SEM of single- and mixed-species biofilms.

For SEM analysis, S. aureus was grown in single species biofilm with and without exogenous supplementation of purified matrix material (0.5 mg/ml) or glucan (0.25 mg/ml) or with C. albicans in mixed biofilms, on coverslips for 48 h. Coverslips were washed twice with PBS and then fixed in 2% paraformaldehyde, 2.5% glutaraldehyde in phosphate buffer, pH 7.4, for 1 h at room temperature and then at 4°C overnight. Following initial fixation, specimens were washed in three changes of 0.1 M PBS for a total of 30 min, postfixed with 1% osmium tetroxide in PBS for 1 h, and washed again in three changes of buffer. Dehydration of specimens was done using a series of graded ethyl alcohol, 30%, 50%, 70%, 90%, and 100% for 10 min each, and two more changes of 100% ethyl alcohol. Specimens were then chemically dried by immersing them sequentially in 2 parts 100% ethyl alcohol-1 part hexamethyldisilazane (HMDS) (Electron Microscopy Sciences, Fort Washington, PA) for 10 min, 1 part 100% ethyl alcohol-1 part HDMS for 10 min, 1 part 100% ethyl alcohol-2 parts HDMS for 10 min, and then 2 changes for 10 min each with 100% HDMS. Specimens were air dried in a hood overnight, mounted on SEM pin mounts, and sputter coated with 10 to 20 nm of platinum-palladium in a sputter coater (EMS 150T ES). SEM images were captured using a Quanta 200 scanning electron microscope (FEI Co., Hillsboro, OR).