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PPDC Nematology lab

Plant Pest Diagnostics Center - Nematology Laboratory

Protocol for Extraction of Plant Parasitic Nematodes from Samples

I. Extraction from soil and root samples (gravity-sieving technique)

  1. Separate loose root pieces from soil in sample, put roots aside temporarily in large bowl/beaker.
  2. Process soil samples in 600 ml increments at the maximum until entire sample is processed.
  3. Place 600 ml soil in stainless steel bowl.  Add enough tap water to cover soil in bowl.  Break soil clods in water and agitate suspension by hand. (Wear disposable gloves).
  4. Immediately pass soil suspension through a 20-mesh (850µm-aperture) sieve held over a second bowl.  Do not pour the heavy settling sediment through the sieve.  Collect suspension in the second bowl
  5. Rinse residue on sieve over second bowl.  Pick out smaller roots from sieve and add to larger roots in bowl/beaker.  Keep roots moist.  Discard residue on 20-mesh sieve.  Thoroughly wash sieve and bowl with scalding hot water.
  6. After about 30 seconds, pass soil suspension through a 60-mesh (250 µm-aperture) sieve held over a clean bowl.  Collect suspension in bowl.
  7. Backwash residue on 60 mesh sieve into beaker.  This will be directly examined microscopically for cysts and large vermiform nematodes by a nematologist.
  8. If necessary, resuspend the soil in suspension by slightly agitating the liquid or swirling the bowl.  Pour suspension through a 200 mesh (74 µm-aperture) sieve* held over a clean bowl.  Collect suspension in clean bowl.
  9. Backwash residue on 200-mesh sieve into a clean 250 ml beaker.  Wash out bowl with scalding hot water.
  10. After about 30 seconds, pour suspension through a 325-mesh (44µm-aperture) sieve* held over a clean bowl.  Wash out bowl with scalding hot water.
  11. Backwash residue on 325-mesh sieve into same 250 ml beaker used in step 9.
  12. After about 30-40 seconds, pour suspension through a 500-mesh (25µm-aperture) sieve.  Do not hold over a bowl.  Allow liquid to pour into sink.
  13. Backwash residue on 500-mesh sieve in 250 ml beaker of steps 9 & 11.  This residue collection is now ready for further processing by sugar centrifugation or mist extraction techniques.
  14. Swirl backwashed suspension obtained from sieving and separate into two equal portions in two separate 250 ml beakers.  One half will be further processed for nematodes extraction by sugar centrifugation, and the other half by misting.
  15. Using hand shears, cut roots into approximately 1-2 cm segments for mist extraction.  Place cut roots in 600 ml beaker. 
  16. Thoroughly wash all bowls and sieves with scalding hot water.  Thoroughly wash countertop, sink and surrounding work area with hot water.  Disinfect with 70% ethanol.  Place bowls and sieves upside down to drain on countertop.

*Note:  Depending on soil type, the number and mesh size of intermediary sieves used in the above technique may vary.  Heavier soils (clays, clay loams) may require the use of all intermediary sieves (i.e., 100-mesh or 147µm-aperture, 200 & 325-mesh sieves), however, with lighter soils (sand, sandy loam) 200 and 325-mesh sieves may not be necessary.  At any length, it is necessary that 20, 60 and 500-mesh sieves are always used.

II. Extraction from soil residues by sugar centrifugation technique

  1. Pour backwashed soil residue in suspension collected through sieving (step I.14), into a 50 ml centrifuge tube. 
  2. Fill eight centrifuge tubes.  One sample suspension per one or more tubes.  Fill all tubes equally, to about 1/4th inch from top.  If necessary, use distilled water in squeeze bottle to match volumetric levels of all tubes.
  3. Place tubes in centrifuge.  If using less than eight tubes, place tubes opposite each other for balance. 
  4. Centrifuge at top speed (2500 rpm) for 5 minutes.  Use automatic timer.
  5. After centrifuge has stopped remove tubes.  Discard supernatant water from tube by pouring water in one steady motion into sink.  Using Kimwipe® paper tissue, wipe out debris attached to the inside top section of each tube.  Place tubes in holder.
  6. Fill each tube to about 1/4th inch from top with sugar solution.  Fill all tubes equally.  For filiform nematodes prepare sugar solution of specific gravity 1.18 (Sucrose = 673 g in 1000 ml distilled water); for cyst nematodes prepare sugar solution of specific gravity 1.23 (1225 g in 1000 ml distilled water).  Prepare solution at room temperature; agitate on mechanical stirrer until sucrose has completely dissolved.  Check specific gravity for accuracy.    
  7. Put tubes in centrifuge and spin at 2500 rpm, for 50–55 seconds.  Rapidly stop spin cycle by using break.  Do not use automatic timer for spin cycle.
  8. Quickly remove tubes from centrifuge and pour nematode-sugar suspension of each tube through a clean 500 mesh sieve.  Rinse immediately and carefully with distilled water. 
  9. Backwash sieve residue with distilled water into a 250 ml beaker.  The suspension is now ready for microscopic examination.
  10. Wash all tubes with soap and hot water.  Wash sieves.  Wash counter top.  Surface sterilize work area with 70% ethanol.

III. Extraction from soil, roots, tubers, bulbs, stems and foliage by misting (mist extraction technique)

  1. Pour backwashed soil residue in suspension collected through sieving (step I.14) through a stainless steel wire basket double-lined with Kimwipe® paper tissue.  Place basket over large beaker and allow liquid to drain into beaker.  Discard filterant liquid.
  2. Place basket over funnel and tube apparatus on rack.  Label sample and place label tag on outside of tube using rubber band.
  3. Place apparatus in mist chamber.  Sample will be misted for 1.5 minutes in a 10 minute cycle.
  4. Cut roots, bulbs, stems and foliage in 1-2 cm segments.  Rinse  shears in scalding hot water or 70% ethanol + hot water rinse between samples.
  5. Place cut roots, tubers, bulbs, stems and foliage separately in baskets.  Roots may be placed in same basket with soil, unless intentioned otherwise.
  6. Extension collars may be used to accommodate these samples within a basket.  Do not tightly pack sample within collar. 
  7. After 3-5 days for soil and roots, and/or 2 days for tubers, bulbs, stems and foliage, carefully remove rack from chamber.
  8. Allow suspensions in tubes to stand undisturbed for 30 minutes on counter top. 
  9. Without disturbing the settled residue at base of tube, carefully decant water up to 4.5 cm (~20 ml) from base, using a vacuum aspirator. 
  10. If suspension is opaque, do not decant.  Instead, pass entire solution through a 500-mesh (25µm-aperture) sieve and collect backwashed residue in beaker, scanning dish or tube.  The suspension is now ready for microscopic examination.
  11. Thoroughly wash apparatus and mist chamber with soapy hot water.  Rinse thoroughly with distilled water.  Clean spray nozzles.  Lubricate orifices of spray nozzles with vacuum grease.
References:
  1. Ayoub, S. M. 1977.  Plant nematology: an agricultural training aid. Department of  Food and Agriculture, Division of Plant Industry, State of California.  157 p.
  2. Cobb, N. A. 1918. Estimating the nema population of soil.  Agricultural Technical Circular 1. Bureau of Plant Industry. United States Department of Agriculture.
  3. Jenkins, W. P. 1964.  A rapid centrifugal-flotation technique for separating nematodes from soil.  Plant Disease Reporter, 48: 692.
  4. Southey, J. F. 1986.  Laboratory methods for work with plant and soil nematodes.  Ministry of Agriculture, Fisheries and Food. Reference Book 402. London: Her Majesty’s Stationery Office.  Sixth edition. 202 p.

prepared by Dr. John Chitambar, January 2007.