DisplayTitle

Materials and Methods: Free-living and Plant-Parasitic
Nematodes (Roundworms)

Materials

Equipment:

• conical glass (preferably) or plastic funnel, at least 100 mm in diameter
• ring stand (to hold funnel)
• flexible rubber or plastic tubing
• spring-action hose clamp
• circular piece of wire screen to fit inside top of funnel
• 2-ply facial tissue (without moisturizing lotion)
• Pasteur pipettes and bulbs
• petri plate or watch glass
• microscope slides and cover slips
• clear nail polish
• alcohol lamp
• stereomicroscope (dissecting microscope)
• compound microscope

Soil samples:

• freshly collected soil, preferably from forest floor or garden \

Materials

To assemble and set up a Baermann funnel to extract nematodes from soil:

Figure 16. Diagram of Baermann funnel for extraction of nematodes. (Courtesy C. Jasalavich).

1. Set up ring stand. Attach hose to funnel and place funnel with hose into ring of ring stand. Secure clamp to hose attached to funnel. Place circular piece of wire screen inside of funnel.
   
2. Add tap water to the funnel until the water surface is barely touching the wire supporting screen. At this time, make sure that water is not leaking from the tube. If tube is leaking, re-adjust hose clamp until leaking stops.
   
3. Place an open sheet of two-ply facial tissue over the supporting screen in the Baermann funnel, letting the edges of tissue drape over the outside edge of the funnel.
   NOTE: Do not use facial tissue that has moisturizing lotion incorporated into the tissue.
   
4. Carefully add approximately ½ to 1 cup of freshly collected soil onto the open facial tissue. Gently spread the soil out in an even layer.
   NOTE: If you want to compare types and numbers of nematodes extracted from soils representing different ecosystems (i.e. forest versus lawn versus garden versus farmer's field), set up several Baermann funnels and carefully measure and add the same amount of soil to each funnel.
   
5. Fold over all four corners of the open tissue onto the soil placed on the tissue.
   
6. Carefully add additional water to the funnel until the water surface is almost above the top of the tissue.
   
7. Let the Baermann funnel sit undisturbed for 24 to 48 hours at room temperature. Add additional water to the funnel periodically (once a day should be plenty) to replace water that evaporates.
   
8. When you are ready to observe nematodes, carefully release the clamp on the hose attached to the funnel, and collect 5 to 10 ml of solution in a Petri plate or watch glass.
   
9. Observe solution with a dissecting microscope for the presence of plant-parasitic and free-living nematodes. You should be able to differentiate between stylet-bearing (most likely plant-parasitic) and non-stylet-bearing (free-living) nematodes at the highest magnification of the dissecting microscope. Plant-parasitic nematodes recovered from soil will be sluggish and slow moving whereas most free-living nematodes will be very active swimmers

NOTE: To see the body structure of a nematode in more detail, you will need to examine it with a compound microscope. Prepare a slide mount as follows. Place three or four drops of clear nail polish on a clean microscope slide so as to form the corners of a triangle or rectangle of a size that will support a cover slip (FIGURE 17). Do not add the cover slip yet. (Once your mount is completed, the nail polish supports will prevent your nematodes from getting crushed.) Next, use a Pasteur pipette or eyedropper to place a drop of water containing nematodes in the center of the dots. Warm this drop of water by passing the slide six to eight times over the flame of an alcohol lamp to relax the nematodes. You want to warm it just enough so that the nematodes stop moving, but not too much or the nematodes will burst; you can check the progress by looking at this slide under your dissecting microscope. After the nematodes have stopped moving, place the cover slip so that it rests on the nail polish supports. The edges of the cover slip should be sealed with clear nail polish to prevent the water underneath it from evaporating. Now it is time to look at your nematodes with a compound microscope.

Please click here for a downloadable powerpoint presentation of the instructions above.