Original Research

Antimicrobial effectiveness of different preparations of

calcium hydroxide
Anshul Gangwar
Department of Pedodontics and
Preventive Dentistry, Faculty of
Dental Sciences, King Georges
Medical University, Lucknow,
India

ABSTRACT

Received
: 22-12-09
Review completed : 03-05-10
Accepted
: 03-10-10

Background: Intracanal medicaments have an antibacterial effect on the root canal flora. The
effectiveness of such vehicles has to be tested and substantiated against the normal bacterial
flora. The various vehicles that were tested for their effectiveness were commercially prepared
calcium hydroxide (Metapex), saline, glycerine, CMCP, and Rexidine-M gel. The bacterial cultures
tested for sensitivity were aerobes (Staphylococcus aureus, Streptococcus viridans, Streptococcus
pyogens, Enterococcus faecalis), anaerobes (Lactobacillus, Bacteroides melaninogenicus) and
a commonly found fungus (Candida albicans). One hundred and twenty-five samples were
divided into five experimental groups consisting of 21 samples each and one control group of
the same size.
Aim: The aim of this study was to investigate in vitro the influence of four different vehicles
on the effectiveness of calcium hydroxide against the commonly found aerobic and anaerobic
bacteria in endodontic infections.
Materials and Methods: The samples were taken and transported using standardized techniques
and pure growth of each bacterium was isolated. Petri dishes were prepared for the various
calcium hydroxide combinations for each bacterium and incubated. The inhibition zone was
recorded at three intervals of 24, 96and 168 hours. Results were tabulated and sent for statistical
analysis.
Results and Conclusions: It was seen that calcium hydroxide and CMCP combination showed
the maximum zone of inhibition, and maximum inhibitory effect was seen at 24 hours. The
bacteria most susceptible was found to be S. aureus and the least susceptible was E. faecalis.
Further clinical studies are required to substantiate these results.
Key words: Calcium hydroxide, in vitro, Staphylococcus aureus, Enterococcus faecalis

The most common cause of dental pulp injury is bacterial

assault. Bacteria or bacterial products may enter the pulp
via various routes.
It has been well established that the endodontic infections
are polymicrobial in nature. The microorganisms most
frequently isolated from the infected root canals are
streptococci and micrococci. The development of various
Address for correspondence:
Dr. Anshul Gangwar
E-mail: [email protected]
Access this article online
Quick Response Code:

Website:
www.ijdr.in
PMID:
***
DOI:
10.4103/0970-9290.79986

Indian Journal of Dental Research, 22(1), 2011

new techniques for the growth and isolation of obligate

anaerobes has shown that anaerobic microorganisms are
much more prevalent root canal species than were earlier
thought to be. The primary goal of an endodontic treatment
is to achieve a bacteria free environment in the root canal
in order to achieve clinical success.
Normally, the infected tissue removal is achieved by
irrigating the canal with proteolytic disinfecting solution and
thorough biomechanical cleaning. After all the infected and
necrotic pulp tissue is removed, the root canal is disinfected,
enlarged and shaped to allow optimal root canal filling.
However, with the use of conventional chemomechanical
technique of root canal treatment preparation, neither the
removal of soft tissue is complete nor does the elimination
of bacteria seem to be sufficient. Inability to completely
eradicate the microorganisms and/or provide bacteria proof
seal can result in root canal therapy failure. Hence, the use
of intracanal medicaments has been advocated to further
reduce the number of microorganisms.
66

Antimicrobial effectiveness of different preparations of calcium hydroxide

Hermann[1] introduced calcium hydroxide and its various

preparations for clinical application. Initially, calcium
hydroxide was used in vital pulp therapy; its emergence
as an intracanal medicament has almost became a panacea
in multibacterial infections of root canals. Its various
effects include: (1) microbial control, (2) healing periapical
inflammation, (3) arresting inflammatory root resorption, (4)
stimulating hard tissue formation in apexification procedure
and (5) serving as a temporary obturating material between
appointments.
An antibacterial intracanal medicament must have a wide
spectrum of activity and a reasonable duration of action
to eliminate all the bacteria in the root canal. However,
till date, no intracanal medicament has proved to be
active against the whole spectrum of microorganisms. The
combination therapy of antimicrobial agents chosen should
cover the most common known or suspected pathogen
in polymicrobial infection in order to obtain enhanced
inhibition.
As per the available literature, various calcium hydroxide
preparations have been tried so as to utilize its antibacterial
potential. The complex synergistic interaction between
microbiota and pathological process has called for scientific
research to study the antimicrobial properties of therapeutic
agent, which would further add to the benefits of therapy.
Keeping the above fact in view, the present in vitro study
was undertaken.
This study aims to evaluate the antimicrobial properties
of different preparations of calcium hydroxide on specific,
most commonly associated microorganisms found within
infected root canal.

MATERIALS AND METHODS

This in vitro study was conducted on 125 samples in the
outpatient Department of Pedodontics and Preventive
Dentistry.

Criteria for collection of samples

1. After thorough oral prophylaxis, the patient was advised

to maintain oral hygiene properly.
2. All the samples were collected under rubber dam
isolation to avoid any contamination in test samples.

Collection of sample

The selection of tooth for sample was made after thorough

history taking, clinical, radiological examination, routine
investigations, and proper oral hygiene maintenance.
None of the patients selected for the study received
antibiotic therapy within previous 15 days or during the
course of treatment so that the antibiotic does not alter
the growth of bacteria in present study. Precaution was
taken to see to that the selected patient did not present
67

Gangwar

with any subjective and objective signs of systemic or

chronic debilitating diseases. Permanent uniradicular
teeth exhibiting no vitality were selected for sampling,
irrespective of sex and socioeconomic status. The patients
age ranged from 10 to 14 years. The whole procedure
was carried out under sterile conditions to avoid any
contamination.
One hundred teeth were selected for the collection of test
samples. Special care was exercised to use aseptic technique
for the access opening and procuring the samples from
the root canals without the use of any irrigant. Access to
all selected cases of root canal treatment was gained using
the standardized technique of root canal preparation. A
sterile paper point was then placed to full length of the
root canal for 60 seconds. The paper point sample from
the root canal was preserved in tight screw capped bottle
containing the transportation media. These samples were
then immediately transferred to the Bacteriology Laboratory
of the Department of Microbiology. The test samples were
inoculated in Robertsons cooked meat broth and incubated
at temperature of 37 1C for 24 hours in aerobic and
anaerobic atmosphere before subjecting them to various
experimental procedures.

Materials and instruments

Media
1. Transportation media
Thioglycollate broth
Robertson's cooked meat broth
2. Calcium hydroxide powder assay 96% (Rolex Chemical
Limited, Mumbai, India)
3. Metapex (Meta Dental New York, Elmhurst, USA)
4. Sterile N-saline sodium chloride injection IP (0.9%
w/v) (Core Health Care Limited, Ahemdabad, Gujrat,
India.)
5. Excela R (Qualigens Fine Chemicals Limited, Mumbai,
India)
6. Camphorated paramonochlorophenol (trade name
Cresophene) (Septodont Saint Maur des Sosses Cedex,
France)
7. Rexidine-M gel (Indoco Remedies Ltd., Maharashtra,
India)
Instruments
1. Sterile paper point (Dentsply India Pvt. Ltd., Delhi,
India)
2. Platinum loop
3. Incubator
4. Test tube stand

Procedure
Incubation of samples
Samples collected from the patient were inoculated in
Robertson's cooked media and incubated at a temperature
of 37 1oC.
Indian Journal of Dental Research, 22(1), 2011

Antimicrobial effectiveness of different preparations of calcium hydroxide

Experimental aerobic and anaerobic bacterial isolates used

in this study were:

Aerobes

Staphylococcus aureus
Streptococcus pyogens
Streptococcus viridans
Enterococcus faecalis
Candida albicans (fungi)

Anaerobes

Lactobacillus species
The aerobes were isolated from clinical trials. The anaerobes
were obtained from Department of Microbiology, Sanjay
Gandhi Postgraduate Institute of Medical Sciences,
Lucknow.
Each microbial strain was evaluated against calcium
hydroxide formulation prepared with different vehicles as
follows [Figure 1]:
a. Metapex
b. Calcium hydroxide + N-saline
c. Calcium hydroxide + glycerine Excela R
d. Calcium hydroxide + camphorated paramonochlorphenol
e. Calcium hydroxide + Rexidine-M gel (chlorhexidine +
metronidazole)
f. Control group (no preparation used)
The antimicrobial effect of various calcium hydroxide
formulations was evaluated at various time intervals, i.e.,
1 hour, 24, 96 and 168 hours. The antimicrobial effect was
evaluated by measuring the zone of inhibition.
The various calcium hydroxide pastes were prepared using
calcium hydroxide powder. The consistency of various
calcium hydroxide pastes was similar to that of tooth paste,
with the viscosity of 3501 cP at 0.1 rpm and pH of 12.5 as
determined by a digital pH meter.

Gangwar

Agar diffusion method

Incubation of trypticase soy blood agar and Muller Hinton
agar: To test the effectiveness of various preparations of
calcium hydroxide by agar diffusion method, respective agar
plates inoculated with aerobic bacteria were incubated at
37C. For anaerobes, respective anaerobic agar plates were
placed in anaerobic jars (CO2 incubator) in an atmosphere of
10% carbon dioxide gas + 10% hydrogen gas + 80% nitrogen
gas and incubated at 37C.
Observations were made to detect the zone of bacterial
inhibition around each medicament at various time intervals,
i.e., 1, 24, 96 and 168 hours. Zones of inhibition were
measured using Vernier Calipers and divider. Inhibitory
growth of 21 microorganisms was evaluated against each
preparation of calcium hydroxide.
Statistical analysis was done using the analysis of variance
(ANOVA) test and the Students t test

RESULTS
Analysis of zone of inhibition around each medicament
against microorganisms was done [Figures 2 and 3].The
zone showed no changes after 1 hour and no inhibition of
bacteria was seen in the control group.
The mean inhibition scores for different groups after 24
hours were as follows:
Group D > Group C > Group E > Group A > Group B
The orders of mean inhibition scores for different groups
after 96 hours were as follows:
Group D > Group C > Group E > Group A > Group B
The mean inhibition scores for different groups after 168
hours were as follows:
Group D > Group C > Group E > Group A > Group B
Figure 4 shows the overall scores for different bacterial strain

Experimental group

Control group

No preparations
Group A

Group B

Group C

Group D

Group E

Metapex

Ca(OH)2 + N saline

Ca(OH)2 +
glycerine

Ca(OH)2 +
CMCP

Ca(OH)2 +
Rexidine-M

21
Microorganisms

21
Microorganisms

21
Microorganisms

21
Microorganisms

(Group F)

21
21
Microorganisms Microorganisms

Figure 1: Distribution of preparations used

Indian Journal of Dental Research, 22(1), 2011

68

Antimicrobial effectiveness of different preparations of calcium hydroxide

Gangwar

inhibition as against all the experimental medicaments.

S. aureus shows the maximum score of inhibition at all
time zones while E. faecalis consistently shows the least
inhibition at all time zones.

A sterile paper point, as per the method of M. Georgopolou

et al.[2] was inserted into canal and sample was taken.
This was done to ensure standardization of technique for
collection of samples.

Figure 5 shows the overall inhibition scores for

different medicaments against all the bacterial
strains under study. Group D showed the maximum
effectiveness at all time periods while Group B showed
the least effectiveness.

For in vitro preparations, the most common method for

antimicrobial activity assessment is agar diffusion method,
as also supported by previous studies.[3]

DISCUSSION
In the past, numerous antimicrobial agents had been
used as root canal medicaments. These included
traditional phenolic and fixative agents such as
camphorated monochlorophenol, formocresol, eugenol,
metacresylacetate and halides (iodine, potassium iodide).
The current intracanal dressing of choice is calcium
hydroxide, a white odorless powder with high pH and
low solubility in water. Delivery of dry calcium hydroxide
powder alone is difficult or impossible to use in smaller
or curved canals. In most cases, calcium hydroxide must
be mixed with a liquid to facilitate placement. Calcium
hydroxide is normally used as slurry of calcium hydroxide
in a water base.

The zones of bacterial inhibition around each medicament

was used as the criteria for comparison of the effectiveness of
the medicament. In control cases, no formulation of calcium
hydroxide preparation was used for bacterial counteraction.
All the preparations of calcium hydroxide showed inhibitory
growth but it was found that S. aureus showed maximum
inhibition, whereas E. faecalis was the least inhibited
bacterial strain. Among the different preparations of
calcium hydroxide, calcium hydroxide with camphorated
paramonochlorophenol showed the best result, while calcium
hydroxide with saline showed minimum zone of inhibition.
In this study, inhibitory effect of calcium hydroxide
preparation was seen optimum at 24 hours with slight
change occurring at 96 hours. However, at 168 hours, some
bacterial growth was observed. Acidic pH favors microbial
growth; however, after application of calcium hydroxide,

Figure 2: Slide showing E. faecalis growth after 24 hours

Figure 3: Slide showing S. aureus growth after 24 hours

Figure 4: Bar graph showing bacterial growth against all calcium

hydroxide combinations

Figure 5: Bar graph showing calcium hydroxide combinations against

bacterial growth

69

Indian Journal of Dental Research, 22(1), 2011

Antimicrobial effectiveness of different preparations of calcium hydroxide

alkaline pH is obtained. Due to disassociation of calcium

hydroxide into Ca++ and OH ions, these ions exert lethal
effect on bacteria. With time, calcium hydroxide diffuses
into agar media to a great extent, thereby resulting in
dilution of Ca++ and OH ions, which in return decreases the
pH of the media. This reduced pH is responsible for further
growth of microorganisms.

Gangwar

Castagnola and Orlay [4] reported that iodoform pastes

destroy the microorganisms in tissue remnants and render
them unsuitable for supporting microbial life.

conducted:
Calcium hydroxide + camphorated paramonochlorophenol showed the best results in inhibiting microbial
growth.
Among the microorganisms tested, S. aureus was found
to be most susceptible to the bactericidal action of
calcium hydroxide preparations, whereas E. faecalis
was found to be least susceptible.
According to the time taken in achieving the
antimicrobial efficacy, maximum inhibitory effect was
noticed after 24 hours.

Result of the present study is in accordance with that

of Tchaou et al[5] They showed that there is no/minimal
antibacterial effect with Metapex/Vitapex. Pabla et al[6]
reported least antimicrobial activity of Metapex/Vitapex
against aerobic and anaerobic bacteria in comparison to zinc
oxide, eugenol, KRI paste and MAISTO paste in their study.

The results of this study are encouraging and we can safely

conclude that although the present study is an in vitro
one, yet taking a clue from this, further in vivo studies
are recommended, the results of which must be carefully
analyzed before their extrapolation to clinical conditions
with caution.

The results of our study showed that combination of calcium

hydroxide and saline provides the minimum inhibition
zones for all strains of microorganisms.
In this study, the inhibitory effect of calcium hydroxide
with glycerine was found to be inferior to that of calcium
hydroxide with camphorated paramonochlorophenol
combination, as also reported by Gomes et al.[7] They
concluded that antibacterial effect of calcium hydroxide
in combination with glycerine was lesser than that of
calcium hydroxide in combination with camphorated
paramonochlorophenol. This may be due to the release
of camphorated paramonochlorophenol from the paste
that increases the calcium hydroxide diffusibility. [8]
Furthermore, when calcium hydroxide is mixed with
camphorated paramonochlorophenol, it yields calcium
paramonochlorophenolate (PMC), which is a weak salt
that progressively releases paramonochlorophenol and
hydroxyl ions to the surrounding medium, which are both
bactericidal. The present experiment showed that the
combination of both (calcium hydroxide + camphorated
paramonochlorophenol) was found to be more lethal for bacteria.
Combination of calcium hydroxide with Rexidine-M
(combination of metronidazole and chlorhexidine) showed
inhibition of bacterial zone in all microbial strains at different
time intervals, but minimal range of inhibition in comparison
to calcium hydroxide + camphorated paramonochlorphenol
and calcium hydroxide + glycerine ExcelaR.

CONCLUSIONS
The following conclusions can be drawn from the study

Indian Journal of Dental Research, 22(1), 2011

ACKNOWLEDGMENTS
The author would like to thank Dr. Pandey, Dr. Loomba, Dr. Amita
Jain and Dr. Vandana Tiwari for all their assistance in thsis endeavor.
The author also would like to acknowledge the help of all her Post
graduate colleagues, and teaching staffs and non-teaching staffs of
Department of Pediatric Dentistry.

REFERENCES
1.

2.

3.
4.
5.

6.

7.
8.

Hermann BW. Calcium hydroxyl als mittel zum behandein und fullen
von wurzelkanalen. Dissertation, Germany: University of Wurzberg,
1920.
Georgopoulou M, Kontakiotis E, Nakau M. In vitro evaluation of
effectiveness of Calcium hydroxide and paramono-chlorophenol
on anaerobic bacteria from the root canal. Endod Dent Traumatol
1993;9:249-53.
Siqueira JF Jr, de Uzeda M. Influence of different vehicles on the
antibacterial effects of calcium hydroxide. J Endod 1998;24:663-5.
Castagnola L, Orlay HG. Treatment of gangrene of the pulp. Br Dent
J 1952;93:93-102.
Tchauo WS, Turing BF, Minan GE, Coll JA. In vitro inhibition of bacteria
from root canals of primary teeth by various dental materials. Pediatr
Dent 1995;17:351-5.
Pabla T, Gulati MS, Mohan U. Evaluation of antimicrobial efficiency
of various root canal filling materials for primary teeth. J Indian Soc
Pedod Prev Dent 1997;15:134 40.
Gomes BP, Ferraz CC, Rosalin PL. Susceptibility of selected microorganisms to intracanal medicaments. Int Endo J 2000;33:729.
Holland R, Souza V, Nery MJ, Filho JA. A histological study of the effect
of Ca(OH)2 in the treatment of Pulpless teeth of dogs. Int Endo J Br
Soc 1979;12:15-23.

How to cite this article: Gangwar A. Antimicrobial effectiveness of different

preparations of calcium hydroxide. Indian J Dent Res 2011;22:66-70.
Source of Support: Nil, Conflict of Interest: None declared.

70

Copyright of Indian Journal of Dental Research is the property of Medknow Publications & Media Pvt. Ltd. and
its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's
express written permission. However, users may print, download, or email articles for individual use.