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.
Nucleocounter yc 100 system
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Sourced in Denmark
The Nucleocounter® YC-100™ system is a laboratory instrument designed for counting and analyzing cell populations. The system utilizes a specialized cartridge and fluorescent dye to determine the total number of cells in a sample.
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2 protocols using nucleocounter yc 100 system
1
Cultivating and Preserving Naumovozyma castellii
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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.
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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.
2
Passaging and Growth Assay of N. castellii
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N. castellii was previously called Saccharomyces castellii or Naumovia castellii. The N. castellii strains used in this study were the parental wild-type strain YMC48 and the derived YMC490-494 strains (Table 1 ). Strains were grown at 25 °C in YPD medium containing 1% (w/v) yeast extract, 2% (w/v) peptone and 2% (w/v) glucose or plates additionally containing 2% (w/v) agar, unless stated otherwise. Ura-dropout plates contained 26.7 g/L minimal SD base, 0.77 g/L ura-DO supplement and 2% agar.
Cell passaging was performed by streaking a single colony onto fresh YPD plates and, after 48 h incubation at 25 °C, single colonies were re-streaked onto new YPD plates. Re-streaking was repeated to obtain up to 16 subsequent passages. Each passage corresponded to 20–25 generations [16 (link),21 (link)].
Total cell counts were performed with the Nucleocounter® YC-100™ system (Chemometec, Allerød, Denmark) following the manufacturer’s instructions. For growth assays, 50 mL liquid YPD medium cultures were started at a cell density of 3 × 106 cells/mL from an overnight culture inoculated with a single colony. The cultures were grown at 25 °C, 200 rpm shaking, for 8 h. Measurements of cell density were taken directly after inoculation and then at two-hour intervals.
Cell passaging was performed by streaking a single colony onto fresh YPD plates and, after 48 h incubation at 25 °C, single colonies were re-streaked onto new YPD plates. Re-streaking was repeated to obtain up to 16 subsequent passages. Each passage corresponded to 20–25 generations [16 (link),21 (link)].
Total cell counts were performed with the Nucleocounter® YC-100™ system (Chemometec, Allerød, Denmark) following the manufacturer’s instructions. For growth assays, 50 mL liquid YPD medium cultures were started at a cell density of 3 × 106 cells/mL from an overnight culture inoculated with a single colony. The cultures were grown at 25 °C, 200 rpm shaking, for 8 h. Measurements of cell density were taken directly after inoculation and then at two-hour intervals.
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