Method: Long Term Storage of Yeast Stocks

August 29 1990

J. Howe and C. Helms

Purpose:

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.

Time required:

2 days to grow the cells; less than 5 minutes bench time for each strain.

Method A Procedure:

Day 1

1. 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.
2. 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.

Day 3

1. 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:

Day 1

1. 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.

Day 2

1. 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).

References:

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.