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Method: Long Term Storage of Yeast Stocks
August 29 1990
J. Howe and C. Helms
Yeast strains may be stored indefinitely at low temperatures (-80 degrees C). It is lab policy to prepare a frozen stock of newly acquired or created strains for the archives as soon as possible after beginning to work with it. Two archiving methods are presented below. In Method A , the cells are grown on a plate, while in Method B the cells are grown in liquid culture.
2 days to grow the cells; less than 5 minutes bench time for each strain.
Method A Procedure:
- On a YPD (or selective medium) plate, streak the cells (from an isolated colony) in one solid line across the plate. You will need enough cells to load the rounded end of a toothpick after the streak has grown up. Several different strains may be streaked onto the same plate; label the back of the plate appropriately. Invert the plate and incubate at 30 degrees C for 2 days.
- For each strain to be stored, prepare a sterile, labeled cryo-vial, containing 1 ml sterile deionized water and 225 µl 80% glycerol (15% glycerol, final concentration). YPD or selective medium may be used instead of water; it is the glycerol concentration which is more important.
- Using the wide, rounded end of a sterile toothpick, scrape up as many yeast cells from the grown streak as can be 'loaded' onto the toothpick. Transfer the cells to the cryo-vial and scrape off as many as possible onto the inside of the prepared cryo-vial. Cap the vial, and shake (vortex if necessary) to suspend the cells evenly within the 15% glycerol solution. Immediately transfer the vial to the -80°C freezer. Yeast cells will settle to the bottom quickly if left too long on the bench, so if preparing many strainsd for long-term stoage, vortex them all again just before putting them into the -80 degrees C freezer.
To recover a strain from the glycerol stock, use a sterile toothpick to scrape some of the ice, then streak out the cells on a medium e.g., YPD agar plate. Do not thaw the frozen stocks, because each freeze-thaw cycle will result in a 50% loss in cell viability.
Method B Procedure:
- Pick an isolated yeast colony from a plate; inoculate into 5 ml YPD in a snap-cap tube and grow overnight at 30 degrees C on roller drum.
- Pipet 812 µl of the yeast culture into a 2 ml cryo-vial. Add 188 µl of 80% sterile glycerol, vortex. Place vial in -8 degrees C freezer (cells lose viability when stored above -55 degrees C).
To revive stocks: Take a sterile toothpick and place into stock in -80šC freezer; streak out onto YPD or AHC plate and incubate for 24-48 hours at 30šC. Alternatively, inoculate into 5 ml AHC or YPD liquid culture and place on roller drum in the incubator for 24 (YPD) to 48 hours (AHC).
Sherman, F., Fink, G. R., and J. B. Hicks. (1986) The Laboratory Course Manual for Methods in Yeast Genetics. Cold Spring Harbor Press, Cold Spring Harbor, NY. p. 179.