> **Recommended USDA-Cyst-Extractor w/Acetone Purification 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. 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

        -   Acetone (optional)

        -   Milk filter sock

    b.  Equipment needed

        -   USDA-Cyst Extractor apparatus

        -   No. 4 (4.75 mm), No. 20 (850 µm) and No. 60 (250 µm) US
            Standard mesh sieves

        -   Jars

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

    a.  Screen thoroughly air-dried soil sample through a No. 4
        (4.75 mm) sieve.

    b.  Record the weight of the sample and add the entire (approx. 5-12
        lb) sample of screened soil into the container.

    c.  Place the nested No. 20 sieve over the No. 60 (250 µm) sieve
        setup under the overflow spout.

    d.  Turn on the fast and slow water control valves, allowing water
        to flow into the container from the bottom. Use sticks or other
        appropriate utensils, which can be cleaned between samples, to
        help mix the soil and water.

    e.  When the soil/water mixture is within five inches of the
        container top, turn off the fast water control valve and let the
        mixture slowly rise until it reaches the overflow spout.

    f.  Floats (cysts and organic material) will be collected on a No.
        20 sieve nested over a No. 60 sieve.

    g.  Rinse the sides of the container to wash any cysts or material
        off of the walls as water continues to overflow into the nested
        sieves.

    h.  Continue to wash until the water runs clear (usually less than
        10 minutes depending on soil type and amount).

    i.  While the water is running, rinse the No. 20 sieve thoroughly to
        completely wash the cysts through the No. 20 sieve.

    j.  Remove the No. 20 sieve and clean thoroughly to prepare for the
        next sample.

    k.  After the water runs clear remove the No. 60 sieve tilt, and
        concentrate by careful washing the collected material and cysts
        on the No. 60 sieve toward the edge of the sieve.

    l.  Carefully rinse the material from the No. 60 sieve into a funnel
        that is attached to a milk filter sock suspended inside a jar.

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

    n.  Thoroughly clean the Extractor apparatus, funnel, and No. 60
        sieve in preparation for the next 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 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 junk 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.

```{=html}
<!-- -->
```
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.  Barker, K.R., Carter, C.C., and Sasser, J.N. An Advanced Treatise on
    Meloidogyne. Raleigh, NC. 1985.

2.  Wang, X. and Thurston, D. PCN Cyst Extraction Using the
    USDA-Cyst-Extractor. May 2006.

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