Adapted from the lab compendium Snowflake Yeast Protocols — Ratcliff Lab / MuLTEE (V2).
Measuring ploidy (propidium iodide DNA staining)
- Grow the strain of interest in YPD to mid-log phase; transfer 250 μL culture to a 1.5 mL tube.
- Pellet cells (5,000 × g, 1 min), wash with 1 mL H₂O.
- Macroscopic strains: break clusters into small pieces by vigorous pipetting with 100 μL regular-bore tips before washing.
- Fix/permeabilize in 1 mL 70% ethanol, room temperature, 2 h, end-over-end rotation (max speed).
- Wash twice with 1 mL 50 mM sodium citrate.
- Resuspend in 200 μL 50 mM sodium citrate + 0.5 mg/mL RNase A; incubate at 37 °C for 2 h, inverting gently every 30 min.
- Add 5 μL of 1 mg/mL propidium iodide; incubate at 30 °C overnight in the dark, gentle rotation (minimum speed).
- PI-stained clusters may be stored at 4 °C for up to 1 week before imaging.
- Crush 5 μL of stained clusters into a single cell layer between slide and coverslip for imaging.
- Image at ×20 magnification, red fluorescent channel (600 ms exposure, 2.2× gain) at the focal plane plus one z-plane 0.3 μm above and below (3 planes total, later max-projected), plus a brightfield image at the focal plane (100 ms exposure, 4.1× gain). Set focal plane in brightfield, then move down 1 μm for nuclear signal.
- Calibrate exposure/gain so the brightest pixels in a tetraploid control strain are ~80% of maximum pixel value.
- Run known-ploidy controls (e.g., engineered “grande” diploid and tetraploid snowflake yeast strains) through the same procedure alongside samples in every experiment.