Sporicidal activity determination using the modified standard protocol
The current improved standard protocol (experiment workflow depicted in Fig. 2) is based on a previously published protocol10 later modified by Khun et al.13. We made several following optimizations to the previous protocols to standardize the comparison of NTP devices. We simplified sample preparation by purchasing a commercial stock spore suspension. The samples were prepared and also evaluated in the same laboratory and only shipped to the respective laboratories for exposure by individual devices. We also introduced controls within the same tested membrane, and automated the analysis of inhibition zones using Aurora software.
Sample Preparation
Spores of the Gram-positive bacterium Bacillus subtilis (ATCC 6633) were deposited to a membrane (Whatman Nuclepore Track-Etched Polycarbonate Membrane Filter, hydrophilic, 25 mm in diameter, circle, 0.1 μm pore size) in a homogeneous monolayer. This bacterium was selected as a suitable non-pathogenic bacterial representative used to determine the sterilizing ability of various agents. A stock suspension of B. subtilis spores (Excelsior scientific, Cell Line 6633, 108 CFU per 0.1 mL) was diluted 1:1000 in sterile Ringer’s solution (8.6 g/L NaCl, 0.3 g/L KCl and 0.33 g/L CaCl2 in Water for Injections). The membrane was inserted into a holder (Whatman Swin-Lok Syringe-Type Reusable Membrane Filter Holders, for 2.5 cm filter, polypropylene holder) and sterilized in an autoclave. The spores were deposited via filtration using a vacuum flask closed with a rubber plug and connected to a vacuum pump and a pressure gauge. The sterilized holder with the membrane was connected to the tube passing through the plug and a 10 mL syringe with the working spore suspension was attached to the top of the filter holder (Fig. 2). The suspension was filtered through the membrane using the vacuum flask at approximately 80 kPa for 10–15 min, and the empty syringe was removed afterwards. To form a homogeneous layer of spores, sterile air was blown through the membrane for another 5 min. The membrane was removed from the holder under sterile conditions and placed in an open Petri dish to dry for about 1 h.
Experimental workflow of the used standard protocol.
NTP exposure
Samples (dried membranes with deposited spores) were cut in half, one half was treated with NTP and the other one served as control. All NTP treatments performed by different devices had a fixed duration of 30 min and were performed at a distance of 1 cm between the electrode and the sample, except for the indirect RF source (1), where the distance was smaller due to its setup, as explained below. For each device, the most efficient configurations determined in preliminary experiments were used for the NTP treatments. Afterwards, surviving spores were detected on both the treated and control samples in parallel. In case NTP exposure and sample evaluation took place in different laboratories, each membrane half was placed in a separate sterile Petri dish and sealed with parafilm, and only the half to be treated was shipped.
CFU reduction
The reduction of CFU after the NTP treatment of spores was analyzed by bacterial cell recovery from the membrane. The membrane half (treated or control) was transferred with sterile tweezers into a sterile 50 mL Falcon test tube containing 10 mL of sterile Ringer’s solution and 5 sterile glass beads. Spores were washed into suspension by vigorous mixing for 60 s using a vortex mixer. To determine the number of viable bacteria, the suspension was decimally diluted, inoculated on Luria–Bertani (LB) agar plates (Miller, Belgium), cultivated for 24 h at 37 °C, and colonies were counted and normalized to corresponding untreated control. All experiments were performed in triplicates (three biological repetitions of one technical replicate), averaged, and depicted in percent +/- standard deviation.
Analysis of the Inhibition zones—Aurora
To analyze the local sporicidal effect of NTP treatment, the size of the inhibition zone (i.e., area without bacterial growth) on the sample was examined as described previously13. Samples were prepared as described above and the complete (uncut) membrane was treated with NTP as described above. Afterwards, treated membranes were placed directly on the LB agar plates (Miller, Belgium) and cultivated for 24 h at 37 °C. After the cultivation, images of the plates with membranes were taken and the area of inhibition zones was analyzed using Aurora software, in collaboration with HexTech s.r.o., Czech Republic. This tool was already used for the same purpose22,23 and is described more in detail by Hrudka24. It uses artificial intelligence (AI) and machine learning for the classification of pixels in the input image according to the intensity values of the RGB channels. This approach enables the software to accurately differentiate between bacteria-free areas (inhibition zones) and microbial cultures and define a percentage of the sample area occupied by the microbial culture with over 98% accuracy. The results were normalized to corresponding untreated controls. All experiments were performed in triplicates, averaged, and depicted in percent +/– standard deviation.
NTP generating devices
Seven different NTP generating devices from five laboratories across Europe were studied, namely indirect RF source (1), PCC (2), RF Ar/He jet (3), vDBD (4), Ar jet (5), CD (6) and MiniJet (7). All of them operate at atmospheric pressure, making them suitable for biological applications. In preliminary experiments, the parameters of individual devices were fine-tuned for the highest sporicidal efficacy while avoiding damage to the sample. These preliminary tests were conducted for a wider range of the parameter combinations (for devices allowing it) in the same way as described above for the main experiments. Configurations that destroyed the membrane due to high or very focused energy deposition were excluded. We selected several configurations that seemed to match the above and used them for comparison study. The configurations are listed in the table of technical parameters (Table 1) together with details on the type of excitation signal, power (measured at the NTP generating device), supply voltage peak to peak (also measured at the NTP generating device) and working gas (flow rate, if there is any). The discharge power of non-DC supplies was determined using Lissajous figures, which depict the charge transferred between the electrodes as a function of the voltage supplying the device. The Lissajous figures were generated by measuring the HV supplying the NTP generating device and the voltage on the measurement capacitor connected in series. The charge generated during the discharge in the NTP generating device (microdischarges) was determined and its dependence on the supply voltage was plotted, enabling discharge power calculation25. Treatment with UV light (254 nm) using a laminar flow box lamp was included as a conventional sterilization method and served as positive control.
Indirect radiofrequency (RF) source (1)
The indirect RF source (1) (Fig. 3) is a special type of plasma jet device developed at Consorzio RFX in Padova, Italy. Compared to traditional plasma jet devices this one does not work with direct contact of NTP with the treated sample26. Its base is made of a copper tube, 12.0 ± 0.5 mm in diameter, closed on one side with a grounded grid electrode made of brass. The second grid electrode is placed 1.0 ± 0.1 mm above the grounded one, inside the tube, and it is excited with a RF voltage of 5.0 ± 0.2 MHz frequency and 1.0 ± 0.1 kV peak-to-peak amplitude. When He flows inside the tube, at a rate of 3.0 ± 0.1 L/min, NTP is formed between the two grids. The total power transferred to the NTP was measured previously, using a Hall current probe27, to be approximately 0.41 ± 0.05 W. To fully exploit the chemical species produced by the NTP which enrich the afterglow, the source shall be placed as close as possible to the sample. Several studies have already been carried out on this device; showing inactivation efficacy on different bacterial species28,29.
Scheme and a photograph of the He indirect radiofrequency source (indirect RF source (1)) developed at Consorzio RFX in Padova, Italy.
Plasma coagulation controller (PCC) (2)
The Plasma Coagulation Controller (PCC (2)) (Fig. 4) is a He plasma jet device developed at Consorzio RFX in Padova, Italy. Although originally developed for enhancing blood coagulation, its antibacterial efficacy has been reported as well20. A central tungsten electrode, powered by voltage micropulses, is covered with a glass capillary (outer diameter of 1.0 ± 0.1 mm) and surrounded by a glass nozzle (inner diameter of 3.5 ± 0.2 mm); and a grounded ring electrode is placed just outside the nozzle. NTP is generated in the gap between the capillary and the nozzle, where He flows at a rate of 2.0 ± 0.1 L/min, and a plasma plume develops behind the nozzle. The two following parameters can be tuned: the frequency and the amplitude of the pulses. In the present study, pulses of 6.1 ± 0.1 kV repeated at 5.00 ± 0.01 kHz frequency were selected for testing. Since the micro-pulsed waveform slightly bounces on the negative side after the initial positive pulse, the peak-to-peak voltage is higher than the peak voltage control parameter. Exact power measurement for this source has not become available to date; however, an upper limit of the power was determined by measuring voltage and current on the low voltage side. This value includes transformer dissipation and therefore cannot precisely determine the power transferred to NTP, but estimates it is below 4 W. More technical details were published previously30.
Scheme and a photograph of the Plasma Coagulation Controller from Milano (PCC (2)), the non-thermal plasma generating device based on He plasma jet, developed at Consorzio RFX in Padova, Italy.
Radiofrequency (RF) plasma jet (3)
The radiofrequency plasma jet (RF Ar/He jet (3)) (Fig. 5) developed at the Università di Milano-Bicocca, Italy, is a device designed for operating with large parameter tunability and enables studies on NTP generation in RF electric fields31. A central tungsten electrode is covered with aluminum oxide, except for a 13 ± 1 mm at the end, which remains exposed. A grounded ring electrode is placed outside the nozzle made of a standard Pasteur pipette (internal diameter of 5.0 ± 0.2 mm, tapered at the end to 3.5 ± 0.5 mm), where the Ar or He gas flows at a rate of 3.0 ± 0.1 L/min and the plasma plume is formed. This configuration is designed to reduce the sensitivity of the device to the surrounding environment. A sinusoidal voltage generator in the MHz range delivers a HV waveform to the central tungsten electrode, amplified by a resonant circuit formed by an inductor and the device capacitance. The power supply is modulated with a 5% duty cycle on a kHz-based time scale. Two tunable parameters impact the operation of the source: i.e., the frequency of the generated voltage waveform, in the range of 1–15 MHz, and the frequency of the modulation, ranging between 0.1 and 10 kHz. The power of the source has been measured using a Rogowski coil, properly correcting for parasitic phase shift32. However, given the high noise typical for the RF range, the measurement is affected by a large error.
Scheme and a photograph of the non-thermal plasma generating device based on the radiofrequency plasma jet (RF Ar/He jet (3)), developed at Università di Milano-Bicocca, Italy.
Volume DBD device (4)
The volume DBD (vDBD (4)) (Fig. 6) is an NTP generating device with a planar electrode configuration developed at Wroclaw University of Science and Technology, Poland. Its antibacterial and antifungal properties were reported previously33,34. The plane-parallel electrodes are made of aluminum with dimensions of 110 × 110 mm and the distance between them is 3 mm. The upper electrode is supplied with a HV AC pulse generator (Dora PS, Wroclaw, Poland), and the bottom one is grounded and covered with a 1 mm thick aluminum oxide dielectric layer. NTP is generated in the air gap between the upper electrode and the dielectric layer. At atmospheric pressure, this type of discharge acquires the form of many independent transient current filaments or microdischarges. Increasing supply voltage generates significantly more microdischarges35. The voltage amplitude was set to 6.2 ± 0.2 kV and the fundamental frequency of the sinusoidal signal forming a voltage pulse of variable period was 38 ± 2 kHz. The treated samples were placed on the dielectric layer and were hence in direct contact with NTP. NTP treatment was carried out at three different discharge powers ((24.2 ± 0.2) W, (14.8 ± 0.2) W, and (5.4 ± 0.2) W) determined from Lissajous curves as described previously36. The power dissipated in the device was regulated by setting the period of the voltage pulse. The power system showed some instability of operation leading to changes in the period of the pulse repetition during the measurements. To overcome this issue, we used the concept of duty cycle (i.e., ratio of pulse duration over pulse repetition time).
Scheme and a photograph of a non-thermal plasma generating device based on the volume dielectric barrier discharge (vDBD (4)) with a planar electrode configuration developed at Wroclaw University of Science and Technology, Poland.
Ar plasma jet (5)
The Ar plasma jet device developed at Wroclaw University of Science and Technology, Poland is a cylindrical plasma jet with additional Ar gas flow (Ar jet (5)) (Fig. 7). Its main element is an aluminum oxide tube with inner and outer diameters of 1.6 and 5.5 mm, respectively. Two ring brass electrodes are placed on its outer surface, 5 mm apart, and supplied with a HV AC pulse generator (Dora PS, Wroclaw, Poland). Electric discharge develops inside the dielectric tube and the generated NTP is carried out with the Ar flow of 2.5 ± 0.2 L/min. This setup allows for direct treatment of sample surfaces with a plasma plume, the diameter of which decreases with distance from the outlet of the dielectric tube. The used supply voltage amplitude was 5.5 ± 0.2 kV and the fundamental frequency of the sinusoidal signal was 38 ± 2 kHz. NTP exposure was carried out at three different discharge powers (i.e., powers dissipated in the device to generate NTP), as determined using Lissajous curves and regulated by varying the period of the voltage pulse. Similarly to the vDBD (4) device, some instability in the operation of the power supply system (i.e., fluctuation of the pulse repetition time) was observed during the NTP exposure. Therefore, we used the duty cycle parameter to set the operating conditions of the device.
Scheme and a photograph of the non-thermal plasma generating device based on the Ar plasma jet (Ar jet (5)) developed at Wroclaw University of Science and Technology, Poland.
Corona discharge device (6)
The Corona discharge CD (6) device (Fig. 8) developed at the University of Chemistry and Technology, Prague, Czech Republic is a “plug and play” NTP generating device operating in the air37. It is based on a DC bipolar corona discharge with a point-to-ring electrode system. The point electrode is a syringe needle (Medoject 0.6 mm × 25 mm needle) and a brass conical ring electrode approximately 11 mm in diameter is 3.3 mm below. When connected to the HV DC power supply, a negative corona discharge is formed on the tip of the point electrode (connected to the negative terminal of a HV source) and a positive one burns at the edge of the ring electrode (connected to the positive terminal of a HV source), together forming the bipolar corona discharge. The current and voltage of the discharge are 150 ± 20 µA and 7 ± 0.1 kV, respectively, as had been set by precise positioning of the electrodes. The electric system is covered by a tubular 3D-printed PETG (polyethylene terephthalate glycol) core, which makes the device safe and easy to manipulate and operate. The overall arrangement and geometry lead to the creation of an ion wind by ions accelerated in the electric field between the electrodes. It carries the reactive species from the discharge towards the sample and then back to the electrode system through the holes around the ring electrode. Therefore, the atmosphere around the sample is closed (no access to fresh ambient air) and accumulates the reactive species inside the sample chamber. NTP treatment by the CD (6) device is indirect, i.e., there is no direct contact between NTP and the sample, the sample is only impacted by the afterglow (the reactive species generated in NTP).
Scheme and a photograph of the non-thermal plasma generating device based on DC bipolar corona discharge (CD (6)) operating in the air developed at University of Chemistry and Technology, Prague.
MiniJet (7)
The MiniJet PM-10.R (MiniJet (7)) device (Fig. 9) is available commercially (Heuermann HF-Technik GmbH, Germany)38, and is operated at the Czech Technical University in Prague in the laboratory PlasmaLab@CTU39. This device is based on the microwave Ar plasma jet. To ignite NTP, a nozzle (6 mm in diameter) is placed inside the starter box, mounted at the side of the chassis, in such a way that the inner electrode gently touches the wire meshwork inserted within. After the power is switched on, the nozzle is slowly pulled out of the box, and the NTP plume forms. The generated NTP is very pure and has a high degree of ionization, as determined by the operating frequency. The MiniJet (7) generator produces a constant and reproducible microwave signal of about 2.45 GHz in the ISM frequency band (reserved for industrial, scientific, and medical use). The working gas is Ar, as guaranteed by the manufacturer. Both the power (ranging between 2 and 10 W) and the flow (up to 3 L/min) can be set at the front panel. NTP temperature and size of the plume are directly proportional to the microwave power and inversely proportional to the gas flow. The schematic of the device is not provided, as it is not available.
Photograph of the commercially available MiniJet (7) PM-10.R (Heuermann HF-Technik GmbH, Germany), the microwave plasma jet device operated at Czech Technical University in Prague, Czech Republic.
Energy deposition (thermograms)
An infrared image (thermogram) was taken for each sample after 30 min of NTP treatment for energy deposition analysis. The infrared camera FLIR E4 2.0 L (Teledyne FLIR), IR resolution of 80 × 60, and accuracy of ± 2 °C, was placed about 20 cm over the sample (facing perpendicular to the sample). The images were processed and the peak temperature was determined using FLIR Tools software (FLIR Systems). Area corresponding to the membrane was selected for the analysis. The temperature scales were adjusted to be identical for all the samples to ensure comparability of the results.
