0099-2399/98/2401-0015503.
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JOURNALOF ENDODONTICS
Copyright © 1998 by The American Association of Endodontists
In Vitro Determination of Direct Antimicrobial
Effect of Calcium Hydroxide
Carlos Estrela, DDS, PhD, Fabiana Cristina Pimenta, MSc, Izabel Yoko Ito, PhD, and
Lili Luschke Bammann, PhD
The objective of this study was to determine in
vitro the time required for calcium hydroxide in
direct contact with microorganisms to express its
antimicrobial effect. The microorganisms used
were: Micrococcus luteus (ATCC-9341), Staphylo-
coccus aureus (ATCC-6538), Fusobacterium nu-
cleatum (ATCC-25586), Pseudomonas aeruginosa
(ATCC-27853), Escherichia coli, and Streptococcus
sp. The strains were cultivated in Brain Heart Infu-
sion (BHI), with the exception of F. nucleatum (BHI-
PRAS). Pure and mixed suspensions of the micro-
organisms were prepared. Paper cones immersed
in these substances were covered with calcium
hydroxide paste, and after 0, 1, 2, 6, 12, 24, 48, and
72 h and 7 days they were transferred to an appro-
priate medium to observe the growth and multipli-
cation of the microorganisms. Incubation was con-
ducted at 37°C for 48 h, according to the
requirements of oxygen of each microorganism.
The antimicrobial effect of calcium hydroxide was
shown to occur after 12 h on M. luteus and F.
nucleatum, 24 h on Streptococcus sp, 48 h on E.
coli, and 72 h on S. aureus and P. aeruginosa.
Mixture II (M. luteus + Streptococcus sp + S. au-
reus) was sensitive to calcium hydroxide antimi-
crobial potential after 48 h, whereas mixture I (M.
luteus + E. coil + P. aeruginosa), mixture III (E.
coli + P. aeruginosa), and mixture IV (S. aureus +
P. aeruginosa) were inactivated after 72 h of expo-
sure.
Bacteria and their subproducts that stimulate organic reactions
are the main causes of pathological alterations in root canals and
the periapical area (1, 2).
Low oxygen tension, nutrient supply, and loss of natural defense
after pulp necrosis benefit microorganism interactions and can lead
to the predominance of the following species: Eubacterium, Pep-
tococcus, Peptostreptococcus, Prevotella, Porphyromonas, and
15
Printed in U.S.A.
VOL. 24, NO. 1, JANUARY1998
Fusobacterium (3-5). Facultative bacteria have been identified in
persistent endodontic infection (6-8).
After cleaning and shapping of infected root canals, a significant
reduction in the number of bacteria can be observed. Nevertheless,
an intracanal dressing is necessary to improve this process (9).
The ionic disassociation of calcium hydroxide into calcium and
hydroxyl ions and their effects on bacteria by inhibiting cytoplas-
mic membrane enzymes, and on tissue through alkaline phos-
phatase activation, confers the antimicrobial and mineralization
properties (10, 11).
Estrela et al. (12) reported that the antimicrobial mechanism of
action of calcium hydroxide is directly influenced by the liberation
of hydroxyl ions and by inactivation of enzymes of the cytoplasmic
membrane of bacteria, which chemically alters the organic com-
ponents and nutrient transport, causing toxic effects on the cells.
Several studies report the antimicrobial action of calcium hy-
droxide on different microorganisms (13-17), however, the ideal
time for complete antimicrobial effectiveness, acting in direct
contact in the root canal or in the dentinal tubules, is still unknown.
The aim of this research was the in vitro determination of the
time required for the antimicrobial effect of calcium hydroxide in
direct contact.
MATERIALS AND M E T H O D S
Six bacterial strains were used: (four standard strains) Micro-
coccus luteus (ATCC-9341), Staphylococcus aureus (ATCC-
6538), Fusobacterium nucleatum (ATCC-25586), Pseudomonas
aeruginosa (ATCC-27853) (two strains isolated and identified at
the Laboratory of Bacteriology of the Institute of Tropical Pathol-
ogy and Public Health, Federal University of Goi~is), Escherichia
coli, and Streptococcus sp.
These strains were plated in 5 ml of Brain Heart Infusion (BHI;
Difco Laboratories, Detroit, MI), with the exception of F. nuclea-
turn that was plated in BHI-PRAS broth (Anaerobe Systems) and
incubated at 37°C for 48 h.
Microorganisms were cultivated in solid medium, and suspen-
sions were prepared and adjusted by scale 1 of MacFarland (3.0 ×
108 cells/ml).
Suspensions of each microorganism to be used to contaminate
the cones were prepared. Mixtures of these suspensions (1:1) were
then prepared: mixture I--M. luteus, Streptococcus sp, S. aureus,
E. coli, and P. aeruginosa; mixture II (coccus)--M. luteus, Strep-
16 E s t r e l a e t al. Journal of EndodonUcs

TABLE 1. E v a l u a t i o n o f t h e e f f e c t o f t h e d i r e c t c o n t a c t o f c a l c i u m h y d r o x i d e on p u r e c u l t u r e s o f m i c r o o r g a n i s m s

Microorganisms 0h 1h 2h 6 h 12 h 24 h 48 h 72 h 7 days
m
M. luteus + + + + - - -
(AGPC)
S. a u r e u s + + + + + + +
(AGPC)
m m
Streptococcus sp + + + + + - -
(AGPC)
E. c o l i + + + + + + -
(AGNC)
m
P, a e r u g i n o s a + + + + + + +
(AGNC)
m
F. n u c l e a t u m + + + + - - -
(ANGNR)
+ = presence of growth; - - absence of g r o w t h ; A G P C = aerobic Gram-positive coccus; AGNC = aerobic Gram-negative coccus; AGNR - anaerobic Gram-negative rods.

TABLE 2. Evaluation o f t h e e f f e c t o f t h e d i r e c t c o n t a c t o f c a l c i u m h y d r o x i d e on m i x e d c u l t u r e s of m i c r o o r g a n i s m s

Microorganisms 0h 1h 2h 6 h 12 h 24 h 48 h 72 h 7 days
Mixture I + + + + + + + - -
Mixture II + + + + + + - - -
Mixture III + + + + + + + - -
Mixture IV + + + + + + + - -
+ = Presence of growth; = absence of g r o w t h ; mixture I = M . l u t e u s + S t r e p t o c o c c u s sp + E. c o i l + P. a e r u g i n o s a ; mixture II - M . l u t e u s + S t r e p t o c o c c u s s p + S. a u r e u s ; mixture
III - E. c o i l + P. a e r u g i n o s a ; mixture IV = S. a u r e u s + P. a e r u g i n o s a .

tococcus sp, and S. aureus; mixture III (rods)--E. coli and P. nucleatum, 24 h on Streptococcus sp, 48 h on E. coli, and 72 h on
aeruginosa; and mixture IV (coccus + rods)--S, aureus and P. S. aureus and P. aeruginosa. Mixture II (M. luteus + Streptococ-
aeruginosa. cus sp S. aureus) was sensitive to calcium hydroxide antimicrobial
One hundred and seventy-one absorbent #50 paper cones (Tan- potential after 48 h whereas mixture I (M. luteus + E. coli + P.
ari-Tanariman Industries, Ltd., Manacaru, Brazil) previously ster- aeruginosa), mixture III (E. coli + P. aeruginosa), and mixture IV
ilized were immersed in the inoculate for 3 rain. After this period, (S. aureus + P. aeruginosa) were inactivated after 72 h of expo-
the cones were placed on Petri dishes and covered with calcium sure.
hydroxide paste (Quimis, USA), using saline as vehicle, with a
viscosity of 3501 cP-0.1 rpm (Reometer Digital Brookfield, model
DV-III-LV), corresponding to the consistency of toothpaste, with DISCUSSION
a pH of 12.6 determined using a pH meter (Analion, pH Digital,
The complex intemal anatomy of root canals offers opportunity
PM 605).
and a good environment for growth, multiplication, and interaction
At intervals of 0, 1, 2, 6, 12, 24, 48, and 72 h and 7 days, two
cones were removed from contact with calcium hydroxide paste. of microorganisms in pulp infections. Difficulties in antimicrobial
One cone was immersed in 10 ml of BHI broth, and the other one control require the use of intracanal dressings that complement
was seeded to a 2% BHI agar surface with rotary movements and chemical and mechanical preparations.
Calcium hydroxide became popular due to its antimicrobial and
incubated at 37°C for 24 to 48 h.
biological properties (10-16). Safavi and Nichols (14, 15) and
The F. nucleatum cones were immersed in BHI-PRAS for 48 h
and incubated in an anaerobic environment. A total of 18 cones Barthel et al. (18) demonstrated that calcium hydroxide had the
were used for each culture, with the exception of 1:. nucleatum, for capacity to hydrolyze the lipid portion of the bacterial lipopoly-
which nine cones were used. The experiment was repeated three saccharide promoting its degradation, and also could alter the
times at different periods. biological properties of the endotoxin. Kontakiotis et al. (16)
An Olympus VMT stereomicroscope (×1, X2, Japan M. showed in vitro that calcium hydroxide, by absorbing carbon
dioxide, can help the indirect action of antimicrobials on anaerobic
421313) was used to observe colony growth.
obligatory and facultative bacteria. Sjogen et al. (13), studying the
The tubes of BHI that presented growth were selected for other
observations. Inocules (0.1 ml) were seeded on the BHI agar antimicrobial effect of calcium hydroxide on bacteriological sam-
surface and in 2.5 ml of BHI, after incubation described previously, pies from 30 root canals with pulp necrosis and apical periodon-
to confirm multiplication of the bacteria. BHI-PRAS was used for titis, showed that bacteria that had survived biomechanical prep-
F. nucleatum.
aration were eliminated in 7 days. However, the process was
ineffective at 10 rain.
Study of the action of the pH of calcium hydroxide in antimi-
RESULTS crobial control (13) allowed Estrela et al. (10) to raise the hypoth-
esis that calcium hydroxide produced reversible and irreversible
The results are shown in Tables 1 and 2. bacterial enzymatic inactivation. Inactivation can be observed in
Significant findings follow. The antimicrobial effect of calcium extreme conditions of pH over a long period of time, during which
hydroxide was shown to occur after 12 h on M. luteus and F. there is a total loss of biological activity of the cytoplasmic
Vol. 24, No. 1, January 1998
membrane. Enzymatic function reversibility occurs when the ideal
pH is achieved. Lehninger (19) reports that extreme pH causes the
uncoiling of many proteins with subsequent loss of biological
activities. For many years, the process of denaturation was thought
to be irreversible. However, if pH returns to normal, there is a
return of native structure and lost biological activity.
Data in Table 1 show that calcium hydroxide is effective against
M. luteus and F. nucleatum after only 12 h of contact. F. nucleatum
is the most prevalent species in root canal samples, and it has been
positively associated with infected root canals, as previously re-
ported by Sundqvist (4). The ineffectiveness of calcium hydroxide
on S. aureus and P. aeruginosa after 24 to 48 h can be explained,
because these microorganisms usually show resistance to chemical
agents. Significantly, the intracanal dressing tested in this study is
also active on all microorganisms after 72 h of exposure.
Calcium hydroxide also acts against pools of microorganisms,
as can be seen in Table 2. Its antimicrobial action started after 48 h
of exposure against mixture II, where usually resistant microor-
ganisms were present, and was completely effective after 72 h.
The antimicrobial effect of calcium hydroxide was shown to
occur in 7 days by Sjogren et al. (13); however, in this in vitro
study, positive effects of the antimicrobial were shown after 3 days
of direct contact. The high pH of this intracanal dressing in direct
contact with the microorganisms analyzed apparently caused in-
activation of the cytoplasmic membrane enzymes in a irreversible
way and toxic effects on the bacterial cells.
Estrela et al. (20), using a colorimetric method and universal
indicating solution, evaluated in vitro the diffusion of hydroxyl
ions of calcium hydroxide through the dentin in an inert atmo-
sphere of nitrogen. They observed small modifications of pH on
the external surface of the apical cementum, as well as in the
interior of the root canal. In the group in which the vehicles were
anesthetic solution and saline solution, the pH of the apical ce-
mentum at 30 days was - 7 to 8, remaining unchanged at 60 days.
In the group with polyethylene glycol vehicle, a pH of 7 to 8 in the
apical cementum was only attained at 45 days and remained the
same at 60 days. In the interior of the root canal, all of the calcium
hydroxide pastes maintained a pH of >12 during the 60 days of
observation.
This value of pH found on the external surface may be respon-
sible for the temporary inactivation of the bacterial enzymes,
causing the differences seen when calcium hydroxide acts in direct
contact in the root canal interior or indirectly in the dentinal tubules
and on the apical cementum surface. The speed of hydroxyl ions
diffusion, the degree of calcification, and denfinal permeability
influence the pH alterations on the apical cementum surface and in
the root canal interior, thus affecting the antimicrobial effect of
calcium hydroxide.
Dr. Estrela is chairman and professor of Endodontics, and Dr. Pimenta is
professor of Microbiology, Institute of Tropical Pathology and Public Health,
Antibacterial Effect of Calcium Hydroxide 17
Federal University of Gel&s, Goi&nia, Brazil. Dr. Ito is chairman and professor
of Microbiology, University of S~.o Paulo, Ribeir~o Preto, Brazil. Dr. Bammann
is chairman and professor of Microbiology, Federal University of Pelotas,
Pelotas, Brazil. Address requests for reprints to Dr. Carlos Estrela, Rua B-l,
Quadra 6, Lote 2, Setor Bueno, Goi&nia, GO, Brazil.
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