Cultivating and Preserving Naumovozyma castellii

Naumovozyma castellii was previously called Saccharomyces castellii or Naumovia castellii. The N. castellii strains used in this study were NRRL Y-12630 (type strain), Y188 (wild type), YMC48 (MATα, hoΔ::hphMX4, ura3) and YMC481 (MATα, hoΔ::hphMX4, ura3, tlc1::kanMX3), ∼90 generations after the loss of telomerase (38 (link),39 (link)). Strains were exclusively grown at 25°C in YPD medium containing 1% (w/v) yeast extract, 2% (w/v) peptone and 2% (w/v) glucose. Single colonies from each strain were grown to mid-logarithmic phase in 50 ml YPD medium until the cell density reached ∼3.30 × 10⁷ cells/ml. Total cell counts were performed with the Nucleocounter^® YC-100™ system (Chemometec) following the manufacturer's instructions. The cells were harvested by centrifugation at 1500xg for 5 min, resuspended in 1 ml of 1× TNE (1 mM Tris–HCl, 10 mM NaCl, 0.1 mM EDTA, pH 7.4), 15% glycerol and stored in −20°C.