SCIENTIFIC ARTICLE
Australian Dental Journal 2007;52:(2):118-121
Antimicrobial action of calcium hydroxide, chlorhexidine
and their combination on endodontic pathogens
V Ballal,* M Kundabala,† S Acharya,† M Ballal‡
Abstract
Background: The common recovery of Candida
albicans and Enterococcus faecalis from failed root
canals of teeth in which previous treatment has
failed is notable. These organisms have been shown
to be resistant to antimicrobial action of calcium
hydroxide but are sensitive to chlorhexidine
gluconate. The aim of the present in vitro study was
to investigate the antimicrobial efficacy of calcium
hydroxide paste, 2% chlorhexidine gel and their
combination against Candida albicans and
Enterococcus faecalis.
Methods: Inoculae of these organisms were used to
make lawn cultures on Sabouraud’s dextrose agar
and blood agar plates. Wells were prepared with
these lawn cultures and filled with calcium
hydroxide paste, 2% chlorhexidine gel and their
combination. The agar plates were kept overnight
for incubation at 37˚C and the zone of inhibition
was examined after 24 and 72 hours.
Results: The results suggest that 2% chlorhexidine
gel alone is more effective at 72 hours than calcium
hydroxide paste alone or in combination with 2%
chlorhexidine gel against both the organisms, even
though calcium hydroxide showed better antifungal
efficacy against Candida albicans at 24 hours.
Conclusion: In failed root canal treatments, 2%
chlorhexidine gel may be a more effective intracanal
medicament than calcium hydroxide paste or their
combination against Candida albicans and
Enterococcus faecalis.
Key words: Culturing techniques, chlorhexidine,
calcium hydroxide, Enterococcus faecalis, Candida
albicans.
(Accepted for publication 19 September 2006.)
*Assistant Professor, Manipal College of Dental Sciences, Manipal,
Karnataka, India.
†Professor, Manipal College of Dental Sciences, Manipal, Karnataka,
India.
‡Professor, Department of Microbiology, Kasturba Medical College,
Manipal, Karnataka, India.
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INTRODUCTION
The primary goal of endodontic therapy is the
reduction or elimination of micro-organisms and their
by-products from the root canal system. Although a
number of instrumentation and irrigation techniques
exist, debris is often left behind in the root canal
system. Studies have shown that proper canal cleaning,
shaping and irrigation, significantly reduce and
sometimes eliminate bacteria from canals.1 However,
complete elimination of bacteria is not always achieved
in clinical practice due to the anatomical complexities
of root canals and consequent limitations of access by
instruments and irrigants.
The use of antimicrobial medication has been
advocated to disinfect the root canal system.1,2 Root
canals of infected teeth have a complex microbial flora
consisting of cocci, rods, spirochetes, filaments and
sometimes fungi. The common recovery of
Enterococcus faecalis from the root canals of teeth in
which previous treatment has failed is notable.3
Enterococcus faecalis have been shown to have an
ability to survive in root canals as a single organism
without the support of the other bacteria.4 Candida
albicans is the most commonly isolated fungi from the
oral cavity of both healthy and medically compromised
individuals.5 The occurrence of fungi in root canal
infections has been reported by culture, molecular
methods and electron microscopy in situ.6 In addition,
recent studies have confirmed the presence of fungi
with therapy-resistant endodontic infections.7 After
studying the prevalence of Candida species in different
oral sites, Najzar-Fleger et al. have concluded that
55 per cent of root canals contain Candida albicans
cells.8 Candida albicans seems to be the most common
species of fungi infecting the root canal, which is
resistant to antimicrobial effect of calcium hydroxide.9
Calcium hydroxide plays an important role in
endodontics by its ability to induce hard tissue
formation and antibacterial property. When it is used
along with sodium hypochlorite, it exaggerates the
tissue-dissolving ability of sodium hypochlorite.10
Calcium hydroxide has been advocated as an intracanal
medicament due to its bactericidal properties. Its high
pH (of about 11–12.5) has a destructive effect on
Australian Dental Journal 2007;52:2.
bacterial cell membrane and protein structure. Table 1. Mean inhibitory zone for Candida albicans
However, the antimicrobial ability of calcium N Mean Std. Deviation F p
hydroxide seems dependent upon direct contact with After 24 hrs
bacteria.3 Moreover, calcium hydroxide is not very Ca(OH)2 45 21.0667 1.42063
effective in eliminating bacteria from the dentinal chlorhexidine 45 20.0889 0.90006 97.03 0.001 vhs
combination 45 18.1556 0.47461
tubules.11 Enterococcus faecalis is small enough to
After 72 hrs
proficiently invade and live within dentinal tubules.12 Ca(OH)2 45 17.4444 1.27128
Previous studies have reported that Enterococcus chlorhexidine 45 21.4444 1.45470 199.85 0.001 vhs
combination 45 17.0889 0.46818
faecalis present in the dentinal tubules are resistant to
calcium hydroxide intracanal dressing over 10 days.13
Chlorhexidine gluconate has been shown to be an Table 2. Mean inhibitory zone for Enterococcus
effective antimicrobial endodontic irrigant.14 faecalis
Chlorhexidine does not have the ability to dissolve the
N Mean Std. Deviation
pulp tissue.15 The substantivity property of
After 24 hrs
chlorhexidine may prevent the microbial colonization Ca(OH)2 45 9.0000 0.0000
on the dentinal surface.16 White et al. demonstrated that chlorhexidine 45 12.0000 0.0000
chlorhexidine irrigation continued to prevent the combination 45 7.0000 0.0000
re-infection of dentine for up to 72 hours. Several After 72 hrs
Ca(OH)2 45 7.0000 0.0000
studies have shown that Enterococcus faecalis and chlorhexidine 45 14.0000 0.0000
Candida albicans were resistant to the antibacterial combination 45 6.0000 0.0000
effect of calcium hydroxide medication, but were
sensitive to chlorhexidine.17-19
The purpose of this study was to investigate the chemical used against a particular isolate were recorded
antimicrobial efficacy of calcium hydroxide paste, using a transparent ruler. The results were statistically
2% chlorhexidine gel and combination of analysed using the one-way ANOVA and Duncan
2% chlorhexidine gel and calcium hydroxide paste multiple range test.
against Enterococcus faecalis and Candida albicans in
vitro. RESULTS
MATERIALS AND METHODS Statistical analyses of differences between mean
inhibitory zones for Candida albicans and
The present investigation was performed against Enterococcus faecalis are shown in Tables 1 and 2.
Candida albicans, a fungi and Enterococcus faecalis, a
For Candida albicans at 24 hours, all the three
facultative anaerobe. Since the organisms used in this
groups were statistically different (p<0.001) (Fig 1).
study are not strict anaerobes, they were incubated
Group 1 (calcium hydroxide) showed the maximum
under aerobic conditions. Sabouraud’s dextrose agar
inhibitory zone (21.0667mm). Group 2 (chlorhexidine)
and blood agar plates were used as culture media.
showed intermediate inhibitory zone (20.0889mm).
These agar plates were prepared in sterile glass petri
Group 3 (combination of calcium hydroxide and
dishes and kept overnight for sterility at 37°C. After
chlorhexidine) showed minimum inhibitory zone
ensuring sterility, the inoculae of the strains were
(18.1556mm). In Group 4, no inhibitory zone was seen
prepared with peptone water and the turbidity was
(control). At 72 hours, Groups 1 and 3 were similar,
compared using the McFarland’s turbidity standard
but Group 2 was statistically different from Group 1
tube No. 0.5. This inoculae was used to make lawn and 3 and showed the maximum inhibitory zone
culture of the organisms using sterile cotton swabs on (21.4444mm; p<0.001).
Sabouraud’s agar and blood agar plates. After making
the lawn culture, wells with 3mm diameter and 4mm
depth were punched in the agar plates with sterile
punch. A total of 135 wells in blood agar for
Enterococcus faecalis were prepared. The first 45 wells
were filled with calcium hydroxide paste prepared with
distilled water (1gm of calcium hydroxide powder
mixed with 1ml of distilled water). The next 45 wells
were filled with 2% chlorhexidine gel (KMC Hospital
Pharmacy) and remaining 45 wells were filled with the
combination of 2% chlorhexidine gel and calcium
hydroxide paste. Carbopol was used as a control. A
similar procedure was conducted for Candida albicans.
The plates were incubated overnight at 37°C. The
specimens were examined after 24 and 72 hours
respectively and the zones of inhibition for each Fig 1. Mean inhibitory zone for Candida albicans.
Australian Dental Journal 2007;52:2. 119

Fig 2. Mean inhibitory zone for Enterococcus faecalis.
For Enterococcus faecalis at 24 hours, all three
groups were statistically different (p<0.001) (Fig 2).
Group 1 (calcium hydroxide) showed an intermediate
inhibitory zone (9.0000mm). Group 2 (chlorhexidine)
showed maximum inhibitory zone (12.0000mm).
Group 3 (combination of calcium hydroxide and
chlorhexidine) showed minimum inhibitory zone
(7.0000mm). In Group 4, no inhibitory zone was seen
(control). At 72 hours, Groups 1 and 3 were almost
similar, but Group 2 was statistically different from
Group 1 and 3 and showed the maximum inhibitory
zone (14.0000mm; p<0.001).
DISCUSSION
The present study investigated the antimicrobial
efficacy of calcium hydroxide and 2% chlorhexidine
and their combination against Enterococcus faecalis
and Candida albicans. Most of the intracanal
medicaments lose their efficacy after 24 hours and they
will be totally ineffective after 72 hours. Therefore, this
study was conducted to check the efficacy of the
materials after 24 hours and 72 hours.
The present study used Sabouraud’s dextrose agar
and blood agar as the culture media, since these media
are easily available and commonly used media for
Enterococcus faecalis and Candida albicans.
Calcium hydroxide was tested in this study because
of its prevalent clinical application as intracanal
medicament. The antimicrobial property of calcium
hydroxide given by its high pH is related to the release
of hydroxyl ions in an aqueous environment. It is
known to effectively kill most endodontic pathogens
with the exception of very few such as Enterococcus
faecalis and Candida albicans.13,20
Unlike the conventional medicaments, the positively
charged molecules of chlorhexidine can adsorb into the
dentine and prevent microbial colonization on the
dentine surface for a longer period due to its
substantive antimicrobial property. Previous studies
have shown that chlorhexidine is very effective against
Candida albicans and Enterococcus faecalis.4,19
When calcium hydroxide was used alone, the efficacy
of the chemical was high in the first 24 hours against
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both Candida albicans and Enterococcus faecalis. But
after 72 hours the efficacy reduced for both the micro-
organisms. This may be attributed to the dilution of
calcium hydroxide as time progressed. But when
chlorhexidine was used alone on these two pathogens,
the efficacy of the chemical increased from 24 to 72
hours, which might be due to its substantivity.
However, a combination of chlorhexidine and
calcium hydroxide did not show comparatively good
results after 24 hours and 72 hours. This might be due
to the difference in pH of chlorhexidine and calcium
hydroxide and also due to the binding of chlorhexidine
molecule to calcium hydroxide ions and hence
inhibiting the free release of chlorhexidine molecule.
Pilot studies were carried out by preparing 2%
chlorhexidine gel with calcium hydroxide paste. The
study was performed for a period of six hours using UV
spectrophotometer (Shimadhu – Japan UV 1601 PC).
Results showed that 2% chlorhexidine gel was not
stable at a pH of 12 when combined with calcium
hydroxide paste. This result is in agreement with the
previous study which showed chlorhexidine
precipitates when mixed with calcium hydroxide.21
In contradiction to the previous study, this study
showed chlorhexidine when used alone was more
effective than calcium hydroxide alone or the
combination of chlorhexidine and calcium hydroxide.22
This finding might be due to the difference in
methodology used in the two studies. Carbopol which
was used as control did not show any inhibitory zone
since it does not have any antibacterial or antifungal
activity. However, endodontic infections are primarily
polymicrobial. The medicament that is effective against
single microbe in vitro may not necessarily be effective
against the same microbe in vivo since the root canal
system contains multiple micro-organisms. Hence,
further studies regarding the use of same medicament in
vivo should be conducted.
CONCLUSION
The present study suggests that 2% chlorhexidine gel
alone as an intracanal medicament has a significant
antimicrobial effect against Enterococcus faecalis and
Candida albicans. It is more effective than calcium
hydroxide paste alone or the combination of calcium
hydroxide paste and chlorhexidine gel at 72 hours.
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Address for correspondence/reprints:
Dr N Vasudev Ballal
Assistant Professor
Manipal College of Dental Sciences
Manipal – 576 104
Karnataka, India
Fax: +90 0820 2570061
Email: drballal@yahoo.com
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