Figure 1. Assay for measuring the response of freely moving C. elegans to odorants: (A) Procedure for preparing worms and measuring their response to a volatile odorant. Select plates with unstarved gravid adults for the addition of bleach to dissolve worms while preserving embryos. Move the hatched L1 worms by washing onto plates with E. coli OP50 and grow to young adulthood (~96 hrs after bleaching). Wash young adults to remove bacteria and move to plates without E. coli to pre-expose them with the vehicle (V; e.g., ethanol) before assaying their response to an odorant in the vehicle (O; e.g., butanone). Transfer pre-exposed worms to the center of each rectangular arena to measure dispersal with no odorants (top) or chemotaxis towards an odorant (
q4) or the vehicle (
q1) (bottom). Count the number of worms in each quadrant (
q1 to
q4) of the arena using a video taken after 1h. (B) A decision chart for interpreting behavior. Results can be interpreted only if sufficient numbers of worms (>100 per plate) of comparable age (young adults) were assayed, and they dispersed uniformly in the absence of odorant (dispersal ~2). In the absence of added odorants, assays where the worms remain in the middle of the arena (dispersal ~1, resulting from attraction to center and/or defective locomotion) or accumulate at one quadrant (dispersal ~0, indicative of response to an unknown gradient in the arena) cannot be interpreted. (C) Chemotaxis of wild-type worms is reproducible when tested using gradients of three different odorants. Apparent chemotaxis on arenas without added odorants (grey) paired with chemotaxis measured using three replicate assays for each odorant (butanone, orange; benzaldehyde, blue; nonanone, magenta) are plotted using both calculated chemotaxis index (top) and proportions of animals in extreme quadrants (1 or 4) or near the origin (O; quadrants 2 & 3) (bottom). Effect sizes (ES, Cohen's d), significance (* for P<0.05, unpaired t-test), populations tested (N), numbers of worms in each population required for interpretation (n), 95% confidence intervals (error bars), calculated dispersals, and calculated response ratios are shown. Assays remained reproducible when performed on different days (3 different days for butanone and benzaldehyde, and 2 different days for nonanone). (D) Summary schematic of the chemotaxis response of C. elegans to the tested volatile odorants (attraction to butanone and benzaldehyde, but aversion to nonanone).