We sought an explanation for the differences in our previously published concentrations and what we have found here using this very sensitive protocol (Davies et al., 2003, 2004; Kapfhamer et al., 2008). Examining our previous protocol, we identified a major confound in the estimation of worm volume in the assay; we made an assumption that, in a pellet of worms that remains following centrifugation and removal of the supernatant, worms made up the vast majority of the volume of the pellet. However, when we calculated the volume of worms in a pellet by counting the worms and determining their volume, we found that this volume could vary by more than 2 fold depending on the size of the pellet and adult age of the worms, and at the most accounted for less than half of the volume (data not shown). When we used a known number of worms of known volume, our original spectrophotometric assay protocol closely recapitulated the internal ethanol concentration determined by gas chromatography (Figure 2b), demonstrating that use of this simple protocol is acceptable for future internal ethanol concentration measurements in C elegans.
