Adapted from the lab compendium Snowflake Yeast Protocols — Ratcliff Lab / MuLTEE (V2).
- Transfer 25 μL of culture to a 1.5 mL microcentrifuge tube.
- Macroscopic strains: sample using 100 μL wide-bore tips, then break clusters into small pieces using 100 μL regular-bore tips to facilitate single-layer crushing later (see Crushing clusters › Breaking up and crushing clusters: collected methods).
- Pellet clusters (5,000 × g, 1 min), wash with 1 mL 1× PBS.
- Stain in 100 μL of 5 μM Calcofluor White in 1× PBS at room temperature.
- Crush 5 μL of stained clusters into a single layer of cells between a slide and coverslip, without shearing.
- Image on a Nikon Eclipse Ti inverted microscope, brightfield and UV/fluorescent channels, ≥5 fields of view per strain at ×40 magnification. Set focus in brightfield to the plane where cells appear grey, then move down 0.7 μm for sharp fluorescent cell-wall signal.
- Cell volume is calculated as an ellipsoid: V = 4/3 × πab², where a and b are the major- and minor-axis lengths measured from segmented cells.
Image analysis pipeline (cell segmentation): same pipeline as Phenotyping & imaging › Measuring cell aspect ratio.