Yeast Growth Protocols

Yeasts are considered model systems for eukaryotic studies as they exhibit fast growth and have dispersed cells. Moreover, replica plating and mutant isolation of yeast cells is done with relative ease, as they have a well-defined genetic system. Most significantly, yeasts have a highly versatile DNA transformation system that can be utilized effectively for protein production.

Yeast is commonly grown in an YPD medium. The “Y” in YPD refers to a yeast extract, containing the water-soluble compounds generated when yeast is forced to self-digest. “P” refers to peptone, a mixture of peptides and amino acids prepared by digesting animal protein with proteases. “D” refers to dextrose, or glucose, source of carbon for yeast. YPD contains essential nutrients to satisfy the metabolic requirements of cells. However, certain media requires a defined composition, to meet the application needs a variety of synthetic media was developed. Individual components of the synthetic media may be modified to suit the needs of an experiment.

• Growth Protocols for Yeast in Different Media
• Yeast Strain Preservation and Revival
• Yeast Cultures
  • Yeast Culture Media
  • Synthetic Drop-Out Medium Supplements
  • Other Supplements for Yeast Cultures

GROWTH PROTOCOLS FOR YEAST IN DIFFERENT MEDIA

The protocols listed below can be used for growing and maintaining yeasts in liquid and solid media:

Yeast Growth in YPD Broth

1. Suspend 50 g of YPD Broth (Product No. Y1375h) in 1 L of distilled water
2. Autoclave for 15 minutes at 121 °C
3. Inoculate yeast cultures (sourced from yeast broths/plates) in detergent-free tubes/flasks containing the prepared liquid medium. The medium should not be more than a third of culture tube volume or a fifth of the total flask volume
4. Vortex the contents briefly to disperse cells
5. Grow the culture in a shaking incubator at 300 rpm (for flasks) or 350 rpm (for culture tubes)

Yeast Growth on YPD Agar

1. Suspend 65 g of YPD Agar (Product No. Y1500) in 1 L of distilled water
2. Heat to boiling while stirring to dissolve all ingredients completely
3. Autoclave for 15 minutes at 121 °C
4. Pour around 25-30 mL of the agar medium on to sterile plates and let it set in a laminar flow chamber
5. Streak (yeast obtained from YPD plate)/spread (yeast sourced from broth cultures) cells on the agar plates and incubate at 30 °C

Note: Single yeast colonies may be observed after around 24 hours, but incubations over 48 hours are needed before they can be used for replica plating purposes. The growth rate of yeast cultures using synthetic drop-out medium supplements is ~50% slower.

YEAST STRAIN PRESERVATION AND REVIVAL

Yeast cells grown using the above methods can be stored frozen in glycerol at -70 °C for more than 3 years or in rich medium slants at 4 °C for 6 months to a year. YPD plates with yeast cells can also be stored at 4 °C for 2-3 months sealed with Parafilm®. The protocols for the preservation and revival of yeast cells are listed below:

Yeast Cells Preservation

1. Prepare a sterile 30% (w/v) glycerol solution (Product No. G5516)
2. Add 1.0 mL of the glycerol solution into 4 mL sterile screwcap vials
3. Take 1.0 mL of early stationary/late log phase yeast broth scrape a large inoculum from a freshly grown plate
4. Re-suspend in 1 mL of sterile 30% glycerol solution
5. Mix, freeze on dry ice, and store at –70 °C

Note: Yeast cells can also be stored similarly using 8% (v/v) dimethylsulfoxide (DMSO, Product No. D8418).

Yeast Strain Revival

1. Scrape frozen stock with a sterile toothpick or bacteriological needle
2. Streak yeast cells on the appropriate plate and incubate at 30 °C

Note: Avoid thawing the stock. In case the stock has thawed, vortex well before refreezing.

YEAST CULTURES

Yeasts are usually grown in YPD or synthetic media at 30 °C. We offer wide range of growth media and supplements for your specific application in both powder and liquid formats.

References

1. Sherman F. 2002. Getting started with yeast.3-41. https://doi.org/10.1016/s0076-6879(02)50954-x

2. MacDonald PN. 2001. Two-Hybrid Systems. https://doi.org/10.1385/1592592104

3. Treco DA, Winston F. 2008. Growth and Manipulation of Yeast. Current Protocols in Molecular Biology. 82(1): https://doi.org/10.1002/0471142727.mb1302s82

4. Schiestl RH, Gietz RD. 1989. High efficiency transformation of intact yeast cells using single stranded nucleic acids as a carrier. Curr Genet. 16(5-6):339-346. https://doi.org/10.1007/bf00340712

5. Li B, Fields S. 1993. Identification of mutations in p53 that affect its binding to SV40 large T antigen by using the yeast two?hybrid system. FASEB j.. 7(10):957-963. https://doi.org/10.1096/fasebj.7.10.8344494