Nakano et al.

Asian Pac J Dent 2012; 12: 21-26.

Microbicidal effect of ozone gas in vitro: interaction with organic
compounds and endodontic irrigation agents
Masako Nakano, DMD, PhD,a Ayuko Takao, DMD, PhD,b Toshiko Ozawa, DMD, PhD,aTomokazu Igarashi, DDS, DDSc,b Nobuko Maeda, DDS, DDSc,b and Noriyasu Hosoya,DMD, PhDaab

Department of Endodontology, and Department of Oral Microbiology, Tsurumi University School ofDental Medicine, Yokohama, Japan
Purpose: The aim of this study was to estimate the utility of an ozone gas generating apparatus (HealOzone) for
endodontic treatment by its bactericidal efficiency in vitro.
Materials and Methods: Water- or TS broth-suspended microorganisms (Streptococcus mutans, Lactobacillus
casei, Enterococcus faecalis, and Candida albicans) were exposed to ozone gas for up to 80 s. Serially diluted
irrigation agents (H2O2, NaOCl) and E. faecalis were incubated at room temperature for a minute with or withoutozone exposure (20 s). In both experiments, survived microbes were determined by semi-quantitative
cultivation.
Results: Eighty second-exposure of ozone gas completely killed the microorganisms suspended in water.
However, this treatment had no effect on the same microorganisms suspended in the culture broth, whichcontained the organic compounds. Combination of ozone gas exposure and H2O2 solution showed synergic
effect to kill E. faecalis in water, but not in the broth. In contrast, ozone inhibited the bactericidal effect ofdiluted NaOCl solution against E. faecalis in water.
Conclusion: Based on the experimental results, probability of sterilization effect of ozone gas either used withor without root canal irrigation agents was doubtful. (Asian Pac J Dent 2012; 12: 21-26.)
Key Words: Healozone, hydrogen peroxide, microbicidal effect, ozone gas, sodium hypochlorite

Introduction
Ozone is one of the strongest oxidizer. It is used in sewage treatment to remove pollutions, and otherindustrial processes as a disinfectant, a bleaching agent, and a deodorant.1-4 While these characteristics of ozone
seem to be also useful for dental field, ozone inhalation at high concentration induces acute toxicity for humanrespiratory tract.5,6 Therefore, a system to avoid leakage of ozone gas to oral cavity is necessary for the dental
treatment apparatus.
HealOzone (KaVo Dental GmbH, Biberach/Riß, Germany) is well known and the most safety one in thiscategory.7 A handpiece of the apparatus releases the gas only when a silicon tip completely attaches to a treated
tooth. After treatment, remaining ozone gas is rapidly exhausted from the area by a vacuum pump to prevent
both patient and dentist from the gas exposure.
The major target of ozone gas therapy by HealOzone is pit and fissure caries.8 Since the traditional invasive
process, “Drill and Fill”, is stressful for patients, ozone and remineralization therapy for primary caries as
minimally invasive dental treatment is very attractive.9
conducted in UK and the evaluation has been discussed.

The clinical trials in this field have been mostly

8,9

For endodontic treatment, on the other hand, the manufacturer of HealOzone also provides a device to get thegas at periapical region. However, root canal has a complex structure and is rich in organic compounds, and thecondition for ozone treatment for endodontics should be different from that for enamel caries on tooth surface.
Diffused ozone gas in root canal might react with the remaining organic compounds that interfere withdisinfectant effect of ozone. Furthermore, penetration efficacy of ozone gas into wet tissues is not established.
Based on the backgrounds, we planned the experiments in vitro to expect effectiveness of ozone gas
application in clinical endodontic field. The purposes of this study were to investigate: a) microbicidal effect of21

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Asian Pac J Dent 2012; 12: 21-26.

ozone gas on the various microorganisms with or without the organic compounds, and b) influence ofcoexistence of irrigation solutions and ozone gas on their bactericidal effect.

Materials and Methods
Ozone generating apparatus
HealOzone was used as the ozone treatment apparatus.
Microorganisms
Streptococcus mutans (S. mutans) ATCC25175, Lactobacillus casei (L. casei) ATCC393, Enterococcus
faecalis (E. faecalis) ATCC19433, and Candida albicans (C. albicans) ATCC18804 were used.

For allexperiments, each test strain was inoculated into Tryptic soy broth (TS broth; Becton, Dickinson and Company,Sparks, MD, USA) and incubated at 37˚C overnight under an aerobic condition.

The culture was thencentrifuged at 10,000 g for 1 minute. The cell pellet was washed twice by sterile water and then suspended inwater as approximately 1010 cfu/mL. The sample was subdivided and stored at -80˚C before use.
Disinfectant efficacy of ozone gas against various microorganisms
The frozen stock of microbial suspension was diluted by either water or TS broth to adjust the concentration toapproximately 106 cfu/mL for bacteria and 105 cfu/mL for C. albicans. Each suspension was dispensed to thewells of a sterilized micro well plate (Sumilon, Tokyo, Japan) as 100 µL per well, and exposed to ozone gas for
0, 20, 40, and 80 s. After the period, each treated sample was diluted by TS broth and spread onto Tryptic soy
agar plates. The amount of bacteria survived was determined by counting colonies grown on the plates after
48-hour incubation at 37˚C.
Oxidation- and bacterial killing-extent of ozone gas exposed from agar layer surface
In all of the following experiments, E. faecalis was used as a test organism, which is frequently isolated fromrefractory apical periodontitis.10 The melting agar (2%) mixed with indigo carmine aqueous solution (0.01%,final) was solidified on a slide glass. Similarly, the E. faecalis (106 cfu/mL, final) suspended-agar layer, with orwithout TS broth, was prepared. Then the surface of each agar layer was exposed to ozone gas for 120 s. The
depth and diameter of decolorized area were measured on the indigo carmine gel immediately after exposure,and those of colony-free area in bacteria-suspended gel were determined after 48-hour incubation at 37˚C. In thecase of the water-prepared gel, melting agar containing two-fold concentrated TS broth was overlaid onto thesurface to support bacterial growth within the gel.
Bactericidal effect of simultaneous exposure of ozone gas and irrigation agents
Solutions of sodium hypochlorite (NaOCl; 1%=10,000 ppm) and hydrogen peroxide (H2O2; 12%) wereserially two-fold diluted either by water or by TS broth. E. faecalis suspension (approximately 105 cfu/10mL
either in water or in TS broth) was prepared from the stock bacterial suspension.

The examinations ofbactericidal effect for each dilution series in a micro well plate were performed under three different conditions
as follows. 1) Neutralization control: Irrigation solution diluted (50 mL) and bacterial suspension (5 mL) weresimultaneously added into 1 mL of 1% sodium thioglycolate solution (STG), which acts as a neutralizer. After aminute-incubation at room temperature, the mixture was serially tenfold diluted and plated onto TS agar plates.
2) Irrigation solution treatment: Bacterial suspension (10 mL) was added to the diluted irrigation solution (100mL) in a well as the final bacterial inoculation size at 106 cfu/mL, and incubated for a minute. After thetreatment period, 50 mL of reaction mixture was added into 1 mL of STG. Following protocol was the same as22

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in 1). 3) Irrigation solution treatment with ozone: Bacterial suspension added in each irrigation solution wasexposed to ozone gas for 20 s. Incubation of bacterium with the solution was continued for a total period of one
minute, following the same protocol as that for 1). All agar plates were incubated at 37˚C for up to 72 hours,and survived bacterial colonies were counted. For a statistic analysis of interaction of each irrigation agent andozone, protocols 2) and 3) using the same chemicals in water were independently repeated six times. Survived
bacterial colony counts after treatments were analyzed by Wilcoxon rank-sum test.

Results
Effect of ozone gas against various microorganisms with or without the organic compounds
Eighty second-exposure of ozone gas completely killed all microbes examined (S. mutans, L. casei, and E.
faecalis; approximately 106 cfu/mL, and C. albicans; approximately 105 cfu/mL) suspended within water, but
not within TS broth (Fig. 1).

Fig. 1. Microbicidal effect of ozone gas against dental
infection-related microbes. Eighty second-exposure ofozone gas killed test organisms suspended in water,
but not those in TS broth.

Fig. 2. Extent of bactericidal and oxidation effects ofozone gas on agar gels. Depths of inhibition zone(D1) and of Indigo carmine-decolorized zone
immediately after exposure (D2) was matched;approximately 2 mm from the gel surfaces.

Oxidation- and bacterial killing-extent of ozone gas exposed from agar layer surface
Ozone gas exposure for 120 s was effective against E. faecalis suspended into the agar gel prepared with water,
but not with TS broth. In the former case, the bactericidal area reached to approximately 2 mm depth from thegel surface, which was almost the same as that of the decolorized zone in Indigo carmine-containing gel afterozone exposure (Fig. 2). Diameters of effective zones of ozone were matched to that of the inner space ofsilicon cap.
Bactericidal effect of simultaneous exposure of ozone gas and irrigation agents against E. faecalis
Contamination of the organic compounds interfered the effect of both irrigation agents, especially of NaOCl
as shown in Fig. 3. Ozone did not influence on the bactericidal effect of both irrigation agents diluted with TS
broth. Under TS-free condition, ozone treatment for 20 s tended to increase the bacterial killing of H 2O2 at 6%,23

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and on the other hand, to decrease that of NaOCl at 1.2 ppm. Results of additional examination performed forstatistical analysis of these interactions were shown in Fig. 4. Ozone enhanced bactericidal effect of H2O2 at 6%(p<0.01) and 12% (p<0.05), and interfered that of NaOCl at 0.6 ppm (p<0.01) and 1.2 ppm (p<0.05).

Fig. 3. Influence of ozone gas exposure on bactericidal effect of endodontic irrigation agents: A) H2O2, B) NaOCl.
Each solution was serially 2-fold diluted using either water or TS broth. Neutralized: irrigants immediatelyneutralized; -ozone: irrgants alone; +ozone: irrigants with ozone.

(A) H2O2(B) NaOCl

Fig. 4. Survived colony counts of E. faecalis after H2O2 (A)- or NaOCl (B)-treatment with or without ozone
exposure. Maximum concentrations examined 12% for H2O2 and 10,000 ppm for NaOCl, respectively.

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Asian Pac J Dent 2012; 12: 21-26.

Discussion
As shown in Fig. 1, ozone gas exposure for 80 s completely killed 105-106 cfu/mL of various dental
infection-related microorganisms suspended in water.

However, coexistence of the organic compounds

extremely interfered its ability as a disinfectant. These results simply reflect a basic characteristic of ozone gas:
it is a non-specific oxidizer. In clinical endodontic treatment, therefore, ozone gas will equally react to viablemicroorganisms and the contaminants in root canal, such as host tissue components, dead bacteria, and bacterial
extrapolysaccharides. It leads to useless consumption of ozone gas, resulting in a decline of the sterilization
efficacy.
Some recent studies reported that ozone treatment had no effect against biofilm of E. faecalis in vitro,11-13 andthese results might be also caused by non-specific reaction of ozone and bacterial extracellular products.
Ultimately, ozone gas itself might not be useful under the organic compound-rich condition, such as root canal.
When applied onto tooth surface, depth of ozone penetration was reported to be approximately 2 mm.14 In ourbasic experiment using 2% agar gel containing indigo carmine as the chemical indicator,15,16 oxidizing depth
after 120 second-exposure of ozone gas was also approximately 2 mm from the surface.

This value

corresponded with the extent of bactericidal effect of ozone under the organic compound-free condition. Ozone
did not show horizontal diffusion from the directly exposed area, and prolonging the exposure time of the gas(up to 180 s) could not extend the regions of either oxidization or bactericidal effects (data not shown). This
result might be partially because of limited penetration of ozone from the exposed surface, and of transfer toaqueous phase.
From the manufacturer’s data, HealOzone supplies ozone gas at a concentration of 2,100 ppm (v/v; 5.4g/Nm3). Under the condition at 25˚C, pH 7.0, 105 Pa (1 atm) of gas pressure, the concentration of ozone in wateris calculated as 0.96 ppm (w/v; 0.96 g/Nm3), which is the maximum value in equilibrium. Nagayoshi et al.
reported that although 2-4 mg/L (2-4 ppm, w/v) of ozonated water killed various oral microorganisms within 10s, 0.5 mg/L (0.5 ppm, w/v) of it was not efficient enough to kill them within 120 s.17 In addition, when once inaqueous phase, ozone will react with various organic/inorganic compounds, resulting in rapid elimination of theactive molecule.18
To get the best performance of ozone gas as a disinfectant, organic compounds in treatment region should beremoved as much as possible before ozone exposure. Therefore, as the next step, simultaneous usage of
irrigation solutions and ozone gas was examined to investigate the interaction. H2O2 (12 and 6% in water) andozone (20 s) showed the synergic bactericidal effect. However, ozone gas interfered with the bactericidal effectof NaOCl at low concentration (1.2 ppm and 0.6 ppm in water). Since NaOCl is a popular and strong remover ofthe organic compounds,19 this interaction must be considered as a demerit for ozone treatment. Ozone may
cause oxidative degradation of active molecules/ions in the NaOCl solution, which is unstable especially at lowconcentration.
If ozone gas is to be applied for endodontic therapy, therefore, a possible procedure before ozone gastreatment may be NaOCl irrigation followed by H2O2 irrigation. However, the synergy of H2O2 and ozone
seems not to be strong enough to change general treatment protocol of root canal.
Within the limitation of the current experiments, the following coclusions can be drawn. Microbicidal effectof ozone gas was strongly interfered by contamination of organic compounds. Since ozone diminished thebactericidal effect of NaOCl at low concentration, co-treatment of ozone with NaOCl is not recommended.

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While H2O2 and ozone showed synergy, however, the effect observed in the present study is not strong enoughfor clinical application.
Acknowledgment
This study was partially supported by a grant from Tsurumi University Dental Society.

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Correspondence to:Dr. Masako Nakano
Department of Endodontology, Tsurumi University School of Dental Medicine
2-1-3, Tsurumi, Tsurumi-ku, Yokohama 230-8501, Japan
Fax: +81-45-573-9599 E-mail: nakano-m@tsurumi-u.ac.jp
Copyright ©2012 by the Asian Pacific Journal of Dentistry.

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Accepted May 29, 2012.
Online ISSN 2185-3487, Print ISSN 2185-3479