Purification of SCN Cysts by Sucrose Centrifugation

Protocol

Procedure:

  • Wash cyst/sediment suspensions from beakers into 50 ml plastic centrifuge tubes.
  • Adjust the volume of liquid in each tube to approximately the same level (to avoid excessive centrifuge vibration).
  • Place the tubes, 2 by 2, into opposite positions in the centrifuge.
  • Centrifuge the tubes at 2000 RPM for 4 minutes. After 4 minutes, turn the centrifuge off and let the rotor coast to a stop.
  • Save the supernatant and pour into the original sample beaker.
  • Wipe the rim of the centrifuge tube to collect cysts stuck to the tube wall, and rinse into beaker.
  • Add sucrose solution (1362 g sucrose / 1L tap water) to tubes until each is about 2/3 full, and stir or agitate the tubes to re-suspend the sediments and cysts. After stirring, add more sucrose solution to level off paired tubes.
  • Centrifuge at 2000 RPM for 2 minutes. After 2 minutes, turn the centrifuge off and let the rotor coast to a stop.
  • For each sample, pour the supernatant through a #60 or #80 mesh sieve. In addition, pour the supernatant from the 1st centrifuge step through the sieve. Rinse thoroughly with tap water.
  • Collect cysts by rinsing them from the sieve back into the original beaker using a water bottle.
  • Wash the plug out of the bottom of the tubes, and clean and rinse the tubes.