2.2. In vitro inhibition assays for P. larvae
Inhibitory activities of the LAB against P. larvae (RIAS No. P1
GIFU-1 strain) were investigated by in vitro inhibition assay as described
previously (Yoshiyama and Kimura, 2009). Briefly, vegetative
forms of P. larvae were cultured at 35 C for 48 h in Brain–
Heart Infusion (BHI) medium (Difco, USA) and adjusted to a concentration
of OD600 = 0.5. These bacterial suspensions of P. larvae
(500 ll) were spread over the surface of each BHI agar plate and
three replicate plates were prepared as previously described by
Evans and Armstrong (2005). Total of 208 LAB were cultured on
MRS agar plates at 35 C for 3 days before being suspended in sterile
distilled water adjusted to a concentration of OD600 = 0.5. Each
LAB suspension (20 ll) was then placed onto sterile paper filter
disks (7 mm diameter, Whatman™ USA), and the disks were
placed on the BHI plates spread with P. larvae. After incubation
at 35 C for 48 h, plates were examined for inhibition halos. For
those plates with inhibition zones, the diameters were measured
(mean ± standard deviation) from three independent experiments.
Sterile distilled water was used as a negative control and tetracycline
(10 lg/ml) was used as a positive control.