Staining Roots for Plant-Parasitic Nematode Visualization

Protocol

Procedure:

  • Place the root samples in separate 100 or 150 ml glass beakers.
  • Prepare 600 ml of a water-bleach solution by adding 100 ml household bleach to 500 ml distilled water.
  • Add approximately 50 ml of water-bleach solution to each beaker containing a root sample.
  • Incubate roots in the water-bleach solution for 4 minutes, stirring with a metal spatula periodically.
  • Rinse roots for 45 seconds or so in running tap water, then soak the roots in tap water for 15 minutes.
  • Pour off the tap water, rinse with tap water one last time, then add approximately 40 ml of distilled water and approximately 7 or 8 drops of the acid fuchsin stain to each beaker.
  • Place beakers containing roots and stain on a hot plate and heat until boiling fairly well.
  • Take the beakers that have boiled from the hot plate and allow them to cool enough so that they are comfortable to touch.
  • After cooling, carefully rinse roots in running tap water for approximately 1 minute, then carefully drain off most of the excess water.
  • Add 10 to 20 ml of acidified glycerine to each beaker. Acidified glycerine is prepared by adding 5 to 10 drops of dilute 1.0 M HCl to regular glycerine.