Microfluidic Maintenance of C. elegans for Long-Term Imaging

When animals were ∼72 h posthatching, they were loaded into the microfluidic devices along with a solution of 100 mg of E. coli ml⁻¹ of liquid NGM (NGM without the agar). For animals continuing to receive the pharmacological treatment after development, the compound was introduced into the E. coli solution at the appropriate concentration before the concentrated bacteria solution was added to the device (Fig. 7B). On each day for the remainder of the experiment, the devices were washed using liquid NGM to remove progeny and debris, and a fresh solution of bacteria was added to the device (Fig. 7C,D). The arena of pillars and barriers in the outlet ports allow for the retention of adult animals and the filtering out of unwanted progeny, as has been previously demonstrated for C. elegans maintenance in microfluidic devices (Hulme et al., 2010 (link); Wen et al., 2012 (link); Xian et al., 2013 (link); Wen et al., 2014 (link)).
After clearing the devices of progeny and debris, and before adding fresh E. coli, animals were imaged in the microfluidic chambers (Fig. 7C,D) for 45-s episodes at a rate of five frames per second. A Nikon Eclipse TI-E microscope with Andor Zyla sCMOS 5.5 camera was used. Any animals that remained stationary during the first image sequence, although few in number, were re-imaged until a movie including sufficient worm locomotion was obtained.