Nematode extraction and preservation

  • Sugar centrifugation used on Antarctic dry valley soils
  • Anhydrobiotic nematode extraction
  • Formalin preservation

Sugar centrifugation method for extraction of nematodes (Freckman, 1997)

    1. Pour 100-200cc of soil in a beaker. Remove rocks greater than 3-4mm in diameter.
    2. Weigh remaining soil. You should have around 100g.
    3. Bring up to 650mL with tap water.
    4. Stir carefully (star stir or figure 8) for 30 seconds.
    5. Immediately pour liquid into wet screens – a stack of 40 mesh on top of a 400 mesh
    6. Rinse gently with cold tap water through the top of the stack, keeping the screens at an angle, as the water filters through. Keep the water on ice.
    7. Remove the top screen.
    8. 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.
    9. Backwash the nematodes into a 50mL plastic centrifuge tube, tipping the screen into the funnel above the tube. Rinse funnel gently.
    10. Put in the centrifuge, making sure to balance the load, for five minutes at 1744 RPM.
    11. Decant off liquid, leaving a few mL on top of the soil.
    12. Fill with the sugar solution which should be made up ahead of time and chilled (454g/L tap water).
    13. Stir gently with spatula until pellet is broken up and suspended.
    14. Centrifuge for one minute at 1744 RPM.
    15. Decant into wet 500 mesh screen.
    16. Rinse well with water and backwash into a centrifuge tube. Try to keep volume below 10mL so sample will fit in counting dish.
    17. Refrigerate (5°C) samples until ready to count.

Modification for soils with large clay content

After stirring for 30 seconds, step 4 above, add 2 ml of Separan solution (2.5g per 500 ml water). Stir again for 5 seconds and let settle for 60 seconds. Continue with step 5.

Anhydrobiotic nematode extraction procedure (1997)

    1. Prepare sugar (regular table sugar) solutions – 2M (684.6 g/L) and 1.25M (427.9 g/L). Make about 20mL 2M per sample to be processed and 400mL 1.25M for each sample. Chill solutions before starting extraction.
    2. Set up a series of centrifuge tubes on ice containing 5mL 2M sugar.
    3. Measure out 100g of soil (less rocks) in a beaker.
    4. Bring up to 250mL with 1.25M sucrose solution.
    5. Star stir for 30 seconds.
    6. Pour into screens that have been prewetted with 1.25M sugar solution.
    7. Rinse through the top screen with 1.25M sugar.
    8. Remove top screen.
    9. Rinse bottom screen from the front only with 1.25M sugar. Squirt only at an angle. Work slowly and get all the soil in the bottom wedge of the screen. Tap gently to reduce sugar volume.
    10. Rinse the bottom screen into a 150mL beaker with 1.25M sugar, from the front, using the funnel. Rinse the funnel.
    11. Using an automatic pipette, slowly pipette the sugar and sediment from the beaker into a centrifuge tube containing 2M sugar. Pipette in slowly, at an angle in order to retain the boundary between the sugar layers. Rinse the beaker with 1.25M sugar and pipette in the dregs. Use as many centrifuge tubes as is necessary per sample.
    12. Even off tubes and centrifuge for five minutes at 1744 RPM.
    13. Decant the liquid contents of the centrifuge tubes through a 500 mesh screen, stopping before the sediment can go in. Be consistent in how each sample is poured, as this will effect clarity when counting, and accuracy.

Formalin preservation (Ed Kuhn, 1997)

Wear all the proper protective gear when using formalin since it is toxic and easily absorbed by the skin.

    1. After counting for nematodes, all samples are stored in a 5°C refrigerator overnight. Do not add a drop of alcohol. Samples are then aspirated below 5mL to the point where the plastic centrifuge tube begins to taper.
    2. Transfer samples to glass vials (pre – labelled on cap and side) with two strong squirts of cold tap water into the tip. Fill the vials no more than 1/3 full. Keep the level consistent on all vials.
    3. Use a hot/stir plate to heat 10% formalin. Suspend a thermometer into the formalin. Heat to the range of 80-90°C, but no higher or the formalin will evaporate.
    4. Draw out 5mL of hot formalin with an automatic pipetter and apply to all samples. Slant the pipette tip off the inside wall of the vial as you add to each one.
    5. Screw the cap on and let samples attain room temperature before applying parafilm seal around cap.