Preparation of mass spores of yeast

Reference:

Rockmill, B., Lambie, E.J., and Roeder, G.S. (1991). Spore enrichment. Methods Enzymol. 194, 146-149.

Ian W. Dawes, and Ian D. Hardie (1974). Selective killing of vegetative cells in sporulated yeast cultures by exposure to diethyl ether. Molec. Gen. Genet. 131, 281-289.

This protocol usually will result in an enrichment for spores over unsporulated cells by at least a factor 1000.

1. Starting with liquid sporulation medium: Collect cells by centrifugation
   (5 min at 3,500 rpm in Mistral 2000 centrifuge)
   Starting with solid sporulation medium: Collect cells from plate by washing with 1 ml of sterile water; transfer cell suspension into vial, and harvest cells by centrifugation
2. Discard supernatant
   Resuspend cell pellet in 1 ml of sterile water
3. Add 50 µl of Yeast Lytic Enzyme solution (0,1 g/ml)
   Incubate at 37°C for 1 hour in shaker
   Vortex cell suspension to get spores free from the asci
4. Optional: Dilute cell suspension (100 x)
   Plate 100 µl out onto (selective) YPD in glass petri dishes
5. Place a filter paper (approximately 4 x 4 cm) in the inverted lid of each petri dish
   Apply approximately 1 ml of diethyl ether to the filters
   Place petri dishes inverted in a glass box
   Place also a small beaker with ether in the box
   Cover the box with a glass plate
   Leave the box for 15 min in the fume hood (at room temperature)
6. Reapply 1 ml of ether to the filters
   Incubate for a second 15 min in the fume hood
7. Remove the filters
   Place petri dishes with lids ajar in the laminar flow hood for 30 min, until the ether odour is gone
8. Incubate petri dishes at appropriate temperature until colonies appear

Yeast Lytic enzyme

From Arthrobacter luteus (ICN) - + 70,000 U/g
Stored at -20°C