Nematode extractions

Why do a nematode extraction?

• Diversity assessment:
  To figure out what nematode diversity is in the soil or compost.
  Within the first 24-48h the free living, free swimming nematodes will be extracted. Root feeding nematodes are bound to the root environment, move much shorter distance and won’t generally cover the distances within this early time span.
  Keep the extraction going for longer, and we get an idea of root feeding nematode populations, too. A total of 4-5 days is recommended to get a feel for the wider community.
  However, since the low oxygen conditions at the bottom of the collection tube are detrimental to many beneficial nematodes, I don’t wait for the whole 4-5 days, but check at roughly 24h intervals (at the moment – still learning!)
  
  
• To add nematodes to a soil or compost.
  This is particularly useful if there are nematodes in one substrate, that are highly prized, such as the elusive predatory nematodes. By extracting them from one substrate we can use them to inoculate other substrates, such as a batch of compost. Or, if we are lucky to have a compost rich in predatory nematodes, we might want to just add these to a soil troubled by high density of root feeding nematodes.

What methods are useful?

There are a number of different nematode extraction methods. The soilfoodweb school teaches the use of the Baermann funnel. It’s advantage is its ease of access, you don’t need much, nor expensive equipment. However, it is also one of the less efficient methods in terms of extraction. Further information comparing methods has been published by Wageningen University.
Wageningen also make available some handy nematode counting dishes.

Learning points:

• 2 clamps on the tube are needed to control air flow when releasing the nematodes that have collected.
  Close the top one first, before opening the bottom clamp.
  If not, nematodes swirl all around with the air bubbles, and fewer are available in the collection dish.
  
• Useful sieves for small-scale nematode extractions are zooplankton sieves.
  Wetting them before use makes them easier to use.
  
• Less is more! Having smaller volume samples increases extraction efficiency (tested were 25 mg/50mg/100mg).
  More information in this paper as well as some other useful tips. Just skip the stage of using formalin for our purposes!
  I now use 10 or 20g (depending on how much substrate I have available).
  
• Look at your filters under the microscope. Are they actually big enough to let nematodes through easefully? If not, you might conclude a sample is deficient in nematodes, when in fact the filter is too efficient in sifting them out!
  
  The above article prompted me to look at the impact of filters in the funnel, too.
  In functionality I am reasonably happy with these. They are made from plastic, however, so am exploring a reusable filter with suitable mesh size.

Finding a balance between filters used in the funnel that are open enough for nematodes yet hold back sufficient debris is tricky.

With the above milk filters, more nematodes make it through, but also more particles.

The soilfoodweb school recommends 10 micrometer filter. If I use them as a stand-alone, significant debris comes through, making counting harder (more visual distractions).

So I am using a series of sieves to try and hold back debris: 125 > 75 > 50 > 25 microns. This might mean that some of the larger nematodes stay on top of the “debris filters” (125 to 50). I check that by putting a sample under the scope. But it helps not trying to playing the game “spot all the nematodes”.

This is what the filters looked like with a wood chip compost nematode extraction. Most nematodes were in the 25 micron filter, 0-1 in the 10 microns for several trials. So for this substrate at least, I will just go down to 25. Think I need to add in a 60 or 50 microns one to keep out some debris.

My process:


Set-up:

• Measure 10 or 20g of soil, record how much
• Add filter to a metal sieve or wire basket, then add soil
• Clamp off tubing
• Add water (non chlorinated) – rainwater or water treated with humics (from compost.
  If using water that’s run through compost, I filter it through a 10 micron filter to ensure no nematodes from the compost get mixed with the substrate I want to assess.
• Add sieve to funnel, ensuring sample is covered with water

Filtration

• Measure how much liquid was in the tubing to determine how many nematodes are present per unit.
• Filter through a set of zooplankton filters (125 > 50 > 25 or 10)
• Wash nematodes into one corner of the smallest filter using a lab bottle
• Pour into rodac plate, can use a pipette to distribute evenly across the plate

Microscopy assessement

• Count for x3 squares, if they are fairly consistent in numbers, you can calculate totals
• If there is high variability, count more squares (up to 5)
• Get average of the squares
• Calculate nematodes/g:
  Take the average count, multiply by the number of squares in the counting dish. This is the total for however many grams we extracted from.
  Then calculate how many nematodes this is per 1g.

Goals for biocomplete compost:

• 100 beneficial nematodes (bacterial & fungal feeders, and predatory nematodes) per 1g of compost
• Use nematode extraction promptly, if need be, put them in the fridge for a few days (use within one week max)