2.5.3. Sample Collection
STERILE
A CONSORT flow diagram illustrating subjects' flow during the clinical trial was followed (After seven days of wearing the appliance, three acrylic tiles of each roughness category were gently removed from their wells using a metal pin through the chimney-like structure without disturbing the biofilm. The tiles were held using a sterile tweezer and immersed twice in a 15 ml bijou tube containing phosphate buffer saline (Sigma-Aldrich) to remove the planktonic bacteria and then inserted into a 1.5 ml bijou tube containing 1 ml PBS and vortex mixed for 1 minute to disseminate the biofilm and create a homogenous solution. A ten-fold serial dilution was carried out using PBS, and 50 Bacteria were distinguished by colony morphology and were characterized by Gram reaction [