Teacher's Guide: Protocol for Collecting Root-Knot eggs

What's Wrong with the Plants?

Protocol for Collecting Root-knot eggs

The following protocol is used to extract the eggs from infected roots so they can be used to inoculate plants. The eggs can be used for infecting plants for future extractions and for student experiments. I will inoculate my ‘sample’ plants with these eggs, and after they are examined I will do an extraction to infect more plants. If you continue the cycle, you will always have access to eggs.

1. Cut the roots from the stem of 2-3 root-knot nematode infected plants. Gently remove the soil from the roots and soak in a water-filled bucket. Rinse in a hand strainer or colander. Cut up the roots and place in a large flask. (2000 ml is ideal, but who has one that large? Use what you have.)

2. Add a 20% bleach solution (1 part bleach/4 parts water), cover, and shake vigorously for 90 sec. Too much time will destroy the eggs.

3. Screen out root material using a hand strainer or colander.

4. Run liquid through a 100 mesh screen. Collect the liquid in a bucket or large container.

5. Run the collected liquid through a 500 mesh screen. Eggs will be on top of screen. Gently add water to rinse the bleach. Screen off water.

6. Rinse eggs off the screen with water bottle into a beaker. Use as little water as possible to get all the eggs off the screen.

7. Centrifuge (low-speed; swinging bucket rotor), ~ 2500 RPM for 5 minutes. Eggs will be on the bottom. Aspirate (or dropper) off the excess water. If the plug is stable, you can pour off the water.

8. Prepare a 47% sucrose solution (47g sugar to 100 ml distilled H₂O). Quickly mix eggs with the sugar solution in centrifuge tube/s. Centrifuge ~ 1500 RPM for 40 sec.

9. Pour the eggs off the top of the tube (the eggs will float on the sugar), into a 500 ml beaker with 250 ml of distilled water.

10. Pour the egg/water solution through a 500-mesh screen. Rinse the eggs off the screen into a beaker with a water bottle. The eggs should be used immediately for inoculation. Eggs used for observation should be refrigerated at 5° C. They will remain viable for about a week. If kept too long, fungus will grow in the container and it will destroy the eggs.

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The University of Arizona
Department of Biochemistry and Molecular Biophysics
General Biology Program for Secondary Teachers
warder@email.arizona.edu