YPD(A) – Yeast

Make YPD growth medium

There are five questions in this handout

This protocol describes how to make 0.5 liters of Yeast extract Peptone Dextrose (YPD) medium. The recipe yields 1% Yeast extract, 2% Peptone, and 2% Dextrose. For solid media, 2% agar is added, yielding a YPDA media.

Each team is responsible to provide media throughout the semester in support of the research projects.

Do not use media made by others — be a good lab citizen!

Background

YPD, also known as YEPD, is a complete medium used to grow Saccharomyces yeast. Researchers use it to support liquid culture, and if agar is added, YPDA is used to provide solid medium for growth of colonies.

Follow general chemistry methods: solutes (eg, the media components) are added to the solvent, not the other way around. Also relevant, basic volumetric preparation. For example, if the final volume will be 1-liter, always add the media power to 80-90% volume (800 – 900) mL (the “wetting ratio”), mix to dissolve, then q.s. to final volume of 1-liter — quantum satis, q.s., meaning add water to bring the final volume up to the 1-liter mark.

Note: if agar is added, it won’t dissolve in solution at room temperature.

Materials needed

Assume 2-4 students per group, 5 groups/stations

For each station:

  • balance, 0.1g
  • stir plate and stir bar
  • 0.5L Corning or Kimax Bottle sterile, with cap
  • Assuming bottle is Pyrex (Corning brand), or related heavy borosilicate glass,known to be thermal shock resistant.
  • Graduated Cylinder (500 – 1000 mL)
    weigh boat and spatula

For whole class:

  • YPD Broth
  • Aluminum foil
  • Type 1 water (eg, Barnstead Nanopure Diamond TOC)

Sterilize media bottles (microwave protocol)

  1. If not already sterilized, rinse glassware thoroughly with tap water.
  2. Rinse again with ~100 mL Type 1 water
  3. Add ~100 mL Type 1 water and place bottle into microwave (without cap)
  4. Set timer to 10 minutes and microwave bottles at full power.
  5. Remove bottles from microwave (HOT!) and dispense whatever water remains.
  6. Place cap loosely onto the bottle. Cover caps with aluminum foil and return to storage.

The preferred method to sterilize is by autoclave.

YPD Protocol

  1. Label sterile bottles:
    • YPD
    • Your initials
    • Date
    • __XX_ Sterile filter
  2. Measure about 400ml Type 1 water and add to 1 liter bottle. Add stir-bar, place the bottle onto the stir plate, and begin stirring at medium speed. Leave the heater off.
  3. For 0.5 liter of media, use 25g YPD (5g Yeast Extract, 10g Peptone, 10g Dextrose). Add the ingredients to the water in the beaker and continue to stir
    • Do not cross-contaminate our stocks by using the same spatula for the different components.
    • Do not contaminate our stocks by returning unused reagents back to the stock bottle. Dispose of excess properly.
  4. Dissolve the ingredients, then pour solution into the graduated cylinder and bring the volume up to 500ml using Type 1 water. Remove and clean the stir bar.
  5. Next steps depend on how you are going to sterilize the media.
    • For autoclave, pour the mixture into a 1 liter bottle, loosely fit the cap to the bottle, cover the cap with aluminum foil, and then add autoclave tape. Proceed to sterilize per autoclave instructions.
      • Note that many folks do not autoclave media containing dextrose — instead they autoclave everything else, then add filtered glucose solution (2% w/v in media; e.g., add 25 mL of 40% dextrose).
    • For vacuum filter sterilization, attach the Nalgene filter assembly (VWR cat no. 28199-303) to a sterile 0.5 liter  bottle, attach the hose to the vacuum, and increase vacuum pressure. Pour the mixture into the filter assembly and continue until the media is in the bottle. Upon completion, turn off the vacuum line, remove the filter assembly (discard), and replace the cap on the bottle.
  6. Reserve a small amount of sterile media to test for contamination. Place in culture tube or Petri dish at 30 °C or at other culture conditions relevant to the experiment.

YPDA Protocol

If making plates, then you need to add agar to YPD. To above, add 10g agar

Alternative Sterilization Procedures

If the autoclave is not available, culture media may be sterilized by use of microwave oven (Baqai and Hafiz 1992, Prijana et al 2016). The procedure I have used is to fix the media as above, but leave the bottle uncapped. Place up to 3 bottles in the microwave and set the timer for 10 minutes and a power setting of 50%. Media temperature rapidly reaches above 50 °C (via infrared surface thermometer) with no boil over. Cap the bottles after heating, and store at 4 °C.

Questions

Question 1. What differences, if any would you expect among the three sterilization media as support for yeast growth?

Question 2. Dextrose is just another name for table sugar, correct?

Question 3. What is “yeast extract” and what does it provide growing yeast cells?

Question 4. What is “peptone” and what does it provide growing yeast cells?

Question 5. What is the concentration, w/v, for Yeast extract __________ ? For Peptone ___________ ?

References

Baqai R, Hafiz S. (1992). Microwave oven in microbiology laboratory. JPMA 42: 2.

Border, B. G., & Rice-Spearman, L. (1999). Microwaves in the laboratory: effective decontamination. Clinical Laboratory Science, 12(3), 156.

Prijana, C., Mulyana, Y., & Hidayat, B. (2016). Roles of microwave oven in preparing microbiological growth media. Althea Medical Journal, 3(1), 1-5.

/MD