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67
Can J Dent Hyg 2017;51(2): 67-72
Bacterial contamination in bristles of used charcoal toothbrushes
Comparison of bacterial
contamination in bristles of
charcoal toothbrushes versus
non-charcoal toothbrushes
Janice Lee*, BDS; Keethadevi Palaniappan*, BDS; Tang Tee Hwai*, BDS; Cheah Wen
Kit*, BDS; Daniel Devaprakash Dicksit§, BDS, MPH; Kalyan CG‡, BDS, MDPH; Datuk Dr.
Khairiyah Abd MuttalibΔ, BDS, DPH(Dent); Srinivas Sulugodu Ramachandra‡, MDS
ABSTRACT
Objective: Charcoal toothbrushes have been marketed widely with manufacturers’ claims of lesser bacterial contamination owing to the presence
of activated charcoal. The aim of this study was to evaluate the bacterial contamination of charcoal bristles compared to non-charcoal bristles in
used toothbrushes. Material and methods: Ninety participants were involved in the study. They were given standard brushing instructions on the
use of a charcoal toothbrush, and were asked to return the used brushes after 1 week of usage. After a 1-week washout period, the participants
were then provided with similar brushing instructions and a non-charcoal toothbrush, and were instructed to return the brush after another
week of usage. Bristles of the used toothbrushes were sectioned and placed in a nutrient broth. A pipette was used to extract 0.1 mL of nutrient
broth to smear on agar plates. A colony counter was used to measure colony forming units (CFU) after 24 hours of incubation. Data collected
were analysed using a paired sample t-test. Results: The mean CFU count for non-charcoal bristles was almost double (106.3; 95% CI 53.39,
159.28) that of charcoal bristles (58.8; 95% CI 15.09, 102.55). However, there was no statistically signicant difference between the two groups
(p = 0.198). Conclusion: This study shows no statistically signicant difference in bacterial counts between bristle types, despite substantially
lower CFUs in the charcoal bristles compared with non-charcoal bristles after 1 week of use.
RÉSUMÉ
Objectif : La mise en marché des brosses à dents au charbon a été largement axée par les fabricants sur la réduction de la contamination
bactérienne en raison de la présence du charbon activé. La présente étude avait pour objectif l’évaluation de la contamination bactérienne des
poils de charbon par rapport aux poils sans charbon des brosses à dents usagées. Matériau et méthodes : Quatre-vingt-dix participants ont pris
part à l’étude. Les participants ont reçu les instructions habituelles de brossage sur l’utilisation d’une brosse à dents à poils de charbon et ont été
invités à retourner les brosses à dents usagées après une semaine d’utilisation. Après une période sans traitement d’une semaine, les participants
ont reçu des instructions de brossage semblables et une brosse à dents à poils sans charbon. Ils ont été invités à retourner la brosse après une
autre semaine d’utilisation. Les poils des brosses à dents usagées ont été sectionnés et placés dans un bouillon de culture. Une pipette a été
utilisée pour extraire 0,1 mL de bouillon de culture et l’étaler sur des plaques de gélose. Un compteur de colonies bactériennes a été utilisé pour
mesurer les unités formatrices de colonies (UFC) après 24 heures d’incubation. Les données recueillies ont été analysées au moyen de test t pour
échantillons appariés. Résultats : La concentration moyenne d’UFC présente sur les poils sans charbon était presque le double (106,3; 95 % CI
53,39, 159,28) de celle présente sur les poils de charbon (58,8; 95 % CI 15,09, 102,55). Toutefois, il n’y avait aucune différence statistiquement
signicative entre les deux groupes (p = 0,198). Conclusion : Cette étude ne révèle aucune différence statistiquement signicative dans le compte
de bactéries entre les types de poils, malgré la présence d’un nombre nettement plus faible d’UFC sur les poils de charbon comparativement aux
poils sans charbon après une semaine d’utilisation.
Key words: bacterial contamination, charcoal bristles, used toothbrushes
*Alumna/Alumnus, Faculty of Dentistry, SEGi University, Selangor, Malaysia
§Lecturer, Faculty of Dentistry, SEGi University, Selangor, Malaysia
‡Associate professor, Faculty of Dentistry, SEGi University, Selangor, Malaysia
ΔDean, Faculty of Dentistry, SEGi University, Selangor, Malaysia
Correspondence: Dr. Srinivas SR; periosrinivas@gmail.com
Submitted 13 July 2016; revised 22 November 2016, 3 January 2017; accepted 15 February 2017
©2017 Canadian Dental Hygienists Association
ORIGINAL RESEARCH
WHY THIS ARTICLE IS IMPORTANT
TO DENTAL HYGIENISTS
• Micro-organisms have been shown to adhere
to and survive on toothbrushes.
• Bacterial contamination of toothbrushes
contributes to oral diseases.
• Identifying materials that reduce bacterial
contamination of toothbrush bristles may
improve oral health.
INTRODUCTION
Toothbrushes become contaminated with pathogenic
bacteria from dental plaque, the environment or a
combination of factors. Mehta et al.1 studied the effectiveness
of various methods of reducing bacterial contamination
of toothbrushes, including covering the toothbrush head
with a plastic cap, overnight immersion of toothbrushes
in Listerine®, and overnight immersion of brushes in
chlorhexidine. Each method was tested for a 1-week
68 Can J Dent Hyg 2017;51(2): 67-72
Lee, Palaniappan, Hwai, et al.
period. The results revealed that overnight immersion of
a toothbrush in 0.2% chlorhexidine gluconate was more
effective than overnight immersion in Listerine or covering
the toothbrush head with a plastic cap.1 This study also
concluded that 70% of the used toothbrushes were heavily
contaminated with different pathogenic microorganisms.1
Several other studies have also investigated various
methods of brush decontamination.2-6
A new variant of toothbrushes, charcoal toothbrushes,
has been introduced into the market; these toothbrushes
are popular in South-East Asian countries like Malaysia,
Singapore, and Indonesia.7 Consumers can also buy these
products through online vendors.7 Bristles of charcoal
toothbrushes are black in colour and are prepared
by blending binchotan charcoal into nylon bristles.
Manufacturers of these toothbrushes claim that they have
antimicrobial properties thanks to the charcoal in them,
resulting in less bacterial contamination.7 However, there
is no scientic evidence to support these claims.
It has been well-established that micro-organisms
adhere, accumulate, and survive on toothbrushes.2
Furthermore, these microbes have been shown to be
capable of transmission to the individual, which in turn
can cause diseases.8 Decontamination of toothbrushes
should be a priority in order to eliminate the transmission
of pathogenic micro-organisms from the oral cavity or
from other toothbrushes stored nearby or from the storage
area itself.9 Various materials have been incorporated into
toothbrush bristles with the aim of reducing bacterial
contamination.2 Since it has been suggested that charcoal
may have bacterial resistant properties, toothbrushes have
been created with charcoal infused into the bristles. The aim
of this study was to evaluate the bacterial contamination
of charcoal bristles compared to non-charcoal bristles in
used toothbrushes by comparing the microbial counts
present in the bristles.
MATERIAL AND METHODS
This crossover clinical trial was approved by the
Institutional Ethics Committee of SEGi University. Students
who attended the SEGi Oral Health Centre from June 2015
to August 2015 formed the sampling frame. Those ages
18–25 years with toothbrushing frequency of 2 times daily
were eligible for inclusion in the study. Students selected
for the study had basic periodontal examination (BPE)
scores of 1 and 2;10 students with BPE scores of 3 and
4 were excluded. Likewise, students with International
Caries Detection and Assessment system (ICDAS)11 scores
of ≥3 were excluded from the study. Students with open
carious lesions, poor plaque scores (plaque index scores
of >2),12 severe gingivitis (gingival index score >2),12
throat infections, irregular brushing frequency, as well as
those unwilling to use a charcoal toothbrush, those using
mouthwash and/or antibacterial toothpastes, smokers or
those medically compromised were excluded from the
study. All the students who participated in the study were
manual brush users. From the name list of 200 students
Figure 1. Non-charcoal and charcoal toothbrushes used in this study
Figure 2. Used non-charcoal and charcoal toothbrushes returned in
sterile pouches
69
Can J Dent Hyg 2017;51(2): 67-72
Bacterial contamination in bristles of used charcoal toothbrushes
(provided by the course coordinator of the university) who
met the inclusion criteria, 90 participants were randomly
chosen. All 90 participants were informed about the study
and signed the consent form prior to participation.
All participants were given standard instructions on
toothbrushing and toothbrush storage to minimize bias
in the study. Standard brushing instructions included
brushing twice daily (once each in the morning and night)
for 2 minutes.13 Students were instructed to place the brush
at a 45-degree angle to the gums and gently move the
brush back and forth in short strokes. Participants were
instructed to brush the outer surfaces, the inner surfaces,
and the chewing surfaces of all teeth. They were also
instructed to clean the inside surface of the front teeth,
tilting the brush vertically and making several up-and-
down strokes.13 They were also advised not to use any
type of mouthwash, to wash the toothbrush bristles under
running water without using their ngers to clean the
bristles, not to cover the toothbrush bristles with a cap, and
to place the toothbrush upright after use with the bristles
on top at least 2 feet away from the toilet. Researchers
from the University of Alabama found that brushes stored
in the bathroom are very likely to have faecal matter
lingering in the bristles.14 Toilet ushing was shown to
produce an aerosol spray of bacterium tainted water which
can contaminate the bristles.14 Thus, study participants
were instructed to keep the toothbrushes at least 2 feet
away from the toilet. Students were asked to document
their daily 2-minute brushings on a standardized recording
sheet provided to them.
Each participant was then given a charcoal toothbrush
and asked to return the toothbrush after 1 week of use. After
a wash-out period of 1 week, non-charcoal toothbrushes
were given to the participants and again, they were asked to
use the brushes for 1 week and to return the non-charcoal
toothbrushes after the week. Both the charcoal and non-
charcoal brushes were similar in design with a compact
head, soft bristles, and a bristle tip that was less than 0.01
mm (Figure 1; Colgate® Slim Soft Charcoal Toothbrush).
The participants received individual sterile pouches into
which to place each used toothbrush for return (Figure 2).
On return of the toothbrushes, one-third of the bristles
were cut and collected on separate sterile petri dishes
(Figure 3). Using sterile forceps, the study assistant placed
the toothbrush bristles in separate test tubes containing a
nutrient broth and swirled. A sterile pipette was used to
extract 0.1 mL of the nutrient broth, which was poured
onto a nutrient agar plate. A sterile cotton bud was used
to smear the solution on the agar plate (Figure 4). The
agar plates were then placed in the incubator for 24
hours (Figure 5), after which colonies of microbial growth
were noted (Figure 6). Colony counters (Fisher Scientic
brand, model F22 0360/10R) were used to measure the
colony forming units (CFU) present on each agar plate
Figure 3. Bristles collected in sterile petri dishes
Figure 4. Nutrient broth containing used toothbrush bristles is
smeared on the nutrient agar plate
Figure 5. Smeared nutrient agar plates placed for incubation
Figure 6. Microbial growth noticed after 24 hours incubation (plates
marked “c” contain charcoal bristles; plates marked “n” contain non-
charcoal bristles)
70 Can J Dent Hyg 2017;51(2): 67-72
Lee, Palaniappan, Hwai, et al.
(Figure 7). Data obtained were tabulated and statistically
analysed using MedCalc ver 12. A paired sample t-test was
conducted to compare the number of CFUs for charcoal
and non-charcoal bristles. The signicance level was set at
p < 0.05. Mean values for CFU counts and 95% condence
intervals for the mean were determined for the 2 groups.
RESULTS
Of the 90 participants, 3 did not return one of their
toothbrushes. Five participants did not properly place their
toothbrushes in the sterile pouches provided and these
(5 x 2 brushes) were excluded from the study. A nal count
of 164 toothbrushes—82 charcoal and 82 non-charcoal—
were collected from participants. Out of 164 agar plates
(82 charcoal and 82 non-charcoal), 102 plates (51 charcoal
and 51 non-charcoal) were seen to have microbial colonies
and included in the analysis. There were no growths seen
in 62 plates after 24 hours of incubation. Using the colony
counters, higher counts of CFUs were seen on the agar
plates from used non-charcoal brushes compared with
those from used charcoal brushes.
Table 1 presents the results of the paired sample t-test
comparing the number of CFUs between the 2 types of
bristles. The mean CFUs for non-charcoal bristles were
almost double (106.3; 95% CI 53.39, 159.28) those of the
charcoal bristles (58.8; 95% CI 15.09, 102.55). However,
there was no signicant difference between the 2 products.
(p = 0.198)
DISCUSSION
Results revealed substantially lower CFU counts in agar
plates for used charcoal bristles compared with used
non-charcoal bristles. This difference, however, was not
statistically signicant. This is most likely due to the
high variability of CFUs demonstrated by the standard
deviations found in both products. A power analysis was
not performed prior to study commencement. A post-study
power analysis revealed a sample size of 209 brushes was
required (alpha value of 0.05, beta value of 0.20) to obtain a
statistically signicant difference between means. To date,
there is a dearth of scientic literature on toothbrushes
with charcoal infused bristles. Manufacturers’ claim that
charcoal toothbrushes control micro-organisms, inhibit
mouth odour, effectively remove plaque, and whiten teeth,
yet such claims are not supported by scientic evidence
on bacterial inhibition. Charcoal in itself has the property
of being absorbent, neutralising toxins, poisons, and
noxious gases.3 However, it continues to be a matter of
speculation as to whether these properties contribute to
lesser contamination of used charcoal-infused bristles in
toothbrushes.
Additions of antiplaque and antimicrobial substances
to toothbrush bristles in attempts to reduce contamination
of used toothbrushes are not a new phenomenon. Turner
et al. conducted a study to determine the effectiveness of
chlorhexidine-coated toothbrush laments in reducing
quantities of bacteria.3 The study concluded that there
was no statistically signicant difference in the quantity
of bacteria surviving on chlorhexidine-coated laments
compared with the control group after 30 days of use.3
The manufacturer of the chlorhexidine-coated toothbrush,
however, suggested that chlorhexidine-coated laments
were only effective for a 30-day period, after which time
the toothbrush should be replaced.3 Al–Ahmad et al.
studied the antimicrobial effect of silver-coated toothbrush
heads in-vitro.4 The organisms investigated were
Streptococcus oralis, Streptococcus mutans, Streptococcus
sanguis, Actinomyces viscosus, Lactobacillus casei and
Candida albicans. The study concluded that there was
no signicant reduction in the CFUs by silver-coated
toothbrushes for the above-mentioned tested organisms.4
On the contrary, the CFU counts for S. sanguis (p = 0.02)
and C. albicans (p = 0.01) were signicantly higher on
silver-coated toothbrushes compared with the controls.4
This current study did not investigate specic organisms;
only microbial counts were made.
In 2014, Tomar et al. evaluated the sanitization potential
of UV-rays and 0.2% chlorhexidine (CHX) solution for
disinfection of used toothbrushes.5 Toothbrushes were
collected after 7 days of use and placed into 3 groups:
Group I brushes were soaked in 0.2% CHX mouthwash
Used charcoal brushes
n = 51
Used non-charcoal brushes
n = 51
CFU mean (SD) 58.8235 (155.48) 106.3333 (188.23)
Standard error of the mean 21.7720 26.3580
Mean difference (SD) 47.5098 (259.92)
95% CI –25.5938 to 120.6134
2-tailed probability p = 0.198
Table 1. CFU differences between charcoal and non-charcoal toothbrush bristles
Paired sample t-test signicant if p < 0.05
71
Can J Dent Hyg 2017;51(2): 67-72
Bacterial contamination in bristles of used charcoal toothbrushes
CONCLUSION
Our study showed the number of CFUs in charcoal
toothbrushes was substantially less when compared
with non-charcoal toothbrushes after 1 week of usage.
However, the difference in these microbial counts was not
statistically signicant between the 2 products. Further
studies should be conducted with a larger sample size,
longer duration of use, and with identication of specic
micro-organisms in the bristles.
ACKNOWLEDGEMENTS
We would like to thank Dr Anitha Ravindran,
Faculty of Medicine, for help and guidance on the
microbiological aspects of the study.
CONFLICT OF INTEREST
The authors have declared no conicts of interest
in connection with this article.
for 12 hours, Group II brushes were placed in UV-light
toothbrush holders for 7 minutes, and Group III brushes
were soaked in normal saline for 12 hours. Microbial
analysis and mean bacterial counts showed that all 3
methods were effective in reducing the bacterial counts
on the toothbrushes tested (p < 0.007). However, UV ray
treatment was more effective (p = 0.001) when compared
with CHX and normal saline.5 The authors suggested that
UV light is capable of deactivating the micro-organisms
by disrupting the chemical bonds that hold the DNA
atom.5 Studies have suggested that longer exposure to
UV light can further lead to complete deactivation of
micro-organisms.5
Basman et al. studied toothbrush disinfection using
0.12% chlorhexidine gluconate, 2% sodium hypochlorite
(NaOCl), a mouthrinse containing essential oils and
alcohol, and 50% white vinegar.6 The most effective
method for elimination of all tested bacterial species was
found to be 50% white vinegar (p = 0.000), followed by 2%
NaOCl, mouthrinse containing essential oils and alcohol,
0.12% chlorhexidine gluconate, dishwasher use, and tap
water (control).6
Some studies in rural populations have reported
abrasion on the labial surfaces of teeth due to use of
charcoal powder for toothbrushing.15 Although no direct
comparison can be made between abrasiveness of charcoal
powder and the charcoal-infused toothbrush bristles used
in this study, further studies could be done over a longer
duration to explore whether charcoal brushes damage
the tooth structure. Toothbrush trauma results in portals
of entry for micro-organisms, leading to infection.16
Contaminated toothbrushes can easily be a source of such
infections.16 As a result, various products that claim lesser
contamination of used toothbrushes have been developed.17
Limitations of the study
One limitation was the lack of analysis of the types
of bacteria present. It is possible that anaerobic bacteria
may be harboured differently from aerobic bacteria. In
future studies, specic types of bacterial growth (aerobic/
anaerobic) should be studied. A major study limitation was
the lack of an initial power analysis which would have
revealed the necessity of using a larger sample size. To
compare the effectiveness of the 2 products, studies with a
larger sample size will need to be conducted. Additionally,
the manufacturers of charcoal toothbrushes have not
provided information regarding the concentration of the
charcoal in the brush. Thus, the concentration of charcoal
at baseline or after a certain period of use cannot be
examined with the currently marketed brushes.
Figure 7. Colony counter (Fisher Scientic brand,
model F22 0360/10R) used to measure the total
colony forming units
72 Can J Dent Hyg 2017;51(2): 67-72
Lee, Palaniappan, Hwai, et al.
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