bailii NCYC 1766 in YEPD corrected to pH 4.0. Resistant of sub-populations were selected of single colonies growing in 8 mM benzoic acid or in 350 mM acetic acid at 14 days, and re-inoculated. The rates of growth Z. bailii (NCYC 1766) in increasing FK228 price concentrations of weak-acid preservatives was monitored in the 96-well microtitre plates by the time required for the yeast colonies to reach 0.5–1 mm in size. In the absence of preservatives, this required only 2–3 days incubation. At higher concentrations of preservatives, the incubation

time required increased up to 12–14 days. As previously described in Section 2.4, single colonies of Z. bailii (NCYC 1766) growing in 6 mM sorbic acid, 8 mM benzoic acid or 350 mM acetic acid after 14 days, were mixed in the microtitre well and accurately counted

by haemocytometer. Each was then serially diluted in YEPD containing the same weak acid concentration to 104 cells/ml. Each was then cross-inoculated into all combinations of weak acids, at 15–30 cells/ml into 20 ml aliquots of YEPD containing sorbic acid (0–8 mM), HDAC cancer benzoic acid 0–8 mM and acetic acid 0–450 mM. These were then dispensed into microtitre plates at 200 μl/well (maximum 3–6 colonies/well). Plates were sealed, lidded, double-bagged to prevent evaporation, and incubated at 25 °C for 14 days. The method used for determination of cellular internal pH by flow cytometry was a modification of the method described in Stratford et al. (2009). Exponentially-growing yeast cells were obtained from shake flasks at OD next 1.65–2.2 (measured OD 0.15–0.2 following an 11-fold dilution in water). Z. bailii (NCYC 1766) and S. cerevisiae (BY4741) were cultured in 40 mls YEPD pH 4.0 in 100 ml conical flasks shaken for 12–16 h at 130 rpm and

25 °C. Sub-populations in 6 mM sorbic acid, 8 mM benzoic acid and 350 mM acetic acid in microtitre plates were inoculated into 40mls of the same media and shaken for 5 days (OD 0.15 × 11). Control samples were tested in microtitre plates at 0, 6 mM sorbic acid, 8 mM benzoic acid or 350 mM acetic acid to confirm that these populations were ~ 100% resistant to preservatives. CFDASE (carboxyfluoresceindiacetate succimidyl ester) was added to yeast in the growth media at 10 μg/ml final concentration and cells were incubated at 25 °C for 30 min for uptake of the CFDASE. Uncharged CFDASE, colourless and non-fluorescent, passes into the cell where it is cleaved intracellularly by esterases. The fluorescent succimidyl ester binds to proteins, ensuring retention of the dye within the cell. The internal pH of populations of individual fluorescent cells was determined from the linear ratio of the 575 nm (largely pH-independent) and 525 nm (pH-dependent) emission signals. Calibration was carried out using cells of defined intracellular pH, permeated using 2 mM 2, 4-dinitrophenol in 0.