PLASMOSE - antimicrobial effects of modular atmospheric plasma sources

dgkh000112 Research Article PLASMOSE - antimicrobial effects of modular atmospheric plasma sources Plasmose - antimikrobielle Wirksamkeit modularer atmosphärischer Plasmaquellen Ehlbeck Ehlbeck Jörg J

INP Greifswald e.V. (Leibniz-Institute for Plasma Science and Technology), Greifswald, GermanyINP Greifswald e.V. (Leibniz-Institute for Plasma Science and Technology), Greifswald, Germany

ehlbeck@inp-greifswald.de author Brandenburg Brandenburg Ronny R

INP Greifswald e.V. (Leibniz-Institute for Plasma Science and Technology), Greifswald, Germany Vanguard Medical Services for Europe AG, Berlin, Germany

author von Woedtke Woedtke Thomas T

INP Greifswald e.V. (Leibniz-Institute for Plasma Science and Technology), Greifswald, Germany

author Krohmann Krohmann Udo U

INP Greifswald e.V. (Leibniz-Institute for Plasma Science and Technology), Greifswald, Germany

author Stieber Stieber Manfred M

INP Greifswald e.V. (Leibniz-Institute for Plasma Science and Technology), Greifswald, Germany

author Weltmann Weltmann Klaus-Dieter KD

INP Greifswald e.V. (Leibniz-Institute for Plasma Science and Technology), Greifswald, Germany

author German Medical Science GMS Publishing House

Düsseldorf

610 plasma jet microwave plasma sterilization decontamination non-thermal plasmas Plasma Jet Mikrowellenplasma Sterilisation Dekontamination nicht-thermisches Plasma 20080311 engl 1863-5245 3 1 GMS Krankenhaushygiene Interdisziplinär GMS Krankenhaushyg Interdiszip Plasma Medicine - its perspective for wound therapy 14 Das technologische Potential von nicht-thermischen Plasmen zur antimikrobiellen Behandlung von thermolabilen Materialien wurde in einer großen Zahl von Forschungsarbeiten dokumentiert, die Realisierung in einem industriellen plasmabasierten Dekontaminationprozess bleibt aber eine große Herausforderung. Einer der Gründe für diese Situation ist die Tatsache, dass ein antimikrobieller Prozess sowohl alle Anforderungen des Produkts als auch die des gesamten Aufbereitungsprozesses, z.B. eines medizinischen Instruments, berücksichtigen muss. Ziel des durch das BMBF geförderten Verbundprojekts PLASMOSE (Plasmagestützte Oberflächenmodifizierung mittels modularer selektiver Plasmaquelle) ist der Nachweis der Anwendbarkeit eines plasmabasierten Prozesses zur antimikrobiellen Behandlung von thermolabilen Produkten. Dazu werden modulare selektive Plasmaquellen bei Atmosphärendruck eingesetzt. Der Ansatz beruht auf der Kombination der technischen Vorteile eines Atmosphärendruckplasmas mit den Vorteilen bzgl. der Flexibilität und der Handhabungseigenschaften von modularen Vorrichtungen. Zwei unterschiedliche Aufgaben wurden als Zielstellung ausgewählt: die Außenbehandlung von Medizinprodukten und die Innenbehandlung von Hohlkörpern als Primärverpackung für pharmazeutische Produkte. Die Behandlung der äußeren Oberflächen von Medizinprodukten, in diesem Fall Kathetern für intrakardiale elektrophysiologische Untersuchungen, wurde mittels eines RF angeregten Plasmajets in Argon durchgeführt. Aufgrund seiner kompakten Ausführung ist er prädestiniert für eine Modularisierung und kann dadurch nahezu an jede dreidimensionale Struktur angepasst werden. Die Realisierung der antimikrobiellen Behandlung der als Primärverpackung dienenden Hohlkörper hat deutlich andere Prozessanforderungen. Dieser Prozess muss in einer mit hohen Taktraten arbeitenden Abfüllmaschine integrierbar sein und darf aus Kostengründen keine teuren Arbeitsgase benötigen. Daher wurde zur Realisierung eine mikrowellenangeregte Laufentladung eingesetzt, die in Umgebungsluft arbeitet. Der Prozess wurde für die Behandlung von 200 ml PET Flaschen optimiert. Hierzu wurden sowohl Simulationen des elektrischen Feldes als auch optische Emissionsspektroskopie durchgeführt. Begleitet wurden diese Untersuchungen durch mikrobiologische Tests zur Bestimmung der Abtötungskinetik. The technological potential of non-thermal plasmas for the antimicrobial treatment of heat sensitive materials is well known and has been documented in a great number of research activities, but the realisation of industrial plasma-based decontamination processes remains a great challenge. One of the reasons for this situation is the fact that an antimicrobial treatment process needs to consider all properties of the product to be treated as well as the requirements of the complete procedure, e.g. a reprocessing of a medical instrument. The aim of the BMBF-funded network project PLASMOSE is to demonstrate the applicability of plasma-based processes for the antimicrobial treatment on selected, heat sensitive products. Modular and selective plasma sources, driven at atmospheric pressure are used. This basic approach shall combine the technological advantages of atmospheric pressure plasmas (avoidance of vacuum devices and batch processing) with the flexibility and handling properties of modular devices. Two different objectives were selected: the outer surface treatment of medical products and the treatment of hollow packaging for pharmaceutical products. The outer surface treatment of medical products, in particular catheters for intracardial electrophysiological studies, is investigated by means of RF-driven plasma jets in argon. Due to its compact design they are predestined for modularisation and can be adapted to nearly any complex 3-dimensional structure as given by the medical products. The realisation of an antimicrobial treatment process of hollow packaging for pharmaceutical products has quite different demands. Such a process is needed to be implemented in in-line filling procedures and to work without additional process gases. The idea is to use an atmospheric air, microwave-driven self propagating discharge. The plasma process is optimized for the decontamination of 200 ml bottles by field simulation studies combined with optical emissions spectroscopy and micro-biological tests. Introduction The technological potential of non-thermal plasmas for the antimicrobial treatment of heat sensitive materials is well known (see e.g. , , , , , . However the realisation of industrial plasma-based decontamination processes is still a challenge for research and development. This intended the BMBF-funded network project “PLASMOSE” (German: Plasmagestützte Oberflächenmodifizierung mittels modularer selektiver Plasmaquelle) consisting of ten partners with the aim to demonstrate the applicability of plasma-based processes for the antimicrobial treatment on selected, heat sensitive products. Modular and selective plasma sources, driven at atmospheric pressure are used, since they combine the technological advantages of atmospheric pressure plasmas (avoidance of vacuum devices and batch processing) with the flexibility and handling properties of modular devices. Since several years an increasing demand for new antimicrobial processes is registered. Among others the following two objects are of great interest: The complexity of medical devices is continuously increasing and contains the use of thermo labile materials (Figure 1 ), which can not be treated by means of classical sterilisation methods. In packaging industry for food and pharmaceutical products biological decontamination is requested in order to reduce or even to omit preservatives. For both above mentioned objectives the antimicrobial process must be able to treat thermo labile materials such as polyethylene (PE) or polyethylenterephthalat (PET). The high complexity of the most medical products, e. g. instruments for micro invasive surgery, requires an antimicrobial process which is capable of the treatment of small gaps and lumina which causes the challenge for most new technologies. The packaging materials are in most cases simple structures as foils or hollow compartments, e. g. bottles, and require a cost efficient and very fast process. In beverage industry, for example, the treatment time for a new technology has to be below 3 s for a single bottle to be competitive to existing processes mainly based on hydrogen peroxide. In order to fulfill the divergent requirements of biological decontamination processes for the two above mentioned selected examples individual solutions have to been investigated. Although low pressure plasma devices are widely examined and the processes causing the antimicrobial effect are fairly well understood the main drawback is the missing ability of treating gaps and lumina with high aspect ratios. The reason for this is, that the main effect is based on UV interaction in combination with erosion processes both needing a direct access to the contaminated surface. In contrast atmospheric pressure plasmas allow due to better handling properties a certain access to gaps and lumina, but they are often difficult to scale and temperature problems occur. Reprocessing of medical products Methods The radio-frequency (rf)-driven plasma jet (see Figure 2) used for first studies on antimicrobial treatment with such plasma sources consists of a nozzle made of ceramics with an inner diameter of the nozzle outlet of about 7 mm</PlainText></TextGroup>. The rf voltage (f=27.12 MHz, P=20 W) is coupled via a matching network to the needle electrode which is mounted in the centre of the nozzle (Figure 2 <ImgLink imgNo="2" imgType="figure"/>). Typically a gas flow of about 20 slm of pure argon is applied.</Pgraph> <Pgraph>The plasma is generated in the nozzle starting from the tip of the needle electrode and expands into the surrounding air outside the nozzle, since the substrate serves as a “virtual grounded electrode” due to the effect of stray capacitances. The plasma jet has a length of about <TextGroup><PlainText>25 mm</PlainText></TextGroup> measured from the nozzle outlet and a diameter of about 8 mm for the conditions being considered. It has been shown by spatially resolved optical emission spectroscopy and short exposure time photos that the plasma is filamentary and “active”, i.e. high energetic electrons are present at the entire length of the jet (Figure 2 <ImgLink imgNo="2" imgType="figure"/>, right) <TextLink reference="7"></TextLink>. Along the plasma jet a gradual mixing of the argon gas with the surrounding air is investigated. </Pgraph> <Pgraph>Instead of using the substrate as virtual grounded electrode a grounded ring electrode can be placed directly at the nozzle outlet. In this configuration the plasma jet is significantly shortened, but the impedance matching of the system is more stable and less sensitive to stray capacitances. However, even in this case the plasma is filamentary, but additionally; a diffuse shine is observed at the nozzle outlet. Due to the reduced length of the plasma outside the nozzle less mixing of the argon gas with the surrounding air is observed.</Pgraph> <Pgraph>Several single plasma jet modules can be arranged in arrays to treat surfaces of objects having complex geometries such as the catheters. Therefore the jets can be mounted in a ring-like structure for example (Figure 3 <ImgLink imgNo="3" imgType="figure"/>).</Pgraph> <Pgraph>For the outer treatment of catheters or cables the jet nozzle can be equipped with a “T-type” adapter made of glass alternatively (Figure 4 <ImgLink imgNo="4" imgType="figure"/>). The plasma is generated inside the glass adapter, while the catheter is guided via the apertures at both sides of its vertical part. The generated plasma surrounds the outer surface of the catheters more or less completely. </Pgraph> <Pgraph>These different jet configurations can be implemented as modules in a treatment device, shown in Figure 5 <ImgLink imgNo="5" imgType="figure"/> and Figure 6 <ImgLink imgNo="6" imgType="figure"/>, to demonstrate the applicability to real medical devices as e. g. catheters (Figure 1 <ImgLink imgNo="1" imgType="figure"/>). In this device the catheter is fixed in horizontal direction and a plasma module is moved along the catheter.</Pgraph> <SubHeadline>Results and discussion</SubHeadline> <Pgraph>Optical emission spectroscopy (details in <TextLink reference="7"></TextLink>, <TextLink reference="8"></TextLink>) has shown that several possible microbicidal components are generated by the plasma jet, namely </Pgraph> <Pgraph> <OrderedList> <ListItem level="1" levelPosition="1" numString="1.">VUV-radiation by means of emissions mainly from Ar<Subscript>2</Subscript>* excimers</ListItem> <ListItem level="1" levelPosition="2" numString="2.">UV-radiation by means of molecular bands emissions mainly from of excited NO, OH and N<Subscript>2</Subscript> </ListItem> <ListItem level="1" levelPosition="3" numString="3.">reactive oxygen species such as O-atoms and OH-radicals and </ListItem> <ListItem level="1" levelPosition="4" numString="4.">low molecular chemical products, e.g. nitric oxide NO. In fact ozone O<Subscript>3</Subscript> is produced as well, but not determined by its optical emissions. </ListItem> </OrderedList> </Pgraph> <Pgraph>To test the antimicrobial effect of the plasma jet, PE-strips (8x32 mm) are inoculated punctually (diameter of contaminated area about 5 mm) with a suspension of vegetative bacteria (Escherichia coli) or endospores (Bacillus atrophaeus), respectively, dried under aseptic conditions and treated by the plasma jet modules, described in Figure 2 <ImgLink imgNo="2" imgType="figure"/>. The results are shown in Figure 7 <ImgLink imgNo="7" imgType="figure"/> and Figure 8 <ImgLink imgNo="8" imgType="figure"/>. </Pgraph> <Pgraph>Plasma jet treatment of spores and vegetative bacteria on PE-strips results in an increasing time-dependent reduction of the microorganisms. Depending on the initial contamination and the detection limit of microorganisms, recovery given by the methods (colony counting method: 100 cfu/strip (cfu – colony forming units), see <TextLink reference="7"></TextLink>; membrane filtration: 1 cfu/strip) a maximum reduction between 4 and 6 orders of magnitude is investigated. </Pgraph> <Pgraph>In further studies discussed elsewhere the best decontamination was achieved by a direct plasma treatment of the PE-samples. It was pointed out that UV-radiation cannot solely be responsible for the inactivation of microorganism <TextLink reference="7"></TextLink>. Thus, a co-action of excited reactive species, partially assisted by UV and VUV radiation, heat (a moderate but significant heating of the jet can give a certain effect on heat-sensitive bacteria) and low-molecular chemical products can be assumed. However, comparing Figure 7 <ImgLink imgNo="7" imgType="figure"/> and Figure 8 <ImgLink imgNo="8" imgType="figure"/>, the plasma jet with the ring electrode (jet 2) seems to be less effective than the jet without ring electrode (jet 1). For example for E. coli after 4 min of treatment with the jet 1 the detection limit of 100 cfu/object is reached. Using jet 2 this value is reached after 7 min. This difference can be understood by taking into account the reduced production of reactive oxygen species and chemical compounds as well as the lower energy input on the contaminated area by using jet 2. Furthermore the initial contamination can hardly be controlled in our procedure and thus, it is slightly different for both experiments. This may have an influence on the reduction of micro organisms, too.</Pgraph> <Pgraph>To investigate the efficiency of antimicrobial plasma treatment by means of plasma jets on catheters they are divided in 6 sections (each 6 cm long). Each section is contaminated with a suspension of vegetative bacteria (Staphylococcus aureus). The first five sections of the catheter are treated with the plasma module (1 or 2 cycles). The 6th section is not treated and used to determine the initial contamination (control). The treatment was performed by the “T-type” jet module (10 W, 20 slm argon with and without air admixture of 0.25 vol.%) are shown in Figure 9 <ImgLink imgNo="9" imgType="figure"/>.</Pgraph> <Pgraph>A reliable reduction of microorganisms between 4 and 6 orders of magnitude on the catheter sections is shown in Figure 9 <ImgLink imgNo="9" imgType="figure"/>. On some catheter sections no viable counts are found after plasma treatment, i.e. the minimum values are at the detection limit for all conditions. But on some objects there are still survivors, thus the maximum and the median values (data points) are somewhat higher. The spread between minimum and maximum values decreases if air is admixed to the argon gas. In this case 3 or more objects of 5 are free of viable microorganisms, thus the median is at the detection limit. This tendency may be explained by a higher number of reactive oxygen species due to the air admixture. </Pgraph> <Pgraph>These results demonstrate that the findings of the treatment of plastic strips can principally be transferred to the treatment on a real medical instrument, but however the stability and reproducibility is not satisfying at the moment. The investigations lead to the assumption that a proper conditioning of the process is necessary. </Pgraph> <Pgraph>To investigate the effect of a hydrated surface or environment of the microorganisms a first preliminary investigation has been performed. About 1 ml of distilled water was filled in a petri dish and treated by the plasma jet (jet 2) for up to 20 min. After several times of treatment the pH of the water was measured by test strips. The results are shown in Figure 10 <ImgLink imgNo="10" imgType="figure"/> (left). </Pgraph> <Pgraph>It is clearly seen, that an acidification is caused by the argon plasma jet treatment. In the experiment, the initial pH of 5.5 decreases to 2. It has to be mentioned that the amount of water in the Petri dish was reduced during the treatment due to evaporation and nebulisation. Therefore the slope of the curve in Figure 10 (left) is not linear. The correlation of acidification and antimicrobial effects is shown in the right diagram of Figure 10. In this experiment an initial number of B. atropheus spores was given into solution with a defined pH for several minutes and the recovery of the microorganisms was checked. For pH <5 a significant reduction effect is investigated. For pH=2 about 6 orders of magnitude reduction is observed. </Pgraph> <Pgraph>It is known that nitride oxides in contact with wet films or droplets lead to an acidification through different mechanisms. In the gas phase nitrogen dioxide NO<Subscript>2</Subscript> can react with OH-radicals forming nitric acid HNO<Subscript>3</Subscript> which dissolves into the aqueous phase. NO<Subscript>2</Subscript> can be dissolved into aqueous phase forming nitrous acid HNO<Subscript>2</Subscript> and HNO<Subscript>3</Subscript> by the reaction with H<Subscript>2</Subscript>O <TextLink reference="9"></TextLink>. In a similar way the formation of HNO<Subscript>3</Subscript> via reactions with nitrogen peroxides N<Subscript>2</Subscript>O<Subscript>4</Subscript> or NO might be possible. Another possibility are reactions or processes involving (hydrated) ions. However, the interaction between a plasma and a liquid is quite complex and further investigation are necessary on this issue. </Pgraph> </TextBlock> <TextBlock linked="yes" name="Decontamination of hollow packaging"> <MainHeadline>Decontamination of hollow packaging</MainHeadline> <SubHeadline>Methods</SubHeadline> <Pgraph>The basic scheme of the plasma source here described and the treatment process is given in Figure 11 <ImgLink imgNo="11" imgType="figure"/>. The device consists of a multimode waveguide structure which serves as process chamber and an ignition device which is mounted on a moveable lance. The microwave radiation is generated by a magnetron (frequency 2.45 GHz; power up to 1.7 kW) and is coupled to the waveguide via a coupling antenna. The alignment of the magnetron frequency to the process chamber geometry is performed via a moveable shorting plunger. The PET bottle is placed in the centre of the process chamber, the lance with the ignition device is driven into the bottle and by applying the microwave field the plasma is ignited in their bottom region (see Figure 11 <ImgLink imgNo="11" imgType="figure"/>, right). After the ignition of the plasma the lance is moved to its initial position outside of the bottle and the plasma moves freely to the neck of the bottle [<TextLink reference="10"></TextLink>. The plasma is generated in ambient air (i.e. with certain humidity) at atmospheric pressure.</Pgraph> <Pgraph>The shape of the plasma is predominantly given by the field configuration in the process chamber, respectively the modes which can propagate in the waveguide. Therefore simulations of the field distribution in the device by means of the CST program MicroWave Studio allow a rapid development and optimization of new designs. </Pgraph> <SubHeadline>Results and discussion</SubHeadline> <Pgraph>A very stable plasma ignition at atmospheric pressure is performed by an ignition pin similar to the electrode microwave discharges, described e.g. in <TextLink reference="11"></TextLink> The main difference of the device presented here is the launching of the plasma from the ignition device. This is mainly driven by convection. After the launching the plasma expands and moves self propagating through the waveguide and thus the hollow compartment. In Figure 12 <ImgLink imgNo="12" imgType="figure"/> the propagation of the plasma is shown. </Pgraph> <Pgraph>The plasma luminosity in the centre of the process chamber as a function of position and time is shown in the left plot of Figure 12 <ImgLink imgNo="12" imgType="figure"/>. At certain times, the snapshot of the full waveguide is shown (plots at the right). As can be seen in the snapshots, the propagating plasma has a diameter of about 40 mm and a length of 50 to 80 mm. The propagation velocity is about 0.8 m/s. The modulation of the plasma propagation and luminosity is clearly seen in the left plot of Figure 12 <ImgLink imgNo="12" imgType="figure"/> and corresponds with the maxima and minima of the standing microwave field distribution in the waveguide which is shown in Figure 13 <ImgLink imgNo="13" imgType="figure"/>. In the vicinity of the ignition pin the plasma is localized and expansion takes place after launching during the movement to the antenna. </Pgraph> <Pgraph>The velocity of the plasma propagation can be controlled by the amount of absorbed power in the plasma which heats the plasma and leads therefore to convection. Beside this movement by material transport there is also a propagation of the plasma state. This is caused by the strong absorption of the microwave field in the front of the plasma and therefore a high ionisation rate in this plasma region is generated <TextLink reference="12"></TextLink>. This effect can be easily demonstrated by coupling the microwave power from the bottom of the process chamber. Then, the plasma starts after ignition to move towards the microwave source. During this movement the plasma expands and as soon as the convection force becomes dominant, the direction of movement changes. The microwave field distribution has to be adapted to the specific compound and purposes of the treatment; otherwise a local thermal deformation of the bottles occurs. </Pgraph> <Pgraph>Optical emission spectroscopy was carried out to characterize the plasma. The radiation emitted by the plasma is coupled to an optical fibre leading to an optical multichannel analyzer (spectrometer with a ICCD-camera as detector). The overview spectrum of the UV-range is shown in Figure 14 <ImgLink imgNo="14" imgType="figure"/>. </Pgraph> <Pgraph>The UV spectrum between 200 nm and 400 nm of the microwave-induced plasma in air is dominated by two molecular bands, namely the γ-system of NO (transition A<Superscript>2</Superscript>∑<Superscript>+</Superscript>→ X<Superscript>2</Superscript>Π) and the Å-system of OH (A<Superscript>2</Superscript>∑<Superscript>+</Superscript>→X<Superscript>2</Superscript>Π). The latter can be used as a “molecular pyrometer” in order to estimate the rotational temperature T<Subscript>rot</Subscript> of the OH-molecules <TextLink reference="13"></TextLink>, <TextLink reference="14"></TextLink>, <TextLink reference="15"></TextLink>, <TextLink reference="16"></TextLink>. For the conditions being considered (high pressure and thus high collision rates) T<Subscript>rot</Subscript> is close to the gas temperature. To estimate T<Subscript>rot</Subscript>, the OH-spectrum at 309 nm is measured with a resolution of about 0.2 nm (see Figure 15 <ImgLink imgNo="15" imgType="figure"/>). From the intensity ratio of the peaks G<Subscript>ref</Subscript>/G<Subscript>0</Subscript> and G<Subscript>ref</Subscript>/G<Subscript>1</Subscript> respectively, T<Subscript>rot</Subscript> can be taken from the values tabulated in <TextLink reference="15"></TextLink>, which are based on the assumption of a Boltzmann equilibrium and the calculation of population densities. Although this method has previously been shown to be unqualified to examine small temperature gradients in a plasma, the described approach is a simple and easy method to get information on the gas temperature <TextLink reference="17"></TextLink>. A temperature of T=3000 K +/- 500 K can be estimated for the microwave induced plasma operated at a dissipated power of about 1.5 kW. This result is in agreement with the observed propagation of the plasma forced by convection. Although the temperature of the microwave excited plasma is such high, the treatment of plastic materials with an upper limit for the treatment temperature of about 330 K is possible. This effect may be explained by the diffuse shape of the plasma with a low heat capacity and the fast movement of the plasma. </Pgraph> <Pgraph>The propagating microwave plasma has many similarities to plasma torches driven at atmospheric pressures <TextLink reference="14"></TextLink>. NO is formed due to the plasma chemical reactions at high power input <TextLink reference="18"></TextLink>. Thus, the following possible lethal components of the plasma can be identified namely <TextGroup><PlainText>(i) UV-radiation</PlainText></TextGroup>, (ii) low molecular chemical reaction products (mainly nitride oxides NO), (iii) reactive oxygen species (O-atoms and OH-radicals) and heat. </Pgraph> <Pgraph>Furthermore the NO quickly forms other species by reaction with oxygen molecules present in the background air gas. This is confirmed by FTIR-analysis of the residual gas in the plastic bottles several minutes after plasma treatment (see Figure 16 <ImgLink imgNo="16" imgType="figure"/>).</Pgraph> <Pgraph>The dominant species present in the residual gas is nitrogen dioxide (NO<Subscript>2</Subscript>), which is formed via reaction of NO and O<Subscript>2</Subscript>. About 1500 ppm of NO<Subscript>2</Subscript> is present and visible as brown gas in the plastic bottle. NO<Subscript>2</Subscript> is one of the most prominent air pollutants, a poisonous gas and therefore another microbicidal component. </Pgraph> <Pgraph> <Indentation>2<Mark2>NO</Mark2> + <Mark2>O</Mark2><Subscript>2</Subscript> → 2<Mark2>NO</Mark2><Subscript>2</Subscript></Indentation> </Pgraph> <Pgraph>NO<Subscript>2</Subscript> is partly converted to nitrogen peroxide N<Subscript>2</Subscript>O<Subscript>4</Subscript>. NO<Subscript>2</Subscript> and N<Subscript>2</Subscript>O<Subscript>4</Subscript> can form nitrous acid HNO<Subscript>2</Subscript> and HNO<Subscript>3</Subscript> by the reaction with H<Subscript>2</Subscript>O. Acidification can be a candidate for antimicrobial effects. The formation of nitrous oxide N<Subscript>2</Subscript>O is of minor importance. </Pgraph> <Pgraph> <Indentation>2<Mark2>NO</Mark2><Subscript>2</Subscript> ↔ <Mark2>N</Mark2><Subscript>2</Subscript><Mark2>O</Mark2><Subscript>4 </Subscript></Indentation> </Pgraph> <Pgraph> <Indentation>4<Mark2>NO</Mark2><Subscript>2</Subscript> + <Mark2>O</Mark2><Subscript>2</Subscript> + 2<Mark2>H</Mark2><Subscript>2</Subscript><Mark2>O</Mark2> → 4<Mark2>HNO</Mark2><Subscript>3</Subscript></Indentation> </Pgraph> <Pgraph>Thus, the microwave discharge has the ability to perform a very effective decontamination. For microbiological tests, microorganism containing dilution (Escheria coli, Staphylococcus aureus or Aspergillus niger) is sprayed into the plastic bottles and dried under aseptic conditions. Contaminated PET-bottles are treated by the plasma three times, while each plasma cycle is about 550 ms long and characterized by a dissipated energy of about 300 J/cycle. After plasma treatment the bottles are scrubbed with 10 ml of a rinsing solution (water with NaCl and Tween 80). The resulting elution undergoes the classical micro-biological test procedures (count plate method or membrane filtration). The antimicrobial effect of the plasma is demonstrated in Figure 17 <ImgLink imgNo="17" imgType="figure"/>.</Pgraph> <Pgraph>Depending on the microorganism, 5 to 7 orders of magnitude of reduction are observed. Note that the total plasma-on time is about 1.5 s only. Therefore, with respect to further needed improvements, the device is applicable in an in-line aseptic filling procedure. </Pgraph> <Pgraph>In order to simulate the filling process the time between plasma treatment and the elution (more precisely between the end of the last plasma cycle and filling of the bottle with scrubber solution) was varied. The results of this experiment are shown in Figure 18 <ImgLink imgNo="18" imgType="figure"/>. </Pgraph> <Pgraph>The reduction of vegetative bacterium S. aureus is independent of the time between plasma treatment and scrubbing. At least 5 orders of magnitude of reduction is observed, even after one plasma cycle and no matter on the fact, that the bottle is eluted immediately (within about 4 s) or after about 30 min. The more resistant endospores of B. atropheus are not significantly reduced by the plasma treatment alone. The reduction is time dependent and even after 15 min there are still survivors in some bottles. This effect may be explained by the action of the NO<Subscript>2</Subscript> residual gas on the spores.</Pgraph> <Pgraph>Furthermore residual nitrous gases can be eluted by the rinsing solution leading to acidification (mainly due to formation of HNO<Subscript>3</Subscript>). This could already affect the reduction of bacteria. To exclude such effects buffered rinsing solution was used instead of water. The results are shown in Figure 19 <ImgLink imgNo="19" imgType="figure"/>.</Pgraph> <Pgraph>The difference between buffered and non-buffered rinsing solutions is clearly seen for both vegetative bacteria under consideration. With buffer the effect of the residual gas is needed for a significant reduction of the microorganisms. This has also been found for B. atropheus endospores. If the bottles are blown off from the residual gas before scrubbing no such a difference is observed. Thus the hypothesis of an acidification of the rinsing solution by residual plasma gas can be confirmed. </Pgraph> <Pgraph>The above mentioned effects need to be considered in the further process design. Furthermore they demonstrate that even the microbiological test procedures can have an influence on the final results or can do a misleading of the process effectiveness.</Pgraph> </TextBlock> <TextBlock linked="yes" name="Conclusion"> <MainHeadline>Conclusion</MainHeadline> <Pgraph>In this work two different types of atmospheric pressure plasma sources are described. In principle both of them can be applied in industrial processes since they are properly adapted on the specific products and process requirements. The products and demanded processes are as different as the plasma sources themselves. On the one side with the PET bottle decontamination a low cost application for in-line treatment with high process rates was presented. On the other side the application of plasma jets in the special reprocessing of expensive medical devices was shown. </Pgraph> <Pgraph>In both cases the principle capability and effectiveness of the plasma sources for biological decontamination up to the level of sterilization (i.e. reduction of germs by 6 orders of magnitude) could be demonstrated. It was also shown that the plasma processes can be adapted to real products, e.g. intracardial catheters. But the investigation has also shown, that indirect plasma chemical induced processes (e.g. acidification due to nitride oxides) can have an influence on the microbicidal effects. Even the microbiological analysis procedure can interfere with the plasma process and contort the microorganism reduction results. Such effects need to be known and considered in the design of industrial processes. </Pgraph> <Pgraph>In the processes a careful conditioning is necessary in order to provide the process stability and reproducibility. In order to improve plasma methods to stable and applicable plasma processes, a detailed understanding of the plasmas and its interaction with biological matter is provided.</Pgraph> </TextBlock> <TextBlock linked="yes" name="Acknowledgement"> <MainHeadline>Acknowledgement</MainHeadline> <Pgraph>This work is part of the BMBF funded network project ”Plasmose” (FKZ 13N8666), The authors are grateful to C. Lösche and A. Becker (Vanguard AG, Berlin) as well as D. Braun and B. Großjohann (Riemser Arzneimitttel AG) for fruitful discussions. The authors are grateful to J. Grundmann, W. Reich and S. Müller (all INP Greifswald) for providing the FTIR-measurements.</Pgraph> </TextBlock> <References linked="yes"> <Reference refNo="1"> <RefAuthor>Moisan M</RefAuthor> <RefAuthor>Barbeau J</RefAuthor> <RefAuthor>Crevier MC</RefAuthor> <RefAuthor>Pelletier J</RefAuthor> <RefAuthor>Philip N</RefAuthor> <RefAuthor>Saoudi B</RefAuthor> <RefTitle>Plasma sterilization. Methods mechanisms</RefTitle> <RefYear>2002</RefYear> <RefJournal>Pure Appl Chem</RefJournal> <RefPage>349-58</RefPage> <RefTotal>Moisan M, Barbeau J, Crevier MC, Pelletier J, Philip N, Saoudi B. Plasma sterilization. Methods mechanisms. Pure Appl Chem. 2002;74(3):349-58.</RefTotal> </Reference> <Reference refNo="2"> <RefAuthor>Stoffels E</RefAuthor> <RefTitle>Biomedical applications of electric gas discharges</RefTitle> <RefYear>2002</RefYear> <RefJournal>High Temp Mater Proc</RefJournal> <RefPage>191-202</RefPage> <RefTotal>Stoffels E. Biomedical applications of electric gas discharges. High Temp Mater Proc. 2002;6(2):191-202.</RefTotal> </Reference> <Reference refNo="3"> <RefAuthor>Laroussi M</RefAuthor> <RefTitle>Low temperature plasma-based sterilization: Overview and state-of-the-art</RefTitle> <RefYear>2005</RefYear> <RefJournal>Plasma Proc Polym</RefJournal> <RefPage>391-400</RefPage> <RefTotal>Laroussi M. Low temperature plasma-based sterilization: Overview and state-of-the-art. Plasma Proc Polym. 2005;2(5):391-400.</RefTotal> </Reference> <Reference refNo="4"> <RefAuthor>Becker K</RefAuthor> <RefAuthor>Koutsospyros A</RefAuthor> <RefAuthor>Yin SM</RefAuthor> <RefAuthor>Christodoulatos C</RefAuthor> <RefAuthor>Abramzon N</RefAuthor> <RefAuthor>Joaquin JC</RefAuthor> <RefAuthor>Brelles-Marino G</RefAuthor> <RefTitle>Environmental and biological applications of microplasmas</RefTitle> <RefYear>2005</RefYear> <RefJournal>Plasma Phys Control Fusion</RefJournal> <RefPage>B513-23</RefPage> <RefTotal>Becker K, Koutsospyros A, Yin SM, Christodoulatos C, Abramzon N, Joaquin JC, Brelles-Marino G. Environmental and biological applications of microplasmas. Plasma Phys Control Fusion. 2005;47:B513-23.</RefTotal> </Reference> <Reference refNo="5"> <RefAuthor>Boudam MK</RefAuthor> <RefAuthor>Moisan M</RefAuthor> <RefAuthor>Saoudi B</RefAuthor> <RefAuthor>Popovici C</RefAuthor> <RefAuthor>Gherardi N</RefAuthor> <RefAuthor>Massines F</RefAuthor> <RefTitle>Bacterial spore inactivation by atmospheric-pressure plasmas in the presence or absence of UV photons as obtained with the same gas mixture</RefTitle> <RefYear>2006</RefYear> <RefJournal>J Phys D Appl Phys</RefJournal> <RefPage>3494-507</RefPage> <RefTotal>Boudam MK, Moisan M, Saoudi B, Popovici C, Gherardi N, Massines F. Bacterial spore inactivation by atmospheric-pressure plasmas in the presence or absence of UV photons as obtained with the same gas mixture. J Phys D Appl Phys. 2006;39(16):3494-507</RefTotal> </Reference> <Reference refNo="6"> <RefAuthor>Lerouge S</RefAuthor> <RefAuthor>Tabrizian M</RefAuthor> <RefAuthor>Wertheimer MR</RefAuthor> <RefAuthor>Marchand R</RefAuthor> <RefAuthor>Yahia L</RefAuthor> <RefTitle>Safety of plasma-based sterilization: Surface modifications of polymeric medical devices induced by Sterrad((R)) and Plazlyte (TM) processes</RefTitle> <RefYear>2002</RefYear> <RefJournal>Bio-Med Mater Eng</RefJournal> <RefPage>3-13</RefPage> <RefTotal>Lerouge S, Tabrizian M, Wertheimer MR, Marchand R, Yahia L. Safety of plasma-based sterilization: Surface modifications of polymeric medical devices induced by Sterrad((R)) and Plazlyte (TM) processes. Bio-Med Mater Eng. 2002;12(1):3-13.</RefTotal> </Reference> <Reference refNo="7"> <RefAuthor>Brandenburg R</RefAuthor> <RefAuthor>Ehlbeck J</RefAuthor> <RefAuthor>Stieber M</RefAuthor> <RefAuthor>von Woedtke T</RefAuthor> <RefAuthor>Zeymer J</RefAuthor> <RefAuthor>Schluter O</RefAuthor> <RefAuthor>Weltmann KD</RefAuthor> <RefTitle>Antimicrobial treatment of heat sensitive materials by means of atmospheric pressure rf-driven plasma jet</RefTitle> <RefYear>2007</RefYear> <RefJournal>Contrib Plasma Phys</RefJournal> <RefPage>72-9</RefPage> <RefTotal>Brandenburg R, Ehlbeck J, Stieber M, von Woedtke T, Zeymer J, Schluter O, Weltmann KD. Antimicrobial treatment of heat sensitive materials by means of atmospheric pressure rf-driven plasma jet. Contrib Plasma Phys. 2007;47(1-2):72-9.</RefTotal> </Reference> <Reference refNo="8"> <RefAuthor>Foest R</RefAuthor> <RefAuthor>Kindel E</RefAuthor> <RefAuthor>Ohl A</RefAuthor> <RefAuthor>Stieber M</RefAuthor> <RefAuthor>Weltmann KD</RefAuthor> <RefTitle>Non-thermal atmospheric pressure discharges for surface modification</RefTitle> <RefYear>2005</RefYear> <RefJournal>Plasma Phys Control Fusion</RefJournal> <RefPage>B525-36</RefPage> <RefTotal>Foest R, Kindel E, Ohl A, Stieber M, Weltmann KD. Non-thermal atmospheric pressure discharges for surface modification. Plasma Phys Control Fusion. 2005;47:B525-36.</RefTotal> </Reference> <Reference refNo="9"> <RefAuthor>Dodet B</RefAuthor> <RefAuthor>Odic E</RefAuthor> <RefAuthor>Salamitou S</RefAuthor> <RefAuthor>Goldman A</RefAuthor> <RefAuthor>Goldman M</RefAuthor> <RefTitle>Biological Applications of Atmospheric Pressure Dielectric Barrier Discharges Hakone X</RefTitle> <RefYear>2006</RefYear> <RefBookTitle>0th Int Symp High Pressure Low Temperature Plasma Chemistry</RefBookTitle> <RefPage>IL-01</RefPage> <RefTotal>Dodet B, Odic E, Salamitou S, Goldman A, Goldman M. Biological Applications of Atmospheric Pressure Dielectric Barrier Discharges Hakone X. In: 10th Int Symp High Pressure Low Temperature Plasma Chemistry. Saga City, Japan; 2006. p. IL-01.</RefTotal> </Reference> <Reference refNo="10"> <RefAuthor>Ehlbeck J</RefAuthor> <RefAuthor>Ohl A</RefAuthor> <RefAuthor>Maass M</RefAuthor> <RefAuthor>Krohmann U</RefAuthor> <RefAuthor>Neumann T</RefAuthor> <RefTitle>Moving atmospheric microwave plasma for surface and volume treatment</RefTitle> <RefYear>2003</RefYear> <RefJournal>Surf Coat Technol</RefJournal> <RefPage>493-7</RefPage> <RefTotal>Ehlbeck J, Ohl A, Maass M, Krohmann U, Neumann T. Moving atmospheric microwave plasma for surface and volume treatment. Surf Coat Technol. 2003;174:493-7.</RefTotal> </Reference> <Reference refNo="11"> <RefAuthor>Lebedev YA</RefAuthor> <RefAuthor>Mokeev MV</RefAuthor> <RefAuthor>Tatarinov AV</RefAuthor> <RefAuthor>Epstein IL</RefAuthor> <RefTitle>Electrode Microwave Discharge: Status and Problems</RefTitle> <RefYear>2003</RefYear> <RefBookTitle>Microwave Discharges: Fundamentals and applications</RefBookTitle> <RefPage>55-64</RefPage> <RefISBN>3-00-011612-5</RefISBN> <RefTotal>Lebedev YA, Mokeev MV, Tatarinov AV, Epstein IL. Electrode Microwave Discharge: Status and Problems. In: Microwave Discharges: Fundamentals and applications. Greifswald; 2003. ISBN 3-00-011612-5. p. 55-64.</RefTotal> </Reference> <Reference refNo="19"> <RefAuthor>Ehlbeck J</RefAuthor> <RefAuthor>Ohl A</RefAuthor> <RefAuthor>Maaß M</RefAuthor> <RefAuthor>Krohmann U</RefAuthor> <RefAuthor>Neumann T</RefAuthor> <RefTitle>Moving Atmospheric Microwave Plasma for Surface and Volume Treatment</RefTitle> <RefYear>2003</RefYear> <RefBookTitle>Microwave Discharges: Fundamentals and applications</RefBookTitle> <RefPage>160-5</RefPage> <RefISBN>3-00-011612-5</RefISBN> <RefTotal>Ehlbeck J, Ohl A, Maaß M, Krohmann U, Neumann T. Moving Atmospheric Microwave Plasma for Surface and Volume Treatment. In: Microwave Discharges: Fundamentals and applications. Greifswald; 2003. ISBN 3-00-011612-5. p. 160-5.</RefTotal> </Reference> <Reference refNo="12"> <RefAuthor>Rackow K</RefAuthor> <RefAuthor>Ehlbeck J</RefAuthor> <RefAuthor>Krohmann U</RefAuthor> <RefAuthor>Brandenburg R</RefAuthor> <RefAuthor>Weltmann KD</RefAuthor> <RefTitle></RefTitle> <RefYear></RefYear> <RefTotal>Rackow K, Ehlbeck J, Krohmann U, Brandenburg R, Weltmann KD. in prep.</RefTotal> </Reference> <Reference refNo="13"> <RefAuthor>Pellerin S</RefAuthor> <RefAuthor>Cormier JM</RefAuthor> <RefAuthor>Richard F</RefAuthor> <RefAuthor>Musiol K</RefAuthor> <RefAuthor>Chapelle J</RefAuthor> <RefTitle>A spectroscopic diagnostic method using UV OH band spectrum</RefTitle> <RefYear>1996</RefYear> <RefJournal>J Phys D Appl Phys</RefJournal> <RefPage>726-39</RefPage> <RefTotal>Pellerin S, Cormier JM, Richard F, Musiol K, Chapelle J. A spectroscopic diagnostic method using UV OH band spectrum. J Phys D Appl Phys. 1996;29(3):726-39.</RefTotal> </Reference> <Reference refNo="14"> <RefAuthor>Laux CO</RefAuthor> <RefAuthor>Spence TG</RefAuthor> <RefAuthor>Kruger CH</RefAuthor> <RefAuthor>Zare RN</RefAuthor> <RefTitle>Optical diagnostics of atmospheric pressure air plasmas</RefTitle> <RefYear>2003</RefYear> <RefJournal>Plasma Sources Sci Technol</RefJournal> <RefPage>125-38</RefPage> <RefTotal>Laux CO, Spence TG, Kruger CH, Zare RN. Optical diagnostics of atmospheric pressure air plasmas. Plasma Sources Sci Technol. 2003;12(2):125-38.</RefTotal> </Reference> <Reference refNo="15"> <RefAuthor>de Izarra C</RefAuthor> <RefTitle>UV OH spectrum used as a molecular pyrometer</RefTitle> <RefYear>2000,33</RefYear> <RefJournal>J Phys D Appl Phys</RefJournal> <RefPage>1697-704</RefPage> <RefTotal>de Izarra C. UV OH spectrum used as a molecular pyrometer. J Phys D Appl Phys. 2000,33:1697-704.</RefTotal> </Reference> <Reference refNo="16"> <RefAuthor>Levin DA</RefAuthor> <RefAuthor>Laux CO</RefAuthor> <RefAuthor>Kruger CH</RefAuthor> <RefTitle>A general model for the spectral calculation of OH radiation in the ultraviolet</RefTitle> <RefYear>1999</RefYear> <RefJournal>J Quant Spectrosc Radiat Transf</RefJournal> <RefPage>377-92</RefPage> <RefTotal>Levin DA, Laux CO, Kruger CH. A general model for the spectral calculation of OH radiation in the ultraviolet. J Quant Spectrosc Radiat Transf. 1999;61(3):377-92.</RefTotal> </Reference> <Reference refNo="17"> <RefAuthor>Happold J</RefAuthor> <RefAuthor>Lindner P</RefAuthor> <RefAuthor>Roth B</RefAuthor> <RefTitle>Spatially resolved temperature measurements in an atmospheric plasma torch using the A(2)Sigma+, nu '=0 -> X-2 Pi, nu ''=0 OH band</RefTitle> <RefYear>2006</RefYear> <RefJournal>J Phys D Appl Phys</RefJournal> <RefPage>3615-20</RefPage> <RefTotal>Happold J, Lindner P, Roth B. Spatially resolved temperature measurements in an atmospheric plasma torch using the A(2)Sigma+, nu '=0 -> X-2 Pi, nu ''=0 OH band. J Phys D Appl Phys. 2006;39(16):3615-20.</RefTotal> </Reference> <Reference refNo="18"> <RefAuthor>Drost H</RefAuthor> <RefTitle></RefTitle> <RefYear>1978</RefYear> <RefBookTitle>Plasmachemie - Prozesse der chemischen Stoffwandlung unter Plasma-Bedingungen</RefBookTitle> <RefPage></RefPage> <RefTotal>Drost H. Plasmachemie - Prozesse der chemischen Stoffwandlung unter Plasma-Bedingungen. Berlin: Akademie-Verlag; 1978.</RefTotal> </Reference> </References> <Media> <Tables> <NoOfTables>0</NoOfTables> </Tables> <Figures> <Figure format="png" height="361" width="707"> <MediaNo>1</MediaNo> <MediaID>1</MediaID> <Caption> <Pgraph> <Mark1>Figure 1: left - Photograph of intracardial catheters used in the experiments as a representative example for the simulation of plasma-process adaptation in the reprocessing, right - detailed view on the electrodes of the catheter tip</Mark1> </Pgraph> </Caption> </Figure> <Figure format="png" height="427" width="948"> <MediaNo>2</MediaNo> <MediaID>2</MediaID> <Caption> <Pgraph> <Mark1>Figure 2: Rf-driven plasma jets (left – schematic, right – photo of the configuration shown at the left and taken with short exposure time)</Mark1> </Pgraph> </Caption> </Figure> <Figure format="png" height="294" width="926"> <MediaNo>3</MediaNo> <MediaID>3</MediaID> <Caption> <Pgraph> <Mark1>Figure 3: Ring-like arrangement of 8 single Rf-driven plasma jets (right – treatment of real catheter)</Mark1> </Pgraph> </Caption> </Figure> <Figure format="png" height="319" width="784"> <MediaNo>4</MediaNo> <MediaID>4</MediaID> <Caption> <Pgraph> <Mark1>Figure 4: “T-type” plasma jets for the outer treatment of catheters or cables</Mark1> </Pgraph> </Caption> </Figure> <Figure format="png" height="384" width="904"> <MediaNo>5</MediaNo> <MediaID>5</MediaID> <Caption> <Pgraph> <Mark1>Figure 5: Principle of the treatment device </Mark1> </Pgraph> </Caption> </Figure> <Figure format="png" height="334" width="950"> <MediaNo>6</MediaNo> <MediaID>6</MediaID> <Caption> <Pgraph> <Mark1>Figure 6: Realisation of treatment device: left: 3D drawing of the construction, right: photograph of the treatment device</Mark1> </Pgraph> </Caption> </Figure> <Figure format="png" height="381" width="519"> <MediaNo>7</MediaNo> <MediaID>7</MediaID> <Caption> <Pgraph> <Mark1>Figure 7: Median number of surviving microorganisms after treatment with plasma jet 1 (jet without ring electrode, 20 W, 20 slm Ar, 25 mm distance nozzle-substrate) of B. atrophaeus spores and E. coli on test strips (dashed line: detection limit of micro organism recovery, 100 cfu/strip)</Mark1> </Pgraph> </Caption> </Figure> <Figure format="png" height="369" width="508"> <MediaNo>8</MediaNo> <MediaID>8</MediaID> <Caption> <Pgraph> <Mark1>Figure 8: Median number of surviving microorganisms after treatment with plasma jet 2 (jet with grounded ring electrode, 20 W, 20 slm Ar, 6 mm distance nozzle-substrate) of B. atrophaeus spores, E. coli and S. aureus on test strips (dashed line: detection limit of micro organism recovery, 1 cfu/strip)</Mark1> </Pgraph> </Caption> </Figure> <Figure format="png" height="415" width="711"> <MediaNo>9</MediaNo> <MediaID>9</MediaID> <Caption> <Pgraph> <Mark1>Figure 9: Median number (n=5) of surviving microorganisms (S. aureus) after treatment with “T-type” plasma jet module (10 W, 20 slm Ar) on catheter parts (error bars: spread between minimum and maximum values; dashed line: detection limit of 1 cfu/object)</Mark1> </Pgraph> </Caption> </Figure> <Figure format="png" height="476" width="928"> <MediaNo>11</MediaNo> <MediaID>11</MediaID> <Caption> <Pgraph> <Mark1>Figure 11: Propagating microwave plasma device (left) and plasma treatment of bottles (right)</Mark1> </Pgraph> </Caption> </Figure> <Figure format="png" height="492" width="659"> <MediaNo>12</MediaNo> <MediaID>12</MediaID> <Caption> <Pgraph> <Mark1>Figure 12: Propagation of an microwave-induced plasma through the waveguide structure (left – luminosity as function of time and position; right – snapshots of the propagation at certain times), see also [19]</Mark1> </Pgraph> </Caption> </Figure> <Figure format="png" height="541" width="746"> <MediaNo>13</MediaNo> <MediaID>13</MediaID> <Caption> <Pgraph> <Mark1>Figure 13: Comparison of the simulated electric field inside the process chamber (simulated by CST MicroWave Studio) with the time averaged, spatial resolved plasma luminosity. The photo at the right shows the corresponding light emission of the plasma inside the bottle.</Mark1> </Pgraph> </Caption> </Figure> <Figure format="png" height="353" width="791"> <MediaNo>14</MediaNo> <MediaID>14</MediaID> <Caption> <Pgraph> <Mark1>Figure 14: UV-part of the measured emission spectrum of the microwave-induced plasma in air at atmospheric pressure</Mark1> </Pgraph> </Caption> </Figure> <Figure format="png" height="388" width="569"> <MediaNo>15</MediaNo> <MediaID>15</MediaID> <Caption> <Pgraph> <Mark1>Figure 15: OH-spectrum of the microwave-induced plasma</Mark1> </Pgraph> </Caption> </Figure> <Figure format="png" height="493" width="904"> <MediaNo>16</MediaNo> <MediaID>16</MediaID> <Caption> <Pgraph> <Mark1>Figure 16: FTIR-spectrum of the residual gas in a plastic bottle after several minutes after plasma treatment</Mark1> </Pgraph> </Caption> </Figure> <Figure format="png" height="526" width="520"> <MediaNo>17</MediaNo> <MediaID>17</MediaID> <Caption> <Pgraph> <Mark1>Figure 17: Median number of surviving microorganism after treatment of PET-bottles with microwave plasma in air (error bars: spread between minimum and maximum values), detection limit: 1 cfu/object</Mark1> </Pgraph> </Caption> </Figure> <Figure format="png" height="354" width="979"> <MediaNo>19</MediaNo> <MediaID>19</MediaID> <Caption> <Pgraph> <Mark1>Figure 19: Median number of surviving microorganism after treatment of PET-bottles with microwave plasma in air with variation of the time between treatment and scrubbing as well as properties of rinsing solution (error bars: spread between minimum and maximum values, dashed red lines: corresponding detection limits)</Mark1> </Pgraph> </Caption> </Figure> <Figure format="png" height="332" width="956"> <MediaNo>18</MediaNo> <MediaID>18</MediaID> <Caption> <Pgraph> <Mark1>Figure 18: Number of surviving microorganism after treatment of PET-bottles with microwave plasma in air with variation of the time between treatment and scrubbing (dashed blue lines: corresponding detection limits)</Mark1> </Pgraph> </Caption> </Figure> <Figure format="png" height="373" width="1013"> <MediaNo>10</MediaNo> <MediaID>10</MediaID> <Caption> <Pgraph> <Mark1>Figure 10: Acidification of water by plasma treatment and the effect on B. atrophaeus spores</Mark1> </Pgraph> </Caption> </Figure> <NoOfPictures>19</NoOfPictures> </Figures> <InlineFigures> <NoOfPictures>0</NoOfPictures> </InlineFigures> <Attachments> <NoOfAttachments>0</NoOfAttachments> </Attachments> </Media> </OrigData> </GmsArticle>