ working gas on various biological objects is urgent. I believe that the manuscript can be published in the status of a brief scientific report after the English language editing and proof-reading. 

Thank you for the report and reviewer's opinion. The discussion was improved and the editing of the English was improved

Round 2

Reviewer 1 Report

no comment

Author Response

no comment

Thank you

Author Response File: Author Response.docx

Reviewer 2 Report

Dear authors of the manuscript, I believe that the answer to the comments of the reviewer was formal and did not touch on key points. I will try once again to formulate the main points of the review:
1. What's new done? The manuscript does not formulate the fundamental novelty of the work. Everything looks like a description of a routine job. They turned on the device, gave pressure X, recorded the readings of the device. Gave pressure X + 1, recorded the readings of the device. The result obtained in this work will not be repeated if you use a different device. Please state the fundamental aspect of what you have done new.
2. Discussion of results improved. The authors added the results of one article to the manuscript. It seems to me that this is not enough. The discussion needs to be seriously improved.
3. The authors wrote that the language was corrected. It is difficult to agree with this. Already in the second sentence of the abstract, hydroxyl radicals sterilize plasma. Further in the text, there are still a lot of similar linguistic constructions. This needs to be corrected, as it is not always clear what is meant. 

Author Response

1. What's new done? The manuscript does not formulate the fundamental novelty of the work. Everything looks like a description of a routine job. They turned on the device, gave pressure X, recorded the readings of the device. Gave pressure X + 1, recorded the readings of the device. The result obtained in this work will not be repeated if you use a different device. Please state the fundamental aspect of what you have done new.

• Reply to the reviewer: Thank you for paying my attention to indicate the importance of the work. The work describes the mechanisms involved in the treatment of skin infecting bacteria which is rarely handled by investigators. The use of cold plasma in the disinfection of pathogenic microorganisms on or in the skin as well as the acceleration of skin regeneration is a promising technique

2. Discussion of results improved. The authors added the results of one article to the manuscript. It seems to me that this is not enough. The discussion needs to be seriously improved.

1. Reply to the reviewer: The discussion of the results cover all the points under consideration involving the different types of reactive species involved, the role and mechanisms of these species as an antibacterial agent as well as the optimization of the produced species in inducing maximum efficiency. Recent references were added.

3. The authors wrote that the language was corrected. It is difficult to agree with this. Already in the second sentence of the abstract, hydroxyl radicals sterilize plasma. Further in the text, there are still a lot of similar linguistic constructions. This needs to be corrected, as it is not always clear what is meant. 

• The manuscript including the abstract was revised again for the language, hoping it is more clear than before. All the formulations of cold plasma were defined as non-thermal APP.

Author Response File: Author Response.docx

Round 3

Reviewer 2 Report

Done