0099-2399/98/2407-0472503.
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JOURNAL OF ENDODONTICS
Copyright © 1998 by The American Association of Endodontists
Antimicrobial Activity of 2.5% Sodium Hypochlorite
and 0,2% Chlorhexidine Gluconate Separately and
Combined, as Endodontic Irrigants
Jane Rachel Kuruvilla, BDS, MDS, and M. Premanand Kamath, BDS, MDS
Sodium hypochlorite irrigant is known to be toxic
to periapical tissues. Chlorhexidine gluconate, a
safer and effective antimicrobial irrigant, is not
known to dissolve pulpal tissues. To obtain their
optimal properties, their combined action within
the root canal was evaluated.
Ten single rooted nonvital anterior teeth were
irrigated using either 2.5% sodium hypochlorite
alone, 0.2% chlorhexidine gluconate alone, 2.5%
sodium hypochlorite and 0.2% chlorhexidine glu-
conate combined within the root canal, or 0.9%
saline, respectively. Microbiological samples for
culture and Gram's staining were taken before and
proceeding irrigation.
This study indicates that the use of sodium hy-
pochlorite and chlorhexidine gluconate combined
within the root canal resulted in the greatest per-
centage reduction of postirrigant positive cultures.
This may be due to formation of "chlorhexidine
chloride," which increases the ionizing capacity of
the chlorhexidine molecule. This reduction was
significant compared to use of sodium hypochlo-
rite alone but not significant compared to use of
chlorhexidine gluconate alone.
The prime objective of root canal therapy is the retention of the
pulpless or pulpally involved tooth, with its associated periapical
tissues in a healthy state. Achievement of this objective requires
that the pulpal spaces and contents are eliminated as sources of
infection (1). Therefore, the introduction of an antimicrobial end-
odontic irrigant during root canal therapy should be given priority
in the hierarchy of root canal treatment.
Sodium hypochlorite solution has been used as an endodontic
irrigant for well over 4 decades. Although it is an effective anti-
microbial agent (2) and an excellent tissue solvent (3), it is known
to be toxic to the periapical tissues (4,5). Hence, the search for
another irrigant with less potential for adverse effect is desirable.
Chlorhexidine gluconate has become recognized as an effective
472
Printed in U.S.A.
VOL. 24, No. 7, JULY1998
antimicrobial agent, and its use as a potential endodontic irrigant
has been demonstrated in the last decade (6,7,8). It possesses a
broad-spectrum antimicrobial action (9), substantivity (10), and a
relative absence of toxicity (9) that are desirable properties of an
ideal root canal irrigant. However, a significant attribute that
chlorhexidine gluconate is not known to possess is a tissue dis-
solving property (6). Hence, until chlorhexidine gluconate is also
shown to be effective in pulp tissue dissolution, the quest for an
ideal irrigant continues.
It has been postulated that the use of sodium hypochlorite and
chlorhexidine gluconate, combined within the root canal, could
gain:
• an additive antimicrobial action,
• a tissue dissolution property that is better than that obtained with
the use of chlorhexidine alone, and
• a solution less toxic than sodium hypochlorite.
The purpose of this study was to compare the antimicrobial
efficacy of 2.5% sodium hypochlorite and 0.2% chlorhexidine
gluconate combined within the root canal to their individual use in
vivo.
M A T E R I A L S AND M E T H O D S
A total of 40 cases, consisting of single rooted, nonvital anterior
teeth, with a definite periapical radiolucency, were selected for this
study (2,11). None of these selected cases were on antibiotic
treatment before or during sample collection. These cases were
then randomly assigned into four groups of ten cases each. The
groups were:
Group l: 2.5% sodium hypochlorite diluted from a stock of 3%
sodium hypochlorite (Novodent Equipments & Materials Ltd.,
Bombay, India)--3 ml.
Group 2: 0.2% chlorhexidine gluconate diluted from a stock of
100% chlorhexidine gluconate solution (Mehta Pharmaceutical
Industry, Thana, India)--3 ml.
Group 3: Use of 2.5% sodium hypochlorite and 0.2% chlorhexi-
dine gluconate (1.5 ml of sodium hypochlorite + 1.5 ml of chlo-
rhexidine gluconate)--3 ml.
Group 4: 0.9% saline (Parenteral Drugs India Pvt. Ltd.) (Con-
trol)--3 ml.
Vol. 24, No. 7, July 1998
Preparation of Access Cavity
The tooth surface was disinfected by the technique outlined by
Moller (12). After rubber-dam isolation, the tooth, its surround-
ings, and the clamp were cleansed with 30% hydrogen peroxide for
1 min. An initial opening was made, 1 to 2 mm into the dentin, with
a sterile tapering fissure bur at high speed. The tooth was again
swabbed with hydrogen peroxide for 10 s, followed by application
of 2% tincture of iodine for 1 min. The iodine was deactivated with
5% sodium thiosulphate.
Proceeding the penetration of the roof of the pulp chamber, a
sterile surgical length no. 2 or no. 4 tungsten carbide round bur was
used at slow speed, working the bur from the inside to the outside,
to expose the entire pulp chamber.
Collection of Sample
Two sterile glass slides and two sterile test tubes containing
Robertson's Cooked Meat Broth (RCM) as culture medium (Hi
media Laboratories Pvt. Ltd., Bombay) were obtained from the
Department of Microbiology, KMC, Mangalore for each case. A
preoperative radiograph was used to assess the approximate length
of the tooth. A presterile paper point (Novo Absorbent Points,
Roeko, Germany) premoistened with sterile distilled water to pro-
vide "pooling effect" for collection of bacteria (13) was introduced
into the root canal and the material smeared across a glass slide.
Similarly, a presterile barbed broach (Pulpadent, Swiss Product)
was inserted into the root canal to obtain material and then dropped
into a test tube containing the culture medium. This constituted the
"preirrigant smear and culture."
The canal was then irrigated with 3 ml of irrigant (depending on
each group) using a sterile syringe and a 26-gauge needle that was
not allowed to bind to the canal walls. The canal was then dried
with a sterile paper point. Following this, two other readings were
taken for smear preparation and culture in an identical manner to
the preirrigant smear and culture methods. This constituted the
"postirrigant smear and culture."
Processing for Anaerobic Culture
The "preirrigant" and "postirrigant" samples were streaked on
separate blood agar plates. These were then incubated at 37°C for
72 h in an anaerobic chamber (Gas Pak system). The number of
organisms on the incubated plates were counted using a Quebec
colony counter. The viable organisms were expressed as the total
colony forming units per ml (CFU/ml).
Criteria for Data Evaluation
The values obtained in this study were graded as follows:
1. Low count: less than 103 CFU/ml
2. Moderate count: 103 to 105 CFU/ml
3. Moderately high count: 10 6 tO 10 8 CFU/ml
4. High count: >108 CFU/ml
Smear Preparation for Gram's Stain
The Gram's stained smears were seen under the light micro-
scope under an oil immersion field (OIF), and from each sample
Microbial Properties of Endodontic Irrigants 473
five insets with highest colonies counts were selected and a mean
score was allotted for each sample.
The findings were graded as follows:
1. Scanty: --<5 microorganisms/OIF +
2. Moderate: 6 to 30 microorganisms/OIF + +
3. Moderately high: 31 to 100 microorganisms/OIF + + +
4. Abundant: > 100 microorganisms/OIF + + + +
To quantitatively analyse the microorganisms before irrigation
and proceeding irrigation, the "Sign test" was used.
To compare the intergroup antimicrobial efficacy, the "Wil-
coxon signed rank test" was used.
To determine if any correlation between the Gram's staining and
culture method existed in the quantitative analysis of microorgan-
isms within the root canal, the "Spearman's Rank Correlation
Co-efficient" was applied.
RESULTS AND A N A L Y S I S
Table 1 represents the quantitative analysis of the microbial
growth within the root canal, before and after irrigation, in all four
groups studied. The results indicate that the number of postirrigant
positive cultures (CFU/ml) was significantly lower in all 4 groups
(p < 0.001) compared with the preirrigant positive cultures using
the sign test.
Teeth treated with sodium hypochlorite and chlorhexidine glu-
conate combined within the root canal (Group 3) showed the
greatest percentage reduction in the number of microorganisms:
84.6%. The chlorhexidine gluconate treated teeth (Group 2)
showed a percentage reduction of 70% in the number of microor-
ganisms. Sodium hypochlorite treated teeth (Group 1) showed a
percentage reduction of 59.4%, while teeth treated with saline
(Group 4) showed a percentage reduction of only 25%.
Comparison of Group 1 with Group 2
Three ml of 2.5% sodium hypochlorite to 3 ml of 0.2% chlo-
rhexidine gluconate.
The percentage reduction in the number of microorganisms after
irrigation in the chlorhexidine gluconate treated teeth was lower
(70%) than that obtained after irrigation in the sodium hypochlorite
treated teeth (59.4%).
This difference when subjected to the Wilcoxon signed rank test
was not statistically significant (p > 0.05).
Comparison of Group 3 with Group 1
Use of 1.5 ml of 2.5% sodium hypochlorite and 1.5 ml of 0.2%
chlorhexidine gluconate combined within the root canal to 3 ml of
2.5% sodium hypochlorite.
The percentage reduction in the number of microorganisms after
irrigation with the use of sodium hypochlorite and chlorhexidine
gluconate combined within the root canal treated teeth was lower
(84.5%) than that obtained after irrigation in the sodium hypochlo-
rite treated teeth (59.4%).
This difference when subjected to Wilcoxon rank test: one tailed
test (z = 1.77) was statistically significant (p < 0.05).
474 Kuruvilla and Kamath Journal of Endodontics

TABLE 1. Microbial culture (CFU/ml)

GROUP 1 GROUP 2 GROUP 3 GROUP 4

Pre irrigant Post irrigant Pre irrigant Post irrigant Pre irrigant Post irrigant Pre irrigant Post irrigant
103-5 - 103-5 - 103-5 - 103-5 103-5
> 108 103-5 < 103 -- 103-5 -- 103-5 < 10 3
10 3-5 - 10 6-8 <10 3 10 5-8 - 10 3-5 1 0 3-5
> 10 8 10 3-5 10 6-8 - 10 6-8 - 10 's~ 10 3-5
10 6-5 10 3-5 > 10 8 1 0 3-5 10 e-5 - > 10 8 10 6-5
> 10 8 10 3-5 10 6-8 10 3-5 > 10 8 < 10 3 10 6-8 1 0 3-5
>108 103-5 >108 103-5 >100 <103 103-5 <103
> 108 103-5 103-5 - 10 e-8 < 103 1 Or'-8 10 3-5
103-5 < 103 10 3-5 - 103-5 - 10 6-8 103-5
105-8 - 10 e-8 < 103 > 108 1034 > 105 10 6-5
Test o f significance
Sign test
Z = 3.163 Z = 3.163 Z = 3.817 Z = 3
p < 0.001 p < 0.001 p < 0.001 p < 0.001
% Reduction
59.4 70 84.6 25
* criteria for data evaluation
Low count: <103 CFU/ml; moderate count: 103 to 105 CFU/ml; Moderately high count: 108 to 108 CFU/ml; high count: >108 CFU/ml

TABLE 2. Correlation between Gram's stain and culture method

<103 CFU/ml 103-5 CFU/ml 106-8 C F U / m l >108 C F U / m l Correlation C o e f f i c i e n t (r)

+ 1 case 4 cases 1 case -- p

++ -- 6 cases 2 cases 1 case < 0.0001
+ + + -- 3 cases 11 c a s e s 7 cases Highly
+ + + + -- -- -- 4 cases significant
* criteria for data evaluation for number of microorganisms using Gram's stain.
Scanty: <5 microorganisms/OIF +
Moderate: 6 to 30 microorganisms/OIF + +
Moderately high: 31-100 microorganisms/OIF + + +
Abundant: >100 microorganisms/OlF + + + +

Comparison of Group 3 with Group 2
Use of 1.5 ml of 2.5% sodium hypochlorite and 1.5 ml of 0.2%
chlorhexidine gluconate combined within the root canal to 3 ml of
0.2% chlorhexidine gluconate.
The percentage reduction in the number of microorganisms after
irrigation with the use of sodium hypochlorite and chlorhexidine
gluconate treated teeth was lower (84.6%) than that obtained after
irrigation in the chlorhexidine gluconate combined within the root
canal treated teeth, however, this difference when subjected to the
Wilcoxon signed rank test was not statistically significant (p >
o.o5).
Table 2 represents the correlation coefficient between Gram
staining and culture method in the quantitative evaluation of mi-
crobial flora within the root canal. This correlation coefficient (r)
was found to be highly significant (p < 0.001).
DISCUSSION
Microorganisms and their end products are considered the major
cause of pulpal and periapical pathosis (14). Bystrom and
Sundqvist (15) demonstrated that mechanical instrumentation re-
duced bacteria from human root canals by only 50%. These authors
concluded that to predictably eliminate bacteria from the root
canals, the supporting action of a disinfecting agent is mandatory.
The solutions used in this study have been accepted as root canal
irrigants for endodontic usage (16). Moreover, the residual organic
matter within the root canal could alter the response to these
chemical agents. Bearing the above factors in mind, it was pro-
posed to conduct an in vivo study that would be clinically relevant.
The cases selected in this study were similar to the standards
followed by previous investigators (2,11). The tooth surface was
disinfected by the technique outlined by Moiler (12). Microbio-
logical samples were obtained by the procedure outlined by Delany
and associates (17) with a few alterations. Mechanical instrumen-
tation of the canal was not performed, as the purpose of this study
was solely to determine the antimicrobial effectiveness of these
chemical irrigants proceeding irrigation. In addition, Robertson's
cooked meat medium was preferred as the transport medium in this
study, as it supports the growth of the most obligate anaerobes
(18), which contribute to as high as 90% of the microorganisms
within the root canal (19). This medium contains meat and is
enriched with yeast extract, heroin, Vitamin K, and glucose. The
meat contain L-cysteine, gutathione, and unsaturated fatty acid,
which further contribute to anaerobic biosis.
The results of this study indicate that the alternate use of sodium
hypochlorite and chlorhexidine gluconate irrigants resulted in a
greater percentage reduction of microbial flora (84.6%) when
compared with the individual use of sodium hypochlorite (59.4%)
or chlorhexidine gluconate (70%). This percentage reduction in
microbial flora was significant (p < 0.05) when compared with the
individual use of sodium hypochlorite but not significant (p >
0.05; Z = 1.57) when compared with the individual use of chlo-
rhexidine gluconate irrigant (where test of significance Z = 1.65,
Vol. 24, No. 7, July 1998
one tailed test). This could possibly be attributed to the difficulty
to assume distribution of data, which indicated the use of nonpara-
metric tests for the data analysis.
Chlorhexidine gluconate was found to be as effective or possi-
bly more effective in its antimicrobial property when compared
with sodium hypochl0rite irrigant. These irrigants significantly
reduced the postirrigant positive cultures and colony forming units
when compared with the saline irrigated teeth.
The results from the individual trial of chlorhexidine gluconate
and sodium hypochlorite indicate that they are equally effective
antibacterial agents. However, when these solutions were com-
bined within the root canal, the antibacterial action was suggestive
of being augmented.
The possible reason for this could be due to the following
reaction:
• Chlorhexidine is a base, itself capable of forming salts with a
number of organic acids.
• Sodium hypochlorite is an oxidizing agent that may be capable
of oxidizing the gluconate part of chlorhexidine gluconate to
gluconic acid. The chloro groups might get added on to the
guanidine component of the chlorhexidine molecule, thereby
forming "chlorhexidine chloride."
This reaction may be depicted as follows:
I. NaOCI + H20 ...... NaOH + HOCI
HOC1................. HC1 + [O]
HCI ................... H+C1-
H NH NH (guanidine group)
I II 11
II. CH3--N-- C --NH---~ --NH-M~ ---CI (Chlorhexidine molecule)
CI
(chloro group)
H N+CI N+C1
r II II
CH3--N --C--NH---<S--NH--~ --C1
If this were to happen, it would increase the ionizing capacity of
the chlorhexidine molecule, and the solution would incline toward
an alkaline pH. This was evident when the pH of sodium hypo-
chlorite solution, chlorhexidine gluconate solution, and their com-
bination were recorded using a pH meter.
The pH was recorded as follows:
• 2.5% NaOC1 9
• 0.2% Chlorhexidine gtuconate 6.5
• Combination 10
It is a known fact that the ionized species exert better antibacterial
action than the unionized species (20). Further studies to validate
the above findings would be of interest.
This study conforms to the findings of others who have previ-
ously evaluated the antibacterial properties of sodium hypochlorite
and chlorhexidine gluconate as endodontic irrigants.
It was Parsons and associates (10) in 1980 who first studied the
uptake and release of 0.02% and 1% solutions of chlorhexidine by
bovine pulp and dentin specimens and concluded that chlorhexi-
dine was a potent antibacterial agent and may serve as an effective
endodontic irrigant. Other investigators such Vahadaty et al. (7),
Jeansonne and White (6), and Yesilsoy et al. (8), have shown
Microbial Properties of Endodontic Irrigants 475
chlorhexidine gluconate to be as effective an antimicrobial agent as
sodium hypochlorite, in vitro, whereas the Loma Linda group of
investigators has reported 0.2% chlorhexidine gluconate to be a
more effective antimicrobial agent than 5.25% sodium hypochlo-
rite on selected anaerobic test microorganisms.
The results of this study are in contrast to the findings of Ringel
and associates (11), who concluded that 2.5 % sodium hypochlorite
was more effective than 0.2% chlorhexidine gluconate as an anti-
microbial endodontiq irrigant in vivo. As pointed out by Vahadaty
and others (7), a close examination of their results indicate that
chlorhexidine gluconate was as effective as sodium hypochlorite
during the first appointment and at the start of the second appoint-
ment. They have described that the statistically significant differ-
ence at the beginning of the third appointment could be attributed
to the increase in the size of the root canal during preparation,
which increased the "tissue dissolution" action of sodium hypo-
chlorite.
There was a generalized decrease in the root canal flora in the
saline-treated teeth obtained in this study. This generalized de-
crease in microorganisms in the saline-treated teeth may be due to
its ability to flush out debris from the root canal rather than having
any antimicrobial property. It was also interesting to note that all
the cultures obtained following saline irrigation remained positive
for microorganisms.
Another important finding to be noted in this study was a highly
significant correlation between Gram's staining and culture meth-
ods in evaluating the microbial flora within the root canal. All the
selected cases in this study revealed a definite periapical radiolu-
cency on radiographic examination. It can thus be concluded that
Gram's method of staining provides an overall impression of the
presence of microorganisms, within the root canal, with the ad-
vantage of being a simple and easy chair-side procedure that can be
performed within a few minutes. Moreover, it is found to be an
economical and useful aid in evaluating the microbial flora within
the root canal during endodontic treatment.
The limitation of this study was the inability to standardize the
preirrigant positive cultures in all four groups studied. The long-
acting effect of chlorhexidine was due to its property to adsorb
anionic substrate and was also not considered in this study. There-
fore, a more extensive study with a definite data distribution and
use of parametric tests is required to confirm the "synergism"
suggestive in this study.
We thank Dr. K. S. Bhat, Dean, Professor and Head, Department of
Conservative Dentistry and Endodontics, College of Dental Surgery, Manipal
Academy of Higher Education, India, for his valuable help, experience, and
support throughout this study.
Dr. Jane Rachel Kuruvilla is a former postgraduate student of the Depart-
ment of Conservative Dentistry and Endodontics, College of Dental Surgery,
Mangalore and is affiliated with the ManipaI Academy of Higher Education,
Manipal. She is now in private practice in Cochin, India. Dr. M. Premanand
Kamath is Professor and Head, Department of Conservative Dentistry and
Endodontics, College of Dental Surgery, Mangalore and is affiliated with the
Manipal Academy of Higher Education, India. Address requests for reprints to
Dr. Jane Rachel Kuruvilla, Apt. #8C, Providence Grove, Providence Road,
Cochin-682 018, India.
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1988;7.

The Way It Was

In our compromise-admiring, consensus-building society we enthrone the person who "doesn't have an
enemy in the world" or is "beloved by all." That's OK I guess but I can't help recalling the accolade to Teddy
Roosevelt by Gifford Pinchot, who recognized that Roosevelt's efforts to establish our national park system
had been bitterly opposed: "He was loved by those who should love him and hated by those who should hate
him."
Could it be that to have some folks love you would be a disgrace?
Zachariah Yeomans