coli O157:H7, and L. monocytogenes able to grow in media supplemented with rifampicin (Rif) (Sigma-Aldrich, St. Louis, MO) at 50 μg/ml were isolated and used in experiments in which the inoculation level was near the indigenous microbiota level. Unless otherwise specified, culture media were obtained from BD (Franklin Lakes, NJ), and were supplemented with Rif. The isolates were stored at − 80 °C in tryptic soy broth (TSB) supplemented with 15% glycerol (Fisher Scientific, Fair Lawn, NJ). The single-strain inocula were prepared as described by Uesugi et al. (2006). Talazoparib research buy 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 Nutlin-3 order above) and then combining equal volumes of each strain to produce the target inoculum. The populations

in the individual and final heptaminol mixed inocula were determined by serial dilution in Butterfield’s phosphate buffer (BPB) and plating onto media as described below. Inshell walnuts were inoculated as described by Uesugi et al. (2006) for almond kernels. Inshell walnuts (400 g) were weighed into a sterile bag, inoculum (25 ml) was added, and the sealed bag was shaken and rubbed by hand for 2 min. Inoculated walnuts were spread onto four layers of filter paper (57 by 46 cm sheets; Qualitative P-5 Grade, Fisher Scientific) that was placed into a lidded plastic container (leaving a 3- to 5-cm gap to allow for air exchange). Walnuts were dried under ambient conditions for 24 ± 2 h. After drying, inshell walnuts were placed in sterile plastic bags and manually mixed by shaking for 2 min. To evaluate pathogen survival on inshell walnuts, inoculated and control nuts were stored in unsealed bags within closed plastic containers held at refrigerator (4 °C) or ambient conditions for periods of 12 weeks to 3 years, depending upon the experiment. Condensate was not observed in the bags or on the walnuts during storage. Data loggers (TempTale 4, Sensitech Inc., Beverly, MA) were placed in each storage area to record temperature and relative humidity (RH).