JOURNAL OF ENDODONTICS
Copyright © 2004 by The American Association of Endodontists
Antimicrobial Effect of Ozonated Water on Bacteria
Invading Dentinal Tubules
Masato Nagayoshi, DDS, Chiaki Kitamura, DDS, PhD, Takaki Fukuizumi, DDS, PhD,
Tatsuji Nishihara, DDS, PhD, and Masamichi Terashita, DDS, PhD
Ozone is known to act as a strong antimicrobial
agent against bacteria, fungi, and viruses. In the
present study, we examined the effect of ozonated
water against Enterococcus faecalis and Strept-
coccus mutans infections in vitro in bovine dentin.
After irrigation with ozonated water, the viability of
E. faecalis and S. mutans invading dentinal tubules
significantly decreased. Notably, when the speci-
men was irrigated with sonication, ozonated water
had nearly the same antimicrobial activity as 2.5%
sodium hypochlorite (NaOCl). We also compared
the cytotoxicity against L-929 mouse fibroblasts
between ozonated water and NaOCl. The meta-
bolic activity of fibroblasts was high when the cells
were treated with ozonated water, whereas that of
fibroblasts significantly decreased when the cells
were treated with 2.5% NaOCl. These results sug-
gest that ozonated water application may be useful
for endodontic therapy.
It is generally accepted that a disinfecting process is essential for
successful root-canal treatment, and irrigation of the root canals to
remove microorganisms and cleanse pulpal debris is an important
step. Siqueira et al. (1) reported the importance of using antimi-
crobial irrigants during the chemomechanical preparations. Effec-
tive endodontic irrigants require a broad spectrum of antimicrobial
activity, as well as a relative absence of toxicity toward periapical
and oral mucosal tissues. Sodium hypochlorite (NaOCl) is the
major endodontic irrigant used, because of its strong antimicrobial
activity and ability to dissolve necrotic pulpal tissue, and is usually
chosen as a root-canal irrigant. However, NaOCl can evoke cyto-
toxicity when it contacts periapical or oral mucosal tissues (2).
Ozonated water is known as a powerful antimicrobial agent
against bacteria, fungi, protozoa, and viruses (3). 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 (4).
Recently, we found that it reduced the viability of oral microor-
ganisms including Gram-positive oral microorganisms, Gram-neg-
ative oral microorganisms, and Candida albicans, suggesting that
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VOL. 30, NO. 11, NOVEMBER 2004
ozonated water might be useful to control oral infectious micro-
organisms. It has been reported that chlorhexidine gluconate is a
safer and effective antimicrobial irrigant (5). However, we are not
aware of any reports on the efficacy of ozonated water used as an
antibacterial agent against microorganisms in the dentin tubules.
In the present study, we prepared in vitro root canal infection
models and examined the antimicrobial efficacy of ozonated water
against oral microorganisms invading root dentinal tubules. We
also determined whether ozonated water demonstrated cytotoxicity
against mammalian cells.
MATERIALS AND METHODS
Preparation of In Vitro Model of Infection of
Dentinal Tubules
Enterococcus faecalis ATCC 29212 and Streptococcus mutans
Ingbritt were cultured in brain-heart infusion (BHI) broth (Difco,
Detroit, MI) at 37°C for 18 h in an atmosphere of 5% CO2 in air.
An in vitro model for the infection of root canal dentinal tubules
was prepared according to a previous study (6), with minor mod-
ifications. Freshly extracted bovine incisors were stored in 0.5%
NaOCl overnight for surface disinfection. The apical 5-mm portion
of the root and crown were cut off using a rotating diamond saw
under water-cooling. After removing nearly all pulpal tissues, the
root was then cut into sliced blocks (4-mm thick) with a diamond
saw. The canals were widened with an ISO 023 round bar and the
root cementum was intentionally removed. The smear layer of each
block was removed in an ultrasonic bath with 17% EDTA (pH 7.8)
for 4 min and 5% NaOCl for 4 min. Dentin blocks with canals were
then sterilized by autoclaving in water for 15 min at 121°C. Blocks
were kept in sterile water to avoid dehydration until use. For
infection, two blocks were kept in 10 ml of BHI broth inoculated
with E. faecalis or S. mutans and incubated for 6 days. The culture
medium was changed daily.
Disinfection Test of Infected Blocks
Disinfection tests of the infected models were performed ac-
cording to previous studies (7, 8). After incubation, infected blocks
were transferred to the bottom of cell culture wells. The canal of
each specimen was irrigated by flushing (flow rate, 30 ml/min) for
10 min with the following solutions: 4 mg/L of ozonated water
Vol. 30, No. 11, November 2004
(O3aq), 4 mg/L of O3aq with ultrasonication (US; SOLFY-ZX, J.
Morita Co., Tokyo, Japan), distilled water (DW), or DW with US.
One specimen was not irrigated as a positive control and another
one was irrigated by flushing (flow rate, 30 ml/min) with 2.5%
NaOCl for 2 min as a negative control. Excess irrigating solution
was removed by suction. After each irrigation, the specimens were
dried with sterile gauze and the pulpal surface of the dentin block
was scraped off with a sterile ISO 024 round bur at low speed.
Dentin chips were then collected from the pulpal surface with a
sterile round bur at a depth of 100 ␮m, and chips attached to the
surface of the specimen and the bur also were collected using 20
ml of BHI broth. Each suspension of dentin chips including bac-
teria was mixed in a vortex for 1 min and diluted 1/100 with
distilled saline, after which 50 ␮l of each sample was cultured in
BHI agar at 37°C for 24 h in an atmosphere of 5% CO2 in air.
Colony-forming units (CFU) were counted using the spread-plate
method. Differences among variables were compared by using
Student’s t test.
Gram Staining of Bacteria
To evaluate the invasion of bacteria into dentinal tubules, spec-
imens were fixed, demineralized, sectioned, and stained using a
modified Gram-staining method, as described by Brown and
Hopps (9).
Fluorescence Microscopic Observation
Dentin blocks were demineralized with K-CX (Falma, Inc.,
Tokyo, Japan) and cultured with S. mutans in BHI broth at 37°C
for 48 h in an atmosphere of 5% CO2 in air. The culture medium
was changed daily. Incubated blocks were irrigated with the above-
described solutions and then embedded in tissue-freezing medi-
um™ (Leica Instruments, Nussloch, Germany), and frozen imme-
diately in isopentane cooled in solid CO2. Frozen sections (20-␮m
thick) were cut with a cryostat CM 1900 (Leica) and stained with
LIVE/DEAD威 BacLight™ Bacterial Viability Kit solution (Mo-
lecular Probes, Eugene, OR) according to the manufacturer’s in-
structions. In this staining system, both viable and nonviable bac-
terial cells exhibit fluorescent green, whereas only nonviable
bacterial cells exhibit fluorescent red (10, 11), which allows bac-
terial cells to be distinguished according to the permeability of the
cytoplasmic membrane (12). 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 fluorescence microscope
(BX-50; Olympus Optical Co., Ltd., Tokyo, Japan).
MTT Assay for Cytotoxicity with Disinfectants
L-929 mouse fibroblasts were cultured in minimum essential
medium (MEM; GIBCO Laboratories, Grand Island, NY) contain-
ing 10% horse serum, penicillin G (100 units/ml), and streptomy-
cin (100 ␮g/ml) at 37°C in an atmosphere of 5% CO2 in air.
Fibroblasts were rinsed, trypsinized, and seeded at density of 2 ⫻
105 cells/100 ␮l of MEM/well in 96-well plates. After incubation
for 3 h at 37°C in an atmosphere of 5% CO2, 66 ␮l of MEM was
aspirated from each well and 66 ␮l of each of the following
solutions was added to the wells: phosphate-buffered saline (PBS),
DW, 6 mg/L of O3aq (final concentration 4 mg/L), or 3.75%
Ozonated Water on Dentin-Invading Bacteria 779
NaOCl (final concentration 2.5%). After incubation for 2 min at
37°C, 100 ␮l of solution was aspirated from each well, and 100 ␮l
of MEM and 20 ␮l of 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphe-
nyltetrazolium bromide (MTT) solution were added, followed by
the additional incubation for 4 h. Then 100 ␮l of isopropanol/HCl
was added to each well for solubilizing the formazandye. The
optical density was read using a Labsystems Multiskan威 BICRO-
MATIC (Dainippon Pharmaceutical Co., Ltd. Osaka, Japan) plate
reader at 540 to 620 nm. Differences among variables were com-
pared using Student’s t test.
RESULTS
Antimicrobial Activity of Ozonated Water in Infected Blocks
A dense invasion of bacteria into dentinal tubules was detected
in the area up to 100 ␮m from the pulpal side [Fig. 1 (A and B)].
To examine antimicrobial activity, infected dentin blocks were
exposed to several irrigants. In Fig. 1 (C and D), the number of
CFU of bacteria from each specimen are shown. The number of
viable E. faecalis and S. mutans cells decreased with all of the
treatments. In particular, a significant decrease of CFU was ob-
served when the bacterial cells were treated with ozonated water,
and the number of CFU remaining in specimens treated by ozon-
ated water with sonication was nearly the same as in those treated
with NaOCl.
Figure 2 shows the viability of S. mutans that had invaded
dentinal tubules after exposure to several of the irrigants. Only a
few dead cells were detected in the nontreated blocks [Fig. 2 (A
and D)]. Furthermore, S. mutans cells were mostly alive after being
treated with distilled water [Fig. 2 (B and E)], and the number of
dead cells increased slightly by treatment with distilled water and
sonication [Fig. 2 (C and F)]. The effect of ozonated water without
sonication was nearly the same as distilled water with sonication
[Fig. 2 (G and J)]. In contrast, nearly all of S. mutans cells were
killed by ozonated water with sonication [Fig. 2 (H and K)].
Neither live nor dead S. mutans cells were detected in the NaOCl-
treated specimens [Fig. 2 (I and L)].
Comparison of Cytotoxicity of Disinfectants
Mitochondrial enzyme activity data was graphed as optical
density from an MTT assay after mouse fibroblasts were treated
with distilled water, 4 mg/L of ozonated water, 2.5% NaOCl, or
PBS (Fig. 3). There were no significant differences in the meta-
bolic activity of the fibroblasts among PBS, distilled water and 4
mg/L of ozonated water, whereas it was significantly decreased
when the cells were treated with 2.5% NaOCl.
DISCUSSION
It has been reported that ozone, in the gaseous or aqueous phase,
has a strong oxidizing power with a reliable microbicidal effect (4,
13, 14), and it is generally accepted that oxidation mediated by
ozone destroys the cell walls and cytoplasmic membranes of bac-
teria and fungi (15). After the membrane is damaged by oxidation,
its permeability increases and ozone molecules can readily enter
the cells (16), causing the microorganism to die. In the present
study, we prepared an in vitro infected model to examine bacterial
invasion into dentinal tubules and the antimicrobial effect of ozo-
780 Nagayoshi et al. Journal of Endodontics

FIG 1. Antimicrobial activity of ozonated water against infected dentin blocks. The invasion of E. faecalis (A) and S. mutans (B) (blue signals)
into dentinal tubules was evaluated by modified Gram staining. The number of CFU from dentin chip specimen cultures with E. faecalis (C) and
S. mutans (D) after irrigation are shown. Data are expressed as the mean ⫾ standard deviation of triplicate determinations. *Significance of
difference, p ⬍ 0.01; **Significance of difference, p ⬍ 0.05. P ⫽ pulpal side; D ⫽ dentin side; Scale bar ⫽ 25 ␮m.

nated water. The invasion of bacteria into dentinal tubules was

confirmed by a Gram-staining method, and our results were con-
sistent with those of a previous study (7). When the infected canal
was treated with the ozonated water, we found a significant de-
crease in the number of CFU of bacteria in the infected dentin
chips (Fig. 1). Furthermore, fluorescence microscope analysis in-
dicated an increase of membrane permeability of S. mutans cells
that had invaded dentinal tubules when treated with ozonated water
(Fig. 2). These results suggest that ozonated water has a strong
bactericidal effect against bacteria invading into dentinal tubules of
root canals. When S. mutans cells were treated with NaOCl, no
cells were detected, suggesting a complete breakage of those cells.
Many studies have shown that chemomechanical methods are
still not able to entirely clean the root-canal space. Among root-
canal preparation techniques, ultrasonic instrumentation with
NaOCl produces a better cleanliness of the root-canal walls (17).
Nontoxicity to periapical tissues is an important requirement of
endodontic irrigants. Although NaOCl has a strong antimicrobial
activity, it also corrodes dental equipment and can damage peria-
pical and other oral tissues. The cytotoxicity of 5.25% NaOCl
toward periapical tissues has been implicated in the case reports
(18, 19). In the present study, L-929 fibroblasts were significantly FIG 2. Viability of S. mutans that had invaded dentinal tubules after
irrigation. Infected dentin blocks were treated with several types of
damaged by 2.5% NaOCl, in contrast to ozonated water. Many
irrigants, and viable and nonviable cells invading into dentinal tu-
clinicians prefer diluted concentrations to reduce the irritation bules were observed using fluorescence microscopy. Both viable
potential of NaOCl, and a 2.5% NaOCl is commonly recom- and nonviable cells exhibited green signals, whereas nonviable cells
mended (20). In our MTT assay system, the L-929 cells were exhibited red signals. Irrigants: nontreatment (A, D); DW (B, E); DW
damaged by 2.5% NaOCl, an effect similar to other reports (19 – ⫹ US (C, F); O3aq (G, J); O3aq ⫹ US (H, K); NaOCl (I, L). P ⫽ pulpal
22). Furthermore, this low cytotoxicity of ozonated water may be side; D ⫽ dentin side; Scale bar ⫽ 50 ␮m.
Vol. 30, No. 11, November 2004
FIG 3. Cytotoxicity of disinfectants toward mouse fibroblasts. Mitochondrial enzyme activity data was graphed as optical density from an MTT
assay when L-929 mouse fibroblasts were treated with DW, O3aq, NaOCl, or PBS. Data are expressed as the mean ⫾ standard deviation of
triplicate determinations. *Significance of difference, p ⬍ 0.01 compared with the DW group by Student’s t test.
caused by a rapid degradation of ozone just after contact with
organic compounds, such as culture media, and is one of its major
environmental advantages. However, to maintain the activity of
ozonated water, a continuous flow is required. Although the in-
strumentation techniques used in this study should be improved,
the ozone sensitivity data obtained in this study may provide
clinical guidelines on the application of ozone during the endodon-
tic treatment.
In conclusion, ozonated water had nearly the same antimicrobial
activity as 2.5% NaOCl during irrigation, especially when com-
bined with sonication, and showed a low level of toxicity against
cultured cells. Much more research is needed to use ozonated water
in clinical or endodontic therapy. However, our results suggested
that the application of ozonated water might be useful for the root
canal irrigation.
Supported in part by Grants-in-Aid for Scientific Research (14207081,
14207082, 15592025) from The Ministry of Education, Culture, Sports, Sci-
ence and Technology of Japan.
Drs. Nagayoshi, Kitamura, and Terashita are affiliated with the Department
of Operative Dentistry and Endodontics, and Drs. Fukuizumi and Nishihara are
affiliated with the Department of Oral Microbiology, Kyushu Dental College,
Kitakyushu, Japan.
Address requests for reprints to Dr. Masamichi Terashita, Department of
Operative Dentistry and Endodontics, Kyushu Dental College, 2-6-1 Manazuru,
Kokurakita-ku, Kitakyushu 803-8580, Japan. E-mail: tera-m@kyu-dent.ac.jp.
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