Salt and acid tolerance assay

Isolates were assessed for salt and acid tolerance using modified versions of published protocols (Cotter et al., 2005; Van Der Veen et al., 2008; Bergholz et al., 2010). In short, overnight cultures grown in BHIB at 30°C were diluted in either BHIB+6% (w/w) NaCl or BHIB adjusted to pH 5 (with 1 M HCl) to achieve a final concentration of 10⁷ CFU/ml. From these cultures, 200 μl was added in duplicate (technical replicates) to 96-well-plates (Costar™ clear polystyrene, Fisher Scientific) that were incubated at 25°C in a microplate reader (Spectramax, V6.3; Molecular Devices, Sunnyvale, CA). A temperature of 25°C was used to assess isolate salt and acid tolerance under non-intracellular or cold stress conditions. The absorbance (A_600nm) of each well was recorded every 30 min until all isolates reached stationary phase (~26 h) and the resulting growth curves were fitted to the Baranyi and Roberts model (Baranyi and Roberts, 1994) using DMfit (v3.5) available on the ComBase browser (http://browser.combase.cc/DMFit.aspx).