Isolation and Identification of Yeasts from Natural ... - Library Science

12 Spencer and Spencer
3.2.2. Storage Under Liquid Nitrogen (3)
1. Ampules:
a. Mix equal quantities and the glycerol solution (or other cryoprotectant)
in a sterile tube, so that the final concentration of glycerol is 5% v/v.
Transfer 1 mL of the mixture to each of the ampules.
b. Freeze the preparations in a domestic freezer or cooling bath, to -30°C,
at a rate of about S”C/min, and allow to dehydrate for 2 h.
c. Transfer the frozen ampules, without thawing, to the liquid nitrogen
refrigerator. Use the chilled Dewar flask if necessary.
d. Maintain the level of liquid nitrogen to where the ampules are com-
pletely submerged. Shelf life is up to 4 yr.
e. Cultures are revived by rapid thawing in a waterbath at 35”C, with shaking.
2. Storage in straws:
a. Inoculum and cryoprotectant are prepared as in step 1 above.
b. Filling of straws. Inoculum and cryoprotectant are mixed as m Section
3. One straw is taken out of the Petri dish with sterile forceps and filled
with moculum mixture using a Pasteur pipet. Place the tip of the pipet
close to the bottom of the straw, and fill the straw to within 3 mm of the
upper end (about 0.1 mL of inoculum mixture).
c. Heat seal the straw as described m Section 2.
d. Freeze the straws in ampules as described in Section 3.
e. Store in the liquid nitrogen refrigerator as before.
f. Revival: Thaw in a water bath at 35OC as before, resuspend the cells by
squeezing the straws, wipe the straws with 95% v/v alcohol and cut off
the end with sterile scissors.
g. Make sure the cells are all resuspended, by repeated pipeting, and trans-
fer two drops to 0.5 mL of sterile water, giving an approx 1: 10 dilution,
Plate out and make isolations as required.
3.2.3. Storage by Freezing in Glycerol Solution (3)
1. Take an aliquot of the inoculum and make up to a glycerol concentration
of 15-50% v/v. Different workers prefer different concentrations of glyc-
erol in the final mixture.
2. This mixture is transferred to a small sterile culture tube (2-5 mL), and is
then slanted and frozen at -70°C, and stored at this temperature.
3. Routine transfers are made by scraping a little of the culture from the sur-
face of the frozen medium, and transferring to fresh medium.
4. If the freezer should fail, the culture can be transferred and a fresh subcul-
ture frozen later.
5. Survival (shelf life) is for several years, cultures stored at -70°C surviving
longer than those kept at -2OOC. Several tubes of each strain should be stored.