Morphological Analysis of Candida Planktonic and Biofilm Cells

Morphological characterization of planktonic cells and biofilms grown in the four media was performed. To this end, ON cultures were grown in the four culture media at 30°C, the standard temperature for yeast growth in laboratory settings, and at 37°C, the natural body temperature. Moreover, the effect of FBS 10% supplementation in planktonic cultures incubated at 37°C was also tested. Cells were recovered and washed twice with PBS (4000 rpm x 5 min). PBS-resuspended yeasts were imaged under a 100x magnification with a Nikon inverted fluorescent microscope ECLIPSE Ti–S/L100 (Nikon, Japan) coupled with a DS-Qi2 Nikon camera (Nikon, Japan) in Bright Field.
On the other hand, C. parapsilosis biofilms formed on the fed-batch condition (ALI and Bottom zone) and the continuous flow system were visualized via chitin staining with 10 µM Calcofluor white (CW) (Biotium, USA) under a Zeiss LSM 800 confocal scanning laser microscope (CSLM). Images were processed and measured using ImageJ Fiji software. Briefly, the area and the Length-to-Width Ratio (LWR, denoted as aspect ratio in ImageJ) were calculated from randomly chosen cells (n=30) from planktonic and biofilm conditions. Pseudohyphae were defined as forming chains of cells with box-shaped ends and LWR greater than three, and blastospores as cells oval-shaped with a lower LWR (Rupert and Rusche, 2022 (link)).