Goal 1: Collection of plaque specimen After isolating the teeth from saliva with cotton rolls and gently drying them to prevent contamination, the supragingival plaque was carefully removed without traumatizing the gingiva, as this would increase the production of gingival crevicular fluid. 52 The plaque-sampling investigator used a standardized protocol to collect specimens at all the three time intervals. Plaque specimens were collected from the labial surfaces immediately surrounding the orthodontic brackets with a sterilized dental scaler with the same tip dimensions (#8/9 Orban DE hoe scaler, Hu-Friedy, Chicago, Ill). Because the area of increased decalcification was generally immediately adjacent to the brackets, four passes, 1 each along the tooth at the bracket interface at the gingival, mesial, distal, and occlusal aspects, were made to avoid overloading the instrument tip. For the invisalign group a single circular stroke around the center of the clinical crown was made. All specimens from each tooth were placed into individual tubes with anonymous coding and sealed for transport to the laboratory. The coding of the specimens ensured blinding of laboratory personnel and help minimize experimental bias.