Instructions for Decapsulating Rotifer Cysts
Our resting cysts can be made bacteria free by decapsulating the cysts with chlorine bleach, instructions below.
- Place appropriate number of resting eggs in a 2mL tube. Use one scoop of resting eggs for every two treatments.
- Add 200uL of 15ppt Artificial Sea Water to tube, and incubate at room temperature for 15 minutes.
- Add 600uL of cold buffered ASW (pH 10) and 500uL bleach to tube.
- 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.
- Use a cut plastic pipette to transfer embryos (floating on top) to a small 44um filter, and rinse with 200mL of ASW.
- Fill filter with 0.2% sodium thiosulfate in ASW and let drain for 1 minute.
- Wash embryos from filter into 6-well plate with ~5mL ASW.
- OPTIONAL- Allow decapsulated resting eggs to sit at 4°C in low light overnight if eggs are not hatching after 18-20 hours