Vahid A, et al Journal of Dentistry, Tehran University of Medical Sciences

An In-vivo Study Comparing Antimicrobial

Activity of Chlorhexidine 0.2% to Sodium
Hypochlorite 0.5% as Canal Irrigant.

Vahid A1, Aligholi M 2, Namazi HR 3

1
Assistant Professor, Dept. of Endodontic, Faculty of Dentistry, Tehran University of Medical Sciences, Tehran, Iran
2
Assistant Professor, Dept. of Microbiology , Faculty of Medicine, Tehran University of Medical Sciences, Tehran, Iran
3
Dentist
Statement of Problem: Bacterial agents are the primary cause of pulpoperiapical diseases.
Therefore, an irrigating solution with proper antibacterial ability should be used to
eliminate the bacterial agents during canal instrumentation.
Purpose: The aim of present study was to compare the antibacterial effect of 0.2%
chlorhexidine to 0.5% sodium hypochlorite as canal irrigating solutions.
Materials and Methods: In this study 20 single canal teeth with necrotic pulps were
selected by vitality tests and divided in two groups each one consists of ten. Each canal
irrigated with 12 ml of 0.2% chlorhexidine in the first group and 0.5% sodium hypochlorite
in the second one. Each canal was instrumented by 6 k- type files by step back procedure
(two minutes for each file). After canal instrumentation, sterile cotton pellet was placed on
the pulp chamber and the canals were filled temporarily by cavit. Samples were selected by
two strile paper points both before the canal instrumentation (after accessing the canal) and
48 hours later. Samples were cultured on Mitis salivarius agar and anaerobic blood agar and
then, the colony counts were reported in terms of CFU/ml.
Results: Elimination percent mean of streptococcus mutans and anaerobics with 0.2%
chlorhexidine were 99.9 and 99.02 respectively and for 0.5% hypochlorite were 99.7, and
92.7 respectively.
Conclusion: There was no significant difference between these two irrigating solutions by
non parameteric Mann-Whitney test.
Key words: Chlorhexidine- Sodium Hypochlorite- Canal Irrigant.
Journal of Dentistry, Tehran University of Medical Sciences, Tehran, Iran (2004; Vol: 1, No. 1)

M echanical debridement and irrigation of

the canal play a fundamental role in
achieving endodontic success. In spite of all
attempts, attaining complete sterility of the
canal system is still extremely difficult.
Microorganisms can penetrate deep into
accessory canals, apical ramifications, isthmi,
tubules and smear layers. Hence, finding ways
to overcome this issue is essential.
The ideal irrigant should be strongly
antibacterial but not toxic to the periapical
tissue (selective toxicity).(1) It should be tissue
solving and lubricate the file during
instrumentation.(1) Also, it has been shown that
surface tension of an irrigating solution and its
wettability on solid dentin are important factors
in penetration into dentin and lateral canals.(2)
Sodium hypochlorite solution has been widely

2004; Vol. 1, No. 1 43

Journal of Dentistry, Tehran University of Medical Sciences
recommended as an irrigant to aid in
chemomechanical debridement of the root
canal, because it has many of the above
mentioned advantages, except its toxic effect
for periapical tissue and wettability.(1) Also, its
smell and discoloring action are other
disadvantages.
Spangberg et al(3) found that all medicaments
are toxic compared to their antibacterial effect.
They said on the basis of a balance between the
cytotoxicity, the antibcaterial effect and the
specific needs, a recommendation for an
adequate irrigating solution is being made. They
believed that sodium hypochlorite at 5%
concentrations is more stronger than it is needed
to kill bacteria.(3)
Huggers et al(4) in their investigation on
antimicrobial activity and tissue toxicity of
different concentrations (0.25%, 0.025% and
0.0125%) of sodium hypochlorite, found that
even 0.25% concentration could be tissue toxic.
Chlorhexidine gluconate has been recognized as
an effective antimicrobial agent in periodontics,
and its use as a potential endodontic irrigating
solution has been demonstrated in the last
decade.(5) It seems chlorhexidine has most of
the mentioned criteria. It has a long term effect
and the reservoir of this agent forms on the
teeth and on the oral mucosa after mouth rinse.
Antimicrobial activity is released from these
locations as the concentration of chlorhexidine
decreases in the environment.(6) The absorption
of chlorhexidine by hydroxyapatite, teeth and
protein demonstrated previously by a chemical
method.(6) This characteristic can also be useful
inside of the canal.
Many in vitro studies have been performed
comparing these two irrigating solutions while
few clinical trials have been done. In vivo
studies are more natural and closer to the actual
specifics of the human. Therefore, the aim of
this study was to compare the antimicrobial
effect of sodium hypochlorite 0.5% and
chlorhexidine 0.2% during root canal therapy of
twenty infected canals in patients.
44
Vahid A, et al
Materials and Methods
Twenty single rooted teeth with necrotic pulps
were chosen. Pulpal necrosis was determined
either by radiographic presence of apical
rarefaction or by the lack of response to pulp
vitality tests. Ages of patients were between 15
to 50 years old. Apices were obviously formed
and pulp chambers and canals were free of pulp
stones, calcifications, or sharp dilacerations.
Teeth were randomly divided into two groups.
Each of the groups received one of the
irrigating solutions, sodium hypochlorite (SH)
0.5% or chlorhexidine gluconate (CHG) 0.2%.
SH had the concentration that could have
antimicrobial activity with least hazard to
periapical tissue.(1) Irrigating solutions used in
this study were chlorhexidine gluconate %0.2
from Shahredaru and sodium hypochlorite
5.25% from Taj Co which diluted to 0.5%.
Study had two phases which will be described:
Clinical phase: After selecting teeth (taking
primary X-ray and making diagnosis by vitality
tests) informed consent was taken. The decay
was removed by sharp excavator without
opening the pulp chamber. Tooth isolated with
rubber dam and access cavity prepared. Before
entering the canal, pulp chamber was cleaned
with sterile distilled water to clean debris
resulted from access preparation. Pulp chamber
was dried with sterile cotton pellets and two
paper points were put into the canal. After one
minute, paper points were withdrawn and
dropped in screw top test tubes containing
transfer media and was sent to microbiologic
laboratory. Taking samples and preparing
dilutions took less than 10 minutes. This was
referred as first sample.
Then, after length measurement, canals
prepared by step back technique for cleaning
and shaping. Between the procedures, canals
were rinsed by 2 cc of any of irrigating
solutions for each file. Finally, canals were
irrigated by sterile distilled water, then, access
cavity closed with ZOE . Two days later, after
isolation, the area surrounding the tooth and
2004; Vol. 1, No. 1
Vahid A, et al Journal of Dentistry, Tehran University of Medical Sciences
clamp was irrigated with SH, and temporary
filling was removed. Two papers were put in
the canal and then dropped into transfer media
and were sent for culture. Sterility of the
process and instruments during study was
axiomatic.
After irrigation, master point with X-ray was
prepared and canals were filled. Temporary
filling was placed back.
Microbiologic phase: For microbiologic phase,
all media autoclaved and distributed into plates.
Thioglicholate (Difco) transferring medium was
poured into vials and autoclaved. Vials
containing paper points agitated on a vortex
mixer until a homogeneous suspension of
microorganism were obtained.(7,8) For ease of
colony counting, 1 ml. of the homogenized
solution transferred to five test tubes of ringer
solution with 1/10 to 1/100000 dilutions.(9,10)
In first sampling (before instrumentation), 0.1
ml from dilutions 1/10, 1/1000, 1/100000 was
inoculated to plates of blood agar (Merk) for
anaerobic organisms and three plates of Mitis
salivarius agar (Difco) for sterptococcus mutans
and spread with sterile bent glass rod.(9) Plates
with gas pack, catalyzer and indicator were put
in anaerobic jar (Oxoid) and incubated under 35
degree centigrade for 7 days. In second
sampling, culture of undiluted sample added to
previous dilutions and culture was performed as
before. After 7 days colony-forming units
(CFU) were counted. The results were analyzed
by nonparametric Mann- Whitney test.
Results
Tables I-IV indicated the CFU results of the
effect of CHG and SH as irrigating solutions on
streptococcus mutans and anaerobe bacteria. A
generalized decrease of flora of the canal from
culture one to culture two was present. (For
streptococcus mutans exact P=0.089 and for
anaerobe bacteria exact P=0.315). But none of
the teeth were totally free of cultivable
microorganisms. According to table V, the
results showed no significant difference
2004; Vol. 1, No. 1
between groups. As shown on tables I-IV, the
elimination rates for streptococcus mutans and
anaerobe bacteria in the presence of C.H.G.
were 99.9% and 99.02%; and in presence of
S.H. were 99.7% and 92.7% respectively.
Discussion
An important adjunct for che momechanical
preparation of the canal system is irrigation
which aids in debridement and flushing debries
from the canal.(11) At present study antimic-
robial activity of sodium hypochlorite (SH)
0.5% and chlorhexidine gluconate (CHG) 0.2%
compared as canal irrigants.
SH as the most common proteolytic irrigating
solution has most of the required characteristics,
except for its surface tension ,(2) its unpleasant
odor and taste, corrosion of endodontic
instruments, ineffectiveness on some micro-
organisms when used at low concentration, and
SH accident. It doesn’t differentiate between
necrotic and vital tissue in contact with apical
and periapical tissue, and is unable to be
dressed within the canal. Studies indicate that
SH dissolves tissue but experiments in clinical
usage are less impressive.(11)
Histologic and scanning electron microscope
studies do not show that tissue dissolution or
sterilization properties are consistently
effective.(12) It seems that in clinical conditions
irrigating solutions have limited surface contact
and are neutralized in the canal.(11) In present
research, SH 0.5% (Dakin solution) was used
because of its lower toxicity for periapical
tissue despite being effective in the canal.(4)
CHG as a mouthwash is well known for few
decades and it is available at 0.12% in the
market at USA. The concentration chosen was
0.2% which is used in many studies (7,10) and
now is available in Iran. CHG has most of the
advantages needed except tissue solving which
hasn’t been proven yet. It is active against a
wide range of microorganisms, such as Gram–
positive, Gram- negative bacteria, bacterial
spores, lipophilic viruses and yeasts at pH of
45
Journal of Dentistry, Tehran University of Medical Sciences Vahid A, et al

5.5-7. However, compared to NaOCl, it seems Table IV- Antimicrobial activity of 0.5% sodium
to have fewer advantages during instrument- hypochlorite solution on anaerobe microorganisms
(CFU/ ml). Elimination mean percent =92.71%
tation.(11)
Before 48 hour after Reduction
Table I- Antimicrobial activity of 0.2% chlorhexidin Case No.
instrumentation instrumentation percentage
solution on Streptococcus mutants with respect to
1 915000 110000 87.98
No. of CFUs/ml.
2 3195000 30000 99.07
Case Before 48 hour after Percentage 3 580000 50400 99.32
No. instrumentation instrumentation of reduction 4 2400000000 1000000 99.59
1 22500 0 100 5 7220000 50 ~100
2 10000 10 99/9 6 6580000 600 ~100
3 0 0 - 7 4455000 3100 99.94
4 700000 0 100 8 670000 2600 99.7
5 100 0 100 9 11000 52 99.52
6 241900 0 100 10 63250 2 ~100
7 100 0 100
Table V- Mann- Whitney test
8 0 0 -
(A): Ranks
9 800 0 100
10 100 0 100 Mean Sum of
Group No.
Mean elimination percent =99..9% Rank Ranks
DPANA Soduime 10 9.05 90.50
Table II- Antimicrobial activity of 0.2% chlorhexidine
hypochlorite 0.5%
solution on anaerobe microorganisms (CFU/ml).
Chlorhexdine 0.2% 10 11.95 119.50
Mean elimination percentage= 99.02%
Total 20
Case Before 48 hour after Percentage DPSM Soduime hyochlorite 10 8.25 82.50
No. instrumentation instrumentation of reduction 0.5%
1 67550 0 100 Chlorhexdine 0.2% 10 12.75 127.50
2 8250 265 96.8 Total 20
3 103000000 40 ~100
(B): Test statistics
4 1300000 0 100
5 16500 50 99.7 Statistical tests DPANA DPSM
6 100000000 13700 99.99 Mann- Whitney U 35.500 27.500
7 30000000 720 ~100 Wilcoxon W 90.500 82.500
8 50145000 26600 99.95 Z -1.120 -1.887
9 2475000 2860 99.88 Asymp . Sig (2-tailed) .263 .059
10 490000 30100 93.9 Exact Sig. [2* (1- tailed Sig.] .280 (a) .089 (a)
a Not corrected for ties.
Table III- Antibacteial activity of 0.5% sodium
hypochlorite solution on Streptococcus mutans (CFU/ml). b Grouping Variable: Group
Mean elimination percentage=%99.7.
Concerning more than one decade that CHG is
Before 48 hour after Percentage
Case recommended as canal irrigant, its antibacterial
instrumentation instrumentati of reduction
No.
on effect is not well evaluated clinically. At
1 0 0 - present study its antibacterial activity evaluated
2 0 0 - on anaerobic bacteria and Streptococcus
3 14950 180 98/8
mutans. Although anaerobics are the most
4 0 0 -
5 8400 0 100 microbial flora of necrotic canals (obligatory
6 700 0 100 and facultative), since studies still aren’t very
7 1500 0 100 clear about a specific species of anaerobic
8 0 0 - bacteria of mix canal flora, the anaerobic
9 0 0 -
10 0 0 -
bacteria of the canal at present research was
studied nonspecifically.

46 2004; Vol. 1, No. 1

Vahid A, et al Journal of Dentistry, Tehran University of Medical Sciences
Streptococcus Mutans is a facultative anaerobe,
which prefers anaerobic condition and is not
always presents in the canal. Since its
susceptibility to CHG is like other pathoge ns in
endodontic (Porphyromonas Gingivalis,
Prevotella Intermedia, and Fusobacterium
Nucleatum), and also it has been used for other
irrigants anti- microbial studies,(13) it was used at
present study.
This microorganism can enter dentinal tubules
of open apices and continues its life. However
in this study at first culture 12 out of 20 canals
(60%), rendered positive results.
The results of this research showed that
antimicrobial activity of CHG 0.2% is more
effective than SH 0.5% in-vivo, although the
difference is not significant. Vahdaty et al (14)
compared 2% and 0.2% of SH to 2% and 0.2%
CHG, and showed that in equal concentration,
CHG is more effective.
Ringel et al(7) Similarly, used 2.5% SH with
0.2% CHG and found out that SH 2.0% is more
effective. It seems that lower concentration of
CHG is less effective compared to higher
concentration of SH. By the way, comparing
0.2% concentration of CHG to 0.5% SH, the
results were like the study of Yesiloy et al. (1)
Regarding the growth of microorganisms in the
canal system between appointments, lots of
studies comparing different antimicrobial
agents as dressing have been performed.(15,16,17)
According to Barthel and others, CHG gel and
calcium hydroxide paste both had good results
in preventing microbial colonization.(17)
Apparently using CHG solution as dressing
after cleaning the canal is easier and takes less
time.
According to Delany et al, when CHG is used
as irrigant and interappointment dressing, its
0.2% solution can reduce 70% bacteria in
multiradicular and 80% in uniradicular teeth.(10)
The study of Leonardo et al(18) resulted in
aerobe bacteria elimination equal to 100% and
for anaerobes 77.75%. Mean elimination
percent in the present study in chemome-
2004; Vol. 1, No. 1
chanical preparation of the canal and by
benefiting of 12 ml of CHG 0.2% were 99.9 for
Streptoccocus mutans and 99.02 for anaerobes.
These results for 0.5% SH were 99.7 and 92.7,
respectively.
Almost in none of the studies the elimination
percent, particularly for anaerobes rendered
100%. Also dressing the canal with CHG, as
mentioned previously, could have positive
results in present study that for similarity in
methods for none of the irrigating solutions was
considered.
Besides all, the issue of SH accident should be
mentioned. It is a complication that some times
can happen. In the first case reported by
Becking, the accident happened through a
perforation at CEJ of buccal wall and it seems
as much more the concentration the accident
would be worse.(19) Thus with everything
discussed, it seems that in spite of SH
effectiveness, CHG can be a more effective
irrigating and antimicrobial agent with much
fewer side effects for irrigating the canal. More
studies for higher concentrations are
recommended.
Conclusions
In clinical experiment antimicrobial effect of
Chlorhexidine Gluconate 0.2% was similar to
Sodium Hypochlorite 0.5% and no significant
statistical difference existed. Thereby it can be
concluded that CHG, an effective mouth wash
for treating oral infections can be used as
irrigation in endodontic therapy and can easily
replace Sodium Hypochlorite that is caustic,
malodorous, toxic for periapical tissue with low
remaining antimicrobial activity and can cause
corrosion on dental instruments.
CHG 0.2% with high antimicrobial effect can
be used for patients sensitive to SH also in open
apices teeth without hazard to periapecal
tissues. CHG can be dressed in the canal
between appointments and it can keep its
antimicrobial activity during this period of time
and thus prohibits bacterial re-growth. Every
47
Journal of Dentistry, Tehran University of Medical Sciences Vahid A, et al

unwanted entering of irrigating solution (CHG) tions of CHG can be more effective comparable
in the mouth can’t cause gagging and bothering to higher concentrations of SH.
the patient. Since it seems, higher concentra-
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