Disinfection by plasma needlefor dental treatment

Bin Liu & John GoreeDepartment of Physics, University of Iowa

in collaboration with:Jeffrey Horst & David Drake

College of Dentistry, University of Iowa

Terminology

Disinfection = killing pathogenic microorganisms

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

Background

How plasmas can sterilize

A plasma can have:

• Energetic charged particles

• UV radiation emission

• Heat

• Radicals

Plasma

+

-

+

+

+

+

+

+

+

- -

-

-

--

-

+

-

OH

OH

O

UV

O

Radical formation in a plasma

Mechanism:

In humid air discharge, electron-impact dissociation

e + O2 O + O + e

e + H2O OH + H + e

Dissociation requires energetic electrons

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

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

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

Sterilization methods for killing S. mutans

• Rinse with chemical solution

• Laser irradiation

• An alternative: plasma treatment This talk

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

Hypothesis

We will demonstrate that:

• Free radicals are present in the plasma

• Plasma exposure kills S. mutans at low temperatures

• Bactericidal effect is reproducible

Plasma needle method

Plasma needle method

Plasma needle:

• small-sized plasma jet

• operates at:

• atmospheric pressure

• low RF power

• low gas temperature T

Petri dish is positioned to applyplasma treatment to a desired spot

Helium gas supply

Needle tip is flush with end of glass tube

Radio-frequency power supply

Plasma needle setup

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

Needle tip

• Pencil-shaped tip

• Tungsten

• Tip dulled somewhat with use

A sharp tip facilitates gas breakdown

0.2 mm

0.6

mm

He flow in air: a turbulent jet

• Reynolds number:

Re = D V / He = 50

D = 0.004 mV = 1.5 m/sHe = 7.6 air

• This mixing is probably turbulent

• The surrounding air, including O2

and H2O vapor, mixes with the He

and electrons.d

air

He flow

D

Analogy to an impinging jet flame

image: cfd.me.umist.ac.uk/tmcfd/gallery.html

Glow: an indicator of energetic electrons

heee He*HeHe

Glow is the result of electron-impactexcitation of the gas (mainly He)

Images of the glow show the locations of:Energetic electrons

But NOT:Slow electronsRadicals

Radical production requires two inputs:Energetic electronsAir (O2 & H2O)

Results of testing for radicals

Verification that radicals are present

Optical spectrum measured in the glow of the plasma needle

visible

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)

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

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

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

Procedure: plasma treatment

Petri dish

separation

Parameters that we varied

• Exposure time 10 –

120 sec

• Separation 2 - 4

mm

• RF peak-to-peak voltage 600 –

900 V

• Gas flow 0.2 – 4

SLPM

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

Results of temperature measurement

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.

Procedure: incubation

After treatment, plates were incubated:

• in a CO2 incubator

• at 37 ˚C

• for 48 h

During incubation:

bacteria reproduce and form colonies that are visible

before after

bacteria cell colony

The bacterial lawn is visible, after incubation

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 bacteriaDark color = no living bacteria, two possible causes:

• Killed• Never present to begin with

black regions indicate killing

Results for killing bacteria

Plasma needle kills bacteria

Plasma needle can kill S. mutans under conditions attractive for dentistry:

• within tens of seconds

• at low temperature

• homogeneously

• reproducibly

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

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 asinsignificant, slight, moderate, or significant

Depressions visible in photo as a pair of bright spots (due to reflections of photographer’s light)

Overview of results

cool condition warm condition hot condition

best resultachieved by either:

• Low RF voltage• Low gas flow• Large separation

Best results

20 mm

exposure 30 sd = 3 mm

• bacteria were significantly killed for the exposed spots• the killing was homogeneous & reproducible• temperature was low

20 mm

1.5 SLPM600 V

0.2 SLPM800 V

Results under various conditions

voltage

gas flow

exposure time

10 s 30 s 60 s 90 s

600 V 800 V 900 V

0.2 SLPM 1.2 SLPM

Samples that look similar are arranged in columns

plasma heating (as judged by depression in agar)cool hot

separation

3.5 mm 3 mm 2.5 mm

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

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

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

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

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)

Images of glow (an indicator of energetic electrons)

0 .0

0 .5

1 .0

1 .5

2 .0

vert

ical

pos

ition

(m

m)

0 .0

0 .5

1 .0

1 .5

2 .0

vert

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pos

ition

(m

m)

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0 1 2 3 4 5 6 7 80 .0

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rad ia l p o s itio n

vert

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Image Abel-inverted image

600 V

700 V

800 V

900 V

1.5 SLPM, d = 3 mm

30 secexposure

Images of glow (an indicator of energetic electrons)

900 V, 1.5 SLPM, d = 3 mm

0 1 2 3 4 5 6 7 80 .0

0 .5

1 .0

1 .5

2 .0

rad ia l p o s itio n

vert

ical

pos

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(m

m)

900 V

Abel-inverted image

30 secexposure

Possible cause of the ring:

Energetic electrons that are responsible for radical formation are concentrated in a ring.

0 .0

0 .5

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1 .5

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vert

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pos

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(m

m)

0 1 2 3 4 5 6 7 8

rad ia l p o s itio n

Images of glow (an indicator of energetic electrons)

600 V, 1.5 SLPM, d = 3 mm

Abel-inverted image

30 secexposure

Test of reproducibility

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 mm1.5 SLPM600 V

d = 3 mm0.2 SLPM800 V

Test of reproducibility: results

• All 20 exposed spots look similar, but reproducibility is less perfect than for “cool” conditions

Dish 3 Dish 7 Dish 19 Dish 24

hot conditions

d = 3 mm1.5 SLPM800 Vexposure time: 30 s

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