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Study of sterilization effect of dielectric barrier discharge on eucaryotic microorganisms
Vojkovská, Hana ; Ing.Hana Grossmannová, Ph.D. (referee) ; Kozáková, Zdenka (advisor)
Nowadays the wide spectrum of decontamination methods are used for the inactivation of microorganism on various materials and subjects. The serious disadvantage of the conventional decontaminations methods is stressing of the exposed material by heat or chemicals. The presented bachelor thesis discusses plasma sterilization, which is more friendly and more effective on the wide spectrum of procaryotic and eucaryotic microorganisms. Basically, the main inactivation factors for cells exposed to plasma are heat, UV radiation and various reactive species The work was focused on studying of the effect of the dielectric barrier discharge (DBD) operating at atmospheric pressure on bioindicator Aspergillus niger. Plasma was generated in nitrogen and argon. Paper and PET-foil wer used as the carrying medium. The influence of various working conditions on the efficiency of plasma sterilization was studied. Namely it was the influence of plasma exposition time, plasma power density, the type of operating gas and type of the medium supporting the microorganism. According to our results the efficiency of the plasma sterilization increases with increasing plasma power density, resp. the plasma exposition time. When comparing the results observed for the same conditions in argon and nitrogen the higher sterilization effect was reached in argon. The sterilization time was 40 – 120 second in dependence to plasma power density, gas and carrying medium. Furthermore the influence of the carrying medium on the sterilization efficiency was prooved. It was shown, that porous materials have a ”shadowing effect” for microorganisms. The microorganism may penetrate into the paper material and embed in pits and cavities. Such penetration could preclude the interaction of plasma with the microorganism, thereby decreasing the efficiency of spore inactivation. Additionaly paper porosity complicates the detachment of spores into solution, so it is reached less microorganisms as from the PET-foil. The discharge parameters were studied by means of the optical emission spectroscopy.
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Study of elementary inactivation processes acting during sterilization of procaryotic systems in dielectric barrier discharge
Bittnerová, Zuzana ; Sťahel, Pavel (referee) ; Kozáková, Zdenka (advisor)
Presented master’s thesis is focused on the study of the elementary inactivation processes acting during sterilization of procaryotic systems in dielectric barrier discharge (DBD). Sterilization is an important biomedical and food-industry application and plasma sterilization is one of the methods, which are suitable for sterilization of heat and chemical sensitive materials. Biologically contaminated samples were treated in dielectric barrier discharge operated at atmospheric pressure. The discharge was generated in argon and in nitrogen. The plasma power density was 2725,93 mW cm–3 in argon and 2325,93 mW cm–3 in nitrogen. Gram positive bacteria Bacillus subtilis and gram negative bacteria Escherichia coli were used as a bioindicator. Bacteria were spread onto the surface of Whatman No.1 filtration paper. The influence of UV radiation, reactive species, heat and plasma discharge where the synergistic function of all of the agents was studied. Effects of UV radiation and temperature were studied separately. In order to separate the effect of UV radiation generated by DBD the quartz glass transmitting UV radiation was employed. During the plasma exposition selected samples were covered with the quartz window while other samples were directly exposed to the plasma. Covered samples were exposed to UV radiation and temperature (which cannot be eliminated), samples without quartz window were directly plasma exposed (treated). Results show that for covered samples the lower inactivation was reached than by the samples directly exposed to plasma. When studying the effect of temperature, the temperature between the DBD electrodes was measured by means of a thermocouple. Afterwards the samples were placed in an oven and exposed to the same temperature as was measured between the electrodes. By comparing the results of heat treated samples and plasma treated samples it can be assumed that the influence of the temperature during the sterilization process in DBD is very low. The discharge parameters were studied by means of the Optical Emission Spectroscopy. Plasma treated samples were assessed employing Scanning Electron Microscopy (SEM). Damage of Bacillus subtilis cell wall due to the effect of plasma was observed while no effect of plasma on the structure of filtration paper was detected.
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Influence of humidity on total sterilisation effect of dielectric barrier discharge
Kramárová, Petra ; Bartlová, Milada (referee) ; Kozáková, Zdenka (advisor)
The main subject of this diploma thesis is the study of the effect of humidity on the total sterilization effect of the dielectric barrier discharge. Sterilization is a process which can eliminate all forms of life. The plasma sterilization is one of the methods that are suitable for sterilization of temperature and chemical sensitive materials. This sterilization method was proved to be effective on the wide spectrum of procaryotic and eucaryotic microorganisms. Basically, the main inactivation factors for cells exposed to plasma are heat, UV radiation and various reactive species. Dielectric barrier discharge (DBD) operating at atmospheric pressure was used for the sterilization of the samples. The discharge was generated in dry air and in humid air. The plasma power densities were 2 160 mW.cm-3, 2 279 mW.cm-3 and 2 760 mW.cm-3 (dry air) or 2 326 mW.cm-3 and 2 850 mW.cm-3 (humid air). Humidity of air was achieved using a wash bottle filled with water through which air flowed into the DBD reactor. Fungi spores of Aspergillus niger were used as model microorganisms. Whatman paper No. 1 was used as the carrying medium. When comparing sterilization efficiency of humid and dry air operating at the same conditions, the higher sterilization effect was observed in humid air. The sterilization effect of the DBD generated in air was compared with results obtained during plasma generation in argon and nitrogen. At the same conditions, the highest sterilization effect was observed in argon, followed by humid air, nitrogen and dry air. It was found out that in our experimental setup the active species are probably the main inactivation mechanism. The influence of temperature on the inactivation of microorganisms was completely negligible. The discharge parameters were studied by means of the optical emission spectroscopy (OES). Plasma treated samples were analyzed employing scanning electron microscopy (SEM). Damage of the microorganisms due to the effect of plasma as well as plasma effect on the structure of the carrying medium was evaluated.
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Study of sterilization effect of dielectric barrier discharge on procaryotic microorganisms
Bittnerová, Zuzana ; Ing.Hana Grossmannová, Ph.D. (referee) ; Kozáková, Zdenka (advisor)
Presented bachelor thesis is focused on sterilization effect of dielectric barrier discharge on procaryotic microorganisms. Sterilization is important biomedical and food-industry application and plasma sterilization is one of the method, which are suitable for sterilization of heat and chemical sensitive materials. Biologicaly contaminated samples were treated in dielectric barrier discharge operated at atmospheric pressure. The discharge was generated in argon and in nitrogen. Gram positive bacteria Bacillus subtilis was used as a bioindicator. Bacteria was spread onto the surface of paper and PET material. Efficiency of sterilization was evaluated according to exposure time, plasma power density and process gas. The positive effect of DBD on inactivation of microorganism was observed. The efficiency of the plasma sterilization increases with increasing treatment time. As regards plasma power density, the higher density was used the more effective the sterilization was and time necessary for inactivation of all microorganisms was shorter. Sterility was observed within 60 s while using plasma power density 2,37 W/cm, when using plasma power density of 1,78 W/cm longer treatment time is neccesary. The sterilization was more effective if argon was used as a process gas. Time necessary for the most reduction of microorganisms in argon was 10 s, while using nitrogen the reduction was only 50 %.
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Study of the sterilization effect of temperature and UV light present in dielectric barrier discharge
Kramárová, Petra ; Grossmannová, Hana (referee) ; Kozáková, Zdenka (advisor)
The main theme of this bachelor thesis is to study the effect of temperature and UV radiation to the total sterilizing effect of dielectric barrier discharge. Sterilization is a process, which can eliminate all forms of life. The presented bachelor thesis discusses plasma sterilization, which is one of the method, which are suitable for sterilization of heat and chemical sensitive materials. This sterilization method is effective on the wide spektrum of procaryotic and eucaryotic microorganisms. Basically, the main inactivation factors for cells exposed to plasma are heat, UV radiation and various reactive species. In my thesis a dielectric barrier discharge (DBD) operating at atmospheric pressure was used for the sterilization of the samples. Plasma was generated in argon and nitrogen. According to the previous measurement the best sterilization results were observed using plasma power input 2 562,96 mW•cm-3 (argon) a 2 044,44 mW•cm-3 (nitrogen), therefore the same plasma power input was applied during our measurement. Fungi spores of Aspergillus niger were used as model organisms. Whatman paper No. 1st was used as the carrying medium. Each sample series was exposed to plasma for 5, 10, 20, 40, 60, 120 and 180 s. In order to separate the effect of UV radiation generated by DBD the quartz glass transmitting UV radiation was used. During the plasma exposition one of the sample was covered with the quartz window and the other sample was directly exposed to the plasma, afterwards the results were compared. The microbial abatement observed for the samples covered by quartz window was much lower than for the the samples directly exposed to the plasma. In first case the UV radiation and temperature is the main inactivation mechanism, while in the latter one the synergistic effect of UV radiation, temperature and active species is employed. Furthermore the effect of plasma sterilization increases with increasing the plasma exposition time. While comparing results observed for the argon and nitrogen, better results were achieved in argon. Effects of UV radiation and temperature were studied separately. The temperature between the DBD electrodes was measured by means of thermocouple. Afterwards the samples were placed in an oven and exposed to the same temperature as was measured between the electrodes. By comparing the results of heat treated samples and plasma treated samples it can be assumed that the influence of the temperature during the sterilization process in DBD was negligeable. The discharge parameters were studied by means of the optical emission spectroscopy.
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Study of Sterilization Effects Initiated by Dielectric Barriere Discharge
Slámová, Jitka ; Pekárek, Stanislav (referee) ; doc. RNDr. Karol Hensel, Ph.D., oponent (referee) ; Krčma, František (advisor)
The overall goal of the presented dissertation thesis was to study the sterilization efficiency of dielectric barrier discharge operated at atmospheric pressure. The fungi Aspergillus niger, gram-positive bacteria Bacillus subtilis and in some experiments also gram-negative bacteria Escherichia coli were used as a bio-indicator enabling to evaluate the effect of plasma assisted microbial inactivation. The samples of microorganism were placed on paper Whatman 1 or PET foil and exposed to plasma. The plasma was generated in argon, nitrogen, synthetic dry/humid air with frequency up to 10 kHz and plasma power density in the range of 1,2-2,9 W/cm3 (according to the process gas). The influence of process gas, plasma power density, plasma exposition time, type of microorganism and material of the substrate on the sterilization effect of dielectric barrier discharge was evaluated. Furthermore the contribution of each single mechanism (UV radiation, temperature and reactive species) to the sterilization effect of plasma and influence of gas humidity was evaluated. The DBD was analysed by means of optical emission spectroscopy, thermocouple was used to measure temperature during a sterilization process. In order to verify the mechanical damage of the microbial cell or the substrates during the plasma process the samples were studied by scanning electron microscopy. Generally, on the basis of experimental results, at increasing treatment times, the remaining number of spores (CFU) decreased. Similarly at increasing the plasma power input, the sterilization rate increased. When sterilising the spores of A. niger in plasma using different process gasses, the efficiency of plasma sterilization decreased as follows: argon, humid synthetic air, nitrogen and dry synthetic air. The results observed in argon plasma using different microorganism demonstrated that the sensitivity of vegetative cells resp. spores to DBD decreased as follows: A. niger spores, B. subtilis vegetative cells, E. coli vegetative cells and B. subtilis spores. Simultaneously results observed for sterilization of spores and vegetative cells of B. subtilis and A. niger demonstrated that the spores are generally more resistant to plasma than are the corresponding vegetative cells. Combining the results of contribution of each single mechanism, optical emission spectroscopy and inactivation characteristic it was found out that the reactive species significantly contribute to the plasma sterilization in all process gasses. Furthermore the inactivation process can be partly assisted by UV radiation and also the temperature can contribute in limited extent to inactivation process in some gasses. The contribution of UV radiation to the plasma sterilization decreased as follows: nitrogen, argon, dry syntetic air and humid syntetic air. Moreover it was found out that the contribution of each single mechanism can be species dependent, this is due to the different response of microorganism to the unfavorable external conditions. SEM analysis of the substrates prooved the etching actions of the plasma generated in all process gasses on the surface of the PET foil. The several minute plasma exposition of the PET foil resulted in the occurence of the „hole corrosion“ on the PET surface. Contrary to these there were no visible changes observed in the paper structure.
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Study of elementary inactivation processes acting during sterilization of eucaryotic systems in dielectric barrier discharge
Vojkovská, Hana ; Machala, Zdenko (referee) ; Kozáková, Zdenka (advisor)
This diploma thesis is focused on studying of the effect of the dielectric barrier discharge (DBD) on eucaryotic microorganisms. Plasma sterilization is considered to be an alternative method to conventional sterilization processes. Contrary to standard decontamination methods it doesn´t stress exposed material by heat, pressure and chemicals. Plasma acts on eucaryotic and procaryotic systems by means of synergy of three inactivation mechanisms. They are various reactive species, UV radiation and heat. The Aspergillus niger has been chosen as a bio-indicator enabling to evaluate the effect of plasma assisted microbial inactivation. Plasma was generated in dielectric barrier discharge (DBD) at atmospheric pressure. Nitrogen and argon were used as working gases, paper and PET foil were used as carrying media. The influence of various working conditions on the sterilization effect was studied. Namely it was the influence of plasma exposition time, plasma power density, the type of operating gas and type of supporting medium. The effect of UV radiation in combination with temperature, temperature and direct plasma were studied separately. According to our results the efficiency of DBD increases with plasma power density, resp. plasma exposition time. When comparing sterilization efficiency of nitrogen and argon operating at the same conditions, the higher sterilization effect was observed in argon. The influence of the carrying medium on sterilization effectiveness was proved. It was caused by the different structure of surface. It was found out, that in our experimental setup the active species are probably the main inactivation mechanism. The influence of temperature on inactivation of microorganisms was negligible. The combination of UV radiation and temperature reached the decontamination level about 2 orders. The discharge parameters were studied by means of optical emmision spectroscopy. Scanning electron microscopy enabled to evaluate possible damage of exposed materials through DBD.
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Use of plasma jet for biomedical application
Doubravová, Anna ; Márová, Ivana (referee) ; Skoumalová, Petra (advisor)
This master´s thesis is focused on the utilization of the sterilization effects of low temperature plasma towards the bacterial microorganisms that occur mainly on the human skin. The plasma sterilization process is fast efficient, non-toxic, environmentally friendly, cost-effective and safe for the operating staff as well as for the patient. Another advantage of using low temperature plasma is to support cell proliferation and wound healing. By combining these advantages, an effective method can be obtained, which would sterilize the wounds sparingly with regard to the surrounding healthy tissue and support the regeneration of the damaged tissue at the same time. In the experimental part, gram positive and gram negative bacteria were used to prove the sterilization effects with respect to different cell wall structure. Staphylococcus epidermidis and Propionibacterium acnes, which cause purulent skin inflammations, were used as gram-positive microorganisms. Serratia marcescens and Escherichia coli were selected from gram positive bacteria. These model organisms were inoculated at various concentrations on culture broths and treated by plasma at a distance of 1 mm from the agar surface. The microwave discharge was generated in argon at a power of 9 W, a gas flow rate of 5 l / min and water cooling to avoid thermal effects on the treated surface. Subsequently, model skin cells of HaCaT were exposed to low temperature plasma and tested for plasma cytotoxicity to demonstrate its healing effects. The obtained results make it possible to state that the sterilizing effects of low-temperature plasma in all tested gram-positive and gram-negative bacterial strains are verified in this work. Finally, tests were demonstrated using a suitable method of the treatment on human skin cells, where the safety and usefulness of the tested low-temperature plasma was demonstrated when applied to shorten the healing process.
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Use of plasma jet for biomedical application
Doubravová, Anna ; Márová, Ivana (referee) ; Skoumalová, Petra (advisor)
This master´s thesis is focused on the utilization of the sterilization effects of low temperature plasma towards the bacterial microorganisms that occur mainly on the human skin. The plasma sterilization process is fast efficient, non-toxic, environmentally friendly, cost-effective and safe for the operating staff as well as for the patient. Another advantage of using low temperature plasma is to support cell proliferation and wound healing. By combining these advantages, an effective method can be obtained, which would sterilize the wounds sparingly with regard to the surrounding healthy tissue and support the regeneration of the damaged tissue at the same time. In the experimental part, gram positive and gram negative bacteria were used to prove the sterilization effects with respect to different cell wall structure. Staphylococcus epidermidis and Propionibacterium acnes, which cause purulent skin inflammations, were used as gram-positive microorganisms. Serratia marcescens and Escherichia coli were selected from gram positive bacteria. These model organisms were inoculated at various concentrations on culture broths and treated by plasma at a distance of 1 mm from the agar surface. The microwave discharge was generated in argon at a power of 9 W, a gas flow rate of 5 l / min and water cooling to avoid thermal effects on the treated surface. Subsequently, model skin cells of HaCaT were exposed to low temperature plasma and tested for plasma cytotoxicity to demonstrate its healing effects. The obtained results make it possible to state that the sterilizing effects of low-temperature plasma in all tested gram-positive and gram-negative bacterial strains are verified in this work. Finally, tests were demonstrated using a suitable method of the treatment on human skin cells, where the safety and usefulness of the tested low-temperature plasma was demonstrated when applied to shorten the healing process.
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Study of Sterilization Effects Initiated by Dielectric Barriere Discharge
Slámová, Jitka ; Pekárek, Stanislav (referee) ; doc. RNDr. Karol Hensel, Ph.D., oponent (referee) ; Krčma, František (advisor)
The overall goal of the presented dissertation thesis was to study the sterilization efficiency of dielectric barrier discharge operated at atmospheric pressure. The fungi Aspergillus niger, gram-positive bacteria Bacillus subtilis and in some experiments also gram-negative bacteria Escherichia coli were used as a bio-indicator enabling to evaluate the effect of plasma assisted microbial inactivation. The samples of microorganism were placed on paper Whatman 1 or PET foil and exposed to plasma. The plasma was generated in argon, nitrogen, synthetic dry/humid air with frequency up to 10 kHz and plasma power density in the range of 1,2-2,9 W/cm3 (according to the process gas). The influence of process gas, plasma power density, plasma exposition time, type of microorganism and material of the substrate on the sterilization effect of dielectric barrier discharge was evaluated. Furthermore the contribution of each single mechanism (UV radiation, temperature and reactive species) to the sterilization effect of plasma and influence of gas humidity was evaluated. The DBD was analysed by means of optical emission spectroscopy, thermocouple was used to measure temperature during a sterilization process. In order to verify the mechanical damage of the microbial cell or the substrates during the plasma process the samples were studied by scanning electron microscopy. Generally, on the basis of experimental results, at increasing treatment times, the remaining number of spores (CFU) decreased. Similarly at increasing the plasma power input, the sterilization rate increased. When sterilising the spores of A. niger in plasma using different process gasses, the efficiency of plasma sterilization decreased as follows: argon, humid synthetic air, nitrogen and dry synthetic air. The results observed in argon plasma using different microorganism demonstrated that the sensitivity of vegetative cells resp. spores to DBD decreased as follows: A. niger spores, B. subtilis vegetative cells, E. coli vegetative cells and B. subtilis spores. Simultaneously results observed for sterilization of spores and vegetative cells of B. subtilis and A. niger demonstrated that the spores are generally more resistant to plasma than are the corresponding vegetative cells. Combining the results of contribution of each single mechanism, optical emission spectroscopy and inactivation characteristic it was found out that the reactive species significantly contribute to the plasma sterilization in all process gasses. Furthermore the inactivation process can be partly assisted by UV radiation and also the temperature can contribute in limited extent to inactivation process in some gasses. The contribution of UV radiation to the plasma sterilization decreased as follows: nitrogen, argon, dry syntetic air and humid syntetic air. Moreover it was found out that the contribution of each single mechanism can be species dependent, this is due to the different response of microorganism to the unfavorable external conditions. SEM analysis of the substrates prooved the etching actions of the plasma generated in all process gasses on the surface of the PET foil. The several minute plasma exposition of the PET foil resulted in the occurence of the „hole corrosion“ on the PET surface. Contrary to these there were no visible changes observed in the paper structure.
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