Journal of Dental Research and Review

: 2021  |  Volume : 8  |  Issue : 4  |  Page : 261--266

Efficacy of two different mouth rinses in inhibition of the growth of streptococcus mutans on toothbrush bristles

Swarali Shah, Sanket Kunte, Chetana Jagtap, Shweta Jajoo, Alok Patel, Preetam Shah 
 Department of Pediatric and Preventive Dentistry, Bharati Vidyapeeth Dental College and Hospital, Pune, Maharashtra, India

Correspondence Address:
Swarali Shah
Department of Pediatric and Preventive Dentistry, Bharati Vidyapeeth Dental College and Hospital, Pune, Maharashtra


Background: Toothbrush serves to be an important mechanical tool in maintaining oral hygiene. In today's era of modern medicine, with a finite knowledge and awareness about toothbrush disinfection, this tool could be reason for cross-contamination. Thus, the study aimed at comparison of the efficacy of two different mouth rinses (chlorhexidine and hiora) in inhibiting the growth of Streptococcus mutans on the bristle of toothbrush used by children. Materials and Methods: A total of 52 children were elected for this study based on the inclusion and exclusion criteria and were divided into subgroups based on the time duration for after which the microbial count was assessed in both the groups. Each of the toothbrushes was placed in the phosphate-buffered saline solution, vortexed and then diluted serially. Mitis salivarius bacitracin agar plates were inoculated following which the incubation was done for 48 h. S. mutans were identified by morphology of the colonies formed, Gram staining and biochemical tests. Results: The difference between the two groups was statistically significant in both scenarios when toothbrushes were processed immediately and when processed after 24 h. Bacterial counts were the least with chlorhexidine group and in those tested after the storage period suggesting the need for drying time for toothbrush to get disinfected before the next use. Conclusion: For disinfection of the toothbrush and avoid further risk of cross contamination, use of mouth rinses and adequate drying time will serve the purpose in an economic and strategic way.

How to cite this article:
Shah S, Kunte S, Jagtap C, Jajoo S, Patel A, Shah P. Efficacy of two different mouth rinses in inhibition of the growth of streptococcus mutans on toothbrush bristles.J Dent Res Rev 2021;8:261-266

How to cite this URL:
Shah S, Kunte S, Jagtap C, Jajoo S, Patel A, Shah P. Efficacy of two different mouth rinses in inhibition of the growth of streptococcus mutans on toothbrush bristles. J Dent Res Rev [serial online] 2021 [cited 2022 May 18 ];8:261-266
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The maintenance of oral health and prevention of dental diseases begin at one simple step – tooth brushing. The mechanical action of toothbrush bristles disrupts the biofilm and removes the organisms adhering to the tooth surfaces.[1] However, it gets infected with microorganisms sucg as Streptococcus mutans right after its first use and its contamination increases with repeated use.[2] The viability on its head ranges from 24 h to 7 days.[1]

There are various sources of contamination of the oral cavity including contaminated fingers, aerosols from toilet flush, bacteria from moist environment in the bathroom, the toothbrush storage containers or when kept in close proximity to each other.[2],[3] Reuse of such toothbrushes increases the risk of re-infection eventually causing various local and systemic diseases.[4],[5] Their decontamination is, therefore, crucial.

Due to the lack of awareness in this field, proper toothbrush care is often neglected.[6] Proper tools and methods of disinfection and patient's education should be brought focused upon to avoid the potential ill-effects.[7] ADA suggests disinfection of toothbrush with antimicrobial agents, especially in patients in high risk groups or having systemic disease.[8]

During 1920s, alcohol was used for disinfection. Later in 1929, Kauffman recommended methods such as drying in sunlight, use of table salt, ultraviolet light, immersion in disinfecting solutions or use of sprays on the bristles, storing in closed container with preparation containing formaldehyde, etc.[9],[10] However, drawbacks of this solution were poor penetration, reduction of its efficacy in the presence of protein, and a nonvolatile residue formation.[11]

The use of chlorhexidine (CHX), sodium hypochlorite, vinegar, hydrogen peroxide, etc., as toothbrush disinfectants has been practiced previously. However, owing to the drawbacks accompanying various chemicals, inclination has been toward herbal medicine in recent times which provides a more holistic approach. Hiora mouth rinse (by Himalaya Herbal Healthcare) is one such herbal mouth rinse which is stated to have anti-oxidant, anti-inflammatory, and anti-microbial properties.

In accordance to available literature, the present study aimed at evaluation and comparison of the antimicrobial efficacy of chlorhexidine mouth rinse and a herbal mouth rinse for toothbrush bristle disinfection to eliminate contamination and aid its prevention.

 Materials and Methods

The sample size was selected after consultation with Statistician. Ethical clearance for the study was obtained from the Institutional Ethics Committee (Registration no. ECR/328/Inst/MH/2016). An informed consent was obtained from the patient's parent or guardian. A signed assent, wherever applicable, from the patient before the investigation began.

Patients on antibiotic medication 30 days before sample collection and those with medical or psychological conditions were excluded from the study. The samples were collected from the patients falling in the age group of 6–9 years having DMF/def scores of more than 5 and all the primary and permanent molars present. Children were asked to not brush their teeth on the day of collection of samples. A skilled operator did perform tooth brushing of all the children. A microbiologist identified S. mutans colonies by their morphology, Gram staining, and confirmatory biochemical tests.

Fifty-two samples were collected in total and divided randomly into two groups based disinfectant solution used. For each child, brushing was done using roll's technique for 1 min using fluoridated toothpaste. After brushing, each toothbrush in Group I was rinsed with 20 mL distilled water followed by immersing in 20 mL chlorhexidine mouth rinse for 1 min. Similarly, each toothbrush in Group II was rinsed with 20 mL distilled water followed by immersing in 20 mL herbal mouth rinse for 1 min.

The used toothbrushes, after the procedure were divided into two subgroups: Subgroup A, wherein each toothbrush was placed shortly (within 3 min) in 20 mL phosphate-buffered saline after which all toothbrushes were transported to the laboratory and the procedure was performed processed within one or 2 h. Subgroup B, wherein the toothbrushes were stored in an upright position, at room temperature, for a day. After 24 h, each toothbrush was placed in 20 ml of phosphate-buffered saline and transported to the laboratory for processing.

Agar preparation was done by mixing 90 g of Mitis Salivarius Dehydrated Agar in one liter of distilled water and 20% w/v sucrose. This mix was then boiled for a minute after which it was autoclaved for 15 min at 121°C. When the temperature of the mix reached 50°C–55°C, 1% of potassium tellurite and 0.2U bacitracin were added. Agar was then poured in plates, sealed and stored at temperature of 4°C for preventing contamination till use.

Each tube containing toothbrush was vortexed for 30 s to disperse the bacteria, after which they were serially diluted from 1:10 to 1:103. Using an automatic pipette, 50 μl of each of this dilution was pipetted onto each agar plate and evenly distributed using sterile spreaders. Incubation was done at 37°C under anaerobic conditions for 48 h. Post that, the isolated colonies were examined and identification was done based on their morphology and Gram staining under the light microscope. Manual counting of the colonies was done. Thioglycollate broth test showed positive results by turning from clear to turbid solution. Change in color also showed positive results in case of Mannitol and Sucrose fermentation tests.

Statistical analysis

Statistical analysis of the obtained data was performed using IBM Statistical Package for the Social Sciences (SPSS) software, version 23.0 (SPSS Inc., Chicago, IL, USA). Descriptive quantitative data were expressed in median and interquartile range. Kolmogorov–Smirnov test was used for testing the normality of the distributions of the quantitative variables. This test showed that data were not normally distributed (P > 0.05). Hence, a nonparametric test (Mann–Whitney test) was used to analyze two independent groups − efficacy of chlorhexidine and herbal mouth rinse for toothbrush disinfection.


Chlorhexidine showed higher disinfection ability than herbal mouth rinse (P < 0.05)* [Table 1]. The comparison between CFU of Groups I and II in immediately processed samples showed higher antimicrobial efficacy of Chlorhexidine than herbal mouth rinse (P < 0.05)* with mean value of Group Ia being 18.38 CFU/50 μl as opposed to 29.62 CFU/50 μl for Group IIa [Table 2]. Statistically significant difference was found in the antimicrobial efficacy of Chlorhexidine than the herbal mouth rinse (P < 0.05)* in samples processed after the storage period with mean value of Group Ib being 2.15 CFU/50 μl as opposed to 5.38 CFU/50 μl of Group IIb [Table 2].{Table 1}{Table 2}


Streptococcal, Neisserial, and Candidal are few of the variety of species that infect the oral cavity immediately after the birth. However, the main etiological agent, that is S. mutans, causes dental caries in humans infecting their oral cavity only after the teeth are erupted.[12] Transmission of the contagious infections such as dental and periodontal diseases could occur directly by means of saliva or indirectly through the sharing of fomites such as spoons, cups, or toothbrushes. This will increase the incidence of dental caries, in especially children.[13]

The advantageous role of tooth brushing as it is archived in the literature is indisputable. However, toothbrush has also been proved to be a major cause of reinfection in the oral cavity.[14] Studies reveal that even in healthy individuals the toothbrush head has microbial load of around 108 CFU.[15],[16],[17] The contamination of toothbrush begins as early as after its first use and further tends to increase with its repeated use.[18] The duration of viability of microbes on a toothbrush differs from 8 to 24 h.[19],[20] Kozai et al. reported their presence 6 h after exposure to the air, along with findings of Wetzel et al. who reported the toothbrushes were still contaminated after an 8 h drying period.[15],[21]

In the era where organ transplantation and the alteration of immune system plays a novel treatment approach, it is important to consider toothbrush as a device that is a potential source for harboring microbes. The microscopic cuts made in gingiva and tongue by sharp bristles of the device may act as portals of entry for microorganisms. Recontamination will further lead to cause secondary infections in individuals repeatedly using it.[2] Thus, it is imperative to come up with certain chemical and physical strategies to diminish the microbial burden on toothbrushes.[9],[22]

The storage conditions of the toothbrush highly influence the survival of bacteria on them. People from lower economic strata live communally, so there is a risk of cross-infection with contamination of the toothbrushes that are kept in close proximity or even with sharing. Studies have revealed that sharing of toothbrush, among sharing of other personal items such as towel, handkerchief, razors, and clothing is one of a risk factor involved in the transmission of hepatitis B.[23]

In clinical practice, there is not yet any standardized protocol observed for the handling, storage, and disinfection of toothbrushes. Few of the commonly employed practices include storing with other bathing/personal supplies, wrapped in a plastic wrapper or a paper towel, or in a brush stand.[2] Dayoub et al. observed that contamination in the toothbrushes that are kept in aerated conditions was much lesser than those stored in plastic bags.[19] This states that wet environment is likely to be an ideal factor concerning the growth of microorganisms thus, the use of a disinfectant, at a regular interval is a must.

American Dental Association recommends renewal of toothbrush every 2–3 months and even more often if any illness supervenes.[20],[24] Glass and Jensen and Denny have advised to change the brushes after every 3 days for those patients undergoing chemotherapy.[24],[25] However, an alternative and more economically acceptable solution is to rather have them decontaminated.

The efficacy of different methods for disinfection has been investigated. Various chemical containing sprays, UV light toothbrush sanitizers, microwave ovens, and dishwashers have been suggested methods for disinfection of the toothbrush.[26],[27] However, it is not always feasible to sterilize toothbrushes in between uses and thus a decontamination procedure might be acceptable.[4] Moreover, as Devine et al. quoted the decontamination method of the toothbrushes must be rapid in action, cost-effective, nontoxic, and could be easily implemented.[28]

Sato et al. observed the rinsing of toothbrushes with tap water had resulted in continued high levels of contamination and biofilm formation.[10] Caudry et al. reported a complete disinfection after keeping the toothbrushes soaked in an antiseptic mouth rinses for 20 min.[16],[29] Similarly, Nelson Filho et al. came to a conclusion that soaking the toothbrush in 0.12% chlorhexidine solution and 1% sodium hypochlorite were effective in achieving disinfection.[30]

Chlorhexidine, a broad-spectrum antimicrobial has been suggested as a decontamination agent since a while. Chlorhexidine digluconate 0.12% inhibits the biofilm formation of S. mutans on the bristles of the toothbrush[30],[31],[32] and has been the most effective against species such as S. mutans, Streptococcus pyogenes, S. aureus, and Candida albicans.[1]

In previous studies, chlorhexidine gluconate has been regarded as a potent antimicrobial agent.[21],[33],[34] Although in another study soaking in phenolic compounds (Listerine) for about 20 min was found sufficient,[16] Mehta et al. observed that an overnight immersion in chlorhexidine gluconate was highly beneficial and more effective than Listerine in reducing the microbial load.[35]

Contrarily, Ayurveda has been gaining momentum in the holistic arena. Herbal medicines have been found to treat various oral diseases. According to Dalirsani et al. and Anupama et al., herbal mouth rinses have the merits of decreased side effects and being more economical.[36],[37]

One such herbal mouth rinse in the name of Hiora has been marketed. The contents of this mouth rinse are Pilu (Salvadora persica) showing antioxidant activity, Bibhitaka (Terminalia bellirica), Nagavalli (Piper betle) showing anti-oxidant, anti-inflammatory, anti-microbial properties. Hence, mouth rinse was utilized in this study as a toothbrush disinfectant and comparison of its antibacterial efficacy against chlorhexidine was determined.[11]

The selection of age group was to ensure good cooperation from individuals and to acquire the highest levels of S. mutans attached to tooth surfaces to all or any primary and first permanent molars. In order to standardize the high levels of S. mutans, only children were included with high caries level (def and DMFT over 5). The exclusion criteria of no antibiotic treatment within the previous month to eliminate possibility of false low bacterial load.[32],[38],[39]

The individuals were informed to not brush their teeth 24 h before the collection of sample to ensure highest level of S. mutans colony counts. Tooth brushing was performed by a single operator to standardize the duration, motion and the force of brushing. Pea-sized amount of a fluoridated toothpaste (according to ADA recommendation) was used.[40],[41],[42] This is in contrast to Nelson Filho et al. and do Nascimento et al. who did not use toothpaste in their studies as they believed its disinfectant quality would affect the bacterial count.[30],[32]

A selective media for S. mutans – Mitis salivarius bacitracin agar was used. Adding Sucrose helped increase the adherence of S. mutans to the agar plate.[43] The anaerobic jar system facilitated the growth of this facultative anaerobic.[44]

The results of this study revealed that the toothbrushes in immediately processed groups loaded quite a high count of S. mutans. This count was, however, significantly decreased when the toothbrushes were kept in clean ventilated area for over 24 h. This explains the requirement of humidity for the growth of S. mutans.[45],[46]

Group I showed a statistically significant difference between bacterial counts, lowest count being in subgroup Ib. Group II also showed a statistically significant difference between the bacterial counts, with the score being the lowest in subgroup IIb.

Our finding agreed with the studies in which bacterial count was found to be decreased by the time when toothbrushes were kept in ventilated area.[45],[46] Overall, Chlorhexidine group shows lower bacterial counts than the Hiora group, the difference being statistically significant. This was in congruity with the findings by Nelson Filho et al. where the effectiveness of Chlorhexidine gluconate surpassed the effectiveness of Brushtox which was a commercially available toothbrush disinfectant.[30] do Nascimento et al. found 8 h immersion in chlorhexidine solution to be highly effective.[32] In alignment with this, the present study determined 1–2 min immersion more economical and convenient than 8 h immersion.

Thus, a simple method of immersing the toothbrush for 2 min in a disinfectant solution will curb the recontamination. This habit can be made up a part of daily routine and the protocol formulated could be advised for the general public to follow.

To our knowledge, this is the only study to compare the toothbrush disinfection ability of Chlorhexidine and Hiora mouth rinses. By comparing these, it was deduced that chlorhexidine is the toothbrush disinfectant of choice as it can achieve microbial decontamination within a short span (3 min) and is much longer lasting compared to Hiora.

This study, however, had a restraint of accurately standardizing the baseline bacterial count among all the groups due to individual variability. To curb this, artificial in vitro contamination of the toothbrushes by standardized S. mutans colonies could be performed in future, followed by the determination of the efficacy of the disinfectant solution or methods.


Modern day dentistry emphasizes that “Prevention is better than cure.” The same can be applied for prevention of various dental problems as well. Tooth brushing is a basic practice for mechanical debridement of the oral cavity. The frequent change of toothbrush, as suggested by ADA, could raise the cost of maintenance which increases financial burden on common man. Instead, decontamination of toothbrushes will prove to be more economical. Mouth rinses which are used as chemical plaque control measure could also serve as a handy economical toothbrush disinfectant. The significance of proper toothbrush care when emphasized will prove to be beneficial in improving oral health practices.

Ethical clearance

Clearance for the study was obtained from the Institutional Ethics committee.

Financial support and sponsorship


Conflicts of interest

There are no conflicts of interest.


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