Bactericidal effects of low temperature oxygen plasma on bacillus stearothermophilus and staphylococcus aureus
NATURA MONTENEGRINA, Podgorica, 10(1): 57-70 57 BACTERICIDAL EFFECTS OF LOW-TEMPERATURE OXYGEN PLASMA ON Bacillus stearothermophilus AND Staphylococcus aureus Danijela VUJOŠEVIĆ 1,3 , Boban MUGOŠA 2 , Uroš CVELBAR 3 , Miran MOZETIČ 3 , Urška REPNIK 4 , Tina ZAVAŠNIK-BERGANT 4 , Danijela RAJKOVIĆ 1 , Sanja MEDENICA 2 1Centre for Medical Microbiology, Institute of Public Health, Ljubljanska bb, 81000 Podgorica, Montenegro. Email: danijela.vujosevic@ijzcg.me. 2 Centre for Prevention and Disease Control, Institute of Public Health, Ljubljanska bb, 81000 Podgorica, Montenegro. Email: boban.mugosa@ijzcg.me. 3 Plasma Laboratory F4, Jožef Stefan Institute, Jamova 39, 1000 Ljubljana, Slovenia. Email: uros.cvelbar@ijs.si. 4 Biochemistry and Molecular Biology Department, Jožef Stefan Institute, Jamova 39, 1000 Ljubljana, Slovenia. Email: urska.repnik@ijs.si Key words: Oxygen plasma, sterilization, bacteria. Synopsis Plasma of different origins has been shown to possess effective anti-microbial characteristics. In this study three complementary techniques: scanning electron microscopy (SEM), fluorescence microscopy and flow cytometry were applied to monitor time-dependent changes in bacterial viability, morphology and nucleic acids content of bacteria Bacillus stearothermophilus and Staphylococcus aureus. The plasma sterilization capability demonstrated through this study indicated the potential of this low-temperature oxygen plasma as a promising alternative sterilization technique. Ključne riječi: Kiseonikova plazma, sterilizacija, bakterije. Sinopsis BAKTERICIDALNI EFEKTI NISKO-TEMPERATURNE KISEONIKOVE PLAZME NA BACILLUS STEAROTHERMOPHILUS I STAPHYLOCOCCUS AUREUS Poznato je da plazma različitog porijekla posjeduje efikasne antimikrobne osobine. U ovoj studiji su korištene tri komplementarne tehnike: elektronska mikroskopija, fluore-scentna mikroskopija, kao i protočna citometrija za praćenje vijabilnosti, morfologije i sadržaja nukleinskih kiselina bakterija Bacillus stearothermophilus i Staphylococcus aureus, nakon obrade plazmom. Mogućnosti sterilizacije plazmom demonstrirane u ovoj studiji ukazuju na mogućnost nisko temperaturne kiseonikove plazme kao obećavajuće alternativne tehnike za sterilizaciju. Vulošević et al: BACTERICIDAL EFFECTS OF LOW-TEMPERATURE OXYGEN PLASMA ON . . . 58 INTRODUCTION A novel sterilization method capable of more rapidly killing microorganisms and less damaging material is low-temperature plasma sterilization (CHAU et al., 1996). The plasma sterilization is safer as opposed to classical sterilization of heat sensitive material with ethylene oxide which leaves highly toxic residues absorbed in the sterilized material, whereas no residuals are left on the surface after plasma treatment (PHILIP et al., 2000). Low-temperature plasma is effective against a broad range of bacteria and killing these microorganisms by producing various reactive species like oxygen, hydroxyl free radicals, and other active species, although these killing mechanisms are still being studied (GADRI et al., 2000; LAROUSSI et al., 2004). Therefore, a relatively simple and inexpensive design and the absence of the toxicity resulting from the treatment itself, give low-temperature plasma the potential to replace conventional sterilization methods of medical devices, such as implants, dental instruments etc (MOISAN et al., 2002; JACOBS & LIN, 2001; SAMUEL, et al., 1998; HELHEL et al., 2005). However, the research of low-temperature plasma sterilization has the complex issue, which is further burdened by a large number of experiment variables, is insufficiently definitive about the selected methodologies and experimental conditions. Certainly, more tests and in-depth study of low-temperature sterilization are needed to elucidate the mechanism of low-temperature plasma sterilization (CHOI et al., 2006). There is a need for reliable and accurate monitoring of plasma sterilization during the process and evaluating after. In the plasma, a large number of variables influence the whole system between physical and chemical process. However, till now no reliable methods have been found to access the sterilization during sample processing in plasma. Monitoring during the process can be done by measuring changes of reactive species in plasma. Reliable and accurate plasma diagnostic techniques are presently being developed to provide real-time information on plasma during system operation (KANAZAWA, et al., 1989). And more, new, faster and more accurate techniques for evaluation of post treated bacteria need to be developed, apart from standard count plate technique. The purpose of this study was to determine the sterilization effects of low- temperature highly dissociated oxygen plasma on two selected Gram positive bacteria, i.e. on temperature resisting Bacillus stearothermophilus and on Staphylococcus aureus, commonly involved in infections and food poisoning. Three complementary techniques apart from plate counting; scanning electron microscopy (SEM), fluorescence microscopy and flow cytometry were applied to monitor time- dependent changes in bacterial viability, morphology and nucleic acids content, all in comparison to heat-treated bacteria at 140 °C. Natura Montenegrina 10(1) 59 MATERIAL AND METHODS Bacteria. Bacillus stearothermophilus (ATCC No. 7953) and Staphylococcus aureus (ATCC No. 25923) were obtained from the MicroBioLogics CE (MN, USA). Bacterial cultures were grown overnight on Colombia agar plates (Difco Laboratories, MI, USA) at 55 o C for Bacillus stearothermophilus and 37 o C for Staphylococcus aureus. Cells were harvested and resuspended in sterile water. 100 µl suspension containing 1 × 10 7 cells of Bacillus stearothermophilus and Staphylococcus aureus were evenly distributed on the surface of sterile glass or aluminum substrate and air dried in a laminar-flow hood. Bacteria on carriers were then either exposed to low-temperature oxygen plasma, or vacuum-treated, or high- temperature dry heated or remained un-treated (control). Carriers. Pyrex glass rectangular substrate and aluminum sheets were used as carriers for bacteria. Glass carriers were used when, later on, fluorescence or flow cytometry were applied, while aluminum sheets were used with SEM, in order to avoid charging effects inside the electron microscope. Carriers’ activation. Both carriers were well activated prior to bacteria deposition to avoid bacteria agglomeration and in order to achieve uniform even distribution of cells across the entire carrier and the formation of thin layer of cells on the carrier’s surface. The activation process was also performed by oxygen plasma treatment for 5 s. Plasma with the same characteristic parameters was used for both, the activation of carriers and for the treatment of bacteria. Heat treatment. For studying of temperature effects on bacteria degradation the samples (bacteria on substrate carriers) were treated with high temperature dry heat at 140 ° C for 20 s, 2 min and 10 min. Plasma treatment. Inductively coupled radio-frequency generator with the output power of about 200 W and the frequency of 27.12 MHz was used to create uniform plasma in a glass tube with the inner diameter of 36 mm and the length of 65 cm (Figure 1). The tube was first evacuated to the pressure of 3 Pa and then filled with oxygen to pressure from 30 Pa to 150 Pa. Plasma parameters (electron temperature, neutral O-atom density, ion density) were measured with a double Langmuir probe and Fiber Optics Catalytic Probes. Samples were exposed for different treatment time to plasma with the electron temperature of 5 eV, the neutral O-atom density of approximately 8.5 × 10 21 m -3 and the ion density of 1.4 × 10 16 m -3 . In another control experiment, bacteria were exposed to vacuum conditions but without plasma in order to detect possible changes caused by evacuation only and not by plasma. Vulošević et al: BACTERICIDAL EFFECTS OF LOW-TEMPERATURE OXYGEN PLASMA ON . . . 60 Figure 1. Schematic of the discharge vessel. Plate counts technique. Before and after each treatment the samples (bacteria on carriers) were placed in sterile containers with 2 ml 0.85% NaCl saline solution. The containers were then briefly vortexed in order to wash bacterial cells from carriers. The suspension was serially diluted (1/10) in saline to the required concentration range. A sample of 100 µl of the diluted suspension was inoculated onto Colombia agar plate. Plates were than incubated at 37 °C (Staphylococcus aureus) and 55 o C (Bacillus stearothermophilus), respectively for 24-48 h. Colony forming units were finally counted to determine the number of survivors of bacteria. Scanning electron microscopy (SEM). Samples were imaged using field- emission scanning electron microscopy at a low kinetic energy of primary electrons. Low-temperature oxygen plasma-treated bacteria, high temperature dry heat-treated bacteria, vacuum-treated bacteria (control) and un-treated bacteria (control) on aluminum carriers were viewed in the Carl Zeiss Supra 35 VP scanning electron microscope. Images were taken at approx. 10 -3 Pa with a beam of primary electrons at 1000 eV and 600 eV. Fluorescence microscopy. Fluorescently labeled low-temperature oxygen plasma-treated bacteria, high-temperature dry heat-treated bacteria, vacuum-treated bacteria and un-treated bacteria on glass carriers were viewed using wide-field fluorescence microscopy. LIVE/DEAD BacLight TM bacterial viability kit L7012 (Molecular Probes, The Netherlands) was used to stain bacteria on glass carriers according to the manufacturer’s procedure. For staining solution two DNA stains SYTO 9 (3.34 mM) and propidium iodide (PI, 20 mM), were mixed together (1.5 µl + 1.5 µl) and diluted with 1 ml of deionised sterile water. Bacteria were incubated with 20 µl of staining solution at room temperature in the dark. After 15 min, cells were washed with deionised sterile water and viewed under the inverted fluorescence microscope Olympus IX71 with digital camera Olympus DP50. Green fluorescence signal of SYTO 9 and red fluorescence signal of PI were detected using U-M41001 (exc. 461 - 500 nm / em. 511 - 560 nm) and U-MWIY2 (exc. 545 – 580 nm / em. > 600 nm) Olympus filter cubes, respectively. Oil objectives × 60 (N.A. = 1.40) and ×100 (N.A. = 1.35) were used. Natura Montenegrina 10(1) 61 Flow cytometry. Fluorescently labeled low-temperature oxygen plasma-treated bacteria, dry heat-treated bacteria and un-treated bacteria on glass carriers were analyzed on a FACSCalibur flow cytometer using CellQuest software, version 3.3 (Becton Dickinson, CA). After treatment, glass carriers were gently washed with sterile 0,85% NaCl in order to prepare a single-cell solution. LIVE/DEAD BacLight TM bacterial viability kit L7012 (Molecular Probes, The Netherlands) was used to stain bacteria. In 500 µl of a cell suspension, 0.75 µl of 3.34 mM SYTO 9 and 0.75 µl on 30 mM PI were added, and the suspension was then incubated at room temperature for 15 min, protected from the light. The analysis gate was set in the untreated sample in dot plots of green (FL1, SYTO 9) and red (FL3, PI) fluorescence versus side scatter. RESULTS WITH DISCUSSION Plasma characteristics and activation of carriers. Prior to experiments performed on bacteria characteristic parameters in low-temperature oxygen plasma were measured with a double Langmuir probe and a Fibre Optics Catalytic Probes. The determined electron temperature was 5 eV, the neutral O-atom density was 8.5 × 10 21 m -3 and the ion density was 1.4 × 10 16 m -3 . As observed, in generated oxygen plasma the degree of dissociation of oxygen (O 2 (g)) to neutral O atoms (O• (g)) exceeded the ionization fraction by more than 5 orders of magnitude, i.e. allowing for almost entire plasma to interact as oxygen radicals with bacteria. These plasma parameters were kept constant during all experiments with cells described in continuation. The activation of carriers was done in the period of 5 s. Taking into account that in plasma the flux of O atoms was about 1 × 10 23 m -2 s -1 , the dose of atoms which was received by carriers exceeded 1 × 10 24 m -2 , i.e. 1 × 10 12 m -2 . This flux of O atoms allowed high surface energy by mostly removing impurities of a particular Al and glass carrier, respectively. In this way, even distribution of bacteria cells on the entire carrier surface was enabled. Furthermore, X-ray photoelectron spectroscopy (XPS) analysis of thus activated Al and glass carriers showed no traces of organic impurities, which could cause a decrease of surface energy and consequently uneven distribution of bacteria later on. Therefore, we have concluded that bacteria were deposited on well activated carriers. Samples were exposed to plasma separately for different periods of time. The density of free O atoms in plasma was the same (i.e. approximately 8.5 × 10 21 m -3 ), as it was used previously for the activation of carriers. The flux of O atoms was 1 × 10 23 m -2 s -1 and the dose of free O atoms which were received by bacteria again exceeded 1 × 10 24 m -2 . Vulošević et al: BACTERICIDAL EFFECTS OF LOW-TEMPERATURE OXYGEN PLASMA ON . . . 62 Morphological changes caused by low-temperature oxygen plasma. To better investigate the effects on the cell structure changes during the oxygen plasma sterilization process, scanning electron microscopy (SEM) was used to obtain the cell image on both: un-treated and treated bacteria. Representative SEM micrographs of the un-treated bacteria (Figure 2), bacteria treated with dry heat at 140 °C (Figure 3 and 4) and bacteria treated with low-temperature highly dissociated oxygen plasma (Figure 5) have been taken. The un-treated bacteria Bacillus stearothermophilus (Figure 2a) and Staphylococcus aureus (Figure 2b) exhibit regular rod-shaped and coccoid form, respectively. Furthermore, no alterations or lesions of the cell wall were observed for the un-treated bacteria. Figure 2. SEM micrographs of un-treated bacteria on aluminium substrate: (a) Bacillus stearothermophilus and (b) Staphylococcus aureus. Figure 3. SEM photographs of heat-treated bacteria at 140 °C for 20 seconds: (a) Bacillus stearothermophilus and (b) Staphylococcus aureus. SEM images of the bacteria treated with dry heat at 140 °C either 20 s (Figs. 3a and 3b) or 10 min (Figure 4a and b) show that Bacillus stearothermophilus still retained its typical rod shaped form (Figure 3a and 4a) and Staphylococcus aureus Natura Montenegrina 10(1) 63 still its typical coccoid form (Figure 3b and 4b). Both thus appeared quite similar to the un-treated cells in the control experiments (Figure 2a and b). Nevertheless, some changes in bacterial cell wall morphology were observed after prolonged treatment time at 140 °C indicating certain effect of high temperature on bacterial cells after 10 min (Figure 4a and b). On the other hand, after 20 s at 140 °C no visible changes in cell wall morphology were observed (Figure 3a and b). Figure 4. SEM photographs of heat-treated bacteria at 140 °C for 10 minutes: (a) Bacillus stearothermophilus and (b) Staphylococcus aureus. Figure 5. SEM photographs of bacteria treated with low-temperature highly dissociated oxygen plasma for 20 seconds: (a) Bacillus stearothermophilus and (b) Staphylococcus aureus. In contrast, treatment with highly dissociated oxygen plasma for 20 s resulted in a significant reduction of cell size and in modified morphology (Figure 5) compared to the un-treated controls (Figure 2) or thermally treated bacteria (Figure 3 and 4). Bacteria Bacillus stearothermophilus (Figure 5a) and Staphylococcus aureus (Figure 5 b) became badly damaged; their cell membrane and cytoplasm were strongly eroded and ruptured. Cells in plasma completely lost their cell integrity Vulošević et al: BACTERICIDAL EFFECTS OF LOW-TEMPERATURE OXYGEN PLASMA ON . . . 64 already after 20 s with cell wall and bacterial cytoplasm becoming barely recognizable (Figure 5). Even longer than 20 s (up to 240 s) were also tested and, expectedly, bacteria Bacillus stearothermophilus (Figure 6) and Staphylococcus aureus (Figure 7) were completely destroyed after the prolonged plasma treatment. Figure 6. SEM photographs of bacteria Bacillus stearothermophilus treated with low- temperature highly dissociated oxygen plasma: (a) for 55 seconds; (b) for 240 seconds. Figure 7. SEM photographs of bacteria Staphylococcus aureus treated with low- temperature highly dissociated oxygen plasma: (a) for 60 seconds; (b) for 240 seconds. Another control experiment was performed with Bacillus stearothermophilus and Staphylococcus aureus in such a way that bacteria were exposed to the vacuum only but without plasma. The size and shape of bacteria did not depend on this pre- treatment under vacuum conditions (Figure 8). Furthermore, SEM did not confirm any notable changes on the surface of bacteria which would be caused directly by vacuum, used in a reactor during plasma experiment. We have thus concluded that, in our experiments, evacuation itself did not cause any visible morphological changes of bacteria. These result demonstrated that a strong etching process of the plasma caused the sterilizing effect on the bacteria, when they were exposed to this low- temperature highly dissociated oxygen plasma. Bacteria have been heavily damaged, reduced to microscopic debris, ruptured with their cellular contents Natura Montenegrina 10(1) 65 released on the substrate surface. In addition, for longer exposure times, total cell fragmentation was observed. This demonstrates that the plasma has direct physical impact on the cells. Figure 8. SEM photographs of vacuum-treated bacteria: (a) Bacillus stearothermophilus and (b) Staphylococcus aureus. Fluorescence labeling. Viability of bacteria was assessed with the DEAD/LIVE BacLight TM Viability Kit. It consists of two dyes, SYTO 9 can enter bacteria with intact cell membranes and emits green fluorescence, whereas propidium iodide (PI) penetrates only into bacterial cells with damaged membranes and exhibits red fluorescence. When bacterial cells are stained with a mixture of the two dyes, viable cells fluorescence bright green, while dead cells exhibit weaker green fluorescence and also fluoresce red. This differential staining was particularly obvious when cells were observed under the fluorescent microscope. In Figure 9 (Bacillus stearothermophilus) and Figure 10 (Staphylococcus aureus) binding of DNA dye SYTO 9 and PI is shown for the un-treated bacteria (a, d), the bacteria treated with high-temperature dry heat at 140 °C for 10 min (b, e) and the bacteria treated with low-temperature highly dissociated oxygen plasma for 20 s (c, f). Binding of DNA dyes to bacteria treated with high-temperature dry heat for 20 s at 140 °C is not shown, because no morphological changes of bacterial cell wall were observed with SEM after 20 s In our experiments, the un-treated bacteria Bacillus stearothermophilus showed strong SYTO 9 staining (green fluorescence, Figure 9a) but very weak PI staining (red fluorescence, Figure 9d). In high-temperature-treated bacteria, the number of PI-stained cells stained increased compared to SYTO 9 only-stained cells (Figure 9b and e). Plasma-treated bacteria show neither SYTO 9 (Figure 9c) nor PI (Figure 9f) fluorescence signal. SYTO 9 and propidium iodide bind to the nucleic acids, then absence of fluorescence could be explained by denaturation and/or fragmentation bacterial DNA. Vulošević et al: BACTERICIDAL EFFECTS OF LOW-TEMPERATURE OXYGEN PLASMA ON . . . 66 Figure 9. Fluorescence microscopy images of bacteria Bacillus stearothermophilus labelled with DNA dyes: SYTO 9 (green fluorescence in a, b and c) and propidium iodide (red fluorescence in d, e and f). (a, d) un-treated bacteria, (b, e) bacteria treated with high-temperature dry heat for 10 minutes at 140 ° C and (c, f) bacteria treated with low-temperature highly dissociated oxygen plasma for 20 seconds. Un-treated bacteria show strong SYTO 9 (a) staining (fluorescence) but weak propidium iodine (PI) staining (d). In high-temperature treated bacteria PI staining (red fluorescence) increased (e) compared to SYTO 9 (b). Plasma treated-bacteria show no SYTO 9 (c) or PI (f) fluorescence signal. Binding of DNA SYTO 9 and PI in bacteria Staphylococcus aureus exhibited quite a similar way of staining (Figure 10). The un-treated bacteria showed strong SYTO 9 staining (Figure 10a, green fluorescence). In contrast to the un-treated bacteria Bacillus stearothermophilus, there were some PI-stained bacteria Staphylococcus aureus cells already in the un-treated sample (Figure 10d). In high- temperature-treated bacteria, PI-stained bacteria dominated (Figure 10e, red fluorescence) and only a few cells remained SYTO 9 positive (Figure 10b, green fluorescence) and PI negative (Figure 10e). Plasma treated-bacteria Staphylococcus aureus showed neither SYTO 9 (Figure 10c) nor PI (Figure 10f) fluorescence, the same as Bacillus stearothermophilus. In the case of plasma-treated bacteria, the original DNA is either heavily modified or fully oxidized, so there is practically no DNA left to assure stain binding. Degradation of bacterial DNA is achieved by oxygen radicals, especially neutral oxygen atoms. The resulted emitted fluorescence is poor: stain is evenly distributed on the carrier showing no preference to the sites where bacteria reside. This [...]... after the plasma treatment CONCLUSION For sterilization effects of low- temperature highly dissociated oxygen plasma, we came to following conclusions: The results of the SEM analyses demonstrated that a strong etching process of the plasma caused the sterilizing effect on the bacteria, when they were exposed to this low- temperature highly dissociated oxygen plasma The results of bacterial destruction obtained... dissociated oxygen plasma for 20 s or 2 min After the treatment, bacteria were gently washed from the carriers in order to obtain a suitable suspension In Figure 11 and 12, results for Bacillus stearothermophilus and Staphylococcus aureus, respectively, are displayed 67 Vuloševi et al: BACTERICIDAL EFFECTS OF LOW- TEMPERATURE OXYGEN PLASMA ON Figure 11 Flow cytometric analysis of Bacillus stearothermophilus. .. Vuloševi et al: BACTERICIDAL EFFECTS OF LOW- TEMPERATURE OXYGEN PLASMA ON REFERENCES: CHAU, T T., KWAN, C K., GREGORY, B., FRANCISCO, M 1996: Microwave Plasmas for Low Temperature Dry Sterilization – Biomaterials, 17: 1273-1277 PHILIP, N., SAOUDI, B., CREVIER, M C., MOISAN, M., BARBEAU, J., PELLETIER, J 2002: The Respective Roles of UV Photons and Oxygen Atoms in Plasma Sterilization at Reduced Gas... case of N2-O2 mixtures - IEEE Transactions on Plasma Science, 30: 1429-14435 GADRI, R B., ROTH, J R., KELLY-WINTENBERG, K., FELDMAN, P., CHEN, Z 2000: Sterilization and Plasma Processing of Room Temperature Surfaces with a One Atmosphere Uniform Glow Discharge Plasma (OAUGDP) – Surface & Coatings Technology, 131: 528–542 LAROUSSI, M., LEIPOLD F 2004: Evaluation of the Roles of Reactive Species, Heat, and. .. Radiation in the Inactivation of Bacterial Cells by Air Plasmas at Atmospheric Pressure - International Journal of Mass Spectrometry, 233: 81-86 MOISAN, M., BARBEAU, J., CREVIER, M C 2002: Plasma Sterilization Methods and Mechanisms - Pure and Applied Chemistry, 74: 349-358 JACOBS, P T., & LIN S M 2001: Sterilization Process Utilizing Low- temperature Plasma In: Block, S S (Eds), Disinfection, Sterilization,... objects being sterilized Reduction in cell viability was achieved on various types of bacteria The plasma sterilization capability demonstrated through this study indicated the potential of this low- temperature highly dissociated oxygen plasma as a promising alternative sterilization technique ACKNOWLEDGEMENT The research was supported by Slovenian Ministry of Science and Montenegro Government, Grant No... treatment, only a few cells were left in the analysis gate The results of this analysis suggest that low- temperature plasma sterilization is not only capable of killing bacteria, but it also capable of removing the dead bacteria cells from the surface of the objects being sterilized This method proved to be very efficient monitoring method as well as quantifying method for detection of dead and live... observations clearly showed that in the case of plasma- treated bacteria the original DNA is either heavily modified or fully oxidized, so there is practically no DNA left to assure stain binding The results of flow cytometric analysis suggest that low- temperature plasma sterilization is not only capable of killing bacteria, but it is also capable of removing the dead bacteria cells from the surface of the... effectiveness of heat and plasma treatments was even more pronounced The viability of the untreated sample was above 95% 20 s exposure to 140°C did not significantly affect the viability, whereas after 2 min, the viability was almost halved, and after 10 min exposure, almost all of the cells left were dead In contrast, already 20 s of plasma treatment was enough to kill almost all bacteria and after 2 min plasma. ..Natura Montenegrina 10(1) confirms also the nature of low temperature plasma sterilization, where neutral atoms interact chemically with bacteria material The atoms homogeneously erode bacteria envelope and destroy its DNA, by etching Figure 10 Fluorescence microscopy images of bacteria Staphylococcus aureus labelled with DNA dyes SYTO 9 (green fluorescence in a, b and c) propidium iodide . NATURA MONTENEGRINA, Podgorica, 10(1): 57-70 57 BACTERICIDAL EFFECTS OF LOW- TEMPERATURE OXYGEN PLASMA ON Bacillus stearothermophilus AND Staphylococcus aureus Danijela. vacuum conditions but without plasma in order to detect possible changes caused by evacuation only and not by plasma. Vulošević et al: BACTERICIDAL EFFECTS OF LOW- TEMPERATURE OXYGEN PLASMA. 11 and 12, results for Bacillus stearothermophilus and Staphylococcus aureus, respectively, are displayed. Vulošević et al: BACTERICIDAL EFFECTS OF LOW- TEMPERATURE OXYGEN PLASMA ON