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ORIGINAL ARTICLE

Antimicrobial Activity of Four Commercially Available Mouthwashes against Streptococcus Mutans: An In Vitro Study
Sujal M. Parkar, Prateek Thakkar, Kavan Shah
Department of Public Health Dentistry, Ahmedabad Dental College and Hospital, Gandhinagar, Gujarat, India

ABSTRACT
Background: Antimicrobial agents have been used as a chemical plaque control agent to prevent oral disease and to improve oral health. The aim of the study was to evaluate the antimicrobial activity against Streptococcus mutans invitro of four commercially available mouthwashes. Materials and Methods: Four commercially available mouthwashes containing chlorhexidine, triclosan, tea tree leaves, and Aloe vera were selected. Two different techniques were used: (1) Agar disc diffusion method to determine the zone of inhibition in mm and (2) contact test method to determine the bacterial count of S.mutans invitro. The samples were incubated anaerobically for 24h. Comparisons among various mouthwashes were tested using analysis of variance. All analyses were done at 5% level of significance. Results: Triclosan mouthwash was most effective in terms on zone of inhibition having mean diameter of 211.41mm. Significant difference(P<0.05) was observed among various mouthwashes when the mean diameter of inhibition zone was compared. Chlorhexidine mouthwash was most effective in reduction of bacterial count from baseline to 24h. Highly significant difference(P<0.001) was observed when various mouthwashes were compared in terms of reduction in mean number of bacterial colony count at full strength of concentration(1:100). Conclusion: Chlorhexidine mouthwash showed excellent antimicrobial activity against S.mutans. Triclosan and tea tree leaves mouthwashes had intermediate level of efficacy. KEY WORDS: Antimicrobial activity, contact test, disc diffusion, mouthwashes, S.mutans, zone of inhibition

INTRODUCTION
Streptococcus mutans is considered one of the most important cariogenic species of the human oral microbial flora.[1] There are ample of evidences from both cross-sectional and longitudinal studies showing the strong association between S.mutans and dental caries.[2] All the available evidence indicates that any preventive strategy should have S.mutans as its principal target.[3] Oral microorganisms organize to form a complex dental biofilm. Dental biofilms are however not easily controlled by mechanical means and also the mechanical control of dental biofilm has a somewhat limited success in part because it is regarded as time-consuming and technically difficult by most individuals. Thus, it seems reasonable to control caries by agents that either prevent the formation of or disrupt biofilms of teeth or inhibit acid formation or stimulate base formation by dental biofilms. Caries control
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using chemical agents has been long standing interest. Mouthwashes have been particularly well-accepted by individuals due to their ease of use. Various carious prophylactic agents had been tried. Three agents have received a particular interest as caries prophylactic agents. These are chlorhexidine, which is cationic antimicrobial agent, triclosan, which is nonionic antimicrobial agent and xylitol, which is a sugar alcohol claimed to have various effects on oral microflora. Some of these substances may have undesirable side effects, such as staining of teeth and taste alteration. Phytotherapic agents with antimicrobial and anti-inflammatory properties have been investigated.[4,5] Hence, there has been increased interest in plants with antibacterial and anti-inflammatory activity. In order to test the antimicrobial activity of antiseptic products and to identify their possible spectrum of action, different invitro tests have been used. Through extensive literatures review it was concluded that there was scanty of information regarding the effect of chemical anti-microbial agents with that of natural occurring agents.
Address for Correspondence:
Dr.Sujal M. Parkar, B-25, Krishna Bunglows-I, Gandhinagar Highway, Motera, Ahmedabad - 380005, Gujarat, India. E-mail:drsujal_pcd@live.com

DOI: 10.4103/2249-9725.123971

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Thus, the purpose of this invitro study was to evaluate the antimicrobial activity of four commercial mouthwash products, containing chlorhexidine, triclosan, tea tree leaves, and aloe vera against S mutans.

10ml of 100% concentration of respective mouthwash. The range of dilution tested was 1:5-1:95, and 100% of mouthwash to be tested.

MATERIALS AND METHODS

Microorganisms
The test microorganism S.mutans(MTCC 890) was selected for the evaluation. The microorganism was subcultured on specific media, tryptic soy broth(Himedia-M011), bacitracin(Himedia-DD00) procured by HiMedia Laboratory Pvt Ltd, Bombay, India and incubated anaerobically at 37C for 24h.

Disc preparation Whatman no.1 filter paper was used to cut the 6mm diameter disc. The discs were autoclaved. The discs were impregnated from each dilution; these impregnated discs were kept over the S.mutans(MTCC 890) seeded blood agar(BA) plates from the standardized bacterial suspension of 1.0105 counts in log phase. Later the incubation of BA plates in anaerobic jar for 24h at 361C was done. The results/observations were taken in form of zone size in mm.
Contact test Dilution of active ingredient Same as disc diffusion process.

Mouthwashes used in the study

In the present study four different commercial brands of mouthwashes were purchased from the drug stores of Ahmedabad city. Out of four mouthwashes two were chemical products and the other two were herbal products. The details of various mouthwashes used are given in Table1.

Screening for antimicrobial activity

Antimicrobial effectiveness of various mouthwashes was assessed by using two techniques: (1) Disc diffusion process[6] and (2) Contact test.[7] Disc diffusion process Dilution of active ingredient Different dilutions were prepared to get the desired concentration of all four commercial preparations along with standard chlorhexidine solution. The range of dilution prepared was consisting 1:5-1:100 ratio of diluting fluid: Commercial product tested. To prepare 1:5 dilution 9.5 part distilled water and 0.5 part of mouthwash was used to start with 1:5 dilution, similar way 9 part distilled water plus 1 part mouthwash was used for 1:10 dilution, and so on dilution was carried out up to 0.5 part distilled water plus 9.5 part mouthwash and last tube was kept of
Table1: Various mouthwashes used in the study
Name of mouthwash Hexidine Colgate Plax Ingredients as listed on packages Chlorhexidine gluconate 0.2%w/v in pleasantly flavored aqueous base, propylene glycol, menthol, flavor, color Ethanol, polyvinyl methyl ether/maleic acid copolymer(0.20%), sodium lauryl sulfate, sodium methyl taurate, sodium saccharin, sorbitol, triclosan(0.03%), red CI 16035, flavoring agents, water Potassium nitrate B.P.1.25%w/v, flavored with tea tree oil 0.22%w/v, ethanol 6%w/v Aloe vera(98%), potassium metabisulfate(preservative)

Preparation of standard bacterial suspension 0.5 McFarland standard(bacterial count 1.5108) from overnight growth of S.mutans(MTCC 890) on tryptic soy broth(Himedia-M011). Further serial dilutions were prepared by taking 1ml of first tube of 0.5 McFarland equivalent tubes to get the final bacterial suspension having final concentration 1.0105. The 1ml of bacterial suspension was taken and it was added to mouthwash containing tube and the contact time was kept constant 1min for all dilutions of mouthwash under test. At this time, 1ml of contact solution was transferred to 9ml of standard neutralizer and 20min was given to transfer the 0.1ml of this suspension on tryptic soy agar(TSA) media. The incubation of all the plates was carried out in anaerobic jar for 24h at 361C. After incubation, the bacterial colonies were counted from the most suitable plates and the dilution series was assessed for congruency.

Statistical analysis
The antimicrobial activity, indicated by an inhibition zone surrounding the well containing the mouthwash was recorded if the zone of inhibition was greater than 6mm(disc diameter). The experiments were performed in duplicate and the mean values of the diameter of inhibition zones withstandard deviations were calculated. The duplicate plate counts derived from each sample were converted to colony forming units(CFU)/ml. The sample CFU scores were logarithmically(base 10) transformed in order to obtain normal distribution. One-way analysis of variance(ANOVA) was performed for determining if significant differences existed between the reductions in CFU for the different agents. For multiple comparisons Bonferroni correction was applied. Statistical Package for Social Sciences(SPSS version17, SPSS Inc., Chicago) was used for analysis. P<0.05 was taken as statistically significant.
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Emoform Extract of Aloe vera

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RESULTS
Inhibition zone
The inhibition zone of S.mutans for all four mouthwashes were observed at various concentrations starting from 1:5 to 1:100(full strength of concentration) for 24h incubation period. The Aloe vera containing mouthwash shows minimal inhibitory activity from the initial concentration of 1:5 to full strength of concentration(1:100) as compared to other three mouthwashes. The maximum inhibitory activity was noticed for mouthwash containing triclosan. There was a statistical significant result(P<0.05) when the mean diameter of inhibition zones for various mouthwashes at concentration of(1:20, 1:30, 1:40, 1:50, and 1:100) while at there was highly significant difference(P<0.001) at concentrations of(1:60-1:90) were compared[Table2]. Further the multiple comparisons for various mouthwashes at full strength of concentration(1:100) were done by using Bonferroni correction test[Table3].

chlorhexidine, triclosan, and tea tree leaves containing mouthwashes individually(P>0.05).

DISCUSSION
In the present study, S.mutans was used due to its direct correlation with the number of colonized oral sites. So decline in the number of this microorganism has been considered to be equivalent to a decrease in dental caries. To assess the antimicrobial potential of mouthwashes disc diffusion and contact test technique were used. Serial dilutions of agar were used in both techniques. The agar method is considered a standardized and reliable technique, allowing the simultaneous evaluation of many substances using a large number of bacterial strains. Furthermore, this method involves a direct contact of the tested substances with the microbial cultures, which is important for the evaluation of mouthwash solutions.[8] The short interval killing test used is a modification of standard contact tests that have been used to determine the killing times of chlorhexidine, as root canal irrigants by DArcangelo and Varvara(1998)[9] as solutions or creams for topical cleansing, such as in wound treatment or antisepsis by Goldenheim (1993)[10] or as mouthwashes by Wilson etal.(1990).[11] However, the contact time should be shorter for a mouthrinse product, since the usual contact time of these products in the oral cavity is around 1min.
Table3: Multiple comparisons for mean diameter of inhibition zones between various mouthwashes at full strength of concentration(1:100) by using Bonferronicorrection test
Mouthwashes Chlorhexidine vs triclosan Chlorhexidine vs tea tree leaves Chlorhexidine vs Aloe vera Triclosan vs tea tree leaves Triclosan vs Aloe vera Tea tree leaves vs Aloe vera
*Mean difference significant at P<0.05

Bacterial colony forming units

The inoculum size at baseline had bacterial load of 1.2105 CFU/ml. Table4 shows the mean number of CFU/ml of S.mutans after 24h of incubation at various concentrations of mouthwashes was determined and found to be statistically significant(P<0.05). No bacterial colonies were detected for chlorhexidine containing mouthwash after concentration of 1:20. While there is a gradual decrease for the colonies count as the concentrations increases for both triclosan and tea tree leaves containing mouthwashes. The mouthwash containing Aloe vera shows the least decrease in the colony count as the concentration increases. Table5 shows the result for multiple comparisons among different mouthwashes. There was a statistical significant mean difference(P<0.05) when the chlorhexidine, triclosan, and tea tree leaves containing mouthwashes were compared with aloe vera. No significant difference was observed when the comparison was made among

Mean difference 2.000 7.500* 11.000* 9.500* 13.000* 3.500

Pvalue 0.795 0.013 0.003 0.005 0.002 0.180

Table2: Inhibition zones at various concentrations of different mouthwashes(meanstandard deviation in mm)

Concentrations Chlorhexidine 1:5 1:10 1:20 1:30 1:40 1:50 1:60 1:70 1:80 1:90 1:95 1:100
n=2, *P<0.05, **P<0.001

Triclosan

Mouthwashes Tea tree leaves 60 60 71.41 80 80 9.50.71 100 100 111.41 111.41 120 11.50.71

Pvalue Aloe vera 60 60 60 60 71.41 71.41 60 60 60 6.50.71 6.50.71 80 0.48 0.013* 0.001* 0.007* 0.012* <0.001** <0.001** <0.001** <0.001** <0.001** <0.001** 0.001*

71.41 100 10.50.71 11.50.71 131.41 131.41 151.41 17.50.71 17.50.71 17.50.71 17.50.71 191.41

60 100 10.50.71 11.50.71 13.50.71 13.50.71 160 18.50.71 18.50.71 180 180 211.41

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Table4: Mean number of colonies(CFU/ml) of S. mutans at various concentrations of mouthwashes after 24 h

Concentrations Chlorhexidine 1:5 1:10 1:20 1:30 1:40 1:50 1:60 1:70 1:80 1:90 1:95 1:100 1.420.08 1.590.04 0.680.12 0 0 0 0 0 0 0 0 0 Triclosan Mouthwashes Tea tree leaves 2.470 2.470 2.420.02 2.380.04 2.270 2.240.02 2.210.04 2.240.02 2.200 2.160.04 2.070.04 1.430.06 Pvalue Aloe vera 5.040 5.000 4.990 4.990 4.990 4.490.71 4.340.70 4.380.74 4.330.69 4.370.73 4.350.73 4.290.74 <0.001** <0.001** <0.001** <0.001** <0.001** 0.001* 0.001* 0.001* 0.001* 0.006* 0.001* 0.001*

2.420.02 2.530.19 2.560.22 2.380 2.380 2.310 2.250 2.190 2.120.02 1.790.77 1.290.13 0.750.21

n=2, *P<0.05, **P<0.001. Numbers presented as log base 10 values. CFU=Colony forming units

Table5: Multiple comparisons for mean number of colonies between various mouthwashes at full strength concentration(1:100) by using Bonferroni correction test
Mouthwashes Chlorhexidine vs triclosan Chlorhexidine vs tea tree leaves Chlorhexidine vs Aloe vera Triclosan vs tea tree leaves Triclosan vs Aloe vera Tea tree leaves vs Aloe vera
*Mean difference significant at P<0.05

Mean difference 0.75 1.42 4.29* 0.68 3.54* 2.87*

Pvalue 0.74 0.12 0.002 0.92 0.005 0.010

All the three mouthwashes(chlorhexidine, triclosan, and tea tree leaves) except Aloe vera had showed excellent antimicrobial activities. The chlorhexidine mouthwash was the most effective among all others mouthwashes, as it is confirmed by the results of this study, showing zero number of bacterial colonies at the full strength of concentration(1:100). Chlorhexidine-based formulas are currently the golden standard for antimicrobial mouthwashes. Chlorhexidine gluconate is a cationic biguanide with broad-spectrum antimicrobial action. Its mechanism of action is that the cationic molecule binds to the negatively-charged cell walls of the microbes, destabilizing their osmotic balance causing concentration-dependent growth inhibition, and cell death.[18] Secondary interactions causing inhibition of proteolytic and glycosidic enzymes may also be significant.[19] Relatively recent information on the literature regarding triclosan;[20] however, has generated interest based not only on its antimicrobial and anti-inflammatory activity, but especially because of the absence of undesirable side effects. Triclosan is nonionic phenol having broad spectrum antimicrobial activity with moderate substativity. It has been formulated with a copolymer(Gantrez) to improve its substativity and 0.03% triclosan/Gantrez mouthwash achieves moderate reduction in plaque.[3] Tea tree oil is effective against a high number of gram-positive and negative bacteria as well as fungi. Its efficacy is due to its ingredients such as terpene hydrocarbons, mainly monoterpenes, sesquiterpenes, and their associated alcohols. These include a terpinen-4-ol chemotype, a terpinolene chemotype, and four 1,8-cineole chemotypes. With biological activity, the antimicrobial activity of tea tree oil is attributed mainly to terpinen-4-ol, a major component of the oil. The antimicrobial activity of tea tree oil is due to hydrocarbons which preferentially partitions the biological membranes and disrupt their vital functions.[21]
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In disc diffusion method the diameter of an inhibition halo of bacterial growth is considered to be directly proportional to the antimicrobial activity of the test solution. The diameter of the halo; however, can be influenced by the thickness and composition of the culture, by the concentration of the antimicrobial agent in the paper disc and by the degree of diffusion of the tested substances, which can be affected by the composition of the mouthwashes and dentifrices.[12] The bacteriological methodology may also differ and this might also influence the results. Most of the studies[13-15] had only assessed anaerobic bacteria; however, the addition of aerobic bacteria can provide valuable information to the results, as shown in the present study and other study conducted by Moran etal.,(1988).[16] The time of incubation also shows some variability. One study conducted by Addy and Harper(1997)[17] employed an anaerobic incubation period of 12h, while other studies[15,16] extended that period up to 72h. For aerobic incubation, 24h were used in the present study; this was same as the study conducted by Moran etal.,(1995).[14] The results of the present study shows that when the mean number of bacterial colonies at full strength of concentration(1:100) were compared, there was a significant difference observed between Aloe vera and other three mouthwashes; however, there was no significant difference among the three mouthwashes containing chlorhexidine, triclosan, and tea tree leaves.

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Little research work had been done to check the effectiveness of aloe vera as mouthwash. However, the antimicrobial effect of a dentifrice containing aloe vera has been demonstrated in an invitro study, in which this phytotherapic agent inhibited the growth of diverse oral microorganisms, such as S.mutans, S.sanguis, Actinomyces viscosus, and Candida albicans.[5] The antimicrobial effects of Aloe vera have been attributed to the plants natural anthraquinones: Aloe emodin, aloetic acid, aloin, anthracine, anthranol, barbaloin, chrysophanic acid, ethereal oil, ester of cinnamonic acid, isobarbaloin, and resistannol.[22] It is important to bear in mind that an experiment conducted invitro has limitations, as it is considered a static system compared to invivo tests, which may reflect the influence of various dynamic factors like systemic conditions, salivary flow, diet, and dental anatomy.[23] Nevertheless, it might be considered that if the antimicrobial agent does not have activity invitro it most likely will not work invivo. In this sense, assessments of antimicrobial activity conducted using monocultures invitro enable a direct contact between the bacterial colonies and the chemical substances tested for a period of 24-48h. Whereas in experiments conducted invivo there is a greater number of microbial species, including indigenous and even protective bacterial species which colonize dental biofilm, thus reducing the accuracy of the antimicrobial testing. One way of circumventing the limitations of invitro studies evaluating the antimicrobial activity of mouthwashes against microorganisms is to make reference to clinical trials invivo assessing their efficacy.[24]

4. 5.

6. 7.

8. 9.

10. 11. 12. 13. 14.

15. 16. 17.

CONCLUSION
Within the limitation of this invitro study, it can be concluded that the chlorhexidine containing mouthwash showed excellent antimicrobial activity against S.mutans. Also the present study demonstrates the intermediate level of efficacy of mouthwash containing triclosan and tea tree leaves. The Aloe vera mouthwash has least antimicrobial activity. The possible explanation may be the active product concentration and its interaction with other constituents, in addition to differences in the formulations, would be responsible for different effects.

18.

19. 20. 21. 22. 23. 24.

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Nunn JH, Steele JG, editors. Prevention of Oral Disease. 4thed. NewYork: Oxford University Press; 2003. p.61-76. LeeSS, ZhangW, LiY. The antimicrobial potential of 14 natural herbal dentifrices: Results of an invitro diffusion method study. J Am Dent Assoc 2004;135:1133-41. SalgadoAD, MaiaJL, PereiraSL, LemosTL, MotaOM. Antiplaque and antigingivitis effects of a gel containing Punica Granatum Linn extract: Adoubleblind clinical study in humans. JAppl Oral Sci 2006; 14:162-6. LeysterCW. An investigation of the levels of antimicrobial efficacy in commercial dentifrices on Streptococcus mutans and Lactobacillus. Saint Martins Univ J 2006;1:15566. HerreraD, RoldanS, SatacruzI, SantosS, MasdevallM, SanzM. Differences in antimicrobial activity of four commercial 0.12% chlorehexidine mouth rinse formulation: In vitro contact test and salivary bacterial counts study. JClin Periodontol 2003;30:30714. WashingtonJA, WoodsGL. Antibacterial susceptibility tests: Dilution and disk diffusion methods. Manual of Clinical Microbiology. 6thed. St. Louis: Mosby; 1995. p.45-50. DArcangelo C, Varvara G. A comparative invitro study of the bactericidal efficacy of sodium hypochlorite and chlorhexidine gluconate plus cetrimide on root canal anaerobic bacterial flora. Minerva Stomatol 1998;47:381-6. GoldenheimPD. In vitro efficacy of povidone-iodine solution and cream against methicillin-resistant Staphylococcusaureus. Postgrad Med J 1993; 69:S62-5. Wilson M, Bansal G, Stanley A, Newman HN. Susceptibility of oral bacteria to phenoxyethanol and phenoxyethanol/chlorhexidine combinations. JPeriodontol 1990;61:53641. Singh SM, Rustogi KN, Volpe AR, Petrone DM, Robinson RS. Effect of a mouthrinse containing triclosan and a copolymer on plaqueformation in a normal oral hygiene regimen. Am J Dent 1990;3:S63-5. Addy M, Jenkins S, Newcombe R. The effect of some chlorhexidine containing mouthrinses on salivary bacterial counts. J Clin Periodontol 1991;18:90-3. MoranJ, AddyM, WadeW, MilsonS, McAndrewR, NewcombeRG. The effect of oxidising mouthrinses compared with chlorhexidine on salivary bacterial counts and plaque regrowth. J Clin Periodontol 1995;22:750-5. ElworthyA, GreenmanJ, DohertyFM, NewcombeRG, AddyM. The substantivity of a number of oral hygiene products determined by the duration of effects on salivary bacteria. JPeriodontol 1996;67:5726. MoranJ, AddyM, NewcombeR. The antibacterial effect of toothpastes on the salivary flora. JClin Periodontol 1988;15:1939. AddyM, HarperP. The role of antiseptic in secondary prevention. In: LangNP, KarringT, LindheJ, editors. Proceedings of the 2ndEuropean Workshop on Periodontology. Chemicals in Periodontics. Berlin: Quintessence Books; 1997. p.152-73. Hugo WB, Longworth AR. The effect of chlorhexidine on the electrophoretic mobility, cytoplasmic constituents, dehydrogenase activity and cell walls of Escherichia coli and Staphylococcus aureus. JPharm Pharmacol 1966;18:569-78. HastingsDC. Non-antibiotic plaque chemotherapy. In: NewmanHN, Wilson, editors. Dental plaque revisited: Oral biofilms in health and disease. Cardiff, UnitedKingdom: Bioline Press; 2000. p.523-48. Moran J, Addy M, Newcombe RG, Marlow I. A study to assess the plaque inhibitory activity of a new triclosan mouth rinse formulation. J Clin Periodontol 2000;27:806-9. CarsonCF, HammerKA, RileyTV. Melaleuca alternifolia(Tea Tree) Oil: Areview of anti microbial and other medicinal properties. Clin Microbiol Rev 2006;19:50-62. WynnRL. Aloe vera gel: Update for dentistry. Gen Dent 2005;53:69. CastroSL, MardeganMA, BandeiraMF, PizzolittoAC, Fontana UF in vitro evaluation of the oral microorganisms sensitivity to antiseptics. Braz J Endo/Perio 2000; 1:6571. Pires JR, Rossa Junior C, Pizzolitto AC. In vitro antimicrobial efficiency of a mouthwash containing triclosan/gantrez and sodium bicarbonate. Braz Oral Res 2007;21:342-7.

How to cite this article: Parkar SM, Thakkar P, Shah K. Antimicrobial activity of four commercially available mouthwashes against streptococcus Mutans: An in vitro study. Univ Res J Dent 2013;3:108-12. Source of Support: Nil, Conflict of Interest: None declared

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