after a single use , they may become contaminated by a wide array of microorganisms present both in the oral cavity and in the external environment .
there is a need for disinfection methods , which are rapidly effective , cost - effective , nontoxic , and that can be easily implemented . with the increasing incidence of drug resistance in the prevalent pathogens and an associated risk with chemotherapeutic agents , it is essential to find an alternative to existing drugs .
the herbals , which have known pharmaceuticals properties could be the best source of these alternative drugs . in recent years
, antimicrobial properties of medicinal plants are being increasingly reported from different parts of the world .
the present investigation was designed to study the antimicrobial efficacy of 3% neem , garlic of concentration 4.15 mg / ml , and green tea of concentration 40 mg / ml on streptococcus mutans and to compare with 0.2% chlorhexidine mouthwash , which is considered as the  gold standard  for oral hygiene .
the toothbrushes were obtained from subjects aged 1821 years staying in the boys hostel of a private engineering college in chennai , tamil nadu , india .
the purpose of the study was explained to the subjects and the hostel warden and the informed consent was obtained . after obtaining ethical clearance ,
a pilot study was conducted with 30 subjects to obtain the feasibility and acceptability and for the sample size . with the help of nmaster software ( developed by the department of biostatistics , cmc vellore for sample size calculation ) keeping the power of the study at 90% and alpha error at 5%
as there were four antimicrobial solutions and distilled water being used as the control , the total number of subjects needed for the study was 75 .
the subjects aged between 18 years and 21 years , having at least three single - rooted and two multirooted functional teeth per quadrant ( third molars excluded ) and with decayed , missing , and filled teeth ( dmft ) score of <3 were included in the study .
subjects using antibiotics or antiseptic mouth washes for at least 3 months prior to the study , undergoing any dental treatment , with orthodontic or with extensive intraoral prosthesis , medically compromised ( e.g. , in diabetic patients ) and subjects using neem , garlic , green tea , chlorhexidine , and fluoride in any form as an oral hygiene aid were excluded from the study . of the total 192 subjects of the age group of 1821 years residing in the hostel , 158 subjects were eligible for the study .
the preparations of test solutions were done with the help of the guidelines given in the literatures .
the 75 subjects were divided into five groups , namely , group i , group ii , group iii , group iv , and group v. each group had 15 subjects .
all of them were instructed to brush twice daily using a horizontal scrub method for 3 min and to rinse the toothbrushes under running tap water for 20 s after brushing .
they were also instructed to keep their toothbrushes in the separately provided disposable glass containers in such a manner that the head of the brush with the bristles should face outside and be left open for drying .
the study was conducted in two stages . in the first stage , for baseline data ,
all 75 subjects were provided with a new set of precoded toothbrushes and nonfluoridated toothpastes .
these toothbrushes were aseptically introduced into test tubes containing thioglycolate medium for 4 h such that only the head of the brushes was immersed in the medium and subjected to microbial analysis .
the 75 subjects were again provided with a new set of toothbrushes and nonfluoridated toothpastes .
the toothbrushes collected from group i were aseptically introduced directly into test tubes containing 5 ml of distilled water ( control ) in which only the head of the toothbrush was immersed in the medium .
similarly , the toothbrushes collected from the subjects in group ii , group iii , group iv , and group v were immersed in the test tubes containing 3% neem , garlic of concentration 4.15 mg / ml , green tea of concentration 40 mg / ml solutions , and 0.2% chlorhexidine mouthwash .
after 12 h of immersion in the solution , the toothbrushes were subjected to microbial analysis .
the toothbrushes were introduced into test tubes containing thioglycolate medium and kept in these for 4 h. the samples were then vortexed for 15 s to dislodge the bacteria from the bristles to the media using a cyclomixer ( remi elektrotechnik limited ) .
10 l of the vortexed sample was being streaked on mitis salivarius agar and incubated in an anaerobic jar with gas pack system ( remi elektrotechnik limited ) for 48 h at 37c in an incubator .
plates were opened after 48 h and the colonies were counted using colony counter ( remi elektrotechnik limited ) and were expressed as number of cfu / ml of the media [ figure 1 ] .
picture of colony counting data were entered in microsoft excel and the analysis was performed using spss software version 16 ( ibm corporation ) .
the t - test was used for comparing the baseline and test values and analysis of variance ( anova ) was used for multiple group comparison . in all the above tests ,
the toothbrushes were introduced into test tubes containing thioglycolate medium and kept in these for 4 h. the samples were then vortexed for 15 s to dislodge the bacteria from the bristles to the media using a cyclomixer ( remi elektrotechnik limited ) .
10 l of the vortexed sample was being streaked on mitis salivarius agar and incubated in an anaerobic jar with gas pack system ( remi elektrotechnik limited ) for 48 h at 37c in an incubator .
plates were opened after 48 h and the colonies were counted using colony counter ( remi elektrotechnik limited ) and were expressed as number of cfu / ml of the media [ figure 1 ] .
data were entered in microsoft excel and the analysis was performed using spss software version 16 ( ibm corporation ) . the t - test was used for comparing the baseline and test values and analysis of variance ( anova )
all the 75 subjects were present from the beginning to the end of the study .
the colony - forming units ( cfus ) in the study ranged 08.8  10 . the mean level of contamination of toothbrush by streptococcus mutans in each group at the baseline and after decontamination with different solutions were given in table 1 .
mean cfus of s. mutans in different groups mean percentage reduction of s. mutans cfus in different groups when comparing the mean percentage reduction in streptococcus mutans , cfus between the control group and each experimental group ( ii , iii , iv , and v ) , the differences noted were all statistically very highly significant ( p < 0.001 ) . when the mean percentage reduction produced by experimental groups ii , iii , and iv were compared with experimental group v , the differences noted were not statistically significant ( p > 0.05 ) [ table 2 ] .
in the study , subjects were taken from the hostel because it is easy to supervise the method of brushing , duration of time , methodology of storing , and it was easy to collect all the toothbrushes as they all stayed under one roof .
toothbrush and toothpaste were used in the study as an oral hygiene aid , which is the most commonly used aid and is most effective , safest device to remove dental plaque .
transmission of streptococcus mutans occurs even through dentifrices . to avoid any such factors confounding the result of this study ,
for sterility control , four new toothbrushes that were freshly removed from the packets were subjected to microbial analysis .
none of the unused toothbrushes had the colonies of streptococcus mutans , suggesting that the origin of these microorganisms is from the oral cavity .
in this study , the number of streptococcus mutans ranged 5  108.8  10 cfus in the baseline .
this shows that the toothbrush that disturbs or reduces the existing oral microflora may at the same time introduce other microorganisms such as streptococcus mutans in the oral cavity .
this disturbance of the existing flora may result in the removal of competitive organisms and thus , increases the possibility for streptococcus mutans to colonize . in this study ,
three herbal disinfectant media were compared with chlorhexidine ( gold standard antimicrobial agent ) to know the efficacy in reducing contamination of the toothbrush .
thioglycolate medium , peptone water , tryptone water , nitrate broth , etc . , are some of the enriched culture media . in this study ,
thioglycolate medium was used for the growth of streptococcus mutans due to its availability and it is also a good transport medium .
however , different study intervals such as 5 h , 6 h , 24 h , and 48 h and 3 days , 5 days , 14 days , 15 days , 24 days , and 28 days , and 3 months have been followed in other studies . in this study , 14 days was selected because in a study conducted by sogi et al . , maximum microbial contamination was found in toothbrushes after 14 days and 28 days . in the study ,
the toothbrushes were kept in antimicrobial solutions for 12 h. various studies showed 5 min , 12 h , 20 h , 24 h , etc .
12 h was selected for this study by considering the fact that in real - life scenario it is advisable to brush twice daily , i.e. , once in every 12 h. hence , the toothbrushes could be dipped in the disinfectant during the interval of tooth brushing .
neem possesses various medicinal properties , and is widely available in most rural and urban areas of developing countries . in this study ,
3% neem was found to be an effective antimicrobial solution against streptococcus mutans on toothbrush bristles ( 87% reduction ) .
this could be due to the presence of polyphenolic tannins present in the extract , which could effectively bind to the surface - associated bacterial proteins , resulting in bacterial aggregation and loss of glucosyltransferase activity .
this bacterial aggregate effectively reduces the count of streptococcus mutans . a study conducted by aarati et al . proved that both aqueous and alcoholic extracts of neem have significant antibacterial activity against streptococcus mutans .
garlic showed the maximum reduction ( 96% ) in the streptococcus mutans count in the present study .
till now , not much studies have been reported as per the literature search , which have used garlic for decontaminating toothbrush .
the result of this study is in concurrence with the study conducted by amin et al .
reported that mouthwash containing garlic could be used for the prevention of dental caries . despite the antimicrobial effects of garlic extract ,
green tea polyphenols have demonstrated significant antioxidant , anticarcinogenic , anti - inflammatory , thermogenic , probiotic , and antimicrobial properties in numerous human , animal , and in vitro studies . in the present investigation ,
green tea showed 84% reduction in the streptococcus mutans count and has been proved to be effective in decontaminating toothbrushes .
this anti - streptococcus mutans property of green tea is due to the presence of polyphenolic compounds in green tea .
not many studies have been reported as per the literature search , which have used green tea for decontaminating toothbrushes .
several studies have indicated that green tea inhibits growth , acid production , metabolism , and glucosyltransferase enzyme activity of streptococcus mutans .
chlorhexidine is a cationic agent that exhibits broad spectrum antimicrobial effect and is a benchmark control in various studies . in the present investigation ,
the study conducted by suma sogi et al . found that chlorhexidine produces 88% bacterial reduction in toothbrushes after 14 days .
but the result of the present study was not in concurrence with the study reported by balappanavar et al . and padma k bhat et al . where the percentages of reduction in streptococcus mutans count were only 64% and 65% , respectively , which were less than the present study .
as per the present results , neem , garlic , and green tea have been proved to be as effective disinfectants as chlorhexidine for toothbrushes .
fani et al . showed that the inhibitory activity of garlic on streptococcus mutans was comparable with chlorhexidine .
in contrast to the present result , priya subramaniam et al . reported that aqueous extract of green tea shows more reduction of streptococcus mutans than chlorhexidine .
in the present investigation , garlic showed the maximum reduction in the streptococcus mutans count .
it produced 96% reduction followed by chlorhexidine ( 92% ) , neem ( 88% ) , and green tea ( 83% ) .
distilled water showed only a 2.2% reduction . when comparing the mean difference and also the mean percentage reduction obtained in streptococcus mutans colony count between the groups , only the comparison with distilled water showed a statistically very high difference .
but when comparing the mean difference or mean percentage reduction in cfus of streptococcus mutans in garlic , neem , and green tea with chlorhexidine , the differences were not statistically significant from each other .
so the present investigation showed that immersion of toothbrush for 12 h in 3% neem , garlic of concentration 4.15 mg / ml , and green tea of concentration 40 mg / ml were as efficacious as 0.2% chlorhexidine for disinfection .
so neem , garlic , and green tea can be used at par with chlorhexidine as a disinfectant for toothbrush .
based on the results of the study , it can be concluded that neem , garlic , and green tea are equally efficacious as chlorhexidine and these herbal products can be used as potent alternatives to chlorhexidine as disinfectants for toothbrushes .
 background : toothbrushes in regular use can become heavily contaminated with microorganisms , which can cause infection or reinfection . there is a need for toothbrush disinfection methods , which are rapidly effective , cost - effective , nontoxic , and that can be easily implemented.aim:to compare the efficacy of 3% neem , garlic of concentration 4.15 mg / ml and green tea of concentration 40 mg / ml with 0.2% chlorhexidine mouthwash as toothbrush disinfectants.materials and methods : the study was a parallel in vitro comparative experimental trial conducted among 75 randomly selected boys aged between 18 years and 21 years . the subjects were divided into five groups , namely , group i , group ii , group iii , group iv , and group v. they were provided with a new set of precoded toothbrushes and nonfluoridated tooth pastes . 
 after 14 days of tooth brushing , the toothbrushes were immersed in antimicrobial solution for 12 h [ group idistilled water ( control ) , group ii3% neem , group iiigarlic of concentration 4.15 mg / ml , group ivgreen tea of concentration 40 mg / ml , and group v0.2% chlorhexidine ] and then subjected to microbial analysis to check the presence of streptococcus mutans . 
 the t - test and analysis of variance ( anova ) were done using statistical package for the social sciences ( spss ) software version 16.results:all test solutions showed a statistically significant reduction of streptococcus mutans count ( p < 0.001 ) . there was no statistical difference between the efficacies of neem , garlic , and green tea when compared with chlorhexidine mouthwash ( p > 0.05).conclusion : neem , garlic , and green tea are equally efficacious as chlorhexidine and these herbal products can be used as potent alternatives to chlorhexidine as disinfectant for toothbrushes . 
INTRODUCTION
MATERIALS AND METHODS
Microbial analysis
Statistical analysis
RESULTS
DISCUSSION
CONCLUSION
Financial support and sponsorship
Conflicts of interest