Evaluation of Bacterial Contamination and Antimicrobial Efficacy on Charcoal-Infused vs, Standard Nylon Toothbrush Bristles.

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.