1. Grow cells in 50 ml YEPD medium until late log phase (OD₆₀₀ = 0.4~1.0).

2. Harvest cells by centrifugation.

3. Wash cells with 20 ml sterile dH₂O.

4. Wash cells with 20 ml 1 M sorbitol.

5. Resuspend cells in 20 ml of 1 M sorbitol, 10 mM sodium phosphate (pH 7.5), 10 mM EDTA, 0.1% beta-mercaptoethanol.

6. Add 1000 units zymolase.

7. Mix and incubate at 30 °C.

8. Check spheroplast formation under microscope. Check cells round up or add 5-10 ml cells to one drop of 1-5% SDS on slide. Speroplasts will lyse in SDS solution.

9. Centrifuge for 5 minutes at 500 g to pellet spheroplasts.