Effect of Plasma Activated Liquid (PAL) on bacteria viability.

<p>A: 1: control bacteria incubated for 2 hours in PBS and % of surviving cells was evaluated by the CFU method. 2: <i>E</i>. <i>coli</i> exposed to He plasma for 10 min with 2 hr storage at 4°C. 3: Bacteria exposed to PAL (PBS treated for 10 min He plasma) for 2 hr at 4°C. 4: Bacteria exposed to He plasma for 10 min and incubated for 2 hours with non-plasma treated PBS at 4°C. 5: Bacteria exposed to plasma for 10 min and then incubated for 2 hours at 4°C with PAL (PBS treated for 10 min He plasma). 6: Bacteria exposed to PAL (PBS treated for 10 min He plasma and left at room temperature for 2 hours) for 2 hours at 4°C. The values are means ± SEM of 3 separate experiments (*p<0.01 and ** p<0.05 vs control). B: Same experimental procedures using He-N₂ plasma. C: Same experimental procedures using He-O₂ plasma.</p

Effect of Plasma Activated Liquid (PAL) on bacteria viability.

<p>A: 1: control bacteria incubated for 2 hours in PBS and % of surviving cells was evaluated by the CFU method. 2: <i>E</i>. <i>coli</i> exposed to He plasma for 10 min with 2 hr storage at 4°C. 3: Bacteria exposed to PAL (PBS treated for 10 min He plasma) for 2 hr at 4°C. 4: Bacteria exposed to He plasma for 10 min and incubated for 2 hours with non-plasma treated PBS at 4°C. 5: Bacteria exposed to plasma for 10 min and then incubated for 2 hours at 4°C with PAL (PBS treated for 10 min He plasma). 6: Bacteria exposed to PAL (PBS treated for 10 min He plasma and left at room temperature for 2 hours) for 2 hours at 4°C. The values are means ± SEM of 3 separate experiments (*p<0.01 and ** p<0.05 vs control). B: Same experimental procedures using He-N₂ plasma. C: Same experimental procedures using He-O₂ plasma.</p

Detection of oxidatively modified proteins following plasma exposure.

<p>A: Bacteria exposed to plasma treatment (He, He-O₂ and He-N₂) for 10 min with 2 hours post-treatment storage. To detect oxidatively, modified protein bacterial extracts were treated with 2,4-dinitorphenylhydrazine to derivatize protein carbonyls and then evaluated by SDS-gel electrophoresis using 2,4-dinitrophenyl antibodies. B: Detection of 4-hydroxy-2-nonenal protein modification by ELISA. The values are means ± SEM of 3 separate experiments. C: Western blot analysis using nitrotyrosine specific antibodies. D: Bacterial extracts collected, lysed and detected by dot blot for lipid A content. Dot blot results were analyzed with a dot calibration curve and relative quantity of bacteria lipid A was estimated. The relative intensity of each spot was quantified (Image J).</p

Effect of plasma exposure on bacteria inactivation and viability.

<p>A: Survival curves obtained by CFU counting method for <i>E</i>. <i>coli</i> bacteria exposed to He plasma for different times (black diamond, control; white square, He gas only; black cross, 1 min He plasma treatment; white circle, 2 min 30 s; white triangle, 5 min and black circle, 10 min) and left for different post-treatment storage times 2, 3, 4, 5, 6 and 24 h in PBS at 4°C. B: Survival curves for <i>E</i>. <i>coli</i> exposed to He-N₂ plasma for different times (black diamond, control; white square, He only; black cross 1 min He-N₂ plasma treatment; white circle, 2 min 30 s; white triangle, 5 min and black circle, 10 min); and left for different post-treatment storage times. C: Survival curves for <i>E</i>. <i>coli</i> exposed to He-O₂ plasma for different times (black diamond, control; white square, He only; black cross, 1 min He-O₂ plasma treatment; white circle, 2 min 30 s; white triangle, 5 min and black circle, 10 min) and left for different post-treatment storage times. D: Survival curves obtained by MPN method for <i>E</i>. <i>coli</i> exposed to He plasma (white square) and He-O₂ plasma (white triangle) for 10 min and different post-treatment storage times (1 hr and 2 hr). Non-treated bacteria (black diamond). The values are means ± SEM of 3 separate experiments. E: Flow cytometry analysis of bacteria after 10 min plasma treatment (He, He-O₂ and He-N₂) and one hour post-treatment storage. Cells were initially gated on an FL2-A vs SSC-A plot (dashes). Simultaneous staining with thiazole orange (TO) and propidium iodide (PI) allowed the distinction between live (TO⁺PI⁻, red circle), dead (TO⁺PI⁺, black circle), and injured (TO⁺PI^int, dark grey) cell populations, revealing increased cell injury and death in the treated sample as expected. The TO⁻PI⁺ population was excluded from the analysis as debris.</p

Effect of plasma exposure on bacterial morphology analyzed by SEM.

<p>A: Control (<i>E</i>. <i>coli</i>). Two pictures for each conditions are presented B: after 10 min He plasma treatment and 2 hour post-treatment storage at 4°C. C: after 10 min He-N₂ plasma treatment and 2 hour post-treatment storage. D: after 10 min He-O₂ plasma treatment and 2 hour post-treatment storage.</p