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Tissue Collection Protocol

To submit fungal tissue to our sequencing service, we require dried tissue to be placed in 1.5 mL (milliliter) tubes. We offer tissue collection tubes in our online store. Watch the video below to learn how to collect tissue for submission.

Organize your Materials

You will need 1.5 mL tubes, tweezers, a lighter, and alcohol swabs. 

Label your Tubes

Label your tubes with observation numbers from Mushroom Observer of iNaturalist. Organization is key!

Collect Tissue

Remove a piece of dried flesh the size of a grain of rice and place it in the appropriate labeled tube.  See detailed instructions below the video.

Tissue Collection Video

This video outlines the procedure to collect tissue using our preferred method in 1.5 milliliter "eppi tubes."

General Instructions

By now you should have taken pictures of the mushroom or other fungus and taken notes in the field (on field data slips or in a notebook); collected the mushroom and brought it back and dried it. Your specimens should be cracker dry. You should have uploaded photos and metadata to iNaturalist of Mushroom Observer for each specimen (observation). 

We will now take a small piece of tissue from the fungus and place it in a small plastic vial: a 1.5 milliliter (mL) tube, called an Eppendorf tube, or "eppi tube" for short. Tubes must be labeled with the corresponding iNaturalist or Mushroom Observer observation number. See instructions for creating and exporting lists at Upload Your Observations. Download and print numbers small enough to fit vertically the height of a tube (no hand writing!); cut and Scotch tape numbers to the tubes.

  • NOTE: Sequencing technology and procedures continually change! Originally we used tubes with a buffer solution. Now we are putting dried tissue into empty eppi tubes.

Tweeze or cut off a small portion of mushroom tissue that is about the size of a large grain of rice using sterilized tweezers or blade and place it in the vial. While it is possible to take dried tissue from any part of the mushroom to obtain a sample of DNA, probability of success depends on selecting parts with high DNA concentrations and low levels of contamination. Avoid cap surfaces to reduce contamination by spores deposited by other fungi and soil. Also avoid fuzzy areas as these may be parasitized by other fungi. The parts of fruiting body with highest concentrations of DNA are usually the spore producing surface, which except for cup fungi and morels, is located on the lower side. Specific recommendations follow:

Mushrooms and Boletes

Preferably tear (rather than cut) a healthy mushroom apart exposing the middle of the stalk (a cut on the margin may be needed to start the tear) to expose uncontaminated surfaces to collect samples from. Tweeze a piece of flesh from the top of the stalk where it joins the cap plus (interior flesh if it is a larger mushroom), place it in the vial, then tweeze a piece of gill or tubes and add it to the vial. This will yield the highest concentrations of DNA and least contamination. Don’t destroy the entire upper stalk as there are important diagnostic structures on the surface that are needed for identification.

Polypores

These fungi are typically tough, so you will need to sterilize a razor blade or scalpel to remove tissue. Healthy looking pore surface should be cut from the underside of the polypore to about 1 mm deep and placed in the vial.

Chanterelles (Cantharellus & Craterellus)

Bag, tag and hold for now. The ITS barcode region is not very helpful for identification in this group, if you are successful in getting a sequence. Other gene regions are useful, but to obtain those sequences, we would have to divert these samples to other labs, or compile a 96 well plate of samples to be sequenced using different primer sets than those used for the ITS barcode.

Crust fungi

There is only a 50%-60% success rate. Success is higher with recently collected specimens than old ones. For thin crusts, take fungal tissues together with some underlying wood.

Stinkhorns

Take pieces of the spore-bearing surface with spores (gleba) for obtaining the highest concentration of DNA.

Puffballs

Young puffballs can be safely dried in a drying oven, and pieces of the spore-bearing interior can be tweezed off and placed in a vial for sequencing. Older specimens should not be placed in a drier with other specimens as they will end up contaminating other collections in the drier. If only mature specimens are available, remove tissue from fresh specimens, place that in a vial, and place the vial with the lid open in the drying oven. Alternatively, place the mature puffball pieces in a paper envelope or sheet of paper folded into a packet and place that in the drying oven.

Fleshy Ascomycetes (Cup fungi, Morels, etc.) 

To remove a piece of the spore producing surface from cup fungi, ensure the inside of the cup is clean. Try to tear the cup in half, and then tweeze a clean piece of tissue from the cup’s inner (spore-producing) surface. For morels, spore-bearing tissue can be used from the indentations on the ‘cap’ or from the upper stalk, but ensure the tissue is clean. False morels (Helvella) are often contaminated by soil, so extra careful cleaning is required when removing a healthy piece of the ‘flap’. Success rate may be low.

Hard (Carbonaceous) Ascomycetes (e.g., Hypoxylon and Dead Man’s Fingers)

Bag, tag and hold for future sequencing. Air-drying is as good or better than oven-drying, and it also prevents the ascospores from being shot out and distributed across all the other specimens in your drying oven. One problem is the ITS barcode region is not helpful for this group. Another problem is separating tissue containing DNA from the hard, carbonaceous parts.

About North American Mycoflora Project

NAMP is a 501(c)(3) non-profit organization expanding the continent-wide community of volunteer citizen scientists and professional mycologists who are documenting the distribution and biodiversity of North American mushrooms and fungi.

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