D and WRHD were tested for antibacterial activity according to
a published methodology (Mongelli et al., 1995). Briefly, 4 mL of D
or WRHD were added to 16 mL of culture medium and homogenized
thoroughly. Vegetative cells of each P. larvae strain grown
in MYPGP agar for 72 h of incubation at 37 C under microaerobic
conditions were suspended in physiological solution and adjusted
to 0.5 of Mac Farland scale. The microorganisms were streaked in
radial patterns on the MYPGP agar plates, four streaks per plate. Finally,
the plates were hatched at 37 C, during 72 h under microaerobic
conditions.
EOs were tested for antibacterial activity according to published
methodology (De Feo et al., 1998). Vegetative cells of each P. larvae
strain grown in MYPGP agar for 72 h of incubation at 37 C under
microaerobic conditions were suspended in physiological solution
and adjusted to 0.5 of Mac Farland scale. An aliquot of 0.10 mL of
this solution was poured into a Petri dish containing MYPGP agar.
This aliquot was spread onto the surface of the agar using a Drigalsky
spatel until the microorganisms were absorbed. Then, a 6 mm
diameter sterilized paper filter disk, previously embedded with 10
lL of the EO under analysis, was put onto the agar surface, and the
plate was incubated at 37 C during 72 h under microaerobic
conditions.
After the incubation period, the zones of growth inhibition of
the bacterium were measured with a caliper. All the experiments
were carried out in duplicate.
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