Ffc 1100

An additional four specimens for each CNC dilution (inoculated with S. mutans for 2 h and 24 h) were obtained as previously described. After rinsing with sterile PBS, specimens were placed into a freshly prepared Karnovsky’s fixative solution for 2 h (2% paraformaldehyde and 2% glutaraldehyde in 0.1 M sodium cacodylate buffer). After an additional rinsing in cacodylate buffer, the specimens were passed through a graded ethanol series (35, 50, 70, 80, 90, and 100 vol%). To remove the ethanol and, at the same time, minimize shrinkage, specimens underwent critical point drying (Critical-Point Dryer, EMS 850, Hatfield, PA, USA). After that, they were mounted on stubs with conductive tape, sputter-coated (JEOL FFC-1100, Tokyo, Japan), and observed with SEM (JSM 840A, JEOL, Tokyo, Japan) at 15 kV acceleration voltage in secondary electrons mode at a magnification of 500×–50,000×.

The specimen surface was observed before and after biofilm formation using scanning electron microscopy (SEM). A set of specimens (n = 3/group) not used for microbiological procedures was mounted on stubs with conductive tape, sputter coated (JEOL FFC-1100, Japan), and observed with SEM (Tabletop SEM TM3030 Plus, Hitachi, Schaumburg, IL, USA) at 15 KV acceleration voltage. Four randomly selected fields at two different magnifications (500X and 5000X) were recorded for each specimen.
Specimens undergoing SEM analysis after biofilm formation (n = 3/group) were placed into a cacodylate-buffered 2% glutaraldehyde fixative solution (pH = 7.4) for 48 h. The specimens were then passed through a graded ethanol series (50, 70, 80, 85, 90, 95, and 100%, v/v). Finally, they were subjected to critical point drying (Critical-Point Dryer, EMS 850, Hatfield, PA, USA), mounted on stubs with conductive tape, sputter coated, and observed with SEM at 15 KV acceleration voltage. Four randomly selected fields at two different magnifications (500X and 5000X) were recorded for each specimen.