Thoma cell counting chamber

hOBs that adhered to the discs were washed with 1X phosphate-buffered saline (PBS; EMD Millipore), followed by the addition of 200 µl trypsin/EDTA (1X; Gibco; Thermo Fisher Scientific, Inc.) for 3 min under aerobic conditions (37°C and 5% CO₂). The hOBs were then mechanically relocated from the discs with a pipette tip (Eppendorf). The solution with the osteoblasts was transferred to a 1.5 ml Eppendorf reaction tube (Eppendorf) centrifuged at 169 × g for 4 min at 4°C, and washed with 1X PBS. Quantification of the number of viable primary osteoblasts on the test samples was determined by trypan blue staining (Sigma-Aldrich; Merck Millipore). Trypan blue enters the damaged membranes of dead cells, leaving viable cells unstained. The viable cells were counted using a Thoma cell counting chamber, obtained from Paul Marienfeld GmbH & Co. KG (Lauda-Königshofen, Germany) according to manufacturer's protocol (19 ) and using an Olympus CKX41SF optical light microscope, (Olympus Soft Imaging Solutions GmbH, Hamburg, Germany). Measurements were performed at 2 and 7 days of co-culture.

Yeasts were grown aerobically at 37°C on Sabouraud agar with 0.4 g/l chloramphenicol and 0.04 g/l gentamycin (BD Diagnostics, Franklin Lakes, NJ, USA). All in vitro investigations were conducted on a third subculture of C. albicans ATCC 10231 (Oxoid; Thermo Fisher Scientific, Inc., Waltham, MA, USA) suspended in Sabouraud broth (cat. no. CM147; Oxoid™; Thermo Fisher Scientific, Inc.) or in distilled water. The suspension was adjusted to 1–20×106 blastoconidia per ml by dilution, following a blastoconidia count using a Thoma cell counting chamber (Marienfeld™, Lauda-Königshofen, Germany). Wild strains were isolated by swabbing from dentures and identified on the basis of their colony aspect on CHROMagar™ medium (BD Diagnostics), by chlamydoconidia formation on BT™ Rice Extract agar (BD Diagnostics) and by the API yeast identification system (bioMérieux, Marcy-l'Etoile, France).