Routine soil processing for SCN egg counts

Protocol

Screening Soil

Procedure:

  • One day before screening soil, place samples out to dry on drying racks, with one-quarter sheet of unused newspaper between the tray and sample. Sample bag and tag should be clipped to the tray used.

Drying rack with sheet of newspaperShelf full of drying racks

  • Before beginning, check that the collection tray and rollers are clean.
  • Turn the machine on using the side-mounted switch.
  • Slowly pour a sample into the feed-chute.

Photograph of feed chute

  • Clean the rollers using the wire brush. It is important to do this before collecting the sample to avoid possible contamination of other samples.
  • Collect the sample from the tray and return to the sample bag. Used newspaper should be discarded and the collection tray should be cleaned before returning it to the machine.

Photograph of output end of feed chute

  • When complete, all samples should be returned to the cold room for storage.

Manual Cyst Extraction

Procedure:

  • Obtain a well mixed soil sample.
  • Fill a bucket with 2 quarts of water (marked on inside of bucket).

Example of bucket used in bucket extraction

  • Pour in the soil, break any clumps with your fingers, and mix the soil suspension well for 15 seconds.
  • Pour the soil suspension through a #20 over a #60 sieve.

Photograph of person filtering cysts and particles out of soil

  • Rinse the debris caught on the top sieve with water, and then discard its contents. Carefully wash the cysts and accompanying sediments from the #60 sieve into a clean beaker or directly into a 100 ml plastic grinding tube using as little water as possible.

Automated Cyst Extraction

Procedure:

  • Add 400 ml tap water to graduated beaker.

400mL tap water in graduated beaker

  • Raise volume to 500 ml with soil.
  • Allow sample to sit for 15 minutes.
  • Thoroughly stir sample.
  • Allow sample to sit for 15 minutes.
  • Add soil/water mixture to the machine beakers and raise volume to 1800 ml.

Photograph of soil mixed with water

  • Open valve to air drills and allow to stir for 15 seconds.

Air drills with soil samples

  • Dump samples 15 seconds after stirring (through a #20 over a #60 sieve.)

Photograph of dumping samples through sieve

  • Collect what is caught on the #60 sieve.

Grinding Cysts to Release Eggs

Procedure:

  • Wash the cyst/sediment suspension (from elutriator or manual extraction method) from the 100 ml beakers into the small PVC #60 sieve.

Washing the sediment/suspension into a sieve

  • Place a #200 over #500 sieve into the funnel below the rubber stopper, and start the drill press using the foot pedal.
  • Bring the spinning rubber stopper into contact with the sieve surface.

Grinding the material in the sieve with a rubber stopper

  • Grind the material on the sieve lightly between the rubber stopper and sieve. Periodically rinse with the wash bottle. This will maintain lubrication between the sieve and pestle and also rinse eggs through the #200 sieve. Grind until nothing remains upon the small PVC sieve except sand.

Occasionally spray the solution with water

  • Release pressure on the foot pedal to stop the drill press, wait for the drill press to stop spinning, and rinse the rubber stopper.
  • Wash the material on the #500 sieve back into the original 100 ml beaker.

Washing the material into the sieve

  • Carefully rinse all sieves under the faucet.
  • Repeat for all remaining samples.
  • Place 4 to 5 drops of 1 M HCl and approximately one eye-dropper full of acid fuchsin stain into each beaker of egg solution.
  • Heat beakers containing eggs and stain in the microwave until the suspension is about to boil (approximately 3 minutes for 12 samples).
  • Remove beakers from microwave and allow to cool.
  • Place beakers in the refrigerator until they are counted.

Counting SCN Eggs

Procedure:

  • Increase the volume of liquid in a sample to 100 ml with tap water.
  • Examine the empty counting slide under the microscope to be certain that there are no eggs remaining in the slide from previous counts.
  • Thoroughly mix the sample.
  • Immediately after mixing the sample, draw sufficient volume into a glass pasteur pipet to fill the counting slide.
  • Fill the counting slide from the pipet.

Sample on slide dyed pink to enable easier viewing of sample

  • Systematically move from cell to cell in the slide grid, counting all SCN eggs.

Microscope view of an egg

  • Record total count and dilution.
  • Pour the liquid from the slide back into the sample, and rinse the slide thoroughly with a water bottle.

Sources for Grinding Cysts to Release Eggs:

  • Standard sieves:
    • Fisher Scientific
      1600 W. Glenlake Avenue
      Itasca, IL 60143
      (800) 223-9114 or
    • VWR Scientific
      P.O. Box 66929, O'Hare AMF
      Chicago, IL 60666
      (800) 932-5000
  • The #60 (PVC) sieve:
    • David Soh
      (515) 294-5094
  • Rubber stopper:
    • David Soh
      (515) 294-5094
  • Motorized stirrer for rubber stopper:
    • Delta 10" drill press running at 2340 RPM (any brand would work).
  • Chemicals:
    • Fisher Scientific
      1600 W. Glenlake Avenue
      Itasca, IL 60143
      (800) 932-5000

Sources for Counting SCN Eggs:

  • Nematode counting slides: Chalex Corporation, 5004 228th Avenue S.E., Issaquah, Washington 98029, (425) 391-1169.