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

Mutagenesis in Yeast 25
14. Conditional mif mutants (temperature-sensitive) can be isolated by the
same procedure, to step 8, after which the grids are replica plated to dupli-
cate YEPG plates and one plate is incubated at the desired restrictive tem-
perature and one at the permissive temperature.
15. Diploid colonies that do not grow at the restrictive temperature but do grow
at the permissive one are noted, and the corresponding haploid clones are
selected on the original grid, after which steps 12 and 13 are followed, to
make the final selection.
3.3.3. Antibiotic-Resistant Mutants (see Notes 10-12)
1. Recover the cells and dilute to lo3 cells/ml with sterile water. Spread 0.1 mL
of this suspension, or streak on plates of YEPD medium. Incubate at
28-30°C for 2-3 d.
2. Pick single colonies, to give as many subclones as the number of mutants
desired.
3. If it is desired to mutagenize the cultures with Mn*+, inoculate the
subclones into individual tubes of YEPD medium, and add 0.1 mL of
M&l2 solution to each tube. If mutagenesis with Mn*+ is not required, use
YEPG and omit the M&l2 solution. Incubate the cultures on a roller drum
operated at about 25-30 rpm, or on a slanted rack on a rotary shaker, for up
to 1 wk, at 28-3O*C.
4. Recover the cells from each tube and resuspend in 1 mL of sterile water.
Plate the cells on YEPG agar containing the antibiotic desired (chloram-
phenicol, erythromycin, oligomycin, or other).
5. Incubate at 28-30°C until resistant colonies appear. This may require at
least 1 wk, depending on the strain and on the antibiotic used.
6. Pick and restreak colonies on the same medium. Ideally, only one colony
should be taken from each yeast culture, to be certain that the mutations
are independent.
7. Grow the cells in 5 mL of YEPG at 28°C for 2 d. Then dilute m sterile
water to a density of lo3 cells/ml, and plate out on YEPG agar. This
ensures that the clone is a genuine resistant mutant.
8. Pick a single colony from each subclone and store on YEP-glycerol agar
containing the antibiotic.
3.3.4. Mutator Mutants (5)
These mutants show an increased rate of spontaneous mutation.
3.3.4.1. ISOLATION OF MUTATOR MUTANTS
1. Wash mutagenized cells thoroughly by centrifugation, about 4000g for
10 min, and resuspend in sterile water.
2. Spread on both media (high- and low-lysine).