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

November 8, 2026 updated by: Özgür Doğan, Afyonkarahisar Health Sciences University

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

Status

Indicates the current recruitment status or the expanded access status.
Completed

Conditions

Conditions

The disease, disorder, syndrome, illness, or injury that is being studied. On ClinicalTrials.gov, conditions may also include other health-related issues, such as lifespan, quality of life, and health risks.

Intervention / Treatment

Intervention / Treatment

A process or action that is the focus of a clinical study. Interventions include drugs, medical devices, procedures, vaccines, and other products that are either investigational or already available. Interventions can also include noninvasive approaches, such as education or modifying diet and exercise.

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

Study Type

Describes the nature of a clinical study. Study types include interventional studies (also called clinical trials), observational studies (including patient registries), and expanded access.
Interventional

Enrollment (Actual)

Enrollment

The number of participants in a clinical study. The "anticipated" enrollment is the target number of participants that the researchers need for the study.
72

Phase

Phase

The stage of a clinical trial studying a drug or biological product, based on definitions developed by the U.S. Food and Drug Administration (FDA). The phase is based on the study's objective, the number of participants, and other characteristics. There are five phases: Early Phase 1 (formerly listed as Phase 0), Phase 1, Phase 2, Phase 3, and Phase 4. Not Applicable is used to describe trials without FDA-defined phases, including trials of devices or behavioral interventions.
  • Not Applicable

Contacts and Locations

This section provides the contact details for those conducting the study, and information on where this study is being conducted.

Study Locations

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

Participation Criteria

Researchers look for people who fit a certain description, called eligibility criteria. Some examples of these criteria are a person's general health condition or prior treatments.

Eligibility Criteria

Eligibility Criteria

The key requirements that people who want to participate in a clinical study must meet or the characteristics they must have. Eligibility criteria consist of both inclusion criteria (which are required for a person to participate in the study) and exclusion criteria (which prevent a person from participating). Types of eligibility criteria include whether a study accepts healthy volunteers, has age or age group requirements, or is limited by sex.

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

This section provides details of the study plan, including how the study is designed and what the study is measuring.

How is the study designed?

Design Details

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

Arms and Interventions

Participant Group / Arm

Participant Group / Arm

A group or subgroup of participants in a clinical trial that receives a specific intervention/treatment, or no intervention, according to the trial's protocol.
Intervention / Treatment

Intervention / Treatment

A process or action that is the focus of a clinical study. Interventions include drugs, medical devices, procedures, vaccines, and other products that are either investigational or already available. Interventions can also include noninvasive approaches, such as education or modifying diet and exercise.
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

Primary Outcome Measures

In a clinical study's protocol, the planned outcome measure that is the most important for evaluating the effect of an intervention/treatment. Most clinical studies have one primary outcome measure, but some have more than one.
Outcome Measure
Measure Description
Time Frame
Presence of Streptococcus mutans on Toothbrushes
Time Frame: 1 month after baseline (end of toothbrush use)

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
Time Frame: 1 month after baseline (end of toothbrush use)

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
Time Frame: 1 month after baseline (end of toothbrush use)

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

Secondary Outcome Measures

In a clinical study's protocol, a planned outcome measure that is not as important as the primary outcome measure for evaluating the effect of an intervention but is still of interest. Most clinical studies have more than one secondary outcome measure.
Outcome Measure
Measure Description
Time Frame
Change in Plaque Index From Baseline to One Month
Time Frame: Baseline and 1 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
Time Frame: Baseline and 1 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
Time Frame: Baseline and 1 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
Time Frame: Baseline and 1 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
Time Frame: Baseline and 1 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

This is where you will find people and organizations involved with this study.

Sponsor

Sponsor

The organization or person who initiates the study and who has authority and control over the study.
Afyonkarahisar Health Sciences University

Publications and helpful links

The person responsible for entering information about the study voluntarily provides these publications. These may be about anything related to the study.

General Publications

Study record dates

These dates track the progress of study record and summary results submissions to ClinicalTrials.gov. Study records and reported results are reviewed by the National Library of Medicine (NLM) to make sure they meet specific quality control standards before being posted on the public website.

Study Major Dates

Study Start (Actual)

Study Start

The actual date on which the first participant was enrolled in a clinical study. The "anticipated" study start date is the date that the researchers think will be the study start date.
March 1, 2021

Primary Completion (Actual)

Primary Completion

The date on which the last participant in a clinical study was examined or received an intervention to collect final data for the primary outcome measure. Whether the clinical study ended according to the protocol or was terminated does not affect this date. For clinical studies with more than one primary outcome measure with different completion dates, this term refers to the date on which data collection is completed for all the primary outcome measures. The "anticipated" primary completion date is the date that the researchers think will be the primary completion date for the study.
August 16, 2022

Study Completion (Actual)

Study Completion

The date on which the last participant in a clinical study was examined or received an intervention/treatment to collect final data for the primary outcome measures, secondary outcome measures, and adverse events (that is, the last participant's last visit). The "anticipated" study completion date is the date that the researchers think will be the study completion date.
September 4, 2022

Study Registration Dates

First Submitted

First Submitted

The date on which the study sponsor or investigator first submitted a study record to ClinicalTrials.gov. There is typically a delay of a few days between the first submitted date and the record's availability on ClinicalTrials.gov (the first posted date).
November 8, 2026

First Submitted That Met QC Criteria

First Submitted That Met QC Criteria

The date on which the study sponsor or investigator first submits a study record that is consistent with National Library of Medicine (NLM) quality control (QC) review criteria. The sponsor or investigator may need to revise and submit a study record one or more times before NLM's QC review criteria are met. It is the responsibility of the sponsor or investigator to ensure that the study record is consistent with the NLM QC review criteria.
November 8, 2026

First Posted (Actual)

First Posted

The date on which the study record was first available on ClinicalTrials.gov after National Library of Medicine (NLM) quality control (QC) review has concluded. There is typically a delay of a few days between the date the study sponsor or investigator submitted the study record and the first posted date.
November 13, 2026

Study Record Updates

Last Update Posted (Actual)

Last Update Posted

The most recent date on which changes to a study record were made available on ClinicalTrials.gov. There may be a delay between when the changes were submitted to ClinicalTrials.gov by the study's sponsor or investigator (the last update submitted date) and the last update posted date.
November 13, 2026

Last Update Submitted That Met QC Criteria

Last Update Submitted That Met QC Criteria

The most recent date on which the study sponsor or investigator submitted changes to a study record that are consistent with National Library of Medicine (NLM) quality control (QC) review criteria. It is the responsibility of the sponsor or investigator to ensure that the study record is consistent with the NLM QC review criteria.
November 8, 2026

Last Verified

Last Verified

The most recent date on which the study sponsor or investigator confirmed the information about a clinical study on ClinicalTrials.gov as accurate and current. If a study with a recruitment status of recruiting; not yet recruiting; or active, not recruiting has not been confirmed within the past 2 years, the study's recruitment status is shown as unknown.
November 1, 2026

More Information

Terms related to this study

Keywords

Other Study ID Numbers

Other Study ID Numbers

Identifiers or ID numbers other than the NCT number that are assigned to a clinical study by the study's sponsor, funders, or others. These numbers may include unique identifiers from other trial registries and National Institutes of Health grant 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

This information was retrieved directly from the website clinicaltrials.gov without any changes. If you have any requests to change, remove or update your study details, please contact register@clinicaltrials.gov. As soon as a change is implemented on clinicaltrials.gov, this will be updated automatically on our website as well.

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