Feg vp supra 35

Scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM) were employed to visualize the ultrastructure of dual-species Candida biofilms treated with IONPs-CS-MCZ nanocarrier and controls. For this, biofilms were formed at the bottom of 24-well plates for SEM, and on sterile coverslips into 24-well plates for CLSM. Biofilm treatment was performed in the absence of an external magnetic field, as described above. For SEM preparation, the samples were serially washed in ethanol for dehydration (70% for 10 min, 95% for 10 min and 100% for 20 min), air-dried in a desiccator, and cut from the bottom of the plates. Samples were then positioned onto aluminum stubs before being coated with gold, and qualitatively analyzed by SEM (FEG-VP Supra 35; Carl Zeiss, Jena, Thüringen, Germany) [39] . As for CLSM analysis, the resulting biofilms were stained with 200 µL of a solution containing 3µg/mL SYTO9 green fluorescent dye and 3µg/mL propidium iodide for 20 to 30 min at room temperature, protected from light [42] . Biofilm samples were then gently rinsed with sterile water and analyzed under a confocal microscope (Nikon C2/C2si, Tokyo, Japan) at 488/500-570 nm for SYTO9 dye and 561/570-1000 nm for propidium iodide.
Fluorescent green and red colors represent living and dead cells, respectively.

The structure of single-and mixed-species biofilms was qualitatively evaluated by scanning electron microscopy (SEM). For this, the biofilms were formed in 24-well plates, during 48 hours, according to the protocol described above. Next, biofilm samples were processed as described by Monteiro et al, 22 and analysed in the FEG-VP Supra 35 electron microscope (Carl Zeiss, Jena, Thuringen, Germany).