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Antimicrobial effect of calcium hydroxide as endo intracanal 
dressing on Streptococcus viridans 

nanik Zubaidah
Department of Conservative Dentistry 
Faculty of Dentistry Airlangga University
Surabaya - Indonesia

abstract 
Calcium	hydroxide	had	been	used	as	the	intra-canal	dressing	in	endodontic	treatment	due	to	its	high	alkaline	and	antimicrobial	

capacity.	It	can	also	dissolve	the	necrotic	tissue,	prevent	dental	root	resorbtion	and	regenerate	a	new	hard	tissue.	The	aim	of	this	
study	was	to	determine	the	concentration	of	calcium	hydroxide	which	had	the	highest	antimicrobial	effect	on	Streptococcus	viridans.	
Samples	were	divided	into	5	groups;	each	group	consisted	of	8	samples	with	different	concentration	of	calcium	hydroxide.	Group	I:	
50%,	group	II:	55,	Group	III:	60%,	Group	IV:	65%,	Group	V:	70%.	The	antimicrobial	testing	was	performed	using	diffusion	method	
against	 Streptococcus	 viridans.	 The	 result	 of	 susceptibility	 test	 was	 showed	 by	 the	 inhibition	 zone	 diameter	 which	 measured	 with	
caliper	(in	millimeter).	We	analyzed	the	data	using	One-Way	ANOVA	test	with	significant	difference	0.05	and	subsequently	LSD	test.	
The	study	showed	that	calcium	hydroxide	with	concentration	60%	has	the	highest	antimicrobial	effect.

Key words:	calcium	hydroxide,	Streptococcus	viridans,	antimicrobial	effect	

Correspondence: Nanik Zubaidah, c/o: Departemen Konservasi Gigi, Fakultas Kedokteran Gigi Universitas Airlangga. Jln. Mayjend. 
Prof. Dr. Moestopo no. 47 Surabaya 60132, Indonesia.

introduction 

Since calcium hydroxide applied in dentistry, it has 
been used for many purposes such as cavity liner; direct 
and indirect pulp capping; apexification; root resorption 
prevention; iatrogenic perforation; improvement in cavity 
base and root canal wall; treatment of horizontal and vertical 
root fracture; main substance of root canal paste (sealer) 
and root canal (filler); and irrigation material.1–3 

Along with science development and modern 
technology, calcium hydroxide as intracanal dressing has 
been introduced in modern endodontic treatment to reach 
sterile root canal. It is due to the capability of calcium 
hydroxide to solve necrotic tissue; stimulate odontoblast 
activity and capable penetrate into accessories root canal 
and dentinal tubule so it would lower permeability of 
dentinal surface; capable to neutralize acid condition 
produced by osteoclast activity therefore it could prevent 
eliminated tissue from further damage; due to presence of 
calcium ion it could promote excessive exudates to dry and 
make permeability of capillary blood vessel decrease; and 
finally calcium hydroxide has very effective antimicrobial 
effect in endodontic treatment.1,4 

Grossman et	al.,5 suggested that a medicine which is 
used as intracanal dressing must fulfill some requirements 
such as: germicide and fungicide; non irritated substance 
on periapical tissue; stable in solution; long antimicrobial 
effect; presence of blood, serum and derivate of tissue 
protein do not decrease the effect; low surface tension; 
does not change tooth color.

Tronstad et	 al.,6 suggested that placing calcium 
hydroxide into root canal would increase pH, contribute 
alkalis environment in the adjacent tissue by diffusing 
hydroxyl ion through dentinal tubule. Increasing pH would 
make calcium hydroxide bactericidal and inhibit osteoclast 
activity. When calcium hydroxide is solved in sterile 
aquabidest, it would decompose in calcium ion (Ca2+) and 
hydroxyl ion (OH--). The occurrence of hydroxyl ion in 
solution would make the environment alkalis (pH = 12.5 
at 37° C) and destroy bacterial membrane. That condition 
will killed bacteria.3,7 

According to Siquira and Lopes8 the antibacterial effect 
of calcium hydroxide used as intra-canal dressing is due to its 
ability to destroyed cytoplasmic membrane cell of bacterial, 
to denaturized protein and destroyed DNA of bacteria. 
The most common bacteria found in infected root canal 
is gram positive bacteria such as: Streptococcus	viridans 
including Streptococcus	 hemolyticus,	 Lactobacillus	 and 
Staphylococcus and followed by gram negative bacteria and 
some fungi group.8,9 Grossman et	al.5 found the domination 
of Streptococcus	 α Hemolitycus	 such as	 Streptococcus	
viridans	 (63%),	 Staphylococcus	 albus (17%), Dipteroid	
bacilli	 (6.5%), Staphylococcus	 aureus,	 Bacillus	 proteus,	
Bacillus	coli. Saifudin10 found Streptococcus	α	hemolyticus 
(76.6%)	and anaerobe obligate bacteria (23.4%) infected 
root canal.

In early 1900, a theory was found on focus infection 
using intracanal dressing with strong antimicrobial effect 
for root canal treatment. It contributes sterile root canal 
and periapical tissue and prevented the possibility of 



�0 Dent. J. (Maj. Ked. Gigi), Vol. 41. No. 1 January-March 2008: 39-42

spreading dangerous bacteria into the body.6 Grossman 
et	 al.5 and Suzuki	 et	 al.11 stated that intracanal dressing 
could destroy pathogenic bacteria and kill microbial flora 
of infected root canal. 

In general the clinical use of the 50% calcium hydroxide 
(50 gram calcium hydroxide powder in 100 ml sterile 
aquabidest) is based on factory procedure.12 Hosoya et	al.2 
and Estrela13 used 44% and 38% calcium hydroxide with 
pH = 11.24 in their study to examine calcium ion (Ca2+) 
and hydroxyl ion (OH–) release in the adjacent apical of 
root canal for 3 days. Sjogren et	al.14 stated that calcium 
hydroxide paste is still effective as long as it is in root canal 
and shows antimicrobial force for seven days. Grossman  
et	al.5 reported that calcium hydroxide is strong disinfectant 
in root canal. In this study, pure calcium hydroxide was 
used not only because its cost is relatively cheap but also 
because it is availability. It is expected that pure calcium 
hydroxide would be able to be applied as intracanal dressing 
in endodontic treatment. The optimum concentration of 
calcium hydroxide as intra-canal dressing which has highest 
antimicrobial effect on Streptococcus	viridans is still need 
to be studied further.

The purpose of this study was to determine the 
concentration of calcium hydroxide which has optimal 
antimicrobial effect against Streptococcus	 viridans. The 
advantage of this study is to determine concentration 
of calcium hydroxide applied as intracanal dressing, so 
optimal endodontic treatment could also be reached. 

material and method 

The study used the post test only controlled group 
design. The study was done at Dentistry and Oral Health 
Department of Dentistry Faculty, Airlangga University and 
antimicrobial test was done in microbiology laboratory of 
Dr. Soetomo General Hospital, Surabaya. The materials 
were pure calcium hydroxide powder (M2047, Merck 
Darmstad, Germany), sterile aquabidest (Kimia Farma), 
Media Brain Heart Infusion (BHI), Media chocolate agar, 
Normal saline (NaOCl 0.85%), Streptococcus	 viridans 
(isolated from the patient).

Calcium hydroxide paste preparation 
Calcium hydroxide paste was made by mixing calcium 

hydroxide powder with sterile aquabidest with concentration 
of 50%, 55%, 60%, 65%, and 70% until paste was formed. 
The mixture was made in appendorf tube, after mixed 
using sterile spatel cement for one minute the mixture was 
homogenized with vortex for 30 seconds.13 

isolation of Streptococcus viridans 
Streptococcus	viridans was isolated from the patient’s 

maxillary anterior teeth with the diagnosis of necrotic pulpa 
and periapical lesion. The procedure was done as follows; 
the working region was isolated by rubber dam and 70% 
alcohol was applied at the surface’s tooth. Cavity entrance 
was made using sterile round bur and removed the pulp 

wall. Sterile paper point was inserted into root canal for 
one minute, and then paper point was put into Brain Heart 
Infusion (BHI) and incubated in 37° C for 24 hours. The 
bacteria culture was re-inoculated by spreading it with ose 
into blood agar media. After 37° C incubation for 24 hours, 
the bacterial growth was examined using light microscope. 
The bacteria was re-cultured using chocolate agar plate and 
incubated 37° C for 24 hours to determine coccus gram 
positive bacterial growth (chain form, α hemolytic). The 
identification was done by gram staining. 

antibacterial examination
Suspension was done by taking colonies of Streptococcus	

viridans and its culture media using ose, and then mixed 
by normal saline (NaCl 0.85%) until turbidity equal to 
standard Mc. Farland 0.5. One ml of S.	viridans suspension 
was taken and put into petridish containing Muller Hinton 
agar media and spread using sterile spreader. Wells were 
made at the surface of agar media by placing platinum 
ring with diameter of 6 mm and 9 mm height. Twenty 
five µl of Calcium Hydroxide with concentration of 50% 
(group I), 55% (group II), 60% (group III), 65% (group 
IV), 70% (group V) were put into the wells in Muller 
Hinton agar media using pippete and incubated at 37° C for 
24 hours. The inhibition zone was measured using caliper  
(0.5 accuracy; in millimeter).16 

result 

The mean and standard deviation of inhibition zone for 
50%, 55%, 60%, 65%, and 70% calcium hydroxide against 
Streptococcus	 viridans showed on table 1 and figure 1. 
The result showed that 60% of calcium hydroxide had the 
highest inhibition zone comparing to other groups.

table1.  The mean and standard deviation of inhibition 
zone calcium hydroxide in various concentration 
against Streptococcus	viridans (mm)

Concentration N X SD

Group I 50%
Group II 55%
Group III 60%
Group IV 65%
Group V 70%

8
8
8
8
8

14.6250
14.8750
15.7500
13.1250
11.350

1.1877
1.6421
1.4880
1.260
1.1877

Note: N = Number of samples, X = Mean of inhibition zone,  
SD = Standard deviation

Statistical analysis using One Direction ANOVA test 
with significance level p = 0.05 was done to determine the 
difference of inhibition zone of calcium hydroxide against 
Streptococcus	 viridans.	 The statistical result showed 
that there was a significant difference of inhibition zone 
of calcium hydroxide in various concentration against 
Streptococcus	 viridans	 (p < 0.05). Least Significant 



��Zubaidah: Antimicrobial effect of calcium hydroxide as endo intracanal dressing 

Difference (LSD) test was done to determine the difference 
of diameter of inhibition zone (table 2).

discussion 

An intra-canal dressing is necessarily given prior to 
root canal preparation in endodontic treatment to sterilize 
root canal either from anaerobe or facultative anaerobe 
bacteria. The dominant bacterial found in root canal are 
Streptococcus	 viridans which is facultative anaerobe. 
Calcium hydroxide used as an intracanal dressing in 
endodontic treatment because it has an alkalis pH (12.5) 
and high anti microbial effect.5,17,22 The antimicrobial effect 
of material or drugs can be examined by sensitivity tests 
against bacteria, such as dilution or diffusion method. 

Agar diffusion method was chosen based on 
several reasons. First, this method could be used for 
activity evaluation of antimicrobial medicine which the 
concentration had been determined. Second, the time 
needed to analyze the inhibition zone was relatively short. 
Third, the equipments were simple and easily obtained. 
Fourth, the cost was relatively inexpensive, and the most 
important was the method could determine the bactericide 
effect of medicine by observing inhibition zone occurred 
in agar media. This method is generally used to examine 
anti microbial effect of materials including material of 
endodontic treatment.17,18

Di Fiore,19 Siqueira and Uzeda,18 Gomes et	al.20 stated 
that the result of anti microbial test using agar diffusion 
method depends on several aspects: the size of material 
molecule, solubility and diffusibility of material in agar 
media, medicine sensitivity, source of bacterial (strain of 
species colony), the number of inoculated bacteria, pH of 
substrate in plate, agar viscosity, condition of agar storage, 
incubation time and metabolic activity of bacteria. The 
higher the solubility and diffusibility of material against 
the media the bigger inhibition zone will be.

Fifty percent of calcium hydroxide is the lowest 
concentration. The result of previous study showed that 
the viscosity of calcium hydroxide mixed with sterile 
aquabidest similar to paste. In this case, it is similar with 
clinical use in which the ratio between calcium hydroxide 
powder and sterile aquabidest with 50% concentration. 
Gomes et	 al.20 using 50% concentration of calcium 
hydroxide paste proved that Gram negative anaerobe 
bacteria was more sensitive to calcium hydroxide than 
Gram positive anaerobe facultative bacteria.

Sixty percent concentration of calcium hydroxide 
showed mean of inhibition zone was 15,7500 mm  
(Table 1) which the highest antimicrobial effect comparing 
to the other groups. It might due to ion hydroxyl released 
(OH–) from calcium hydroxide. Ion hydroxyl (OH–) is 
highly free radical oxidant shows strong reactivity against 
bacterial cell.21 Effect of ion hydroxyl (OH–) is very reactive 
and quickly combines itself with lipid, protein and nucleate 
acid resulting lipid peroxides. It would increase membrane 
permeability of bacterial cells, and followed by protein 
denaturation, inactivating enzyme and DNA destruction 
which kill the bacteria.1,12 In this concentration calcium 
hydroxide has not reached the saturation point so calcium 
hydroxide is still capable to diffuse into Muller Hinton agar 
culture media which has been exposed by Streptococcus	
viridans.

Table 2 showed the antimicrobial test result of calcium 
hydroxide against Streptococcus	 viridans. Calcium 
hydroxide 55%, and 60% concentration compare to group 
50%, 60% concentration compare to 55%, did not show 
significant difference. It might due to viscosity of the 
mixture and the number of released hydroxyl ion was the 
same. In 65% concentration compare to 50%, 55%, and 
60% concentration of calcium hydroxide, and also 70% 
concentration comparing to 50%, 55%, 60%, 65% calcium 

table 2.  The result of LSD test in diameter of inhibition zone of calcium hydroxide in various concentration against 
Streptococcus	viridans

Concentration Group I Group II Group III Group IV Group V

Group I
Group II
Group III
Group IV
Group V

--- 0.712
---

0.102
0.201

---

0.032*
0.013*
0.000*

---

0.000*
0.000*
0.000*
0.013*

---

*) significant difference (p < 0.05)

figure 1.  Mean of inhibition zone for 50%, 55%, 60%, 65%, 
and 70% calcium hydroxide against Streptococcus	
viridans.

14.625
14.875 15.75

13.12511.375

0

5

10

15

20

50% 55% 60% 65% 70%

Concentration Ca (OH)2



�� Dent. J. (Maj. Ked. Gigi), Vol. 41. No. 1 January-March 2008: 39-42

hydroxide showed significant difference in inhibition zone 
against Streptococcus	viridans. It might due to the viscosity 
of calcium hydroxide powder and sterile aquabidest has 
reached saturation point in 65% and 70% concentration, 
consequently, hydroxyl ion is difficult to release into agar 
media resulting the decrease of number of hydroxyl ion 
and small inhibition zone. The diffusibility and solubility 
of calcium hydroxide in agar media was very low due 
to high viscosity of material. Safavi and Nakayama7 
suggested that the effect of water insoluble solvent would 
decrease the affectivity of calcium hydroxide. It is also 
proved by Suzuki11 by mixing calcium hydroxide and pure 
glycerin or propylene glycol (including calcium hydroxide 
vehicle). Both materials are non-polar therefore it does not 
show inhibition zone of bacteria. Since the solution has 
reached saturation point, so, hydroxyl ion (OH–) can not 
diffuse into agar culture media. The number of hydroxyl 
(OH–) which has been released will be less and the anti 
microbial effect will decrease. There is other factor such 
as buffer capacity of culture media which lowering the 
pH. It makes the antimicrobial of calcium hydroxide  
reduce.23, 26 The study showed that calcium hydroxide with 
60% concentration showed the highest antimicrobial effect 
compared to calcium hydroxide with 50%, 55%, 65%, and 
70% concentration. 

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references no 10 tidak ada, no 12 dobel ??