2.3. Inoculum preparation
The single-strain inocula were prepared as described by Uesugi et al.
(2006). The frozen stock culture was streaked for isolation onto tryptic
soy agar (TSA: tryptic soy broth plus 1.5% granulated agar) and incubated
at 37 ± 2 °C for 24 ± 3 h. A 10-μl sterile loop of this culture was transferred
into 10 ml of TSB and incubated at 37 ± 2 °C for 24 ± 3 h; this
transfer procedure into TSB was repeated once. An aliquot (1 ml) of
the second overnight culture was spread over large TSA plates (150 by
15 mm) and incubated at 37 ± 2 °C for 24 ± 3 h. The resulting bacterial
lawn was collected by adding 9 ml of a 0.1% peptone to each plate
and scraping the surface of the plate with a sterile spreader (Lazy-L
Spreader, Andwin Scientific, Tryon, NC). The harvested cells (11 log
CFU/ml) were diluted, as appropriate, with 0.1% peptone to inoculum
levels ranging from 4 to 11 log CFU/ml. The five-strain mixtures of
Salmonella, E. coli O157:H7, or L. monocytogenes were prepared by
growing each strain separately (under the conditions described
above) and then combining equal volumes of each strain to produce
the target inoculum. The populations in the individual and final mixed
inocula were determined by serial dilution in Butterfield's phosphate
buffer (BPB) and plating onto media as described below