1. Disinfection by plasma needle for dental treatment Bin Liu & John Goree Department of Physics, University of Iowa in collaboration with: Jeffrey Horst & David Drake College of Dentistry, University of Iowa

2. Terminology Disinfection = killing pathogenic microorganisms

3. Plasma needle treatment tests were performed 1 July 2005, in 501Van Allen Hall

4. Background

5. How plasmas can sterilize A plasma can have: Energetic charged particles UV radiation emission Heat Radicals O H O H O UV O

6. Radical formation in a plasma Mechanism: In humid air discharge, electron-impact dissociation e + O 2  O + O + e e + H 2 O  OH + H + e Dissociation requires energetic electrons

7. Plasma needle Image credit: E Stoffels et al. 2003 J. Phys. D: Appl. Phys. 36, 2908 Plasma needle: a low-power atmospheric plasma jet developed by Eva Stoffels proposed for using in sterilizing teeth and treating burns or wounds on skin

8. Dental applications where sterilization would be useful Bacteria must be eliminated when: Treating caries (cavities on a tooth’s surface) Treating peridiodontal infections (under the gum) Photo: www.db.od.mah.se/car/data/cariesser.html

9. Streptococcus mutans (S. mutans) S. mutans is: the leading cause of dental caries carried by virtually everyone a gram-positive bacteria ©Todar’s online texbook of bacteriology Growth: Anaerobe, it can grow without oxygen, but prefers oxygen- poor conditions Optimum temperature for growth is 37 ˚C

10. Sterilization methods for killing S. mutans Rinse with chemical solution Laser irradiation An alternative: plasma treatment This talk

11. What’s special about plasma treatment? Pros: Produces OH and O, which have a bactericidal effect Radicals are short-lived, do not remain in the body Cons: Cannot be used on surfaces not exposed to air Can produce excessive heat For dentistry: pulpal necrosis when tooth is heated > 5.5 ˚C

12. Hypothesis We will demonstrate that: Free radicals are present in the plasma Plasma exposure kills S. mutans at low temperatures Bactericidal effect is reproducible

13. Plasma needle method

14. Plasma needle: small-sized plasma jet operates at: atmospheric pressure low RF power low gas temperature T

15. Petri dish is positioned to apply plasma treatment to a desired spot Helium gas supply Needle tip is flush with end of glass tube Radio-frequency power supply Plasma needle setup

16. Principle of plasma needle glass tube insulation He flow hand grip Needle tungsten wire with sharp tip concentric with glass tube powered at radio frequency Helium flows between needle and glass tube

17. Needle tip Pencil-shaped tip Tungsten Tip dulled somewhat with use A sharp tip facilitates gas breakdown 0.2 mm 0.6 mm

18. He flow in air: a turbulent jet Reynolds number: Re = D V / He = 50 D= 0.004 m V = 1.5 m/s He = 7.6 air This mixing is probably turbulent The surrounding air, including O 2 and H 2 O vapor, mixes with the He and electrons. d air He flow D

19. Analogy to an impinging jet flame image: cfd.me.umist.ac.uk/tmcfd/gallery.html

20. Glow: an indicator of energetic electrons Glow is the result of electron-impact excitation of the gas (mainly He) Images of the glow show the locations of: Energetic electrons But NOT: Slow electrons Radicals Radical production requires two inputs: Energetic electrons Air (O 2 & H 2 O)

21. Results of testing for radicals

22. Verification that radicals are present Optical spectrum measured in the glow of the plasma needle visible

23. Procedure 2.Treatment of samples (Goree’s lab) 3. Temperature measurement (Goree’s lab) 1. Sample preparation (Dr. David Drake’s lab) 4. Incubation (Dr. David Drake’s lab) 5. Photography (Eric Corbin)

24. Procedure: sample preparation Agar plates: Petri dishes were filled with agar and nutrient materials Each dish was filled to the same depth Culture medium = nutrient + agar

25. Procedure: sample preparation Central spot (~ 12 mm diameter) was not plated Spiral plating technique: bacteria culture are deposited on rotating agar surface creates a bacterial lawn that is spiral-shaped Inoculation

26. Procedure: plasma treatment #1 #4 #2 #3 #5 #6 For each plate: Spots #1 – 5 were treated with plasma Spot #6 was the control: gas flow plasma off

27. Procedure: plasma treatment Petri dish separation Parameters that we varied Exposure time10 – 120 sec Separation 2 - 4 mm RF peak-to-peak voltage600 – 900 V Gas flow0.2 – 4 SLPM

28. Procedure: temperature measurement Temperature-sensitive indicator strips located a few mm below the surface of the agar Dark spots indicate when the treatment exceeded the indicated temperature Temperature-test dishes

29. Results of temperature measurement

30. Temperature measurement results Limitation of our temperature measurements: temperature sensitive strips were not on the surface where the bacteria would be, but a few mm below the surface  actual temperature on surface might be higher than we measured High temperatures T > 40 ˚C were only observed for these conditions: small separation large voltage large gas flow long exposure time  It is possible to operate a plasma needle so that there is no killing due to heat.

31. Procedure: incubation After treatment, plates were incubated: in a CO 2 incubator at 37 ˚C for 48 h During incubation: bacteria reproduce and form colonies that are visible beforeafter bacteria cell colony The bacterial lawn is visible, after incubation

32. Procedure: method of visualizing bacteria colonies living colonies central black spot was never inoculated 0.048 mm/pixel After incubation, the dishes were photographed, showing the bacterial lawn: Light color = living bacteria Dark color = no living bacteria, two possible causes: Killed Never present to begin with black regions indicate killing

33. Results for killing bacteria

34. Plasma needle kills bacteria Plasma needle can kill S. mutans under conditions attractive for dentistry: within tens of seconds at low temperature homogeneously reproducibly

35. Interpretation of treated spots Spots #1 ~ #5 are dark, indicating a significant killing #1 #2 #3 #4 #5 #6 Spot #6 (control) looks the same as the untreated area

36. Depression of agar Plasma needle treatment causes some agar to disappear (due to evaporation?) This leads to a visible depression. During the experiment, we characterized depression as insignificant, slight, moderate, or significant Depressions visible in photo as a pair of bright spots (due to reflections of photographer’s light)

37. Overview of results cool conditionwarm conditionhot condition best result achieved by either: Low RF voltage Low gas flow Large separation

38. Best results 20 mm exposure 30 s d = 3 mm bacteria were significantly killed for the exposed spots the killing was homogeneous & reproducible temperature was low 20 mm 1.5 SLPM 600 V 0.2 SLPM 800 V

39. Results under various conditions voltage gas flow exposure time 10 s30 s60 s90 s 600 V800 V900 V 0.2 SLPM1.2 SLPM Samples that look similar are arranged in columns plasma heating (as judged by depression in agar) cool hot separation 3.5 mm3 mm2.5 mm

40. Conclusion on plasma bactericidal effect Plasma needle can homogeneously kill S. mutans at low temperature Plasma needle bactericidal effect can be regulated by parameters such as exposure time, gas flow, RF voltage, and needle-to-agar separation

41. Shape of the killing region What can cause these two shapes for the killing region? We propose that : A ring-shaped killing region is not consistent with heat as the killing mechanism (next slide) Bacteria are killed by radicals The spatial distribution of radicals is different, for these two cases

42. Argument that heat is not the killing mechanism, when a ring is observed 20 mm Interpreting the ring-shaped killing region: We expect that heat would have its greatest effect at the center of a spot. In this image: killing was greatest outside the spot’s center, suggesting that killing was not due to heat. We speculate that killing was mainly due to free radicals that were concentrated in the perimeter of the plasma glow. exposure 10 s

43. Central spot: unknown cause Central spot: Its cause is unknown It occurs when the plasma is near the glow-to-arc transition exposure 30 s 20 mm

44. Summary of speculation on killing mechanism 600 V 700 V 800 V 900 V cool conditions: killing by free radicals warm conditions: killing by free radicals hot conditions: killing by: free radicals (ring) unknown cause (central spot)

45. Images of glow (an indicator of energetic electrons) ImageAbel-inverted image 600 V 700 V 800 V 900 V 1.5 SLPM, d = 3 mm 30 sec exposure

46. Images of glow (an indicator of energetic electrons) 900 V, 1.5 SLPM, d = 3 mm 900 V Abel-inverted image 30 sec exposure Possible cause of the ring: Energetic electrons that are responsible for radical formation are concentrated in a ring.

47. Images of glow (an indicator of energetic electrons) 600 V, 1.5 SLPM, d = 3 mm Abel-inverted image 30 sec exposure

48. Test of reproducibility

49. Test of reproducibility: results All 5 exposed spots look similar  bactericidal effect is reproducible 20 mm exposure 30 s low RF voltage 20 mm exposure 30 s low gas flow Cool conditions d = 3 mm 1.5 SLPM 600 V d = 3 mm 0.2 SLPM 800 V

50. Test of reproducibility: results All 20 exposed spots look similar, but reproducibility is less perfect than for “cool” conditions Dish 3 Dish 7 Dish 19Dish 24 hot conditions d = 3 mm 1.5 SLPM 800 V exposure time: 30 s

51. Summary Plasma needle can disinfect S. mutans Plasma needle can be operated so that it kills bacteria: by free radicals at low temperature homogeneously reproducibly Plasma needle bactericidal effect varies with these parameters: exposure time gas flow RF voltage needle-to-agar separation