Maheshri:Liquid Sporulation

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Sporulation in liquid media

Douglas A. Treco and Fred Winston, "Growth and Manipulation of Yeast" Curr Protoc Mol Biol. 2001 May;Chapter 13:Unit13.2.

1. Pick a single colony of the diploid and grow it overnight in YPD.
2. Inoculate 3 ml of fresh YPD with the overnight culture, diluting the cells ~50-fold.
3. Grow the culture at 30°C until the cells are at a concentration of 1–2 × [math]\displaystyle{ 10^7 }[/math] cells/ml.
4. Centrifuge the cells 5 min at 1200 × g and wash twice with sterile water.
5. Resuspend the cells in 2 ml liquid sporulation medium and transfer to a small glass tube. Liquid sporulation medium contains 1% (w/v) potassium acetate.
6. Add any required amino acids to the sporulation medium and incubate the tube with shaking for 3 days at room temperature. For many strains, the cultures can be moved to 30°C after the first day to speed up sporulation.

If a strain appears refractory to induction of sporulation, try pregrowing cells in YPA medium. The use of acetate as a carbon source requires respiration, which is a requirement for sporulation. This requirement for respiratory competence is reflected by the fact that cells that have lost mitochondrial function are unable to sporulate.