| 1) |
In a sterile fume hood pour approximately 100 g of soil into a clean beaker |
| 2) |
Add approximately 650 mL of COLD tap water |
| 3) |
Stir carefully in a figure eight pattern for exactly 30 seconds |
| 4) |
Immediately pour liquid onto a stack of wet soil screens- 40 mesh (425micro-m) on top of a 400 mesh (38 micro-m) soil screen |
| 5) |
Rinse gently with cold tap water through the top of the stack, keeping the screens at an angle, as the water filters through. |
| 6) |
Remove the top screen. |
| 7) |
Rinse top down, never directly on top of nematodes, but at the top of the screen and from behind. Let the water cascade down and carry the nematodes into the bottom wedge of the angled screen. Tap the side of the screen gently to filter all the water through. Rinse from the front and the back, keeping the screen at an angle and not allowing the water to overflow the edge of the screen. |
| 8) |
Backwash the nematodes into a 50 mL plastic centrifuge tube, tipping the screen into the funnel above the tube. Rinse funnel gently. |
| 9) |
Put in the centrifuge, making sure to balance the load, for five minutes at 1750 RPM. |
| 10) |
Decant off liquid, leaving a few mL on top of the soil. |
| 11) |
Fill with chilled 1.33M sugar solution (454g/L tap water). |
| 12) |
Stir gently with spatula until pellet is broken up and suspended. |
| 13) |
Centrifuge for one minute at 1750 RPM. |
| 14) |
Decant into wet 500 mesh (25micro-m) screen which collects the invertebrates while allowing the sugar solution to drain away. |
| 15) |
Rinse well with tap water and backwash into a centrifuge tube. Try to keep volume below 10mL so sample will fit in counting dish.
16) Refrigerate samples at 4-5°C until ready to count. |
