bailii strain NCYC 1766 ( Fig. 2) using cell viability in liquid media. Results from populations of > 1000 cells showed that all Z. bailii cells were able to grow in sorbic acid over

the range of 0–3 mM. However, a declining proportion of cells were able to grow at concentrations up to 7 mM, forming a long “tail” of sorbic-acid-resistant cells. Only ~ 1 cell in 8000 was able to grow in 7 mM sorbic acid. This is in close-agreement with the sorbic acid MIC of 7.62 mM for inocula of 104 cells of strain NCYC 1766 ( Table 1). In contrast, the S. cerevisiae cell population was 100% resistant up to 2 mM sorbic acid but with only a short “tail” of resistance up to 3 mM. Similar results were obtained for both benzoic acid ABT 737 and acetic

acid, showing that extreme acid resistance in Z. bailii was most probably due to a small proportion of the population. It was noted that the resistant “tail” in acetic acid was substantially longer, than that formed in sorbic acid or benzoic acid. The existence of CX 5461 a resistant sub-population may explain why tests on whole Z. bailii populations would fail to reveal the causes of resistance in Z. bailii. Cell suspensions were prepared of the sub-populations of Z. bailii from the 6 mM sorbic acid microtitre plates. These were directly re-inoculated, without washing or sorbic acid removal, into media containing increasing levels of sorbic acid, and the percentage of the population able to grow was again determined at

each level of preservative. It was found that near 100% of the cell population was now able to grow in sorbic acid up to 8 mM ( Fig. 3A). These experiments were repeated using cells cultured from Z. bailii sub-populations growing in 8 mM benzoic acid and from 350 mM acetic acid. Again, near 100% of the cell populations were now able to grow in 9 mM benzoic acid or 450 mM acetic acid respectively ( Fig. 3B; C). It was noted that sub-populations from 350 mM acetic acid showed 100% viability in high levels of acetic acid, but that a proportion, ~ 20%, failed to grow when inoculated into media lacking acetic secondly acid. Since the proportion of cells that grew was expressed as a percentage of the cell population in the absence of sorbic acid, this caused an apparent 120% cell viability at higher acetic acid concentrations. We speculate that this loss of viability was due to cytoplasmic alkalinisation caused by the large acetic acid efflux. Extreme resistance in the sub-populations was shown not to be genetically heritable, since if these sub-populations were grown overnight in YEPD pH 4.0 containing no preservatives and were then re-inoculated into media containing preservative, all populations reverted back to the original population profile of resistance (data not shown).