> **Recommended Fenwick Can Protocol for Extraction of *Globodera* spp.
> Cysts.**
>
> **Introduction:**
>
> The following protocol is recommended by APHIS-PPQ-CPHST for use in
> extracting potato cyst nematodes, *Globodera* spp., from soil samples.
> The protocol should be used as a guidance document with the
> understanding that it may be necessary to modify the procedure
> depending upon soil type, equipment, personnel and other mitigating
> factors specific to your laboratory. Laboratory directors should use
> extant knowledge of their unique system and environment when making
> modifications; noting and recording the changes. Any soil should be
> thoroughly mixed prior to extraction procedures to ensure sample
> homogeneity if the entire sample is not being processed. Furthermore,
> this procedure is specific only to mature nematode cysts and not to
> vermiform nematodes. Additional extraction procedures will need to be
> utilized to extract and collect vermiform nematodes.

A.  **[Materials and Equipment]{.underline}**

    a.  Materials needed

        -   Parafilm

        -   Acetone (optional)

        -   Milk filter sock

    b.  Equipment needed

        -   Fenwick can apparatus

        -   No. 8 (236 mm) and No. 60 (250 µm) US Standard sieves

        -   Jars

B.  **[Extraction for each sample]{.underline}**

    a.  Screen thoroughly air-dried soil through a No. 8 sieve (236 mm).

    b.  Add 500cc of screened soil that has been thoroughly mixed and is
        a representative homogeneous sample into the top
        container/screen (1 mm) of the Fenwick can.

    c.  Place the No. 60 (250 µm) sieve under the outflow spout.

    d.  Wash the soil sample through the top screen via the funnel
        apparatus into the Fenwick.

    e.  The majority of the cysts and organic material will float up and
        over the lip of the apparatus while soil and other heavy
        particles settle to the bottom of the apparatus. The floating
        particles will collect on the No. 60 (250 µm) sieve located
        below the outflow. Continue to wash until the water exiting the
        can runs clear.

    f.  Conduct a final thorough wash of the top container through the
        screen into the middle funnel apparatus. (Make sure, to
        completely clean out the screen mesh between samples)

    g.  Remove the top screen and wash the middle funnel thoroughly.

    h.  Remove the No. 60 sieve with the collected material from the
        area of the Fenwick can and thoroughly clean the Fenwick can
        apparatus in preparation for the next sample. Remove the stopper
        near the base of the Fenwick at this point if it has one.
        **Replace stopper after cleaning**.

    i.  Gently tilt the No. 60 sieve and using a water stream preferable
        from a plastic squeeze bottle rinse the cysts and collected
        material down to a concentration point on the lower section of
        the sieve in order to prepare the sample for step i.

    j.  Carefully wash the cysts and organic layer from the No. 60 sieve
        into a funnel that is attached to a milk filter sock suspended
        inside a jar.

    k.  Rinse the cysts and organics into the bottom of the filter sock,
        squeeze off excess water and place a label inside the sock.

    l.  Thoroughly clean the funnel and No. 60 sieve in preparation for
        the next sample.

    m.  Repeat steps a-l as necessary to process the entire soil sample,
        if required.

    n.  Being careful to record the total volume of soil processed for
        the entire sample.

    o.  Let the filter sock air dry overnight.

    p.  Break apart the clumps of extract using hands or a spatula.

    q.  Pay strict attention to maintaining sample identification and
        record keeping throughout the process.

> Optional, to further separate the cysts from the collected material.

r.  Under a fume hood, slowly place the milk sock into a jar of acetone.
    Cysts and some organic debris will float to the perimeter of the
    sock.

s.  Mix the sediments with a spatula and slowly lift the sock up and
    down several times (time in acetone should not exceed 4 minutes).

t.  Squeeze off excess acetone and carefully cut off the bottom of the
    sock containing the waste material.

u.  Dry the extract in the remaining section of the sock under a fume
    hood.

v.  Once dry, cysts may be counted directly on the milk sock using a
    dissecting microscope or stored until reading occurs.

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C.  Storage

    a.  Extracted samples should be stored at 4^o^C.

D.  Disposal

    a.  Soil

        -   According to permit requirements for regulated samples.

        -   Otherwise disposal as per routine samples.

    b.  Water

        -   According to permit requirements for regulated samples.

        -   Otherwise dispose of as per routine samples.

References:

1.  Wang, X., and Thurston, D. Method for PCN Cyst Extraction Using the
    Fenwick Can. May 2006.

2.  Wang, X. and Thurston, D. PCN/GN Cysts and Acetone Purification. May
    2006.

3.  Barker, K.R., Carter, C.C., and Sasser, J.N. An Advanced Treatise on
    Meloidogyne. Raleigh, NC. 1985.