Protocol 3: Sporulation in liquid medium (Codón et al., 1995)

Reference: Codón, A.C., Gasent-Ramírez, J.M. and Benítez, A. 1995. Factors which affect the frequency of sporulation and tetrad formation in Saccharomyces cerevisiae baker’s yeasts. Appl. Envir. Microbiol. 61, 630-638.

1. Transfer a single yeast colony into 4 ml YPD
   Incubate at 30°C up to early stationary growth fase (approx. 1 day)

1. Transfer preculture into 3 ml PRE5 up to initial OD₆₆₀ of 0,05 (30 µl of a cell suspension with OD₆₀₀ of 2;0)
   Incubate at 22°C up to mid-exponential fase of growth (OD₆₀₀ of 0,5 to 0,7)
   ! It is absolutely required to keep cells in early stage of exponential growth
2. Collect cells by centrifugation (5 min at 3500 rpm in Mistral 2000 centrifuge)
   Wash cells twice with sterile milli-Q water
   ! It is absolutely necessary to remove residual glucose completely by start of sporulation
   Resuspend cells into 3 ml SPO2 medium
   Incubate at 22°C for at least 4 days
3. Check presence of asci with phase contrast microscopy at maximal magnification

Pre-sporulation medium (PRE5)

• 0,8 % Yeast Extract (Gibco)
• 0,3 % Bacto Peptone
• 10 % D-glucose

Yeast extract + Bacto-peptone are autoclaved separately in 50 % of the final volume
Glucose is added from a 20 % stock solution.

Sporulation medium (SPO2)

0,5 % KOAc pH 6,5