Instructions for Decapsulating Rotifer Cysts

Our resting cysts can be made bacteria free by decapsulating the cysts with chlorine bleach, instructions below. 

1. Place appropriate number of resting eggs in a 2mL tube. Use one scoop of resting eggs for every two treatments.
2. Add 200uL of 15ppt Artificial Sea Water to tube, and incubate at room temperature for 15 minutes.
3. Add 600uL of cold buffered ASW (pH 10) and 500uL bleach to tube.
4. Vortex for 10 seconds, incubate for one minute, vortex for another 10 seconds, and incubate for one more minute. Do not allow eggs to stay in bleach for more than 2 minutes total.
5. Use a cut plastic pipette to transfer embryos (floating on top) to a small 44um filter, and rinse with 200mL of ASW.
6. Fill filter with 0.2% sodium thiosulfate in ASW and let drain for 1 minute.
7. Wash embryos from filter into 6-well plate with ~5mL ASW.
8. OPTIONAL- Allow decapsulated resting eggs to sit at 4°C in low light overnight if eggs are not hatching after 18-20 hours