Journal of Indian Association of Public Health Dentistry

: 2018  |  Volume : 16  |  Issue : 2  |  Page : 109--115

Effects of sodium fluoride solution, chlorhexidine gel and fluoride varnish on the microbiology of dental plaque – A randomised controlled trial

Sharath Reddy Pocha1, Dandi Kiran Kumar2, Muralidharan Dhanya3, Kaipa Sudhakar4,  
1 Department of Public Health Dentistry, CKS Teja Institute of Dental Sciences and Research, Tirupati, Andhra Pradesh, India
2 Department of Public Health Dentistry, PMS College of Dental Sciences, Thiruvananthapuram, Kerala, India
3 Department of Public Health Dentistry, Department of Public Health Dentistry, KMCT Dental College, Kozhikode, Kerala, India
4 Department of Public Health Dentistry, Sree Sai Dental College and Research Institute, Srikakulam, Andhra Pradesh, India

Correspondence Address:
Dr. Sharath Reddy Pocha
Department of Public Health Dentistry, CKS Teja Institute of Dental Sciences and Research, Renigunta Road, Tirupati, Andhra Pradesh


Background: Reducing microbial load in the oral cavity will provide an additional rationale for the prevention of dental diseases. Antibacterial properties of topical fluorides and chlorhexidine (CHX) are well documented with certain limitations. However, effects of topical applications of 2% sodium fl uoride solution (NaF soln), 1% CHX gel, and 2.26% NaF varnish on plaque microfl ora and following its termination are less explored. Hence, there is a need to study the comparative effects on these agents in vivo. Aim: The aim was to assess quantitative and qualitative changes in the plaque microflora following topical application of 2% NaF soln, 1% CHX gel, and 2.26% NaF varnish. Materials and Methods: This was a double-blind, randomized, parallel-group clinical trial. A total of willing sixty schoolchildren of Nellore city with apparently good health aged 9–13 years were randomly allocated to three interventional groups as follows: Group A (NaF soln, 20), Group B (CHX gel, 20), and Group C (NaF varnish, 20). NaF soln and CHX gel were applied four times on a weekly interval and NaF varnish was applied only once. Once patients enrolled to participate in the clinical trial, an identification number was sequentially assigned, allowing maintenance of a double-blind, randomized study design. Plaque samples were collected at baseline (P0) and at seven different time intervals (P1: 1st week, P2: 2nd week, P3: 3rd week, P4: 4th week, P5: 8th week, P6: 12th week, and P7: 16th week) and cultured on nutrient agar medium. The number of microbial colony-forming units (CFUs) was calculated using a digital colony counter. Statistical significance within and between groups was calculated using Chi-square test, paired t-test, ANOVA, Dunnett's multiple comparison test, and Tukey's multiple comparison test. Results: There was reduction in the CFU in all the three groups at P2, P3, and P4 when compared to P0 (P < 0.05), but following termination of application, the CFU showed subsequent increase of CFU at P5 and P6, reaching similar to baseline values at P7 (P > 0.05). Single application of NaF varnish was equally potent as four times application of NaF soln and CHX gel, but there was no statistical significance in-between groups in terms of CFU reduction (P > 0.05). P7 sample estimation showed Lactobacilli species with 100% resistance followed by other organisms. Conclusion: All the three agents used demonstrated significant reduction of microbial load in plaque but allowed recolonization following termination of intervention.

How to cite this article:
Pocha SR, Kumar DK, Dhanya M, Sudhakar K. Effects of sodium fluoride solution, chlorhexidine gel and fluoride varnish on the microbiology of dental plaque – A randomised controlled trial.J Indian Assoc Public Health Dent 2018;16:109-115

How to cite this URL:
Pocha SR, Kumar DK, Dhanya M, Sudhakar K. Effects of sodium fluoride solution, chlorhexidine gel and fluoride varnish on the microbiology of dental plaque – A randomised controlled trial. J Indian Assoc Public Health Dent [serial online] 2018 [cited 2023 Sep 26 ];16:109-115
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Full Text


Dental plaque is considered to be a primary etiologic agent for most of the common oral diseases. It is highly specific in nature; its specificity depends on the microorganisms that colonize it.[1],[2] Microbiota of the dental plaque also include bacteria which under certain environmental conditions will cause demineralization of enamel and root surface promoting dental caries, as well as destruction of periodontal tissues in humans.[2]

According to the World Oral Health Report 2003, despite great achievements in the oral health of populations globally, problems still remain in many communities around the world, particularly among underprivileged groups in developed and developing countries.[3] Restorative dentistry becomes restricted to fewer patients because of the increased time taken and of course the considerable costs involved. Preventive dentistry, thus, is the only solution to the problem by which most of the common dental diseases can be prevented. The judicious use of chemoprophylactic and chemotherapeutic agents is one of the most sought out measures used in preventive dentistry and they continue to be a cornerstone in combating oral diseases.[3]

Reducing microbial load in the oral cavity will provide an additional rationale for the prevention of dental diseases. While it is assumed that reductions in numbers of microorganisms in plaque will lead to caries reduction, it has led to the development of many antimicrobial agents to be used in altering the plaque microflora.[4]

Chlorhexidine (CHX) is one among the most tested compounds and its antiplaque properties are well known. At high concentrations, the agent is bactericidal and acts as a detergent by damaging the bacterial cell membrane.[5] The antibacterial activities against cariogenic bacteria are well known.[6] There is evidence that fluoride can interfere with enzyme activity and reduce acid production by oral bacteria, thereby inhibiting the enrichment of cariogenic species within dental plaque. Studies suggest that fluoride also has antiplaque properties, although the mechanisms are not well understood.[4],[7] Comparative analysis of these active agents upon both qualitative and quantitative changes in plaque microflora in similar clinical settings is not reported in literature. This study was thus undertaken to assess both qualitative and quantitative changes of the plaque microflora following topical application of 2% sodium fluoride (NaF soln) solution, 1% CHX gel, and single application of 2.26% NaF varnish.

 Materials and Methods

Study design and population

The present study is a randomized, parallel-design, double-blind, controlled, repeated-measures trial to assess the effects of topical application of NaF solution, CHX gel, and NaF varnish on the microbial load of dental plaque among children aged 9–13 years from a school in Nellore city of Andhra Pradesh. The study was conducted over a period of 3 months from June 2015 to September 2015. Ethical clearance for the study was obtained by the institute's ethics committee. Permission to conduct the study was obtained by the Block Education Officer and the school authorities. Informed consent was obtained from the parents of the participating schoolchildren. All willing participants were briefed about the procedure and written informed consent was obtained from their parents/guardians.

Inclusion criteria

Willing children with apparently good health and agreement to comply with the study visits were included in the study.

Exclusion criteria

Children with clinically evident carious lesion, undergoing orthodontic treatment, having intraoral prosthesis, who have undergone any preventive dental procedures about a year prior to the commencement of the study, and children with medical or pharmacological history that could compromise the conduct of the study were excluded from the study.

Sample size

Pilot study was conducted in order to estimate the required sample size and standardize the methodology in terms of sample collection, culturing, data recording, treatment application procedure, etc. The minimum estimated sample size for observed difference was found to be 17 children for each group, an additional 10% of the estimated sample size was added to compensate for sampling loss if any, and the final sample size was rounded off to 20 children for each group. The sample size was estimated based on the pilot study data using the following formula:

n = (α + β) 2/d2/SA2 + SB2

Where α= 1.96, β =0.80, by pilot study, SA2 + SB2 = 133,459,300, d = 4540.

Primary outcome

The primary outcome was to evaluate changes in bacterial load of plaque following application of topical agents and following its termination in comparison to baseline plaque colony-forming unit (CFU).

Secondary outcomes

The secondary outcomes were:

To compare the three agents in reducing the microbial load of plaque following its applicationsTo assess whether single application of NaF varnish is equally potent as 4-time application of NaF soln and CHX gelTo assess which organisms showed resistance to application of these three agents.


A total of 270 participants were assessed for eligibility and finally 60 willing children satisfying the eligibility criteria were included in the trial. A sequential identification number was provided to the included participants and were then randomly allocated into three different groups using a two-digit random number table; further, the interventions were also assigned a sequential number and were randomly allocated to the three groups of participants by the investigator. At baseline, basic demographic characteristics of the children were recorded and the plaque samples were collected. All materials were provided in their original containers, which were overwrapped and provided a unique code that remained unidentified until the conclusion of the study. The study investigator conducted participant allocation to treatment groups and test product distribution in a separate area so that all other study personnel were unaware of the specific product assigned to each participant.

All the microbiological procedures were performed by the investigator, who was blinded with respect to culture plates of different groups throughout the entire course of the study. The identification number of the culture plates was maintained by the independent microbiologist who concealed the details till the end of the study.

The investigator underwent a calibration session. The aim of this session was to train the investigator in counting and recording the CFUs. The kappa coefficient value for intraexaminer variability was 0.89.

Plaque sample collection

Dental plaque collection was performed by means of sterile, unwaxed dental floss which was passed interproximally between either the mandibular or maxillary second premolar and the respective first molar. The floss (approximately 1' in length), carrying the plaque sample, was immediately transferred into a sterile tube containing reduced transport fluid (RTF), which was prepared as per the guidelines.[7]

Treatment application

Thorough oral prophylaxis was performed for all the participants 1 day prior to topical application. Freshly prepared 2% NaF solution, 1% CHX gel (Hexigel, ICPA, Mumbai, India) in disposable trays, and 2.26% NaF varnish (Durphat, colgate Duraphat varnish 50mg/ml,, UK) were applied according to the standard recommended procedures by the investigator.[8],[9],[10]

Microbial analysis

The dispersion and culturing process was accomplished within a few minutes so that there was a minimal delay between collection and culture. A 0.1-ml volume was aseptically drawn from each sample and transferred into one sterile test tube containing 0.9-ml saline. This procedure was repeated twice, establishing dilutions of 1:10 and 1:100. A corresponding volume of 10 μl of each dilution was plated onto nutrient agar medium. The plates were then incubated at 37°C for 48 h in jars under microaerophilic conditions. Bacterial counts were expressed as CFU/ml of plaque. Representative colonies were confirmed by morphological characteristics, Gram staining, and biochemical analysis. The same procedure of plaque sample collection, culture, and microbial analysis was repeated at intervals of 1st, 2nd, 3rd, 4th, 8th, 12th, and 16th weeks of the study as shown in [Figure 1].{Figure 1}

Statistical analysis

Statistical analysis was performed using the Statistical package for the social science (IBM SPSS Statistics 20.0, Chicago).. Descriptive statistics were computed and are expressed as means, standard deviations, and percentages. Statistical significance within and between groups was calculated using Chi-square test, paired t-test, ANOVA, Dunnett's multiple comparison test, and Tukey's multiple comparison test employing SPSS package version 14. Data are presented as median, lower-to-upper quartiles, mean values, and standard deviation (SD). Significance was set at P < 0.05.


Analysis of demographic data at baseline data revealed no significant differences in relation to age and gender distribution of the schoolchildren among the three intervention groups. Similarly, there was no statistically significant difference in the mean CFUs in plaque samples from the three intervention groups. A qualitative analysis of the baseline plaque samples performed through biochemical analysis revealed more of aerobic microorganisms in comparison to the anaerobic organisms among all the three interventional groups [Table 1]. Mean CFU of the three intervention groups at the different stages of evaluation are mentioned in [Table 2].{Table 1}{Table 2}

Statistically significant differences in colony counts among the three groups were observed during the 2nd, 3rd, and 4th weeks (P < 0.05) when compared to that of baseline. However, the 1st, 8th, 12th, and 16th week evaluation showed nonsignificance (P > 0.05), indicating that there was no significant effect during the 1st week of application and recolonization started from the 5th week onward, i.e., colonies reaching close to its baseline values at 8th week and continuing to remain the same thereafter [Table 3]. Analysis of the effects of intervention at different stages of the study revealed that, though CHX gel application yielded a count as low as 116 mean CFUs compared to 137 and 170 mean CFUs for NaF varnish and NaF solution, respectively, there was no statistically significant difference in the effect of any of the used agents on microbial population, suggesting an almost equal performance in reducing the CFUs [Table 4] and [Figure 2].{Table 3}{Table 4}{Figure 2}

In addition, qualitative analysis was performed on the 16th-week sample. It was found that Lactobacilli species showed 100% resistance to all the treatment agents in plaque. This was followed by streptococci 47% (NaF soln), 38% (CHX), 12% (NaF varnish) Staphylococci 19% (NaF soln), 41% (CHX), 29% (NaF varnish); and Micrococcus 37% (NaF soln), 10% (CHX), 16% (NaF varnish) [Table 5].{Table 5}


The use of antimicrobial agents in the treatment of dental diseases has been studied for over five decades.[11],[12] Topical application of CHX within the oral cavity in the form of gel and varnish has proved effective in Streptococcus Mutans (MS) reduction in both saliva and dental plaque.[13] Fluoride, however, has been experimentally demonstrated to selectively reduce oral bacteria in artificial plaque models,[14],[15],[16],[17] as well as to exert specific effect on the metabolism and acid tolerance of oral bacteria.[18] However, the clinical antimicrobial value of these formulations has been questioned.[19],[20]

Further, only few in vivo bacteriological studies of qualitative and quantitative changes that may occur in dental plaque following topical application of 1% CHX gel, 2% NaF soln, and NaF varnish have been reported in literature.

The present study was designed in such a way so as to consider the natural oral conditions; therefore, oral hygiene instructions were not given. Oral prophylaxis was performed to all the children prior to commencement of the intervention so as to keep the bacterial levels as uniform as possible. Hence, in the present study, it was found that there was no significant difference in the microbial load of dental plaque at baseline.

Qualitative analysis of the baseline plaque sample showed more of aerobic organisms compared with their anaerobic counterparts, possibly due to the use of aerobic technique of inoculation and could be because of including apparently healthy children with no dental caries.[21],[22]

There was a significant reduction of colony counts during application period (P < 0.05) among all the three groups. The reduction in bacterial load was observed after the 1st week of application and this difference was consistently observed till the 4th week of evaluation (P2, P3, and P4). However, after the 5th week, recolonization was observed among all the three groups. Intergroup comparison yielded no significant difference in bacterial load, indicating a similar antimicrobial action of the three agents.

Our reported findings are supported in published literature which emphatically supports that administration of CHX to the oral cavity has been shown to prevent the formation of bacterial plaque.[23],[24] This effect may be the result of a general depression of the total oral flora as suggested by a marked reduction in the number of oral bacteria [24] or CHX may exert a local antibacterial effect by reacting reversibly with the tooth surface.[24],[25]

NaF is reported to be bactericidal at high concentrations and the inhibitory activity of NaF appears to be because of the fluoride ion.[26] It was reported by Mandell (1983)[27] that bacterial susceptibility ranged from 128 to 2048 μg/ml. Commercial fluoride preparations can deliver fluoride ion concentrations to the tooth ranging from 225 to 12,300 μg/ml. These concentrations reflect bactericidal levels of NaF to many oral organisms, but differences exist between reported findings and the present study probably due to variation in exposure time which was higher in reported studies (in vitro system). If the in vivo plaque fluoride levels parallel the in vitro concentration, then the possibility of selectively controlling suspected pathogenic bacteria is suggested. Hamilton and Bowden, 1988,[28] showed that fluoride accumulates in dental plaque and inhibits the metabolism of a wide range of oral microorganisms in vitro.

Single application of fluoride varnish was found to be equally potent as NaF soln and CHX gel which were applied four times in the present study. It can be explained based on the fact that varnish releases a low level of NaF over long period.[15],[29],[30] Reports from a study showed that increased length of exposure time, as well as increased fluoride ion concentration, increases growth suppression of the organisms.[30] Dijkman et al., 1983[31] reported that for NaF varnish-treated surfaces CaF2 will be leached away after about 1 week. This may be partly a reason why varnish is successful in reducing bacterial growth. Further, this can also be explained on the basis of “Hawthorne effect” or “Participation effect” because even these participants were constantly monitored for 4 weeks for collection of sample with other groups.

It is clear from our study that any intervention that is capable of producing significant microbial load reduction generated a substantial bacterial rise once the intervention was suspended and reached almost close to its baseline values, suggesting that continuance of the intervention is the key for maintaining adequate suppression of bacteria. De Paola et al. in 1977[12] reported that regardless of the substance tested once treatment is suspended, MS overgrowth has been a common pattern in clinical trials with both adults and children. It was previously shown that a single 5-min application of 1% NaF gel produces an immediate 8-fold increase in plaque fluoride concentration that rapidly returns to prefluoride levels within 6 h.[32] In contrast, Brown et al. 1983[33] showed that consecutive daily fluoride gel use for 12 weeks increased plaque fluoride retention that required 2 weeks rather than 6 h to diminish appreciably. Loe and Rindom Schiott in 1970[24] showed that, upon discontinuation of CHX treatment, plaque formed at normal rates, suggesting that there are no appreciable effects beyond a 24-h period. Gallagher and Cutress [20] suggested that the lack of effect of 1% CHX gel on plaque bacteria may be due to changes in susceptibility of the oral flora surviving in the mouth, and that the re-emergence of MS is most probably due to re-growth of bacteria which have not been eradicated, allowing subsequent recolonization.[32] Continuity of the intervention would therefore be a determining factor in the final outcome, and the lack of it in the present short clinical trial may be another explanation for the microbial load increase observed with the use of these agents.

Biochemical conformation of colonies showing resistance to CHX gel, NaF soln, and NaF varnish in the present study was identified as Lactobacilli and Streptococci, but the former being 100%. This hypothesis is supported by the fact that some oral streptococci strains show variable susceptibility to CHX and NaF.[26],[34],[35],[36] Oral Lactobacilli levels were not reduced by NaF and CHX probably because these organisms were hidden in habitats not affected by these products as observed among irradiated patients.[37] The final week sample also showed colonies of Streptococcus sanguis and Micrococcus conforming recolonization.


The possible role of “Hawthorne effect” as the children were constantly monitored could have resulted in better oral care and reduced microbial loadMicrobial analysis of plaque was restricted to supragingival plaque only and the possible role of subgingival plaque microorganisms was not assessed. Our short clinical trial lacked treatment continuity which would be a determining factor in the final outcome.


The three agents used demonstrated significant reduction of microbial load in plaque but allowed re colonization following termination of intervention, i.e., from 5th week onward. The colony counts reached close to their baseline values during the 8th week of evaluation. There was no significant difference among the three groups as an antimicrobial agent though single application of varnish proved equally potent compared to four applications of 2% NaF soln and 1% CHX gel. Lactobacilli (100%) and some Streptococcus strains showed resistance to 2% NaF soln, NaF varnish, and 1% CHX gel.

NaF soln, CHX gel, and NaF varnish though highly effective in plaque microflora reduction require repeated applications to maintain long-term suppression. Resistance to these agents on long-term application needs to be studied further.


The authors are grateful to Dr. Nusrath Fareed and Dr. Shanthi Margabandhu for contribution toward the conception to the study and drafting the paper.

Financial support and sponsorship


Conflicts of interest

There are no conflicts of interest.


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