This study compares two types of toothbrushes to see which one stays cleaner after use. Researchers want to know if toothbrushes with charcoal-infused bristles have fewer bacteria than standard toothbrushes with nylon bristles.

Participants in this study are adults and children who do not have gum disease or other major health issues. Everyone in the study will be assigned one of two types of toothbrushes:

• An eco-friendly toothbrush with charcoal bristles.
• A standard toothbrush with nylon bristles. Participants will use their assigned toothbrush at home for two specific time periods (one day and one week). They will follow simple instructions on how to brush and store the toothbrush. After each time period, they will return the toothbrush to the researchers.

The research team will test the bristles in a lab to count the bacteria. They will also measure how well the charcoal bristles stop bacteria from growing compared to the nylon bristles. This will help determine if charcoal toothbrushes are a more sanitary and eco-friendly option for daily oral care.

Study Design and Randomization This study employs a randomized, parallel-group, ex-vivo microbiological design to assess bacterial retention and antimicrobial efficacy. Participants are stratified by age (children and adults) and randomized to receive either an eco-friendly charcoal-infused toothbrush or a standard nylon toothbrush. Allocation is managed using computer-generated random sequences concealed in sequentially numbered, opaque envelopes. To maintain blinding, laboratory outcome assessors are unaware of group assignment, and participants are instructed not to disclose their intervention to the assessment team.

Standardized Usage Protocol

To minimize variability in bacterial load due to behavioral factors, all participants adhere to a strict hygiene protocol:

• Brushing Technique: Participants brush twice daily (morning and evening) for two minutes using a standardized method (45-degree angle to the gingiva with short back-and-forth strokes, and vertical strokes for anterior lingual surfaces).
• Storage and Maintenance: Post-brushing, bristles are rinsed under running water without digital manipulation. Toothbrushes must be stored upright, uncovered, and positioned at least two feet away from sanitary fixtures (e.g., toilets) to minimize aerosol contamination.
• Restrictions: The use of antimicrobial mouthrinses, medicated toothpastes, or bristle covers is prohibited during the active study phases.

Sample Collection Timeline

The study involves two distinct usage periods separated by a washout phase:

1. Phase 1 (24-Hour Assessment): Participants use the assigned toothbrush for one day, followed by immediate return in a sterile pouch.
2. Washout: A 7-day period occurs where participants revert to their non-study oral hygiene routine.
3. Phase 2 (7-Day Assessment): Participants receive a new toothbrush of the same allocation, use it for 7 days, and return it in a sterile pouch for analysis.

Microbiological Analysis Returned samples are processed under aseptic laboratory conditions. Approximately one-third of the bristle field is excised using sterile instrumentation and transferred to test tubes containing 5 mL of sterile saline. The tubes are agitated to elute adherent microorganisms into suspension. A 0.1 mL aliquot is inoculated onto blood agar plates using a sterile spreader.

Plates are incubated in anaerobic gas pack jars at 37°C. Colony Forming Units (CFUs) are quantified after 48 hours of incubation to determine total bacterial load. Additionally, the intrinsic antimicrobial activity of the bristles is evaluated by measuring the zone of inhibition (in millimeters) surrounding bristle samples placed on agar plates after 7days of incubation.

Inclusion Criteria:

• Participants free of any systemic diseases.
• Participants falling into one of two specific age groups:

A) Children aged 8 to 11 years.

B) Adults aged 18 to 35 years.

• Willingness to use the assigned toothbrush type (Eco-friendly charcoal or standard nylon).
• Ability to follow study instructions regarding brushing technique and storage.

Exclusion Criteria:

• Patients with open carious lesions.
• Participants with poor oral hygiene, defined as plaque index scores greater than 2.
• Participants with severe gingivitis, defined as gingival index score greater than 2.
• Current throat infections.
• Irregular brushing frequency (not brushing twice daily).
• Current use of mouthwash or antibacterial toothpastes.
• Current smokers.
• Medically compromised individuals.
• Unwillingness to use a charcoal Eco-friendly toothbrush.

age, gender

october 2025 to october 2026