Microbial Contamination of Different Toothbrush Types Stored in Bathrooms and Rooms in Adolescents

Microbial Contamination of Silver Nanoparticle, Bamboo Charcoal and Standard Toothbrushes Stored in Bathroom Versus Room Environments: A Randomized Clinical Study in Adolescents

This randomized clinical study aimed to compare microbial contamination of three different toothbrush types (standard plastic, silver nanoparticle, and bamboo charcoal) and to investigate whether storing toothbrushes in a bathroom or a room outside the bathroom affects microbial contamination.

The study included 72 healthy adolescents aged 11-18 years who were undergoing removable orthodontic appliance treatment. Participants were randomly assigned to one of three toothbrush groups and two storage conditions (bathroom or room). All participants received standardized oral hygiene instructions and used their assigned toothbrushes twice daily for one month.

After one month, the toothbrushes were collected and examined for the presence of three microorganisms: *Streptococcus mutans*, *Lactobacillus* spp., and *Candida albicans*. In addition, plaque index, gingival index, probing depth, and bleeding on probing were assessed at baseline and after one month.

The study was designed to determine whether toothbrushes marketed as having antimicrobial properties and different storage environments influence microbial contamination and short-term periodontal health.

Study Overview

• Status: Completed
• Conditions: Toothbrush Microbial Contamination
• Intervention / Treatment: Device: Standard Plastic Toothbrush; Device: Silver Nanoparticle Toothbrush; Device: Bamboo Charcoal Toothbrush

Detailed Description

This randomized, parallel-group clinical study was conducted at Afyonkarahisar Health Sciences University, Türkiye, between March 2021 and July 2022. The study investigated the effects of toothbrush type and storage environment on microbial contamination under routine home-use conditions.

Participants were randomly allocated to three toothbrush groups: standard plastic (Colgate Extra Clean), silver nanoparticle (Splat Sensitive Silver), and bamboo charcoal (Colgate Bamboo Charcoal). Each group was further divided into two storage subgroups: bathroom storage and room storage outside the bathroom. Randomization was performed using colored balls drawn by an investigator who was not involved in clinical or microbiological assessments. The microbiologist was blinded to group allocation.

At baseline, all participants received professional prophylaxis and standardized oral hygiene instructions. Participants were instructed to brush twice daily for two minutes using the modified Stillman technique and the same fluoride toothpaste. After brushing, toothbrushes were rinsed under tap water and stored upright and uncovered in the assigned environment. Toothbrushes were not shared or stored in contact with other toothbrushes.

After one month, the used toothbrushes were collected in sterile bags. Under aseptic conditions, one-third of the bristles were removed and transferred into thioglycolate broth. Samples were transported under refrigerated conditions to the microbiology laboratory. Following broth enrichment, samples were cultured on selective media to detect *Streptococcus mutans*, *Lactobacillus* spp., and *Candida albicans*. Suspected isolates were identified using the VITEK 2 Compact system.

Microbial contamination was evaluated based on the presence or absence of each target microorganism rather than quantitative bacterial counts.

Clinical periodontal assessments were performed at baseline and after one month using standardized measurements of plaque index, gingival index, probing depth, and bleeding on probing.

The study was approved by the Clinical Research Ethics Committee of Afyonkarahisar Health Sciences University (approval number: 2020/13; November 6, 2020). Written informed consent was obtained from parents or legal guardians, and assent was obtained from participants.

• Study Type: Interventional
• Enrollment (Actual): 72
• Phase: Not Applicable

Contacts and Locations

Study Locations

• Turkey (Türkiye)
  • Afyonkarahisar, Turkey (Türkiye)
    • Afyonkarahisar Health Sciences University

Participation Criteria

Eligibility Criteria

• Ages Eligible for Study: 11 years to 18 years (Child, Adult)
• Accepts Healthy Volunteers: Yes
• Genders Eligible for Study: All

Description

Inclusion Criteria:

• Adolescents aged 11-18 years.
• Systemically healthy individuals.
• Patients undergoing dentofacial orthopedic treatment with removable functional appliances.

Exclusion Criteria:

• Use of systemic antibiotics within the previous 3 months.
• Use of antimicrobial mouthwash within the previous 3 months.
• Presence of systemic disease.
• Smoking.

Study Plan

How is the study designed?

Design Details

• Primary Purpose: Prevention
• Allocation: Randomized
• Interventional Model: Factorial Assignment
• Masking: Single

Number of Arms

6

Arms and Interventions

Participant Group / Arm	Intervention / Treatment
Active Comparator: Standard Toothbrush - Bathroom; Participants assigned to this arm used a standard plastic toothbrush (Colgate Extra Clean) twice daily for two minutes using the modified Stillman brushing technique and standardized fluoride toothpaste. After each use, the toothbrush was rinsed under tap water and stored upright and uncovered in the bathroom. The intervention lasted one month, after which the toothbrush was collected for microbiological analysis.	Device: Standard Plastic Toothbrush; A commercially available standard plastic toothbrush (Colgate Extra Clean, Colgate-Palmolive Company, New York, NY, USA) was used as the active comparator. Participants brushed their teeth twice daily for two minutes using the modified Stillman technique and standardized fluoride toothpaste for one month. After each use, the toothbrush was rinsed under tap water and stored upright and uncovered in the assigned storage environment (bathroom or room outside the bathroom). At the end of the intervention period, toothbrushes were collected for microbiological analysis.
Active Comparator: Standard Toothbrush - Room; Participants assigned to this arm used a standard plastic toothbrush (Colgate Extra Clean) twice daily for two minutes using the modified Stillman brushing technique and standardized fluoride toothpaste. After each use, the toothbrush was rinsed under tap water and stored upright and uncovered in a room outside the bathroom. The intervention lasted one month, after which the toothbrush was collected for microbiological analysis.	Device: Standard Plastic Toothbrush; A commercially available standard plastic toothbrush (Colgate Extra Clean, Colgate-Palmolive Company, New York, NY, USA) was used as the active comparator. Participants brushed their teeth twice daily for two minutes using the modified Stillman technique and standardized fluoride toothpaste for one month. After each use, the toothbrush was rinsed under tap water and stored upright and uncovered in the assigned storage environment (bathroom or room outside the bathroom). At the end of the intervention period, toothbrushes were collected for microbiological analysis.
Experimental: Silver Nanoparticle Toothbrush - Bathroom; Participants assigned to this arm used a silver nanoparticle-containing toothbrush (Splat Sensitive Silver) twice daily for two minutes using the modified Stillman brushing technique and standardized fluoride toothpaste. After each use, the toothbrush was rinsed under tap water and stored upright and uncovered in the bathroom. The intervention lasted one month, after which the toothbrush was collected for microbiological analysis.	Device: Silver Nanoparticle Toothbrush; A commercially available silver nanoparticle-containing toothbrush (Splat Sensitive Silver, SPLAT-COSMETICA, Moscow, Russia) was used to evaluate microbial contamination compared with a standard plastic toothbrush. Participants brushed their teeth twice daily for two minutes using the modified Stillman technique and standardized fluoride toothpaste for one month. After each use, the toothbrush was rinsed under tap water and stored upright and uncovered in the assigned storage environment (bathroom or room outside the bathroom). At the end of the intervention period, toothbrushes were collected for microbiological analysis.
Experimental: Silver Nanoparticle Toothbrush - Room; Participants assigned to this arm used a silver nanoparticle-containing toothbrush (Splat Sensitive Silver) twice daily for two minutes using the modified Stillman brushing technique and standardized fluoride toothpaste. After each use, the toothbrush was rinsed under tap water and stored upright and uncovered in a room outside the bathroom. The intervention lasted one month, after which the toothbrush was collected for microbiological analysis.	Device: Silver Nanoparticle Toothbrush; A commercially available silver nanoparticle-containing toothbrush (Splat Sensitive Silver, SPLAT-COSMETICA, Moscow, Russia) was used to evaluate microbial contamination compared with a standard plastic toothbrush. Participants brushed their teeth twice daily for two minutes using the modified Stillman technique and standardized fluoride toothpaste for one month. After each use, the toothbrush was rinsed under tap water and stored upright and uncovered in the assigned storage environment (bathroom or room outside the bathroom). At the end of the intervention period, toothbrushes were collected for microbiological analysis.
Experimental: Bamboo Charcoal Toothbrush - Bathroom; Participants assigned to this arm used a bamboo charcoal toothbrush (Colgate Bamboo Charcoal) twice daily for two minutes using the modified Stillman brushing technique and standardized fluoride toothpaste. After each use, the toothbrush was rinsed under tap water and stored upright and uncovered in the bathroom. The intervention lasted one month, after which the toothbrush was collected for microbiological analysis.	Device: Bamboo Charcoal Toothbrush; A commercially available bamboo charcoal toothbrush (Colgate Bamboo Charcoal, Colgate-Palmolive Company, New York, NY, USA) was used to evaluate microbial contamination compared with a standard plastic toothbrush. Participants brushed their teeth twice daily for two minutes using the modified Stillman technique and standardized fluoride toothpaste for one month. After each use, the toothbrush was rinsed under tap water and stored upright and uncovered in the assigned storage environment (bathroom or room outside the bathroom). At the end of the intervention period, toothbrushes were collected for microbiological analysis.
Experimental: Bamboo Charcoal Toothbrush - Room; Participants assigned to this arm used a bamboo charcoal toothbrush (Colgate Bamboo Charcoal) twice daily for two minutes using the modified Stillman brushing technique and standardized fluoride toothpaste. After each use, the toothbrush was rinsed under tap water and stored upright and uncovered in a room outside the bathroom. The intervention lasted one month, after which the toothbrush was collected for microbiological analysis.	Device: Bamboo Charcoal Toothbrush; A commercially available bamboo charcoal toothbrush (Colgate Bamboo Charcoal, Colgate-Palmolive Company, New York, NY, USA) was used to evaluate microbial contamination compared with a standard plastic toothbrush. Participants brushed their teeth twice daily for two minutes using the modified Stillman technique and standardized fluoride toothpaste for one month. After each use, the toothbrush was rinsed under tap water and stored upright and uncovered in the assigned storage environment (bathroom or room outside the bathroom). At the end of the intervention period, toothbrushes were collected for microbiological analysis.

What is the study measuring?

Primary Outcome Measures

Outcome Measure	Measure Description	Time Frame
Presence of Streptococcus mutans on Toothbrushes	The presence or absence of Streptococcus mutans on toothbrush bristles was assessed after one month of use. Bristle samples were cultured on selective Mitis Salivarius agar supplemented with bacitracin and potassium tellurite following broth enrichment. Suspected isolates were identified using the VITEK 2 Compact system. The outcome was recorded as a binary variable (detected/not detected), and the proportion of contaminated toothbrushes was compared across toothbrush types and storage environments.	1 month after baseline (end of toothbrush use)
Presence of Lactobacillus spp. on Toothbrushes	The presence or absence of Lactobacillus spp. on toothbrush bristles was assessed after one month of use. Bristle samples were cultured on Rogosa agar adjusted to pH 5.5 following broth enrichment. Suspected isolates were identified using the VITEK 2 Compact system. The outcome was recorded as a binary variable (detected/not detected), and the proportion of contaminated toothbrushes was compared across toothbrush types and storage environments.	1 month after baseline (end of toothbrush use)
Presence of Candida albicans on Toothbrushes	The presence or absence of Candida albicans on toothbrush bristles was assessed after one month of use. Bristle samples were cultured on Sabouraud dextrose agar supplemented with chloramphenicol following broth enrichment. Suspected isolates were identified using the VITEK 2 Compact system. The outcome was recorded as a binary variable (detected/not detected), and the proportion of contaminated toothbrushes was compared across toothbrush types and storage environments.	1 month after baseline (end of toothbrush use)

Secondary Outcome Measures

Outcome Measure	Measure Description	Time Frame
Change in Plaque Index From Baseline to One Month	Dental plaque accumulation was assessed using the Silness and Löe Plaque Index at baseline and after one month of toothbrush use. The change in plaque index was calculated by subtracting the baseline score from the one-month score. Changes were compared across toothbrush types and storage environments. Lower scores indicate less plaque accumulation.	Baseline and 1 month
Change in Gingival Index From Baseline to One Month	Gingival inflammation was assessed using the Löe and Silness Gingival Index at baseline and after one month of toothbrush use. The change in gingival index was calculated by subtracting the baseline score from the one-month score. Changes were compared across toothbrush types and storage environments. Lower scores indicate less gingival inflammation.	Baseline and 1 month
Change in Buccal Probing Depth From Baseline to One Month	Buccal periodontal probing depth was measured in millimeters using a periodontal probe (Hu-Friedy No. 15) at baseline and after one month of toothbrush use. Measurements were obtained as part of a standardized six-site periodontal examination of all teeth present. The change in buccal probing depth was calculated by subtracting the baseline value from the one-month value. Changes were compared across toothbrush types and storage environments. Positive values indicate an increase in probing depth.	Baseline and 1 month
Change in Lingual Probing Depth From Baseline to One Month	Lingual periodontal probing depth was measured in millimeters using a periodontal probe (Hu-Friedy No. 15) at baseline and after one month of toothbrush use. Measurements were obtained as part of a standardized six-site periodontal examination of all teeth present. The change in lingual probing depth was calculated by subtracting the baseline value from the one-month value. Changes were compared across toothbrush types and storage environments. Positive values indicate an increase in probing depth.	Baseline and 1 month
Change in Bleeding on Probing From Baseline to One Month	Bleeding on probing (BOP) was assessed at six sites per tooth using a periodontal probe (Hu-Friedy No. 15) at baseline and after one month of toothbrush use. BOP was expressed as the percentage of examined sites exhibiting bleeding. The change in BOP was calculated by subtracting the baseline percentage from the one-month percentage and was expressed in percentage points. Changes were compared across toothbrush types and storage environments. Negative values indicate a reduction in gingival bleeding.	Baseline and 1 month

Collaborators and Investigators

• Sponsor: Afyonkarahisar Health Sciences University

Publications and helpful links

General Publications

• Frazelle MR, Munro CL. Toothbrush contamination: a review of the literature. Nurs Res Pract. 2012;2012:420630. doi: 10.1155/2012/420630. Epub 2012 Jan 24.
• Thamke MV, Beldar A, Thakkar P, Murkute S, Ranmare V, Hudwekar A. Comparison of Bacterial Contamination and Antibacterial Efficacy in Bristles of Charcoal Toothbrushes versus Noncharcoal Toothbrushes: A Microbiological Study. Contemp Clin Dent. 2018 Jul-Sep;9(3):463-467. doi: 10.4103/ccd.ccd_309_18.
• Baygin O, Tuzuner T, Yilmaz N, Aksoy S. Short-term antibacterial efficacy of a new silver nanoparticle-containing toothbrush. J Pak Med Assoc. 2017 May;67(5):818-819. No abstract available.

Study record dates

Study Major Dates

• Study Start (Actual): March 1, 2021
• Primary Completion (Actual): August 16, 2022
• Study Completion (Actual): September 4, 2022

Study Registration Dates

• First Submitted: November 8, 2026
• First Submitted That Met QC Criteria: November 8, 2026
• First Posted (Actual): November 13, 2026

Study Record Updates

• Last Update Posted (Actual): November 13, 2026
• Last Update Submitted That Met QC Criteria: November 8, 2026
• Last Verified: November 1, 2026

More Information

Terms related to this study

• Keywords: Adolescents; Lactobacillus; Randomized Clinical Trial; Oral Hygiene; Streptococcus mutans; Candida albicans; Silver Nanoparticles; Toothbrush Contamination; Bamboo Charcoal Toothbrush; Toothbrush Storage
• Other Study ID Numbers: Seher Gözde Uğur; 21.DUS.001 (Other Grant/Funding Number: Afyonkarahisar Health Sciences University - Scientific Research Projects Coordination Unit)

Plan for Individual participant data (IPD)

• Plan to Share Individual Participant Data (IPD)?: NO

IPD Plan Description

Individual participant data (IPD) will not be made publicly available due to participant confidentiality and privacy considerations, as the study involved adolescents. The study was conducted under institutional ethical approval, and no formal IPD sharing plan was established.

Drug and device information, study documents

• Studies a U.S. FDA-regulated drug product: No
• Studies a U.S. FDA-regulated device product: No