from the worms, and all ethanol that was brought with the worms should be captured in the solution, so by determining the volume of the worms we could calculate the ethanol concentration of the worms. This calculation of internal ethanol concentration is dependent on the size of the worms tested and animals of different genotypes differed considerably in size. We estimated the volume of the worms by calculating the volume of a cylinder of the length and average diameter of the worms we tested (Figure 2a). Consistent with our previously reported results, we found that internal ethanol concentration remains much lower than the exogenous dose. However, using this very precise method, we determined that the internal concentration of ethanol in these experiments is approximately 2x what we have observed in the past using different tissue preparation methods. For wild-type N2 animals exposed to 500 mM exogenous ethanol, at 10 minutes of exposure the tissue concentration was 67.5 ± 7.1 mM. The tissue concentration of ethanol increased over time; at 50 minutes of exposure, the tissue concentration was 89.3 ± 8.8 mM (P = 0.02 vs. 10 minutes; Figure 2b). This indicates that the development of acute tolerance that we observe
