EXTRACTION EQUIPMENT & PROCEDURES:

COMBINED PROCEDURES:

This is only a partial listing of methods that have been developed to extract nematodes from soil and plant tissues. It contains those most popular in California. The technique(s) chosen for a particular sample will depend on the nematodes of interest and the type of sample material (soil, plant tissue, etc.). Because of this it is important that the lab know the previous, current and subsequent crop to help them determine the extraction method. Different procedures will extract different proportions of particular nematode genera that might be present. Therefore, it is important to know which extraction procedure has been used by a particular lab in order to properly interpret a laboratory report. Ectoparasitic nematodes which spend their entire lives outside of roots in the soil will not be found in root samples. Therefore the techniques for extracting nematodes from plant tissue will be unlikely to detect the presence of ectoparasitic nematodes. Some ectoparasites such as the dagger nematode (Xiphinema index) and the needle nematode (Longidorus africanus) will cause root galling which may be mistaken for root-knot nematode. In this situation, nematodes will not be recovered from root samples. With root-knot nematode, root galling will become evident within a few days after root-knot nematode juveniles enter roots. It takes additional time, depending on soil temperatures, for those juveniles to become mature, egg laying adults. In this circumstance, nematodes will only be found by direct microscopic examination of roots.

Other methods include the "salad bowl" technique developed at Auburn University and the "pie pan" technique developed at Cornell University. Many of these techniques utilize various sizes of brass or stainless steel sieves originally developed for dry sieving soils known as Tyler screens. They are available in many scientific catalogues.

Sieving is the most basic technique and consists of mixing soil (the volume varies from one lab to another but one pint is a popular amount) with a large volume of water (several quarts to several gallons), allowing a brief time for heavy particles to settle, and then pouring the mixture through one or more sieves of a mesh sizes expected to retain large debris or nematodes. The sizes of screens used varies from one lab to another depending on the type of nematodes expected to be recovered and soil characteristics. Some begin with a course sieve of 10 to 20 mesh/inch which will catch large debris but allow nematodes to pass through. The solution is then passed through sieves of 60 to 500 mesh/inch to catch nematodes. Nematodes and soil particles caught on the sieves are "backwashed" into containers. If not too murky, this solution can then be viewed under a microscope, or subjected to an additional technique to further purify the sample.

A Fenwick can is a metal container with a spout at the top and a threaded fitting at the bottom to which a hose can be connected. Soil is placed in the bottom of the can, water is turned on and enters through a device that creatse a swirling action in the bottom of the can. As the can fills, lighter soil particles and nematodes flow over the spout and onto one or more sieves from which nematodes are "backwashed"after a set period of time (typically 2 to 5 minutes).

A number of types of Elutriators have been developed (originally in North Carolina) in order to further automate the sieving process. A mixture of air and water enters the metal funnels of the elutriator into which soil has been placed and the process proceeds similarly to that described for the Fenwick can. The events of the elutriation process are typically controlled by a timer. Automated techniques are not necessarily faster than hand sieving, but can provide a greater degree of standardization.

The basic Baermann funnel technique which has a large number of modifications utilizes a glass funnel with a wire mesh basket nested on top. A piece of rubber tubing is slipped over the stem and sealed with a clamp. The funnel is filled with water to a level that will cover soil or plant tissue to be placed in the basket at the top of the funnel. A piece of tissue is used to line the basket and minimize the amount of soil that passes through. For reasons that are not understood, nematodes leave the soil or plant tissue, pass through the tissue liner, and accumulate at the constriction of the tube created by the clamp. After a period of time, the clamp is loosened slightly to allow a few milliliters of solution to pass into a container, leaving a fairly clean solution to view under a microscope. Laboratories have developed variations for every component of this technique.

UC Davis lines the funnel with a Kimwipe, UC Riverside uses blue shopwipes cut into circles, and another lab uses white Scottie facial tissues. Another UC Riverside modification eliminates the clamp and utilizes small glass vials held by the rubber tubing.

Samples are left on the funnels for lengths of time varying from a few hours to several days depending on the preferences of the laboratory and the type of nematodes expected to be present.

Small, motile nematodes including juveniles of citrus, cyst and root-knot nematode, and adults and juveniles of lesion nematode seem to be readily extractable via this technique. Another UC Riverside technique is to line the basket with cheesecloth which allows the passage of larger nematodes such as dagger and needle nematode. Ring nematode is not readily extracted by this technique. Several thousand citrus nematode juveniles can be obtained from 50 ml of soil placed in the top of the funnel. For most other species, soil is usually put through a sieving process to concentrate nematodes and the funnel is utilized as a second step to further clean up the sample.

Plant tissue is typically weighed before placing in the funnel and the nematodes reported per gram of tissue. Some labs dry and weigh the roots after extraction and report nematode numbers on a dry weight basis.

Mist chambers were developed at UC Riverside as a means to obtain increased recovery of nematodes from plant tissue placed in still another modification of the Baermann funnel. The stem of the funnel (lengthened with rubber tubing) is placed in a large glass culture tube or test tube. A fine, heated mist is sprayed intermittently (e.g. 30 seconds every 5 minutes) over weighed plant tissue placed in an unlined basket in the top of the funnel. Overflow water passes over the sides of the culture tube and down the drain. The length of time materials are left in the chamber varies from lab to lab, typically from 3 to 5 days. Recovery tends to increase (perhaps partly due to egg hatch) the longer the time of misting. With highly motile nematodes such as Aphelenchoides fragariae, it is possible to lose a portion of the sample in the overflow water. At least one laboratory has experienced the problem of clogging of the overflow drain leading to overnight flooding of labs on the floor below.

An alternative to misting is jar incubation in which plant tissue is placed in a jar with a small volume of water to keep it moist. This is a simpler procedure but may not recover as many nematodes as misting.

Sieving, a Fenwick can, or elutriation are often used as a first step to concentrate nematodes followed by misting, sugar flotation or centrifugation, or a Baermann funnel to obtain a cleaner sample.

The sieve-mist technique as the name imples consists of soil sieving followed by placing the concentrate on a tissue lined funnel in a mist chamber.

Sugar flotation and centrifugation utilize a concentrated sugar solution to float nematodes away from soil particles. Typically, these procedures are used following a sieving type procedure (e.g. elutriation-sugar centrifugation). The concentration of the sugar solution varies from lab to lab and can be adjusted to facilitate recovery of different sized nematodes. The length and speed of the centrifugation also varies from lab to lab. A typical procedure consists of elutriation, followed by centrifugation of the material retained on the screen, after which the pellet is suspended in a sugar solution, recentrifuged during which nematodes float and soil particles sink. The supernatant is poured through a sieve and retained nematodes are "backwashed" and saved for identification. This procedure greatly increases the recovery of ring nematode relative to Baermann funnels, and works well for smaller nematodes such as lesion, and juveniles of root-knot and cyst nematode. Recovery of larger nematodes such as dagger and needle is typically lower with this technique than Baermann funnels lined with cheesecloth. Increasing the sugar concentration or adjusting the length and speed of centrifugation can increase the recovery of larger nematodes but there may be a trade off with respect to nematode survival or identification because of the increased osmosis.

Several techniques have been developed to stain roots so that nematodes will be visible within.

Greenhouse bioassays in which soil is potted with a plant susceptible to the nematodes of interest is utilized in some situations. This technique typically requires several weeks to complete and a place to maintain the plants during this time, but can be more sensitve than other extraction methods and may not require use of a microscope. One common method is to use tomato to bioassay for root-knot nematode.

A specialized procedure has been developed to extract cysts and eggs of sugarbeet cyst nematode. It utilizes a sieving procedure followed by an alcohol-glycerin flotation to extract cysts, followed by homogenization to release eggs from cysts for counting.

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