Journal of Indian Association of Public Health Dentistry

: 2022  |  Volume : 20  |  Issue : 2  |  Page : 200--205

Evaluation of microbial contamination of toothbrushes and their decontamination using various disinfectants: An in vitro study

Tom Joy1, Sanjay Venugopal2, Sowmya Sadanandan3, Melvin Mathew4,  
1 ECHS Polyclinic, Thrissur, Kerala, India
2 Department of Periodontics, Sri Siddhartha Dental College, Tumakuru, Karnataka, India
3 Department of Periodontics, Sri Hasanamba Dental College, Hassan, Karnataka, India
4 Department of Periodontics, Manipal University College, Malaysia

Correspondence Address:
Sowmya Sadanandan
Department of Periodontics, Sri Hasanamba Dental College, Vidyanagar, Hassan - 573 201, Karnataka


Background: Toothbrush often gets contaminated with use and also during storage. Although many toothbrush disinfection methods are mentioned in the past literature. To date there no absolute consensus on the topic. Aims: This study aimed to evaluate the microbial contamination of toothbrushes and their decontamination using various disinfectants. Subjects and Methods: An in vitro study was carried out on 80 subjects with a gingival index (Loe and Silness, 1963) score of two or three received toothbrush and paste with complete oral hygiene instructions. After 1 month, toothbrushes were collected and checked for microbial contamination by aerobic culture. Toothbrushes were divided into four groups (n = 20) and treated with one of the following agents: Group A – 0.2% chlorhexidine gluconate, Group B – Listerine, Group C – Dettol, and Group D – tap water for 1 h. After an hour, the brushes were further cultured to evaluate the efficacy of each agent. Statistical procedures were performed using Statistical Package for the Social Sciences (SPSS) 16 software. Inferential statistics were performed using Chi-square analysis. P < 0.05 was set as statistically significant. Results: All the sampled toothbrushes had significant (P < 0.001) bacterial growth after 1 month of use. Most of the brushes were contaminated with Escherichia coli (17.1%). All the tested disinfectants significantly (P < 0.001) reduced bacterial growth and Dettol showed maximum effectiveness (95.3%). Conclusions: Toothbrushes significantly get contaminated after use, which is reduced after disinfecting the brushes. Although not 100% effective, all the disinfectants significantly reduced the bacteria, and Dettol was found to be comparatively better than other agents.

How to cite this article:
Joy T, Venugopal S, Sadanandan S, Mathew M. Evaluation of microbial contamination of toothbrushes and their decontamination using various disinfectants: An in vitro study.J Indian Assoc Public Health Dent 2022;20:200-205

How to cite this URL:
Joy T, Venugopal S, Sadanandan S, Mathew M. Evaluation of microbial contamination of toothbrushes and their decontamination using various disinfectants: An in vitro study. J Indian Assoc Public Health Dent [serial online] 2022 [cited 2022 Aug 11 ];20:200-205
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Oral health is an essential part of systemic health and the overall well-being of an individual.[1] Oral microbiome consists of various bacteria, viruses, and fungi that are responsible for causing a number of oral diseases.[2] Proper oral hygiene maintenance can greatly reduce these microbes and help in achieving oral health. Toothbrush is the most widely used tool used for oral care maintenance.[3],[4] However, toothbrushes often get contaminated with use and improper storage.[5],[6] Toothbrushes are generally stored in a common container in the bathroom, which can introduce the microbes to the toothbrush. Moreover, the moist and humid conditions in a bathroom are conducive for bacterial growth and cross-contamination through aerosols from toilet flushing, contaminated fingers, and skin commensals.[7] Microbial load increases with every further use and serves as a reservoir to reintroduce the pathogenic microbes leading to repeated infections.[8],[9],[10] Contaminated toothbrushes may disseminate microorganisms, which could be detrimental to oral and systemic health.[11] Hence, toothbrush decontamination plays a vital role in preventing oral and systemic diseases.

Unfortunately, there is ignorance and lack of awareness among the general public regarding the proper care and maintenance of toothbrush. Although previously numerous methods were described for toothbrush decontamination, this topic has gotten little attention and still, there is no uniform consensus regarding the ideal and cost-effective method. Hence, in this study, we intended to assess the microbial contamination of toothbrushes after 1 month of use and test the efficacy of various common chemical agents as toothbrush disinfectants.

 Subjects and Methods

The subjects reporting to the outpatient department of periodontics, between March 2016 and April 2017, were enrolled in the study. The study protocol was approved by the institutional ethical committee (IEC-2014-2015/07) and written informed consent was obtained from the subjects.

A total of 80 participants were selected, which is obtained by the following formula:


Where Z∝ is the standard normal variate (at 5% Type I error it is 1.96). Zβ is the standard normal variate for power (at 80% power it is 0.84).

p̄ is the mean proportion of reduction is considered as 70% and d̄2 is the mean difference to be detected as 35%.


[INLINE:3] per each group

Where P is 70%, which is derived with reference from a previous study.[9]

Inclusion and exclusion criteria

Subjects between the age of 18–45 years with a gingival index score of two or three (Loe and Silness, 1963) with at least 20 natural teeth were included in the study. Subjects on antibiotics and antimicrobials, any ongoing dental treatment, smoking, systemic diseases, and subjects lacking dexterity who cannot follow brushing techniques were excluded from the study.

All the selected subjects were given a medium tufted toothbrush (Manufactured by ICPA Health Products Ltd, Gujarat, India) and toothpaste (Manufactured by Colgate Palmolive India Ltd., Himachal Pradesh, India) along with complete oral hygiene instructions. The study participants were recalled after 1 month for the collection of used toothbrushes. The toothbrushes were collected and placed in sterile boxes along with five unused new toothbrushes (negative control). It was transported immediately for microbial analysis.

Microbial analysis

The toothbrushes were divided into four equal groups depending on the disinfectant used with 20 brushes in each group (n = 20): Group A – 0.2% chlorhexidine gluconate (control group), Group B: Listerine, Group C: Dettol, and Group D: tap water, negative control: five unused new toothbrushes.

Following aseptic precautions, first, the brushes were placed in test tubes containing 5 ml of normal saline for an hour. Further samples from the test tube were cultured in Mueller–Hinton-based blood agar and MacConkey agar and incubated aerobically at 37°C for 24 h. The isolates were identified by Gram staining and standard microbiological protocol. Further, 1 ml of sample was taken from the same test tube with the help of a disposable syringe and inoculated into Robertson's cooked meat (RCM) medium followed by incubation at 37°C for 24 h. The sample from RCM was further cultured in Mitis Salivarius agar.

Each toothbrush was then disinfected by immersing in one of the four disinfectants for 1 h [Figure 1]. After 1 h, these toothbrushes were placed in 5 ml of neutralizer broth and then the samples from neutralizer broth were collected and cultured to evaluate the efficacy of the four disinfectants used in the study.{Figure 1}

All the data were analyzed using statistical package for social science (SPSS) version 23 software (SPSS 23.0, IBM, Armonk, NY, USA). Inferential statistics were performed using Chi-square analysis. For all analyses P < 0.05 was considered to be statistically significant.


The distribution of aerobic bacterial growth among the four test groups before disinfection demonstrated that toothbrushes in all four groups were contaminated after using it for a month. Of 60 tested agar plates in each group, there was statistically significant (P < 0.001) aerobic bacterial growth noted, i.e., 51 plates (85%) in Group A, 48 plates (80%) in Group B, 43 plates (71.7%) in Group C, and 60 plates (100%) in Group D [Table 1]. All the five unused brushes showed negative growth.{Table 1}

After disinfection of the toothbrushes, there was a significant reduction (P < 0.001) in bacterial growth in all the groups except Group D (Tap water). Among 60 cultured agar plates in each group, 10 plates in Group A, 15 plates in Group B, and only four plates in Group C (Dettol) had bacterial growth. Dettol showed maximum efficacy (93.3%) among the disinfectants in reducing bacterial growth [Table 2] and [Figure 2].{Table 2}{Figure 2}

The microbes recovered from the culture before and after disinfection include Escherichia coli, Klebsiella, Pseudomonas, beta-hemolytic streptococci, Streptococcus mitis, Viridans streptococci, Coagulase-negative staphylococci, Bacillus species, and micrococci. The microorganisms were present either alone or as a mixture of species. The most prevalent microorganism before disinfection presenting alone in culture was E. coli (17.1%) and after disinfection was Klebsiella species (7.1%) [Table 3] and [Table 4].{Table 3}{Table 4}


Toothbrush contamination is an inevitable outcome of use and on improper storage leading to many systemic and oral infections.[12] With the regular use, the bristles often get worn out. Hence the American Dental Association recommends replacing toothbrushes at an interval of every three to four months. But this recommendation does not clearly mention if replacement of the toothbrush could also help avoid microbial contamination.[1] Secondly, more frequent changes of the toothbrushes could pose an economic burden. Routinely household procedures such as rinsing and drying seem to be a good method, but might not be sufficient to reduce the microbes.[13] Hence, an economical and easily accessible material is necessary for toothbrush disinfection.

In the present study, we investigated the efficacy of a few of the commonly available antimicrobial agents such as 0.2% chlorhexidine gluconate, Listerine, and Dettol as a toothbrush disinfectant in comparison to plain tap water.

Chlorhexidine gluconate is a cationic bisbiguanide that is effective against an array of microorganisms. Depending on the concentration it serves as a bacteriostatic as well as a bactericidal agent. Listerine is a phenol precipitate that contains thymol, eucalyptol, menthol, methyl salicylate, benzoic acid, and boric acid. Most phenols exert a nonspecific antibacterial action which is dependent upon the ability of the drug in its nonionized form to penetrate the lipid component of the cell walls of microbes and cause structural damage.[14] The active ingredient in Dettol is chloroxylenol. The other ingredients include isopropyl alcohol, pine oil, castor oil, soap, caramel, and water. These ingredients make Dettol effective against Gram-positive and Gram-negative organisms.[15]

In the present study, we found that all the used toothbrushes were contaminated with numerous bacterial isolates, while the unused toothbrushes which served as negative controls had no bacterial growth. The contamination of the used toothbrushes by bacteria may have come from the oral cavity, storage containers, storage environments, and the water used for rinsing.

In the study, after 1 month of use of the toothbrushes were found to be contaminated with E. coli, Klebsiella, Pseudomonas, beta-hemolytic streptococci, S. mitis, V. streptococci, Coagulase-negative staphylococci, Bacillus species, and micrococci [Table 3]. This result is somewhat similar to the study done by Sogi et al.[15] where they reported the presence of microorganisms such as Staphylococcus pyogenes, Klebsiella, E. coli, Proteus species, and beta-hemolytic Streptococcus on used toothbrushes. In another study, Grewal and Kaur isolated Klebsiella, E. coli, and Streptococcus faecalis and reported 100% of the growth of microorganisms on toothbrush after 1 month that was maintained for up to 3 months.[16] Sammons et al.[17] reported the presence of Staphylococci, presumptive coliforms, and Pseudomonas from the toothbrushes they examined, while Osho et al.[18] isolated E. coli, Enterobacter, Staphylococcus aureus, Staphylococcus saprophyticus, and Pseudomonas aeruginosa from toothbrushes after use. In addition, Malmberg et al.[19] isolated Streptococci and Staphylococcus epidermidis from toothbrushes after use while toothbrushes from both healthy patients and patients with oral disease contained potentially pathogenic bacteria such as Staphylococcus species, E. coli, and Pseudomonas species.[20]

The most frequently found species of bacterial isolates in the present study were E. coli (17.1%) followed by Klebsiella (8%) and Pseudomonas (8%). E. coli are coliforms and members of the family Enterobacteriaceae. They are pathogenic to humans in significant numbers. Klebsiella causes pyogenic infections, septicemia, pneumonia, diarrhea, and urinary tract infections.[1],[21]

The presence of enteric rods on the toothbrushes was indicative of possible fecal contamination. The used toothbrushes must have been stored in unhygienic environments such as toilets and bathroom sinks. The bathroom provides a humid environment that encourages the growth of microbes. The bacteria may also have entered the toothbrushes through the rinsing water as Pseudomonas aeruginosa is ubiquitous in nature, including water.[21] Oral commensals could also have contributed for contamination of toothbrushes as enteric rods are found as a part of oral flora.[1] Toothbrushes contaminated with microorganisms which are potent pathogens such as Pseudomonas, S. aureus, and Klebsiella will definitely pose a serious threat to oral as well as general health.[12]

Disinfectants are generally regarded as chemically formulated liquids that are used to kill germs.[22],[23] Several studies have stressed the importance and effectiveness of toothbrush disinfection.[9],[24],[25]

In the present study, various disinfectants were used to assess the efficacy in reducing the contamination of toothbrushes. Disinfectants used were 0.2% chlorhexidine gluconate, Listerine, and Dettol because of their easy availability in the market, nontoxic, economical, and effective against microorganisms.

In our study, immersing toothbrushes in 0.2% chlorhexidine reduced bacterial growth up to 83.3%. In accordance with our study Saleh, reported an 87.5% reduction of microbial growth after disinfecting toothbrushes with chlorhexidine.[26] Nanjunda-Swamy et al.[27] got 100% bacterial reduction after immersing in chlorhexidine for 20 h, the results of which are in partial agreement with our study. The reason for this variation from our study could be due to the time difference in immersion and different concentrations of chlorhexidine used.

The results of another study conducted by Konidala et al.[21] showed that Hexidine and Listerine presented a 100% reduction of microorganisms, whereas Dettol showed 40% efficacy in reducing the microbial load on toothbrushes. In contrast to this report interestingly in our study Dettol showed maximum efficacy (93.3%) compared to chlorhexidine (83.3%) and Listerine (75%).

Various studies have recommended different immersion times of disinfectants to achieve 100% effectiveness.[9],[15],[16],[21],[23],[28] In the present study we found, that at an immersion time of 1 h all the tested antimicrobials were effective in decreasing microbial growth. However, surprisingly none of the agents showed 100% efficacy. This result warrants the need for further randomized studies with larger samples to find the exact immersion time and the best-suited antimicrobial for toothbrush disinfection.

The present study has certain limitations. The study period was only for 1 month and as only aerobic bacterial culture was carried out, more pathogenic organisms such as the anaerobes were not taken into consideration. In addition, the effect of frequency of brushing and storage location was not taken into account which would have influenced the findings, whereas we have included 80 participants and looked for various microbes that could cause toothbrush contamination which can aid in planning preventive methods. The present study also put forth ideas on how toothbrushes can be sterilized using simple and easily available disinfectants.


Used toothbrushes serve as reservoirs for microorganisms and may play a major role in disease transmission in humans. All the toothbrushes used in the study were found to be contaminated with different microorganisms after their use for 1 month. 0.2% chlorhexidine gluconate (83.3%), Listerine (75%), and Dettol (93.3%) were found to be effective in decontaminating the toothbrushes after its application for 1 h, whereas tap water was found to be ineffective. As a preventive measure, it is recommended for everyone in daily life to disinfect their toothbrushes between uses and store in a clean and dry place separately to optimize oral hygiene and systemic health.

Within the limitations of the present study, future randomized studies are recommended to evaluate the effect of different duration of use of the toothbrush, among different age groups, in systemically healthy and diseased to assess the further impact of toothbrush contamination. Furthermore, future studies should focus on different agents including natural and home remedial products.


We would like to acknowledge the help of Dr. Sharada, Professor, Department of Microbiology, Sri Siddhartha Medical College, Tumkur, Karnataka for aiding in microbial analysis and Mr. Jagannath, Statistician, Bangalore, Karnataka for carrying out the statistical analysis for the study.

Financial support and sponsorship


Conflicts of interest

There are no conflicts of interest.


1Karibasappa GN, Nagesh L, Sujatha BK. Assessment of microbial contamination of toothbrush head: An in vitro study. Indian J Dent Res 2011;22:2-5.
2Paster BJ, Boches SK, Galvin JL, Ericson RE, Lau CN, Levanos VA, et al. Bacterial diversity in human subgingival plaque. J Bacteriol 2001;183:3770-83.
3Löe H. Oral hygiene in the prevention of caries and periodontal disease. Int Dent J 2000;50:129-39.
4Choi YK, Choi JS, Lim KO, Park DY, Kim YH. A study on the number of brushing strokes in toothbrushing education. J Dent Hyg Sci 2018;18:105-12.
5Nelson-Filho P, Isper AR, Assed S, Faria G, Ito IY. Effect of triclosan dentifrice on toothbrush contamination. Pediatr Dent 2004;26:11-6.
6Talib RA, Alnaimi RJ, Mustafa EA. The microbial contamination of toothbrushes and their disinfection by antimicrobial solutions. Al Rafidain Dent J 2008;8:144-50.
7Taji SS, Rogers AH. ADRF Trebitsch Scholarship. The microbial contamination of toothbrushes. A pilot study. Aust Dent J 1998;43:128-30.
8Cobb CM. Toothbrushes as a cause of repeated infections of the mouth. Boston Med Surg J 1920;183:263-4.
9Bhat SS, Hegde KS, George RM. Microbial contamination of tooth brushes and their decontamination. J Indian Soc Pedod Prev Dent 2003;21:108-12.
10Bonten MJ, Hayden MK, Nathan C, van Voorhis J, Matushek M, Slaughter S, et al. Epidemiology of colonisation of patients and environment with vancomycin-resistant enterococci. Lancet 1996;348:1615-9.
11Nascimento AP, Watanabe E, Ito IY. Toothbrush contamination by Candida spp. and efficacy of mouthrinse spray for their disinfection. Mycopathologia 2010;169:133-8.
12Sato S, Ito IY, Lara EH, Panzeri H, Albuquerque Junior RF, Pedrazzi V. Bacterial survival rate on toothbrushes and their decontamination with antimicrobial solutions. J Appl Oral Sci 2004;12:99-103.
13Tomar P, Hongal S, Saxena V, Jain M, Rana K, Ganavadiya R. Evaluating sanitization of toothbrushes using ultra violet rays and 0.2% chlorhexidine solution: A comparative clinical study. J Basic Clin Pharm 2014;6:12-8.
14Reddy S. Essentials of Clinical Periodontology and Periodontics. 2nd ed. New Delhi: Jaypee Brothers Medical Publishers (P) Ltd; 2008. p. 288.
15Sogi SH, Subbareddy VV, Kiran SN. Contamination of toothbrush at different time intervals and effectiveness of various disinfecting solutions in reducing the contamination of toothbrush. J Indian Soc Pedod Prev Dent 2002;20:81-5.
16Grewal N, Swaranjit K. A study of toothbrush contamination at different time intervals and comparative effectiveness of various disinfecting solutions in reducing toothbrush contamination. J Indian Soc Pedod Prev Dent 1996;14:10-3.
17Sammons RL, Kaur D, Neal P. Bacterial survival and biofilm formation on conventional and antibacterial toothbrushes. Biofilms 2004;1:123-30.
18Osho A, Thomas BT, Akande YA, Udor RD. Toothbrushes as fomites. J Dent Oral Hyg 2013;5:92-4.
19Malmberg E, Birkhed D, Norvenius G, Norén JG, Dahlén G. Microorganisms on toothbrushes at day-care centers. Acta Odontol Scand 1994;52:93-8.
20Glass RT, Lare MM. Toothbrush contamination: A potential health risk? Quintessence Int 1986;17:39-42.
21Konidala U, Nuvvula S, Mohapatra A, Nirmala SV. Efficacy of various disinfectants on microbially contaminated toothbrushes due to brushing. Contemp Clin Dent 2011;2:302-7.
22Verran J, Leahy-Gilmartin AA. Investigations into the microbial contamination of toothbrushes. Microbios 1996;85:231-8.
23Nelson Filho P, Macari S, Faria G, Assed S, Ito IY. Microbial contamination of toothbrushes and their decontamination. Pediatr Dent 2000;22:381-4.
24Mehta A, Sequeira PS, Bhat G. Bacterial contamination and decontamination of toothbrushes after use. N Y State Dent J 2007;73:20-2.
25Ankola AV, Hebbal M, Eshwar S. How clean is the toothbrush that cleans your tooth? Int J Dent Hyg 2009;7:237-40.
26Saleh DM. Effectiveness of different cleanser solutions on the microbial contamination of toothbrushes. J Kerb Univ 2011;9:302-7.
27Nanjunda-Swamy K, Madihalli AU, Prashanth MB. Evaluation of Streptococcus mutans contamination of tooth brushes and their decontamination using various disinfectants – An in vitro study. J Adv Oral Res 2011;2:23-30.
28Ayşegül O, Elgin IE, Gulcin A, Nedim S. The efficacy of chlorhexidine spray vs. mouthwash in the microbial contamination of child toothbrushes. J Dent Child (Chic) 2007;74:177-81.