4.6. Biofilm Inhibition and Eradication Evaluation by Confocal Microscopy

For biofilm inhibition and eradication, 8-wells µ-Slide assays plates (ibidi, Gräfelfing, Germany) were used. Cell concentrations and incubation times were determined by the biofilm formation assay described in 4.5, but with 10⁴ CFU/mL for C. albicans ATCC 90028 and 10⁷ CFU/mL for the clinical isolate, and an incubation time of 72 h. To perform the inhibition assays, 300 µL of each cell diluted in RPMI with 2% glucose was treated with Ctn[15–34] at the MIC, 10 × MIC, and 100 × MIC. For the eradication test, once the biofilm was formed, peptide was also added at the MIC, 10 × MIC, and 100 × MIC. After treatment, microplates were incubated for 24 h, at 37 °C. All plates were washed 10 × with sterile 10 mM HEPES, 150 mM NaCl, pH 7.4. To quantify peptide action on cells, the Live/Dead FungaLight Yeast Viability Kit (Life Technologies, Carlsbad, CA, USA) was used. Optical microscopy measurements with an inverted Zeiss LSM 710 confocal point-scanning microscope (Jena, Germany) were carried out in order to examine the architecture and the viability of Candida cells before and after exposure to antifungal agents. Argon (488 nm; 45 mW) and diode-pumped solid-state (561 nm; 15 mW) lasers were used with a 40 × dry-objective. After incubation, biofilms were stained with 2 µL SYTO 9 and propidium iodide (PI) dye probes (1:1) and incubated for 15 min. Images were acquired and analyzed with ImageJ 1.47v (rsbweb.nih.gov/ij/).