2.5. Saliva Collection and Microbiological Analysis

Each patient initially chewed one piece of a 3 × 3 cm plastic film (Parafilm®) for 60 s to stimulate saliva production and release the bacteria from the dental biofilm. Saliva was collected using a plastic device and stored in sterile microcentrifuge tubes (Eppendorf®), which were stored in a polystyrene box containing ice and analyzed in the microbiology laboratory within 2 hours of collection.

The saliva from each patient was collected in two moments (baseline and 28 days after starting treatment). Participants were instructed not to eat or drink at least 2 hours before saliva collection, and the samples were collected under the same conditions operated between 9 : 00 and 11 : 00 a.m, so that the circadian influence was minimized.

A volume of 0.1 mL of each sample was transferred to a sterile hemolysis tube containing0.9 mL of saline. This procedure was repeated twice, establishing dilutions of 1 : 10 and 1 : 100. A volume corresponding to 10 μL of each dilution was seeded in Agar mitis bacitracin (MSB) and MacConkey Agar in triplicate for evidence of S. mutans and Gram-negative bacteria, respectively.

The plates were incubated at 37°C for 48 hours in microaerophilic jars and placed in an oven. Colonies with Gram-morphological characteristics were then counted after this period. Bacteria were expressed as CFU/mL of saliva.