2.3. Bacterial Strains and Cultivation Media. The standard
strains of three Gram-positive bacteria Enterococcus faecalis
ATCC 29212, Staphylococcus aureus ATCC 29213, and
S. epidermidis ATCC 12228, three Gram-negative bacteria
Escherichia coli ATCC 25922, Klebsiella pneumoniae ATCC
700603, and Pseudomonas aeruginosa ATCC 27853, and
one yeast Candida albicans ATCC 10231 were obtained
from Oxoid (Basingstoke, United Kingdom). Cation adjusted
Mueller-Hinton broth (MHB) and Sabouraud dextrose
broth (SDB) were used as cultivation media for antibacterial
and antifungal microdilution assay, respectively, and
were equilibrated with Tris-buffered saline (Sigma-Aldrich,
Prague, Czech Republic). Mueller-Hinton agar (MHA) and
Sabouraud dextrose agar (SDA) were used for subsequent
determination of bactericidal and fungicidal concentrations,
respectively. All media were purchased from Oxoid (Basingstoke,
United Kingdom).
2.4. Minimum Inhibitory Concentration (MIC) Determination.
The MICs were determined using the in vitro broth
microdilution method following the guidelines of Clinical
and Laboratory Standards Institute (CLSI) [14, 15] modified
according to the recommendations proposed for effective
assessment of the anti-infective potential of natural products
[16] using 96-well microtiter plates. Briefly, the EO was
dissolved in DMSO with addition of Tween 80 and two-fold
serial dilutions were prepared in MHB for bacteria and in
SDB for the yeast whereas the concentrations tested ranged
from 4 to 2048