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

2004; Vol. 1, No. 1 43

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

recommended as an irrigant to aid in Materials and Methods

chemomechanical debridement of the root Twenty single rooted teeth with necrotic pulps
canal, because it has many of the above were chosen. Pulpal necrosis was determined
mentioned advantages, except its toxic effect either by radiographic presence of apical
for periapical tissue and wettability.(1) Also, its rarefaction or by the lack of response to pulp
smell and discoloring action are other vitality tests. Ages of patients were between 15
disadvantages. to 50 years old. Apices were obviously formed
Spangberg et al(3) found that all medicaments and pulp chambers and canals were free of pulp
are toxic compared to their antibacterial effect. stones, calcifications, or sharp dilacerations.
They said on the basis of a balance between the Teeth were randomly divided into two groups.
cytotoxicity, the antibcaterial effect and the Each of the groups received one of the
specific needs, a recommendation for an irrigating solutions, sodium hypochlorite (SH)
adequate irrigating solution is being made. They 0.5% or chlorhexidine gluconate (CHG) 0.2%.
believed that sodium hypochlorite at 5% SH had the concentration that could have
concentrations is more stronger than it is needed antimicrobial activity with least hazard to
to kill bacteria.(3) periapical tissue.(1) Irrigating solutions used in
Huggers et al(4) in their investigation on this study were chlorhexidine gluconate %0.2
antimicrobial activity and tissue toxicity of from Shahredaru and sodium hypochlorite
different concentrations (0.25%, 0.025% and 5.25% from Taj Co which diluted to 0.5%.
0.0125%) of sodium hypochlorite, found that Study had two phases which will be described:
even 0.25% concentration could be tissue toxic. Clinical phase: After selecting teeth (taking
Chlorhexidine gluconate has been recognized as primary X-ray and making diagnosis by vitality
an effective antimicrobial agent in periodontics, tests) informed consent was taken. The decay
and its use as a potential endodontic irrigating was removed by sharp excavator without
solution has been demonstrated in the last opening the pulp chamber. Tooth isolated with
decade.(5) It seems chlorhexidine has most of rubber dam and access cavity prepared. Before
the mentioned criteria. It has a long term effect entering the canal, pulp chamber was cleaned
and the reservoir of this agent forms on the with sterile distilled water to clean debris
teeth and on the oral mucosa after mouth rinse. resulted from access preparation. Pulp chamber
Antimicrobial activity is released from these was dried with sterile cotton pellets and two
locations as the concentration of chlorhexidine paper points were put into the canal. After one
decreases in the environment.(6) The absorption minute, paper points were withdrawn and
of chlorhexidine by hydroxyapatite, teeth and dropped in screw top test tubes containing
protein demonstrated previously by a chemical transfer media and was sent to microbiologic
method.(6) This characteristic can also be useful laboratory. Taking samples and preparing
inside of the canal. dilutions took less than 10 minutes. This was
Many in vitro studies have been performed referred as first sample.
comparing these two irrigating solutions while Then, after length measurement, canals
few clinical trials have been done. In vivo prepared by step back technique for cleaning
studies are more natural and closer to the actual and shaping. Between the procedures, canals
specifics of the human. Therefore, the aim of were rinsed by 2 cc of any of irrigating
this study was to compare the antimicrobial solutions for each file. Finally, canals were
effect of sodium hypochlorite 0.5% and irrigated by sterile distilled water, then, access
chlorhexidine 0.2% during root canal therapy of cavity closed with ZOE . Two days later, after
twenty infected canals in patients. isolation, the area surrounding the tooth and

44 2004; Vol. 1, No. 1

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

clamp was irrigated with SH, and temporary between groups. As shown on tables I-IV, the
filling was removed. Two papers were put in elimination rates for streptococcus mutans and
the canal and then dropped into transfer media anaerobe bacteria in the presence of C.H.G.
and were sent for culture. Sterility of the were 99.9% and 99.02%; and in presence of
process and instruments during study was S.H. were 99.7% and 92.7% respectively.
axiomatic.
After irrigation, master point with X-ray was Discussion
prepared and canals were filled. Temporary An important adjunct for che momechanical
filling was placed back. preparation of the canal system is irrigation
Microbiologic phase: For microbiologic phase, which aids in debridement and flushing debries
all media autoclaved and distributed into plates. from the canal.(11) At present study antimic-
Thioglicholate (Difco) transferring medium was robial activity of sodium hypochlorite (SH)
poured into vials and autoclaved. Vials 0.5% and chlorhexidine gluconate (CHG) 0.2%
containing paper points agitated on a vortex compared as canal irrigants.
mixer until a homogeneous suspension of SH as the most common proteolytic irrigating
microorganism were obtained.(7,8) For ease of solution has most of the required characteristics,
colony counting, 1 ml. of the homogenized except for its surface tension ,(2) its unpleasant
solution transferred to five test tubes of ringer odor and taste, corrosion of endodontic
solution with 1/10 to 1/100000 dilutions.(9,10) instruments, ineffectiveness on some micro-
In first sampling (before instrumentation), 0.1 organisms when used at low concentration, and
ml from dilutions 1/10, 1/1000, 1/100000 was SH accident. It doesn’t differentiate between
inoculated to plates of blood agar (Merk) for necrotic and vital tissue in contact with apical
anaerobic organisms and three plates of Mitis and periapical tissue, and is unable to be
salivarius agar (Difco) for sterptococcus mutans dressed within the canal. Studies indicate that
and spread with sterile bent glass rod.(9) Plates SH dissolves tissue but experiments in clinical
with gas pack, catalyzer and indicator were put usage are less impressive.(11)
in anaerobic jar (Oxoid) and incubated under 35 Histologic and scanning electron microscope
degree centigrade for 7 days. In second studies do not show that tissue dissolution or
sampling, culture of undiluted sample added to sterilization properties are consistently
previous dilutions and culture was performed as effective.(12) It seems that in clinical conditions
before. After 7 days colony-forming units irrigating solutions have limited surface contact
(CFU) were counted. The results were analyzed and are neutralized in the canal.(11) In present
by nonparametric Mann- Whitney test. research, SH 0.5% (Dakin solution) was used
because of its lower toxicity for periapical
Results tissue despite being effective in the canal.(4)
Tables I-IV indicated the CFU results of the CHG as a mouthwash is well known for few
effect of CHG and SH as irrigating solutions on decades and it is available at 0.12% in the
streptococcus mutans and anaerobe bacteria. A market at USA. The concentration chosen was
generalized decrease of flora of the canal from 0.2% which is used in many studies (7,10) and
culture one to culture two was present. (For now is available in Iran. CHG has most of the
streptococcus mutans exact P=0.089 and for advantages needed except tissue solving which
anaerobe bacteria exact P=0.315). But none of hasn’t been proven yet. It is active against a
the teeth were totally free of cultivable wide range of microorganisms, such as Gram–
microorganisms. According to table V, the positive, Gram- negative bacteria, bacterial
results showed no significant difference spores, lipophilic viruses and yeasts at pH of

2004; Vol. 1, No. 1 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, chanical preparation of the canal and by

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

2004; Vol. 1, No. 1 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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