The isolates were tested to verify the presence/absence of killer
phenotype. Killer activity tests were performed on medium, containingmalt
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 plates were 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
strain was surrounded by a clear zone of growth inhibition, the tested
strain was designated as sensitive. If there was no clear zone of
growth inhibition, the tested strain was designated as neutral.