• Microbiological analysis: Samples will be vortexed for 15 s, diluted, and measured aliquots of the dilutions will be cultivated in duplicate in 3 different culture media. Mitis Salivarius agar supplemented with sucrose and bacitracin will be used for counting of mutans streptococci . For isolation of lactobacilli, Rogosa selective Lactobacillus agar will be used. Counts of total colony forming units (CFU) will be obtained by culture in brain heart infusion agar supplemented with 5% sheep blood and enriched with k-hemin vitamin. The Mitis Salivarius will be incubated under CO 2 conditions at 37 0C for 48 h. Colonies of mutans streptococci will be counted based on their morphology and confirmed with catalase test. Rogosa SL Agar and the BHI will be incubated anaerobically at 37 8C for 72 h. The BHI will be also incubated aerobically at 37 8C for 48 h to determine the total aerobic counts (AE).