Mrd00025

Flasks were sampled each hour beginning one hour after feeding and continuing for 72 consecutive hours. At each hour, 500 µL was removed from each flask and transferred to a well of a deep 96-well plate. Each flask was sampled at each time point. Fluorescent polychromatic beads (Polysciences, 19507-5 ) with a 0.5 μm particle size were added to each well at a final concentration of 3.64x10 8 beads/mL and incubated at 20°C for 10 minutes with shaking. Following the bead incubation, 30 µL from each well of the deep 96-well plate was aliquoted to a 96-well microtiter plate. The process was repeated 11 times to 11 separate wells of the same microtiter plate with pipetting to mix the well contents from the deep 96-well plate. Animals were then treated with sodium azide at a final concentration of 50 mM to paralyze and prevent defecation of the ingested beads.
The 96-well plate was imaged with an ImageXpress Nano (Molecular Devices, SanJose, CA) using both 2x (Nikon MRD00025) and 10x (Nikon MRH00101) objectives. The ImageXpress Nano acquires brightfield images using a 4.7 megaPixel CMOS camera. Images are stored in 16-bit TIFF format. Finally, animals were scored using a large-particle flow cytometer (COPAS BIOSORT, Union Biometrica, Holliston MA). The COPAS BIOSORT sheath flow rate was kept at a constant 10.3 ±0.1 mL per minute to reduce variability in length measurements.