Propidium iodide

Single and mixed biofilms were developed as described above on 12-well polystyrene plates covered with a sterile coverslip (Velab, Mexico City, Mexico).
Coverslips were recovered and placed in contact with a mixture of fluorochromes. The following fluorochromes were applied: calcofluor white 1 g/L (Sigma-Aldrich St. Louis, MO, USA) for chitin; 10 mM FUN®1 (Life Technologies, Gaithersburg MD, USA) for metabolic activity; 1 mg/mL concanvalin A (conA) (Sigma-Aldrich St. Louis, MO, USA) for glucose and mannose residues; and 1.5 μg/mL DAPI (Vector Laboratories, CA, USA) and propidium iodide (PI) 10 mg/mL (AbD Serotec, Raleigh, NC, USA) for nucleic acids. Samples were observed under CLSM (Carl Zeiss, Germany) with filters: 480-530 nm (FUN®1), 360-460 nm (DAPI), 543-560 nm (PI), 355-433 nm (calcofluor white), and 495-519 (conA). Images were processed with Zeiss LSM Image Brower ver. 4.0 software (Carl Zeiss, Germany).

For the detection of biofilm component by epifluorescence microscopy (EFM), HLFCs were grown over sterile glass coverslips (Velab, Mexico City, Mexico) on 12-well plates. Cells were infected as previously described; fungal biofilm, bacterial biofilm, and mixed biofilm were included. After a biofilm maturation period of 24 h, biofilms were stained with calcofluor white (CW) (1 g/L) (Sigma-Aldrich, St. Louis, MO, USA) for the detection of chitin and N-acetylglucosamine, with Concanavalin A-Alexafluor 488 (ConA) (1 mg/ml) (Sigma-Aldrich St. Louis, MO, USA) for the detection of glucose and mannose residues, while propidium iodide (PI) (10 mg/ml) (AbD Serotec, Raleigh, NC, USA) was used to stain nucleic acids and extracellular DNA. The stained cells were observed under an epifluorescence microscope (Imager.Z2, Apotome 2.0, Carl Zeiss, Germany) at the following wavelengths: CW (λ_Excitation = 355 nm; λ_Emission = 433 nm); ConA (λ_Excitation = 495 nm; λ_Emission = 519 nm); PI (λ_Excitation = 535 nm; λ_Emission = 617 nm). Images were analyzed with the LSM Image Brower version 4.0 software (Carl Zeiss, Germany).