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

Mutagenesis in Yeast 29
2. To 3 mL molten 0.6% agar at 50°C, add 2 mL APE solution, mix, and pour
over an individual plate.
3. When the agar has solidified (10 min), gently pour over 3.5-4 mL of GBC
solution. Within a few minutes, wild-type colonies turn red, whereas
mutant colonies turn red more slowly.
4. Pour off the GBC solution as soon as the colonies start to turn red.
5. Stab mutant columns with a sterile toothpick, streak on YEPD, and retest.
3.3.10. Membrane Mutants (12)
These include inositol-requiring mutants, inositol-secreting mutants,
and sterol-requiring mutants, and are obtained by standard methods for
mutagenizing the desired yeast strain. Inositol-secreting mutants may be
detected by replica plating the colonies, after mutagenesis, to an inositol-
deficient medium covered with a lawn of an inositol-requiring, adel or
ade2 strain. Colonies secreting inositol are surrounded by a halo of red
satellite colonies. Inositol is a precursor of a number of membrane phos-
pholipids, essential for metabolism and growth of yeasts.
3.3.10.1. INOSITOL-REQUIRING MUTANTS
1. Dilute suspension appropriately and spread an amount sufficient to give
100-l 50 colonies per plate, on several plates of YBP-glucose agar.
2. Incubate at 30°C for 3-5 d.
3. Replica plate to minimal medium with and without inositol, and incubate for L7 d.
4. Select colonies growing on minimal medium with inositol but not on mini-
mal medium without inositol.
3.3.10.2. INOSITOGEXCRETING MUTANTS (SEE NOTE 21)
1. Spread mutagenized, diluted yeast strain on YEP-glucose agar as before,
and incubate for 3-5 d at 30°C.
2. Spread a lawn of the indicator strain on plates of the minimal medium
and let dry.
3. Replica plate the colonies from the master plate (YEP-glucose) to the plates
having the lawn, Incubate for 3-7 d at 3OOC.
4. Look for white colonies on the selective medium, surrounded by a red
halo, formed by the cells of the inositol- and adenine-requiring mutant.
3.3.10.3. STEROL-REQUIRING MUTANTS (SEE NOTES 22-24)
Sterol-requiting mutants (13) are obtained by mutagenizing with UV-light,
and selecting for nystatin-resistant colonies that are temperature-sensi-
tive at 36”C, by selecting for strains resistant to nystatin (15-28 mg/L) in the
presence of cholesterol (40 mg/L).