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Year : 2010  |  Volume : 21  |  Issue : 4  |  Page : 512-514
An in vitro study of antibacterial effect of calcium hydroxide and chlorhexidine on Enterococcus faecalis

1 Department of Conservative Dentistry and Endodontics, Dr. H S Judge Institute of Dental Sciences, Chandigarh, India
2 Department of Conservative Dentistry and Endodontics, DAV (C) Dental College, Yamuna Nagar, Haryana, India

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Date of Submission03-Jul-2009
Date of Decision11-Nov-2009
Date of Acceptance16-Jun-2010
Date of Web Publication24-Dec-2010


Aim: To evaluate and compare the antibacterial effects of chlorhexidine and calcium hydroxide on Enterococcus faecalis.
Materials and Methods: Root canal treatment involves a number of steps. In spite of all the steps done thoroughly, root canal treatment might fail due to the remnant microbes. Of all such bacteria, E. faecalis is found in failed root canals. The study tests the antibacterial activity of various intracanal medicaments. Agar diffusion test was used to evaluate the antibacterial effects of the following antibacterial agents: i. hexidine:0.2% chlorhexidine gluconate; ii. periogard:0.12% chlorhexidine gluconate; iii. calcium hydroxide powder plus sterile water; iv. metapaste plus sterile water; v. calcium hydroxide plus hexidine; vi. calcium hydroxide plus periogard; vii. metapaste plus hexidine; viii. metapaste plus periogard. The size of zones of inhibition was measured.
Results: The average size of zones of inhibition after 72 hours were hexidine: 5 mm; periogard: 4.25 mm; calcium hydroxide plus sterile water: 0.5 mm; metapaste plus sterile water: 0.5 mm; calcium hydroxide plus hexidine: 4.7 mm; calcium hydroxide plus periogard: 4 mm; metapaste plus hexidine: 4.65 mm; metapaste plus periogard: 4 mm. Results were subjected to statistical analysis using one way analysis of variance and Tukey tests.
Conclusion: Chlorhexidine and its preparations are more potent antibacterial agents againstE. faecalis in comparison to calcium hydroxide.

Keywords: Agar diffusion test, chlorhexidine, metapaste, periogard, trypticase soy agar

How to cite this article:
Jhamb S, Nikhil V, Singh V. An in vitro study of antibacterial effect of calcium hydroxide and chlorhexidine on Enterococcus faecalis. Indian J Dent Res 2010;21:512-4

How to cite this URL:
Jhamb S, Nikhil V, Singh V. An in vitro study of antibacterial effect of calcium hydroxide and chlorhexidine on Enterococcus faecalis. Indian J Dent Res [serial online] 2010 [cited 2019 Aug 20];21:512-4. Available from:
After endodontic therapy, root canal failure from persistent infection is a possibility. Of all the bacteria isolated from the root canals at the time of obturation,  Enterococcus faecalis Scientific Name Search e most commonly isolated bacteria. E. faecalis is found both in primary and retreatment groups and it appears as a monoinfection. [1],[2] One of the methods of reducing the risk of failure from infection is the chemical disinfection of the root canal. This is done using antimicrobial agents such as calcium hydroxide, chlorhexidine, etc. Calcium hydroxide is used due to its various biological properties like antimicrobial action, tissue dissolution, inhibition of root resorption and induction of hard tissue formation. Chlorhexidine is also recognized as a promising root canal disinfectant due to its microbial substantivity. [3]

   Materials and Methods Top

For the in vitro study, a single standard strain (not isolated from the patient) of E. faecalis was selected. E. faecalis is a non-motile, gram positive and spherical bacterium. It can be observed singly, in pairs or in short chains. This bacterium is facultatively anaerobic, ferments glucose without gas production and does not produce a catalase reaction with hydrogen peroxide. It can produce a pseudocatalase reaction if grown on blood agar. The reaction is usually weak. E. faecalis displays gamma hemolysis. It produces a reduction of litmus milk, but does not liquefy gelatin. These bacteria are susceptible to Vancomycin, and against these bacteria, Gentamicin, Streptomycin and cell-wall active agents have synergistic kill activity. The strain of bacteria was inoculated in trypticase soy broth for 12 hours at 36.5°C. Trypticase soy broth turned into a turbid suspension that indicated active growth of bacterium. Before doing the culture test, a microscopic examination of the bacteria was done to confirm the presence of a single strain. A slide was made and gram stained. The procedure involved applying a primary stain Crystal Violet to a heat-fixed smear of bacterial culture. This was followed by the addition of a trapping agent Gram's iodine and then rapid decolorization with alcohol and counterstaining with basic fuschin. The slide was examined under oil immersion microscopic lens at a power of 100×. Enterococci were confirmed by their purple color with rounded appearance in either chains or in bunch. Trypticase soy agar plates were swabbed with E. faecalis. Sterile filter paper points were saturated with test agents and placed on these plates. The test plates were incubated for 72 hours. The test agents were placed into following experimental groups:

  • Hexidine: 0.2% chlorhexidine gluconate
  • Periogard: 0.12% chlorhexidine gluconate
  • Calcium hydroxide powder plus sterile water
  • Metapaste plus sterile water
  • Calcium hydroxide plus hexidine
  • Calcium hydroxide plus periogard
  • Metapaste plus hexidine
  • Metapaste plus periogard

All the combinations were mixed in 1:1 ratio. In all these cases, Ampicillin served as a positive control. Trypticase soy agar plates were then incubated for 72 hours at 36.5°C. The entire experiment was done in duplicate for all the above medicaments used. The entire procedure was performed in extremely sterilized conditions under UV light. The zones of inhibition (clear, circular halo without bacteria surrounding the sample of material) were measured from the periphery of disk after 24 and 72 hours. Zone diameters were measured to the nearest whole millimeter by holding a ruler on the back of  Petri dish More Details illuminated with reflected light. This was done in accordance with the criteria laid down by National Committee for Clinical Laboratory Standards.

   Results Top

There was no change in the size of inhibition zones after 24 hours. The average sizes of zones of inhibition after 72 hours were: hexidine: 5 mm; periogard: 4.25 mm; calcium hydroxide plus sterile water: 0.5 mm; metapaste plus sterile water: 0.5 mm; calcium hydroxide plus hexidine: 4.7 mm; calcium hydroxide plus periogard: 4 mm; metapaste plus hexidine: 4.65 mm; metapaste plus periogard: 4 mm [Table 1]. The greater the size of inhibition zones, the greater is the antibacterial potency of the medicament. The mean and standard deviation was calculated for complete statistical analysis [Table 2] .
Table 1: Size of zones of inhibition (mm)

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Table 2: Mean and standard deviation

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   Discussion Top

E. faecalis was chosen as a test organism since it is associated with failure of root canal therapy. [4] It is shown to infect tubules rapidly and persist within the tubules for at least 10 days without nutrient supply. [5],[6],[7] A 2% and 0.12% of chlorhexidine was used in this study because chlorhexidine has been demonstrated to be antimicrobial when used as an irrigant or an intracanal medicament. [8],[9],[10],[11],[12],[13],[14] Agar diffusion test is the most commonly used test to access the antimicrobial activity of dental materials. [12],[13],[15] The size of zones of inhibition depend on the toxicity of the materials tested and the diffusibility of different components of the material through agar.

In this study, hexidine, i.e. 0.2% chlorhexidine, had the highest antimicrobial activity. Also, 0.12% chlorhexidine, i.e. Periogard, demonstrated lesser antibacterial activity. This could be due to the fact that it is less concentrated in comparison to 0.2% hexidine. Chlorhexidine had a better antibacterial activity in comparison to calcium hydroxide, but loses its property, when used for a long time. [16]

Chlorhexidine in combination with calcium hydroxide preparations (calcium hydroxide and metapaste) showed antibacterial properties similar to that of chlorhexidine alone. This might be due to its high pH (12.8), suggesting an increase of the ionized capacity of the chlorhexidine molecule. [17]

Calcium hydroxide powder and metapaste had least antibacterial action. Calcium hydroxide did not show significant antibacterial activity. This may be due to the reason that calcium hydroxide could not diffuse through agar plates very well or could be a result of buffering agents in the culture medium. The antibacterial effect of calcium hydroxide is mainly obtained from its high alkalinity. In the presence of buffering agents, even though calcium hydroxide can diffuse through agar plates very well, the pH level may not be sufficient to give antibacterial activity.

There is no evidence that special measures should be taken to kill bacteria in the dentinal tubules. It can be argued that bacteria located within tubules after obturation become "entombed" and are of a little consequence unless they have access to nutrients. However, in cases that are resistant to therapy, bacteria and their byproducts are the likely causes for lack of healing. [18] Healing rates rose when intracanal calcium hydroxide was used. [19] The study demonstrated that chlorhexidine or chlorhexidine in combination with calcium hydroxide showed higher antibacterial efficacy in comparison to that of calcium hydroxide alone.

   Conclusions Top

  • Hexidine, i.e. 0.2% chlorhexidine gluconate, is more antibacterial than periogard, i.e. 0.12% chlorhexidine.
  • Calcium hydroxide and metapaste show similar antibacterial properties.
  • The antibacterial activity of calcium hydroxide and metapaste is less than that of chlorhexidine preparations, i.e. 0.2 and 0.12%.
  • Chlorhexidine in combination with calcium hydroxide preparations shows antibacterial activity similar to that of chlorhexidine alone.

   References Top

1.Lin Y, Mickel A, Chogle S. Effectiveness of selected materials against Enterococcus faecalis: Part 3. J Endod 2003;29565-6.  Back to cited text no. 1
2.McHugh CP, Zhang P, Michalek S, Eleazer PD. pH required to kill Enterococcus faecalis in vivo. J Endod 2004;30:218-9.  Back to cited text no. 2
3.Komorowski R, Grad H, Wu XY, Friedman S. Antimicrobial substantivity of Chlorhexidine treated bovine root dentin. J Endod 2005;26:315-7.  Back to cited text no. 3
4.Hancock HH 3rd, Sigurdsson A, Trope M, Moiseiwitsch J. Bacteria isolated after unsuccessful endodontic treatment in North America population. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2001;91:579-86.  Back to cited text no. 4
5.Bystrom A, Claesson R, Sundqvist G. The antibacterial effects of camphorated paramonochlorophenol, Camphorated Phenol and calcium hydroxide in the treatment of infected root canals. Endod Dent Traumatol 1985;1:170-5.  Back to cited text no. 5
6.Safavi KE, Spangberg LS, Langeland K. Root canal dentinal tubule disinfection. J Endod 1990;16:207-10.  Back to cited text no. 6
7.Siqueira JF Jr. Aetiology of root canal treatment failure: Why well treated teeth can fail. Int Endod J 2001;34:1-10.  Back to cited text no. 7
8.Ohara P, Torabinejad M, Kettering JD. Antibacterial effects of various endodontic irrigants on selected anaerobic bacteria. Endod Dent Traumatol 1993:9:95-100.  Back to cited text no. 8
9.Lindskog S, Pierce A, Blomlof L. Chlorhexidine as a root canal medicament for treating inflammatory lesions in the periodontal space. Endod Dent Traumatol 1998:14;186-90.  Back to cited text no. 9
10.White RR, Hays GL, Janer LR. Residual antimicrobial activity after canal irrigation with chlorhexidine. J Endod 1997;23:229-31.  Back to cited text no. 10
11.Leonardo MR, Filho MT, Silva LA, Filho PN, Bonifacio KC, Ito IY. In vivo antimicrobial activity of 2% chlorhexidine used as a root canal irrigating solution. J Endod 1999;25:167-71.  Back to cited text no. 11
12.Lenet BJ, Komorowski R, Huang J, Lawrence HP, Friedman S. Antimicrobial substantivity of bovine root dentin exposed to different chlorhexidine delivery vehicles. J Endod 2000;26:652-5.  Back to cited text no. 12
13.Orstavik D, Hapasalo M. Disinfection by endodontic irrigants and dressings of experimentally infected dentinal tubules. Endod Dent Traumatol 1990:6;142-9.  Back to cited text no. 13
14.Ayhan H, Sultan N, Cirak M, Ruhi MZ, Bodur H. Antimicrobial effects of various endodontic irrigants on selected microorganisms. Int endod J 1999;32:99-102.  Back to cited text no. 14
15.Milosevic A. Antimicrobial activity of calcium hydroxide cements. J Endod 2003;29:50-3.  Back to cited text no. 15
16.Gomes BP, Souza SF, Ferraz CC, Teixeira FB, Zaia AA, Valdrighi L, et al. Effectiveness of 2% chlorhexidine gel and calcium hydroxide against Enterococcus faecalis in bovine root dentin in vitro. Int Endod J 2003;36:267-75.  Back to cited text no. 16
17.Teixeira FB, Levin LG, Trope M. Investigation of pH at different dentinal sites after placement of calcium hydroxide by two methods. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2005;99:511-6.  Back to cited text no. 17
18.Peters LB, Weselink PR, Moorer WR. The fate and role of bacteria left in dentinal tubules. Int Endod J 1995;28:95-9.  Back to cited text no. 18
19.Trope M, Delano EO, Orstavik D. Endodontic treatment of teeth with apical periodontitis: Single vs multivisit treatment. J Endod 1999;25:345-50.  Back to cited text no. 19

Correspondence Address:
Swaty Jhamb
Department of Conservative Dentistry and Endodontics, Dr. H S Judge Institute of Dental Sciences, Chandigarh
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/0970-9290.74222

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  [Table 1], [Table 2]

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