Stress and timing associated with Caenorhabditis elegans immobilization methods

Background: Caenorhabditis elegans is a model organism used to study gene, protein, and cell influence on function and behavior. These studies frequently require C. elegans to be immobilized for imaging or laser ablation experiments. There are a number of known techniques for immobilizing worms, but to our knowledge, there are no comprehensive studies of the various agents in common use today. New method: This study determines the relationship between concentration, immobilization time, exposure time, and recovery likelihood for several immobilization agents. The agents used in this study are 1-Phenoxy-2-propanol, levamisole, sodium azide, polystyrene beads, and environmental cold shock. These tests are conducted using a humidified chamber to keep chemical concentrations consistent. Each of these agents is also tested to determine if they exhibit stress-related after effects using the gcs-1, daf-16, hsp-4, hif-1, hsp-16.2, and tmem-135 stress reporters. Results: We present a range of quick mount immobilization and recovery conditions for each agent tested. This study shows that, under controlled conditions, 1-Phenoxy-2-propanol shows significant stress from the daf-16 reporter. While 1-Phenoxy-2-propanol and sodium azide both create stress related after effects with long term recovery in the case of the hsp-16.2 reporter. Comparison with existing method(s): This study shows that commonly used concentrations of immobilizing agents are ineffective when evaporation is prevented. Conclusions: To improve reproducibility of results it is essential to use consistent concentrations of immobilizing agents. It is also critically important to account for stress-related after effects elicited by immobilization agents when designing any experiment.