2.6.3. SYTO 9/PI staining of live and dead bacteria

The size of the specimens adhered to the dimensions specified in Section 2.5.1. Specimens from each group were positioned in 48-well plates, with each well receiving 500 μL of a working bacterial suspension of S. mutans and E. coli for a 24-hour incubation period. Subsequently, the bacterial solution from each well was aspirated and the specimens were rinsed with 0.85% NaCl solution. Specimens from each group were then transferred to centrifuge tubes, combined with 3 ml of 0.85% NaCl solution, and the bacteria from the specimen surfaces were collected following an ultrasonic shock for 5 minutes. Bacterial solutions for each group were centrifuged at 10000 g for 10 minutes; thereafter, the supernatant was discarded, and 200 μL of staining solution was added to suspend the bacterial pellet, followed by incubation in the dark at room temperature for 15 minutes. Finally, 5 μL of the bacterial solution was extracted from each tube, applied to a slide, and observed via a fluorescent inverted microscope.