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

Rare-Mating in S. cerevisiae 41
2.4. Cytoduction
1. Media as in Section 2.3.
2. A yeast strain carrying the karl-1 mutation and, preferably, other mutations
as markers, especially on adel or ade2 mutation to give a red color to the
colonies. This can be constructed (3), but it is usually quicker and easier to
obtain a strain from a private or public commercial collection.
3. YEP-glucose plates freshly inoculated with a killer-sensitive strain of 5’.
cerevisiae, so as to produce continuous lawns on incubation.
3. Methods
3.1. Obtaining Petites
by Acrifavine Treatment
1. Add one loopful of acriflavin solution to 5 mL of YEP-glucose broth, then
inoculate with the industrial yeast strain.
2. Incubate the culture without agitation at 30°C for 2-3 d in the dark, and
streak out a loopful on YEP-glucose agar.
3. Pick small colonies and test for respiratory deficiency: Do this by
subculturing similar sized inocula (sterile toothpick or loop) into
known positions on YEP-glucose and YEP-glycerol plates. Incubate c.
2 d at 30°C and compare the growth of the inocula on the two plates:
True cytoplasmicpetites are unable to grow on a nonfermentable (e.g.,
YEP-glycerol) medium. Petite cultures on YEP-glucose plates are very
white in color, whereas respiratory-competent wt cultures tend to be
creamy-colored.
3.2. Obtaining Petites
by Ethidium Bromide Treatment
(for Use in Transfer of Subcellular Organelles
by Cytoduction) (see Note 2)
1. Inoculate liquid YEP-glucose medium containing 20 pg/mL of ethidium
bromide, and grow at 3O”C, in the dark (vessels can be foil-wrapped, to
exclude light), as in the method for induction ofpetites with acriflavin, but
with agitation (4).
2. Reinoculate the culture into fresh medium containing ethidium bromide,
and reincubate.
3. Repeat step 2 to ensure that 100% of the cells are converted topetites.
4. Streak out the culture after 2-3 d and pick and test small colonies (see
Section 3.1.3.).