1. Nematode Extraction Methods: Root Extraction Techniques
Kavitha Govindasamy
Jimmy R.Rich
Maria L .Mendes
Respectively, Formerly Graduate Assistant, Professor, and Senior Biological Scientist, Department of Entomology and Nematology, University of Florida, Gainesville, FL 32611, U.S.A.

2. A Plant-Parasitic Nematode
Nematodes are minute, worm-like animals that may damage plants but are often difficult or impossible to detect with the unaided eye." The word "nematode" when literally translated means "thread-like." Other names commonly used include "roundworm," "eelworm," or "nema.“ Plant parasitic nematodes cause economic damage to all annual and perennial crops grown for food as well as landscape ornamentals and turfgrass.
For more information on plant nematodes, go to: and

3. Sampling for Nematodes
Sampling and extracting for nematodes serves two purposes:
Diagnosis a current problem
Predict a future problem
Nematodes cannot be detected or quantified without being extracted from the soil and identified under a microscope.

4. Objectives of sampling and extracting nematodes
Identify the nematode
Population estimation
Diagnose a nematode disease
Make management decisions
Regulatory purposes
Research and surveys
Nematodes cannot be detected or quantified without being extracted from the soil and identified under a microscope.

5. Some Common Nematode Extraction Methods
Root Samples
Jar incubation and sieving
Blending and sieving
Soil samples
Baermann funnel
Modified Baermann funnel
Foliar samples
Punching and incubating
Scissors technique
Other extraction methods are also used including the common Centrifugation-Sugar Flotation method for soil extraction and the Mist Chamber method for extraction of nematodes from roots.

6. Root Extraction Techniques: Jar Method
Principle
Nematodes are extracted based on their active migration from roots
Migratory endoparasitic nematodes like the lesion and burrowing nematodes are collected from root samples.

7. Materials required Glass jars and caps 325 or finer mesh sieve
Labels and rubber bands
Dark chamber
Sieves used to catch nematodes must be at least 325 mesh or finer in mesh. A coarse sieve (200 mesh or coarser) placed over the finer sieve is useful to separate plant debris from the nematodes.

8. Sample ready for processing
Root samples must be kept moist and cool until processing. When collecting roots, mixing these with moist soil will help preserve the roots until laboratory extraction.

9. Wash the roots
Wash the roots thoroughly to remove the soil adhering to it. In the above case, a coarse sieve is being used.

10. Damp dry the roots
After washing, the roots are damp dried with tissue paper.

11. Weigh the damp-dried roots
Weigh roots to eventually quantify the nematodes present on a per gram basis. Ten grams or more of roots is a good quantity to use.

12. Place roots in jars, add water
Damp dry roots are weighed and placed into jars. A small amount of water is added to cover about 75% of the roots. Leave jar lids loosened to allow for some air exchange.

13. Place jars in tray
Jars containing the roots sample are placed in a tray which will be later placed in an incubator or dark area in the laboratory.

14. Store in the dark for 72 hours
Trays are placed inside the dark storage chamber and incubated for 72 h. During the incubation period the nematodes in the roots migrate to the water due the low oxygen levels.

15. Following incubation, remove from storage and add water
After 72 hours, the jar is filled with water to enable the nematodes to float.

16. Collect nematodes on sieve
The water and the roots are passed through a 20 mesh screen placed on top of a 325 mesh screen. The roots are collected in the 20 mesh screen and nematodes in the 325 mesh screen.

17. Rinse nematodes from sieve
Nematodes from the 325 mesh screen are collected in a container and labeled.

18. Nematode samples ready for observation
The nematodes are collected in the containers and taken to the microscope for observation or stored in a refrigerator until counting.

19. Advantages and Limitations
Extracts migratory endoparasitic nematodes
Few materials needed
Simple procedure to follow
Limitations
Not useful for ectoparasitic nematodes
Lack of aeration can be a problem
Sieves are needed to concentrate nematodes

20. Root Extraction Techniques: Blending and Sieving
Principle
Nematodes are released from roots due to maceration.
Root samples are washed free of water and blended for seconds depending upon the hardiness of the roots. Due to the maceration the nematodes are released from the roots.

21. Materials required Blender 40 and 325 or 500 mesh sieves Pan or bowl
Wire mesh
Filter paper
Squeeze bottle
Weighing balance
Scissors
Several sieve sizes can be successfully used. Coarse sieve can range in size from while finer meshes of 325, 400, and 500 can be used.

22. Wash roots well
Wash the roots thoroughly to remove the soil adhering to it. In the above case, a coarse sieve is being used.

23. Cut roots
The roots are cut into small pieces.

24. Damp dry the roots
After washing the roots are damp dried in the tissue paper.

25. Weigh roots
A 10 gram roots samples is usually used to quantify the nematodes present.

26. Place roots into blender, cover roots with water
Add enough water to cover roots placed in the blender.

27. Blend at low speed for 10-20 seconds
Blend the roots for seconds at low speed depending upon the hardness of the roots.

28. Pour the roots into 325 or finer mesh sieve
After blending, pour the blended roots in a 500 mesh sieve.

29. Set up a Baermann tray
The Baermann tray is a dish with a wire mesh placed inside for holding the root sample. Typically, the wire mesh is made from window screen (hardware cloth) and glued between two PVC pipe rings.

30. Place filter paper on the Baermann tray
Most coffee filters, coarse mesh laboratory filter paper or even facial tissue can be used.

31. Add water to Baermann tray
The filter paper should be moistened and water added to slightly above the level of the screen.

32. Transfer the blended roots into the filter paper

33. Incubate for 24 hours
Only partially cover roots with water. After transferring the roots, they are incubated for 24 hours.

34. Remove roots from the pan

35. Pour the water in the dish through a 325 or fine mesh sieve
After 24 hours, the nematodes migrate from the tissue paper to the water in the pan.

36. Concentrate nematodes for observation
Nematodes from the 325 or finer sieve are transferred to a vial which is stored in a refrigerator until observed under microscope.

37. Advantages and Limitations
Fast method to extract nematodes from roots
Foliar nematodes can also be easily extracted
Limitations
Chemicals released from the roots can be
injurious to nematodes
Blending for long time may damage
nematodes

38. Acknowledgments
Janete Brito, Department of Plant Industry, Gainesville, Florida
Frank Woods, Nematode Assay Lab, University of Florida, Gainesville, Florida
Joey Orajay, Entomology and Nematology Department, University of Florida, Gainesville, FL

39. For additional information or comments, please contact: