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

Mutagenesis in Yeast 35
9. Obtaining spontaneous petites by starvation: Cultures are inoculated into 5
mL of Schopfer’s medmm (containing, per L, 30 g glucose, 1 g asparagine,
1.5 g KHzP04 , and 0.5 g MgS04*7H20), in culture tubes or small bottles, and
incubated 6-10 d at 30°C. The cells are then recovered by centrifugation and
plated on YEP-glucose agar. Small colomes are picked to YEP-glucose and
YEP-glycerol agars, and any petite mutants found are isolated and retained.
4.4. Antibiotic-Resistant Mutants
10. These mutants usually occur spontaneously at a relatively high frequency
and mutagenesis is not necessary. However, mutagenesis with M.n2+ or
other agents can be used if a higher mutation frequency is desired.
11. These mutants are resistant to relatively high levels of antibiotic, and can
be used to demonstrate recombination in mtDNA.
12. Where possible, the strains should be tested genetically to be sure that the
mutation IS mitochondrial and not nuclear. This cannot be done with
nonsporulating industrial yeast strains. However, since these strains are
not haploid, the mutations are unlikely to be nuclear.
4.5. Temperature-Sensitive Mutants
13. The method has been used in the isolation of cell division cycle mutants,
which may be detected by Giemsa staining of cells grown at the restrictive
temperature to show the point at which nuclear division and other events in
the cell drvisron cycle have been halted.
14. Osmotrc-remedial mutants may be isolated by rephca plating to a third
plate containing elevated concentrations of KC1 (l-3M) and incubating at
the restrictive temperature along with the second plate, isolating colonies
that grow on high-KC1 medium and not on normal medium.
4.6. Mutants Defective
in Nuclear Fusion (Icarl- Mutants)
15. Other strains carrymg the karl-1 mutation, of both mating types and with
other auxotrophic requirements, are in the collection of the Carlsberg Labo-
ratories (Copenhagen, Denmark).
4.7. Mutants Having Easily Lysed
or Easily Digested Cell Walls
16. These mutants are often unstable and revert rapidly to growth on nor-
mal media.
17. They may also show increased sensitivity to increased or decreased tem-
peratures, antibiotics, or other drugs.