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

Mutagenesis in Yeast 23
7. Place the samples m a series of water blanks, and when the irradiation
procedure is complete, dilute the samples appropriately, to give 100-150
cells m 0.25 mL, for spreading on the YEPD plates. (Record all dilutions.)
Alternatively, the samples may be placed in tubes or flasks of YEPD
broth and incubated for 2-3 h, to allow a round of cell division to take
place, to fix the mutations, and the culture may then be diluted and plated.
8. Incubate the plates at 25-3O”C, for 4-5 d, until the colonies have grown well.
9. Screen the colonies for the desired mutant phenotype. For example, to iso-
late auxotrophic mutants, replica plate the colonies to minimal medium
and again incubate. Retain the master plates. Examine the plates of mini-
mal medium and look for colonies that have not grown. Locate these colo-
nies on the master plates, pick and restreak, and repick to complete media
and to dropout and other diagnostic media as desired.
3.2. General Method for Mutation Induction
Using Chemical Mutagens (see Note 3)
1. Prepare culture as for UV nradiatlon (see Section 3.1.2.).
2. Add desired mutagen (ethylmethane sulfonate, EMS; methyl methane
sulfonate, MMS; MNNG, nitrous acid, NA) typically, to 3% v/v.
3. Incubate for desired time (1MO min).
4. If EMS, MMS, or MNNG are used, dilute the culture into 5% thiosulfate solu-
tion to stop the reaction. For NA, dilute into 0. Wphosphate buffer (pH 7.1).
5. Make dilutions, plate out, and isolate mutants as for mutagenesis by irra-
diation, accordmg to phenotype (see Notes 4-6).
3.3. Mitochondrial ‘Mutations: Petite Mutants
(see Notes 7-9)
3.3.1. AcrifZavin-Induced Petites
1. Add one loopml of the acriflavin solution to a tube of YEPD broth and mix.
2. Inoculate with a light inoculum of the desired yeast strain. This may be hap-
loid, diploid, or of higher ploidy, or an industrial yeast of unknown ploidy.
3. Incubate m the dark at 3O”C, without agitation.
4. After 2-4 d, streak culture out on a plate of YEPD agar and incubate at the
same temperature for 3-5 d.
5. Pick small colonies (restreak if desired) to YEPD and YEPG agar, and
again incubate. Retain those colomes that do not grow on glycerol as the
sole carbon source.
6. Ethidium bromide-induced petites may be induced using the same pro-
cedure, using a final concentration of 20 pg/mL of ethidium bromide m
the medium.