2.3. Yeast Cell Growth Assay

Wild-type BY4741 yeast cells were pre-grown overnight in SRaf then diluted in fresh SRaf medium to density 5 × 10⁵ cells/mL. Cells were grown to 1 × 10⁶ cells/mL then shifted to MM/Gal for proliferation assay under various conditions. The growth conditions presented above were applied to WT for the sake of uniformity, as strains expressing MTs had to be grown in media containing galactose as carbon source, for transgene induction. Yeast expressing recombinant MTs were grown overnight in SRaf-Ura and inoculated in fresh MM/Raf-Ura to 5 × 10⁵ cells/mL. Cells were grown to 1 × 10⁶ cells/mL. At this point (considered time 0), cells were harvested, and shifted to minimal media containing galactose (MM/Gal-Ura) for transgene induction. Cell growth in liquid media was monitored by measuring culture turbidity at 660 nm (OD₆₆₀₎ [22], recorded by a plate reader equipped with thermostat and shaker (Varioskan, Thermo Fisher Scientific, Vantaa, Finland). When used, surplus metal ions were added from sterile stocks of ultra-pure 0.1 M AgNO₃ or 0.1 M CuSO₄. Unless otherwise stated, all incubation was done with agitation (200 rpm) at 30 °C. Before shifting to galactose-media, the cell viability was checked by staining with methylene blue (Roth, Karsruhe, Germany) and only populations with viability >99% were used further.

For growth on solid media, the transformed cells pre-grown in SRaf-Ura were shifted to SGal-Ura and grown for 6 h for transgene induction. Cells were 10-fold serially diluted in a 48-well microtiter plate and stamped on SGal-Ura agar plates using a pin replicator (approximately 4 μL/spot). Plates were photographed after incubation at 30 °C for 3 days.

The cell viability, expressed as percentage of live cells within a whole population, was assessed by staining with methylene blue. Viability was examined for at least 300 cells from one biological replicate. Viable cells were colorless, and dead cells were blue. Original cell suspensions (grown in normal medium) had a viability higher than 99%. Lethal concentration 50 (LC₅₀) represents the Ag(I) concentration eliciting a 50% reduction in cell proliferation after 24 h of incubation in the presence of various Ag(I) concentrations and it was determined by probit analysis for each strain.