Okada et al.

Int Chin J Dent 2007; 7: 79-85.

Potency of ozonated alkali-ion water in inactivating cariogenic
bacteria
Ayako Okada, DDS,a Toru Nikaido, DDS, PhD,a Mariko Gyo, DDS,a Kanako Shida,
DDS,a Junji Tagami, DDS, PhD,a,b and Khairul Matin, BDS, PhD,a,bab

Cariology and Operative Dentistry, Department of Restorative Sciences, and Center of Excellence
Program for Frontier Research on Molecular Destruction and Reconstruction of Tooth and Bone,
Graduate School, Tokyo Medical and Dental University, Tokyo, Japan

Purpose: This study assessed the effects of ozonated alkali-ion water (AW-OZ) on the caries inducing bacteria
Streptococcus sobrinus (S. sobrinus), Lactobacillus casei (L. casei), and Actinomyces naeslundii (A. naeslundii)
in their planktonic states.
Materials and Methods: Pellets of the above bacteria were prepared from fresh cultures and resuspended.
Tokyo Metropolitan City tap water (TW) was used as the source of all experimental solutions and alkali-ion
water (AW) was obtained by electrolyzing TW. An ozone delivery device was used to produce ozonated TW(TW-OZ) and AW-OZ. Phosphate buffered saline (PBS) and 0.05% sodium hypochlorite (NaOCl) were
included as controls. The bacterial pellets were then inoculated with the solutions and stored at room
temperature for 30 minutes. Viability of the bacteria was subsequently measured.
Results: Examining viability following staining with a BacLight viability kit, it was observed that all three
species of cariogenic bacteria died in significantly larger numbers with TW-OZ and AW-OZ treatment compared
to PBS, TW or AW. For S. sobrinus and L. casei, the above data were confirmed by viability counting and was
also verified by turbidimetric analysis for A. naeslundii. However, sparging ozone inevitably reduces the pH
levels of the solutions and that this reduction is very significant for TW-OZ. However, in AW-OZ, the pH could
be maintained with low alkali levels (8.40).
Conclusion: Both TW-OZ and AW-OZ had an antibacterial effect on the three cariogenic biofilm forming
bacteria and AW-OZ might be considered as the safer for human usage.
(Int Chin J Dent 2007; 7: 79-85.)
Key Words: cariogenic bacteria, ozone, ozonated alkali-ion water.

Introduction
Ozone, in either the gaseous or aqueous phases, has strong oxidizing power with a reliable microbicidal
effect.1,2 The advantages of ozone in the aqueous phase are its potency, ease of handling, lack of mutagenicity,
rapid microbicidal effects, and suitability for use as a soaking solution for medical and dental instruments.3 The
use of ozonated water for treatment of endodontic infections, sterilizing of cavities, root canals, and periodontal
pockets has been suggested.4-6 Nagayoshi et al.7 observed that ozonated water produced significant reduction inEnterococcus faecalis ATCC 29212 and Streptococcus mutans Ingbritt viability compared with control samples
and have shown nearly the same antimicrobial activity as 2.5% NaOCl during irrigation, especially whencombined with ultrasonication. Moreover, one of the major environmental advantages of ozone is its low
cytotoxicity, which, in clinical situations, can be caused by a rapid degradation of ozone just after contact withorganic compounds.
The oral cavity, one of the most anatomically complex parts of the human body consists of teeth, periodontal
tissue, tongue, mucosa as well as secretary organelles and harbors a heterogeneous microbial community. To
maintain a healthy oral environment, it is important to keep the normal oral flora predominant relative topathogenic microorganisms. There are several well established oral care methods now being employed inaddition to routine tooth brushing in maintaining good oral health. These approaches primarily target pathogenic
microbes in the oral cavity. The most prevalent caries inducing pathogens have now being identified after nearlya century of continuous research.8 Namely, the mutans streptococci group of oral spreptococci as well as

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Int Chin J Dent 2007; 7: 79-85.

lactobacillus species have been primarily associated with human dental caries. More recently, actinomyces
species have been associated with root caries and root surface biofilm formation.9,10 However, a simple practicalprocedure has not yet been developed to specifically control these pathogens.
As ozone has been shown to be an effective disinfectant in both gaseous and diluted forms, it is of interest to
develop safe ozonation procedures for use in the treatment of medical conditions. Among the potential risk
factors involved in using ozonated water for oral irrigation or mouth rinses is the alteration of pH. Althoughseveral reports have discussed toxicity of ozone for soft tissue,11 only a few reports have evaluated the effects ofozonated water on pH and on hard tooth structures. The pH at which tooth demineralization beings is known as
the clinical pH and is in the vicinity of pH 5.0 to 5.5.12,13 It was also reported that low pH mouth rinses and softdrinks have variable potential in inducing erosion of enamel.14-16 Therefore, it is important to control the pH in
the oral environment.14 From our experience, it was observed that the pH falls instantly when ozone is dissolvedin water. In many instances the pH falls bellow 4.0 when large amounts of ozone are diluted in aqueous
solutions, i.e., tap water (TW) or Milli-Q water. An alkali-ion-water solution with a high alkaline pH range (7.5
to 10.5) is being used as drinking water to minimize acidity in the stomach, to maintain normal blood oxygen
levels as well as for other health maintenance purposes. An alkali-ion water electrolyzer is a commercial device
which is designed to prevent the generation of health-hazardous substances when tap water is used as anelectrolytic solution during water electrolysis.

For example, trichloromethane, acetoaldehyde andtributylmethane produce so-called alkali-ion-water (potable alkaline electrolyzed water), a catholyte that
contains supersaturated hydrogen. Recently, medical and biochemical studies have clearly indicated that thedrinking of alkali-ion–water is good for one’s health.17,18

It was expected that ozonated alkali-ion water(AW-OZ) would have nearly the same effects on planktocnic bacteria as ozonated water (TW-OZ). Therefore,
the present study was designed to evaluate the effects of AW-OZ on caries inducing bacteria including
representatives of the mutans streptococci, lactobacilli and actiomyces in their planktonic states.

Materials and Methods
Preparation of bacterial suspensions
Laboratory strains of three cariogenic bacteria Streptococcus sobrinus 6715 (S. sobrinus), Actinomyces
naeslundii ATCC12104 (A. naeslundii), and Lactobacillus casei IAM12473 (L. casei) were used in this study.
The baseline suspensions of S. sobrinus in phosphate buffered saline (PBS) at OD500=2 were prepared from 16
hours fresh cultures in Brain Heart Infusion (BHI, Becton Dickinson, Sparks, MD, USA) broth after washing
three times with PBS, and stored at 4˚C with gentle stirring. The suspensions of A. naeslundii in PBS at
OD500=2 were prepared after 48 hours anaerobic culture in BHI broth and washed three times with PBS for
storage at 4˚C with gentle stirring. Suspensions of L. casei in PBS at OD500=2 were prepared from 48 hours
fresh cultures in Lactobacilli MRS Broth (MRS, Becton Dickinson, Sparks, MD, USA) after washing three times
with PBS and stored at 4˚C with gentle stirring.
Solution preparation
Solutions used in this study are listed in Table 1. TW was collected from Tokyo Medical and Dental
University COE laboratory tap supplied by the Bureau of Waterworks, Tokyo Metropolitan Government. AW
was produced by using a water electrolysis device (TK7705, National, Osaka, Japan) using the same TW. The
solutions (5 mL each) were sparged with ozone gas from an ozone-generating device (HealOzone, Kavo,

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Int Chin J Dent 2007; 7: 79-85.

Biberach, Germany) with a range of 300-360 s (approximately 1000 mg/L) and ozonated TW (TW-OZ) and
AW-OZ were obtained. PBS, which maintains bacteria at a static condition for several hours and a solution of
sodium hypochlorite (NaOCl), a strong antibacterial agent, were also used as controls in this study. The pH andoxidation-reduction potential (ORP) data of the experimental solutions (Table 1) were measured following
filtration with a 0.22 µm Millipore filter.
Table 1.

Materials investigated.

Solution

Abbreviation

pH

SD

ORP

SD

Phosphate buffered saline
Tap water
Alkali-ion water
Ozonated tap water
Ozonated alkali-ion water
0.05% Sodium hypochlorite aq.

PBS
TW
AW
TW-OZ
AW-OZ
NaOCl

7.30
7.26
10.54
3.50
8.40
10.31

0.00
0.25
0.08
0.32
0.26
0.05

N.D.
12.67
-171.67204.67-62.33207.00

11.72
4.70
15.63
10.97
0.00

Inoculation of bacteria with the solutions
From the above suspensions of S. sobrinus, A. naeslundii, and L. casei, 1.0 mL aliquots were prepared justbefore use. After centrifugation, the supernatants were discarded and the pellets were inoculated with 1.0 mL ofthe each of the test solutions. After 30 minutes incubation, the microtubes were centrifuged, the supernatants
discarded, and the pellets resuspended in 1.0 mL of fresh and filtered PBS to analyze the inoculation effects ofthe different solutions.
Bacterial viability testing after incubation using BacLight straining.
A LIVE/DEAD BacLight Bacterial Viability Kit (Molecular Probes, Invitrogen Detection Technologies,
Carlsbad, CA, USA)19 was used to verify potential inoculation effects of the solutions on bacterial cells. One
half (500 µL) of each bacterial sample was resuspended in PBS and transferred into dark microtubes followed bystaining with 0.5 µL BacLight stain (a mixture of SYTO 9 and propidium iodide). In this staining system, viablebacterial cells exhibit green fluorescence, whereas only nonviable bacterial cells exhibit red fluorescence,20,21
which allows bacterial cells to be distinguished according to the permeability of the cytoplasmic membrane.22
The excitation/emission wavelengths of the dyes were approximately 480/530 nm for SYTO 9 (green signals)
and 520/580 nm for propidium iodide (red signals). The bacterial cells were observed under a fluorescencemicroscope (CKX41, Olympus, Tokyo, Japan) using an objective lens (x60).
Colony forming units of S. sobrinus and L casei
Each bacterial cell suspension (500 µL) was serially diluted (x1,000) and homogenized (UP50H, Dr.
Hielscher GmbH, Baden-Wuerttemberg, Germany) before plating with a spiral plating instrument (Eddy Jet, IUL,
Barcelona, Spain). S. sobrinus samples were plated on Mitis Salivarius (MS) agar medium (Becton Dickinson,
Sparks, MD, USA). L. casei samples were plated on Rogosa agar medium (Becton Dickinson). All plates wereincubated for 48 hours under anaerobic conditions at 37˚C. After 48 hours, the number of colony-forming units(CFUs) was counted with the aid of a microscope.
Turbidimetric analysis of viable A. naeslundii
The remaining 500 µL of fluid inoculated with A. naeslundii samples were initially homogenized, 100 µL
from each sample suspension was then inoculated into 4 mL of BHI broth medium and cultured under anaerobic
conditions for 48 hours at 37˚C. They were then vortexed vigorously and 100 µL of each bacterial cell81

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Int Chin J Dent 2007; 7: 79-85.

suspension was transferred into separate wells of 96-well flat-bottom microplate to quantify bacteria byturbidimetric analysis (OD500nm) with a Biotrak II plate reader (Biochrom, Cambridge, UK).

A baseline

enumeration and controls were carried out as described above.
Statistical analysis
All numerical data were analyzed using the Statistical Package for the Medical Science (SPSS Ver.11 forWindows, Chicago, IL, USA) for statistical procedures. Bacterial viability tests were analyzed using two-way
ANOVA and Bonffroni test at 95% level of confidence. Colony forming units and turbidimetric analysis of
viable cells were analyzed using Mann-Whitney’s U test. All experiments were repeated three times under thesame conditions to insure reproducibility.

Results
Fluorescence microscopic observations
BacLight viability data for each sample are summarized in Table 2 and representative photomicrographs areshown in Fig.	
 1. Clearly, more live cells are visible than dead cells in all groups except for the bacterial cells
treated with NaOCl. Essentially all of the cells were killed following NaOCl treatment. Only 1.97% S. sobrinus,
2.85% L. casei, and 6.98% A. naeslundii died after 30 minutes incubation with PBS.

Almost the same

percentages with similar ratios of dead cells were detected in the case of ozone-free TW. Compared to that,
there was a significant increase in the percentage (an average of 10%, p<0.05) of dead cells when TW-OZ wasused. A similar pattern was also observed in the case of AW-OZ treated bacteria and significantly more bacteria
were killed compared to TW and AW (p<0.05). Interestingly, more live cells were counted when the bacterial
cells treated with AW compared to TW (except for A. naeslundii) as well as for bacterial cells treated with
AW-OZ compared to TW-OZ (p<0.05).
Live bacterial cells are visualized as green and dead cells are red in the same microscopic image following
excitation with blue light (Fig. 1). Microscopic images clearly show that large numbers of S. sobrinus cells
remain alive after 30 minutes inoculation with AW and TW compared to AW-OZ and TW-OZ, indicating morebacteria died following ozone treatment.
Table 2.

Mean averages (%) of dead (red) cells, counted from four micro-photographic images taken
by a fluorescent microscope stained with LIVE/DEAD BacLight viability kit.

Group

S. sobrinus
SD Category

Solution

Mean

PBS

1.97

0.66

No-OZ group

TW
AW

3.31
2.51

1.85
0.90

Oz group

TW-OZ
AW-OZ

12.81
7.60

1.96
3.87

NaOCl

101.97 0.00

Mean

L. caseiSD Category

2.85

1.60

b, c
a, c

2.74
1.39

1.11
1.08

b, d
a, d

12.90
6.05

1.95
2.31

102.85 0.00

Mean

A. naeslundii
SD Category

6.98

1.34

g
e

7.86
11.50

3.53
2.01

i
h

f, g
e, f

20.78
16.61

3.50
2.82

i
h

101.78 3.74

The same letters indicate statistically significant differences (n=4, p<0.05).

S. sobrinus and L. casei viable cell counts (CFU)
The viability of bacterial cells recovered from each sample is summarized in Table 3.

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Int Chin J Dent 2007; 7: 79-85.

remarkable differences can be observed in the results between the bacterial species S. sobrinus and L. casei. The
number of viable bacteria cells after incubation with all solutions used in this study compared to the baseline
viable cell counts suspended in PBS or 30 minutes inoculation in PBS were similar. However, significantly
more bacteria were viable when the cells were treated with AW and TW compared to AW-OZ and TW-OZ(p<0.05). The number of viable bacterial cells treated with NaOCl was lowest among all of the solutions, andalmost no bacteria survived (positive control).
Table 3.

Number of S. sobrinus and L. casei cells surviving after treatment.
Solution

S. sobrinus

L. casei

PBS

3.99

5.44

No-OZ group

TW
AW

3.88
3.99

a
b

5.30
5.42

c
d

Oz group

TW-OZ
AW-OZ

2.63
3.69

a
b

3.50
4.08

c
d

NaOCl

0.00

0.00

Data are expressed as themedian log10 ofcolony-forming units per mL(CFU/mL).
The same letters indicate
statistically significant
differences as analyzed byMann-Whitney’s U test
(p<0.05), n=3.
Data of TW-OZ and AW-OZ
were compared with AW
and TW, respectively.

Fig. 2.
The amounts of viable A. naeslundii cells are shown in thegraph. Data are expressed as the median. Horizontal
bars indicates significant differences (p<0.05), n=4. Data
of TW-OZ and AW-OZ were compared with TW and AW,
respectively. Mann-Whitney’s U tests revealed that both
TW-OZ and AW-OZ had significantly less viable bacteria
compared to TW and AW, respectively.
Fig. 1. Fluorescence photomicrography of S. sobrinus cells stained with a BacLight bacterial viability kit. Live cells are visible as
fluorescing green and dead cells visible as red after 30 minutes inoculation in different solutions (PBS, NaOCl, TW, TW-OZ, AW,
and AW-OZ).

Turbidimetric analysis of viable A. naeslundii
The amounts of viable A. naeslundii cells are after 30 minutes incubation are shown in the graph (Fig. 2).
Mann-Whitney’s U tests revealed that both TW-OZ and AW-OZ had significantly less viable bacteria compared
to TW and AW, respectively. However, the difference between AW and AW-OZ was very small, while thedifference between TW and TW-OZ was large. At the same time the median of NaOCl was 0.022.

Discussion
Ozone has been applied for water disinfection purposes for almost a century. There is frequent debate on the83

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Int Chin J Dent 2007; 7: 79-85.

role of OH radicals and ozone for disinfection processes in drinking water within the research community. Someauthors state that ozone is the main disinfectant20,21 while others suggest that OH radicals may play an importantrole for disinfection.22,23 However, Von Gunten critically reviewed the role of OH radicals in the inactivation of
C. parvum oocysts and B. subtilis spores and concluded that the effects of OH radicals in disinfection processes
can generally be neglected.24 Data in the present study indicated some induction of bacterial inactivation tosome extent by AW water, which contains significant amounts of free OH radicals compared to the TW. On theother hand, ozone’s inactivation potency has been well established after nearly a century of investigations.
Although ozone is quite unstable when it is sparged into any solution, as was done in TW and AW in the present
study, both ozonated solutions showed significant disinfection activity on three cariogenic bacteria. These
results definitely indicate that incorporation of ozone into these solutions made a significant difference. To ourknowledge, inactivation of A. naeslundii by ozonated water was observed for the first time in this study.
However, the antibacterial effect of ozonated water was still far less than that of NaOCl. According toprevious reports, direct application of gaseous ozone appeared to be more effective than dissolved ozone insolutions, mainly because ozone decay begins to occur instantly when mixing is done. On the other hand,
although in the oral cavity gaseous ozone could be used, obtaining sufficient access to specific target sites
becomes difficult in many cases. Bocci has suggested that ozone can be toxic but, when properly used, can bemedically useful.25 The toxic effects of ozone are a function of the gas concentration and exposure duration.
Risks always exist in delivering ozone gas directly to inner portions of the oral cavity because gaseous ozone cancause damage to the lung lobes if inhaled in large amounts. Instead, AW-OZ might be much safer for delivery tomost parts of the oral cavity.
Clearly, TW-OZ had more antibacterial ability than AW-OZ, especially in case of A. naeslundii. Here, one
important issue should be discussed; i.e., the pH and ORP levels of the ozonated water. In the present study, thepH and ORP of TW-OZ were below 4 (3.5 on average) and above 200 mV (204.67), respectively. These
conditions are definitely problematic for use in the oral cavity. In contrast, the pH and ORP for AW-OZ wereabove 8 (average of 8.40) and below 1 (average of -62.33), respectively. These pH and ORP levels are
considered to be much safer for periodic use in the oral cavity. Moreover, an unexpected characteristic of AW
was explored recently (Gyo et al., 84th IADR, Brisbane, 2006; Abst. #1267), i.e., it can effectively dissolve
glucan from the cariogenic biofilms. Therefore, a double benefit may be achieved from AW-OZ if it candissolve glucan as well as mildly inactivate oral pathogens. This may be also important for maintaining thenormal oral flora by not drastically reducing the levels of beneficial oral bacteria.
The results of the present study do not confidently indicate any possibilities of therapeutic use in the clinics,
neither for TW-OZ nor AW-OZ. If considered for prophylactic use for gargling at home it can be suggested that
AW-OZ should be preferred instead of using TW-OZ. Further evaluation will be necessary to draw firm
conclusions in this regard.
Acknowledgments
This work was supported by the Center of Excellence (COE) Program for Frontier Research on Molecular Destruction andReconstruction of Tooth and Bone at Tokyo Medical and Dental University, Tokyo, Japan, and a grant from Matsushita
Electric Works, Ltd. (R & D Center). We would like to express our gratitude to Prof. Howard H. Kuramitsu, State Universityof New York at Buffalo for his kind contribution in editing the manuscript.

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Correspondence to:
Dr. Ayako Okada
Cariology and Operative Dentistry, Department of Restorative Sciences, Graduate School,
Tokyo Medical Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8549, Japan
Fax: +81-3-5803-0195 E-mail: hisoppu2003@yahoo.co.jp
Received November 14, 2007. Accepted November 27, 2007.
Copyright ©2007 by the International Chinese Journal of Dentistry.

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