Goal 2: Microbial sampling Each specimen was diluted in 2 ml of phosphate buffered saline (PBS) dilute yeast extract and the samples will be sonicated for 10 sec before doing serial dilutions. Then 1 ml was submitted to tenfold serial dilution in 9 ml of yeast extract. After each serial dilution the test tubes were vortexed for 10 sec. Ten microlitre (10 μl) of supernatant was then plated onto Tripticase soy agar supplemented with 5% sheep bovine blood (BBL) for total bacterial evaluation and mitis salivarius-bacitracin agar specific for S. mutans. The samples were processed within 2 hours and the plates incubated for 24 hours at 37°C. The number of total CFU (Colony forming unit), specific to S. mutans was identified and counted by the blinded investigator which represented the total recoverable facultative flora.