Worms generate a cuticle that is impermeable to many pharmacological agents (Burns et al., 2010; Cox et al., 1981), and we have previously measured internal concentrations approximately 20x lower than the exogenous dose (Davies et al., 2003, 2004; Kapfhamer et al., 2008). However, the determination of internal ethanol concentration has been the subject of some conflicting reports; using different methodology, others have reported that more than half of the exogenous ethanol accumulates in internal tissues within 20 minutes (Mitchell et al., 2007). To unambiguously determine the internal concentration of ethanol that animals achieve in our behavioral assays, we exposed animals to ethanol on plates as we would for behavioral assays, picked exactly 200 worms from the plate into a known volume of liquid, homogenized them, and performed gas chromatography analysis on the homogenate. All ethanol in the solution would come 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
