2.3. Killer activity
The isolates were tested to verify the presence/absence of killer
phenotype. Killer activity tests were performed on medium, containing malt
extract broth (2%), agar (2%), methylene blue (0.0003%),
buffered at pH 4.6 with 0.1 M citric acidephosphate buffer. As sensitive
reference strain, S. cerevisiae DBVPG 6500 (NCYC 1006; National
Collection of Yeast Cultures, Norwich, England) was used, by
suspending its cells in sterile water and incorporating a concentration
of about 106 CFU/ml into the medium. All the studied colonies
were inoculated on the plates, by using the killer S. cerevisiae strains
K1 and K2 (DBVPG 6497and DBVPG 6499, respectively) as positive
controls; the plateswere incubated at 26 C for three days. The tested
isolates were designated as killer strain when the colony was surrounded
by a clear zone in which no growth of the inoculated sensitive
strain had occurred. The non-killer yeasts were suspended in
sterile water and inoculated in the medium previously described.
The killer reference strains were inoculated on the seeded plates,
which were incubated at 26 C for three days. If the killer reference
strainwas surrounded bya clear zone of growth inhibition, the tested
strain was designated as sensitive. If there was no clear zozone of
growth inhibition, the tested strain was designated as neutral.