Nematode Extraction Method

In a previous post, I described how we sample when we are in the field. Now I want to describe how we remove the nematodes from the soil sample. This extraction method is meant primarily for nematodes; however, we also extract rotifers, tardigrades, and some protozoans. I will just use "nematodes" below instead of listing all of the animals we extract though. We call this the sugar centrifugation extraction method.

The chlorophyll A samples I mentioned in the previous post can be processed later. However, we do need to extract nematodes right away or they will start dying. Ideally, we only store the soil samples in a refrigerator for a couple days.

The first step is to weigh out 50 grams of soil so that we can figure out the moisture content. We record the initial mass, then stick the samples in an oven for a couple of days and record the final weight. This soil can't be used for anything else after it has been cooked for so long.

While we have the soil bag open, we take out 100 grams for our nematode extraction.

Me weighing out 100 grams of soil for nematode extraction under a laminar flow hood (a hood designed to keep the work surface sterile while still allowing us to work under it)

The next step is to add some water to the container with our 100 grams of soil. We mix the soil up in the water then pour the resulting murky water through a series of two sieves. The first sieve catches all the big rocks but allows the nematodes to flow through it. The second sieve catches all the nematodes (as well as quite a bit of dirt that is similar in size) but allows the really fine particles to flow through into the sink.

Zach running the sieves

We recover all of the nematodes along with the trapped dirt into a tube by squirting water backwards onto the sieve. Next, we centrifuge the tubes for 5 minutes to pull all the dirt and nematodes down to the bottom of the tubes. We pour off most of the water, leaving a small amount of water with the dirt in the bottom of the tube. Now we can add a sugar solution (a very thick sugar solution of normal granulated sugar. It is almost half sugar/half water) to the tubes and stir the dirt and nematodes up. All of the nematodes float to the top of the tube in the sugar solution but the dirt falls to the bottom. We centrifuge the mixture for a minute to speed up the separation.

Martijn running the centrifuge

The last step is to pour off the sugar carrying the nematodes onto the last sieve, taking care to avoid pouring the dirt onto the sieve as well. The final sieve is very fine and catches all the nematodes while allowing the sugar and fine silt to wash away. We wash the nematodes with some water (nematodes don't do very well in sugar if they are left there for too long) then rinse them off the sieve into a final tube.

And that's it! If all went well, the nematodes are swimming around with a minimal amount of water and soil. We usually wait about 30 minutes for the nematodes to start moving around before we examine a sample. Some of the living nematodes are dried up in the soil but will begin moving when they have had enough time in water. I plan to explain the next steps (including pictures of the typical nematodes we find) in a later blog post.

We're in the lab today running this extraction method on the samples we collected 2 days ago from the Elevational Transect.