Protocols >

Yeast Bud Scar Staining with Calcofluor

(protocol from Sandy Vandoninck)

• Grow cells overnight under the desired conditions.
• Subculture cells (1:10) in fresh media, grow up cells for 4 hours.
• Fix cells with formaldehyde (final concentration 3.7%) for 30 minutes at growth temperature.
• Aliquot 200 µL of cells into eppendorf tubes, add 20 µL calcofluor (stock at 1mg/ml in water) to each tube. Incubate for 30 minutes at growth temperature.
• Wash cells 2x with water, maintaining samples in the dark.
• Take up cells in 200 µL water.

Note: Cells can be kept for about one week in the cold room in the dark.

The criteria we use for determining budding pattern is:

• Count only cells with 4 to 6 bud scars
• Bipolar: at least 2 scars on opposite ends of cell
• Unipolar: 1 scar on one end, 3 or more on opposite end
• Axial: 4 or more scars grouped together on one side
• Random: scars occuring randomly and ungrouped around cell