Extraction Methods

     Rev:  10/30/2019
  Decanting and Sieving Method 1 Decanting and Sieving Method 2  
  Baermann Funnel    
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Extraction of Nematodes by Decanting and Sieving - Method 1

Materials

Source
  1. 20-mesh sieve (833-µm aperture)
  2. 200-mesh sieve (74-µm aperture)
  3. 325-mesh sieve (43-µm aperture)
  4. Coarse sieve (1 cm aperture)
  5. Two stainless steel bowls or plastic buckets
  6. 250 ml beaker:
  7. 600 ml beaker
  8. Coarse spray wash bottle or tube attached to faucet
Howard Ferris
Department of Entomology and Nematology
University of California
Davis, CA 95616
(530)-752-8432
hferris@ucdavis.edu

Procedure

1.  Review potential extraction errors.
 
2.  Mix soil sample and pass through coarse sieve to remove rocks, roots, etc.
 
3. Take a 600 cc subsample of soil: pack lightly into beaker for uniformity.
 
4. Place soil in one of the buckets or pans; half fill with water.
 
5. Sieving and decanting process (various combinations of the following):
a) Mix soil and water by stirring with hand or paddle; allow to stand until water almost stops swirling.
b) Pour all but heavy sediment through 20-mesh sieve into second bucket; discard residue in first bucket; discard material caught on sieve.
c) Stir material in second bucket; allow to stand until water almost stops swirling.
d) Pour all but heavy sediment through 200-mesh sieve into first bucket; discard residue in second bucket.
e) Backwash material caught on 200-mesh sieve (which includes large nematodes) into 250-ml beaker.
f) Stir material in first bucket; allow to stand until water almost stops swirling.
g) Pour all but heavy sediment through 325-mesh sieve into second bucket; discard residue in first bucket.
h) Backwash material caught on 325-mesh sieve (which includes small to mid-sized nematodes and silty material) into 250-ml beaker.
 
6. Sample in 250 ml beaker will probably be too dirty for direct viewing; sample may be placed on Baermann Funnel or subjected to sucrose-centrifugation. The combined procedure allows extraction of nematodes from larger volumes of soil.
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Extraction of Nematodes by Decanting and Sieving - Method 2

Materials

Source
  1. 60-mesh sieve (246-µm aperture)
  2. 325-mesh sieve (43-µm aperture)
  3. Coarse sieve (1 cm aperture)
  4. Two plastic or metal pitchers (2 liter volume)
  5. Plastic or metal pitcher (3 liter volume)
  6. Two 250 ml beakers
  7. Coarse spray wash bottle or tube attached to faucet
Howard Ferris
Department of Entomology and Nematology
University of California
Davis, CA 95616
(530)-752-8432
hferris@ucdavis.edu

Procedure

1.  Review potential extraction errors.
 
2.  Mix soil sample and pass through coarse sieve to remove rocks, roots, etc.
 
3. Take a 250 cc subsample of soil: pack lightly into beaker for uniformity.
 
4. Place soil in one of the 2 liter pitchers; half fill with water.
 
5. Sieving and decanting process (various combinations of the following):
a) Mix soil and water by stirring.
          b) Pour the soil and water slurry back and forth between the two 2-liter pitchers five times to separate nematodes from soil particles.
c) Pour all but heavy sediment through 60-mesh sieve into the 3-liter pitcher.
          d) Half-fill the 2-liter pitcher with water and repeat steps b and c twice.
c) Stir material in 3-liter pitcher; allow to stand until water almost stops swirling.
d) Pour all but heavy sediment through 325-mesh sieve into first bucket; discard residue in pitcher.
e) Backwash material caught on 325-mesh sieve into 250-ml beaker.
 
6. Sample in 250 ml beaker will probably be too dirty for direct viewing; sample may be placed on Baermann Funnel or subjected to sucrose-centrifugation. The combined procedure allows extraction of nematodes from larger volumes of soil.
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Extraction of Nematodes by the Baermann Funnel Technique

Materials

Source
  1. Glass funnels (12.5 cm diam.)
  2. Wire-mesh baskets (10 cm diam., 5-10-mm aperture)
  3. Rubber tubing
  4. Kimwipes or facial tissue
  5. Ring stand
  6. Tubing clamps
  7. Coarse sieve (1 cm aperture)
  8. 250 ml beakers
  9. 50 ml beakers
  10. Two water bottles, coarse and fine spray
Howard Ferris
Department of Entomology and Nematology
University of California
Davis, CA 95616
(530)-752-8432
hferris@ucdavis.edu

Procedure

1.  Review potential extraction errors.
 
2. Attach 10-cm length of rubber tubing to funnel stem and clamp tubing.
 
3. Mount funnel on ring stand.
 
4. Fill funnel two-thirds full with water.
 
5. Place wire-mesh basket in top of funnel and use it to support tissue.
 
6. Mix soil sample and pass through coarse sieve to remove rocks, roots, etc.
 
7. Spread soil subsample (50 cc soil) evenly on tissue; fold in edges of tissue.
 
8. Complete filling funnel with water so that water level is about 5 mm above wire-mesh; do not let water and soil lose contact during extraction period - add water as needed.
 
9. Maintain temperature at 22-25 C so that it is conducive to nematode movement.
Note nematodes move through tissue and settle in funnel; only active stages are recovered.
 
10. After 48 h, recover extracted nematodes by releasing 20 ml of water from stem of funnel into a counting dish.
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Extraction of Nematodes by Sieving and Sucrose-Centrifugation

Materials

Materials

Source
  1. 20-mesh sieve (833-µm aperture)
  2. 500-mesh sieve (25-µm aperture)
  3. 635-mesh sieve (20-µm aperture)
  4. Coarse sieve (1 cm aperture)
  5. Two 600-ml plastic beakers
  6. Centrifuge tubes (must be a set)
  7. Funnel
  8. Spatula
  9. Test tube rack
  10. Sugar
  11. Two water bottles, coarse and fine spray
Howard Ferris
Department of Entomology and Nematology
University of California
Davis, CA 95616
(530)-752-8432
hferris@ucdavis.edu

Procedure

1.  Review potential extraction errors.

2. Prepare sucrose solution: to 454 g sugar add de-ionized water to bring total volume to 1 liter. Stir until sugar is completely dissolved.

3. Mix soil sample and pass through coarse sieve to remove rocks, roots, etc.

4. Take a 100 cc subsample of soil: pack lightly into beaker for uniformity.

5. Remove sand and organic material:

a) Mix soil subsample in 500 ml water by pouring between beakers ten times.
b) Rinse residues in second beaker into beaker with sample.
c) Swirl beaker with sample; allow to stand for 15 seconds (sand settles).
d) Pour supernatant through 20/500-mesh stacked sieves.
e) Gently tap side of 500-mesh sieve to facilitate drainage.
Note: Larger particles will remain in beaker; organic debris is caught on 20-mesh sieve; nematodes and silt are retained on 500-mesh sieve.
f) Using the coarse-spray water bottle, gently wash nematodes, etc. into one sector of the 500-mesh sieve.
g) Using the fine spray water bottle, wash sample into a centrifuge tube.

6. Centrifuge: add water to centrifuge tubes to equalize volumes; place tubes in centrifuge in balanced pairs.

a) Spin at 1700 rpm (810 g) for 5 minutes without using the brake.
b) Allow to settle for 5 minutes.
c) Aspirate supernatant to approximately 1 cm above pellet.
d) Fill tubes with sucrose solution at room temperature.
e) Stir with a spatula to break up pellet (must be completely dispersed).
f) Spin sample: bring centrifuge up to 1000 rpm ( 280 g) in 30 sec, then apply brake.
Note: Nematodes and clay are suspended in sucrose supernatant; silt and larger particles are in the pellet.

7. Pour supernatant through 635-mesh sieve. Rinse gently with water and transfer to labeled vials using the fine spray water bottle.

(Method modified by Dr. Bruce Jaffee, Department of Nematology, UC Davis, from Jenkins 1964, Pl. Dis. Reptr. 48:692.)

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General Reference on extraction methods:

Barker, K.R. 1985. Nematode extraction and bioassays. Pp 19-35 in K.R. Barker, C.C. Carter and J.N. Sasser (eds) An Advanced Treatise on Meloidogyne, Volume 2. Methodology. North Carolina State University Graphics. 

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