Tissue Collection Protocol

There are a number of different methods that can come into play for people to preserve mushrooms for future analysis. We will discuss several of them, but our preferred protocol is to save fresh tissue from the mushroom in a 1.5 mL aliquot of Nuclei Lysis Solution.

Organize your Materials

You will need 1.5 mL tubes, tweezers, a lighter, and alcohol swabs. You can order supplies directly from our store.

Retrieve your Tissue

Tear your specimen open to expose the inner tissue. Remove a piece of flesh the size of a grain of rice, and place it in your tube.

Label your Tube

Label your tube with the correct collection number. Organization is key!

Tissue Collection Video Protocol

This video outlines the procedure to collect tissue using our preferred method - 1.5 mL microcentrifuge tubes. 

Saving the Tissue – Collection Tube (Microcentifuge tube or Eppendorf [eppi] tube)

By now you should have taken pictures of the mushroom in situ, collected the mushroom, and brought it back for preservation. Your voucher data card should be completely filled out and your sample should have a collection number. We will now be taking a small piece of tissue from the mushroom and preserving it in our 1.5 mL eppi tube containing Nuclei Lysis Solution.

Lay the species that you are going to be working with out on a clean table. Sterilize a scalpel and tweezers with an alcohol swab, and lay them on a piece of paper towel next to your work area. Label the eppi tube with the collection number of your specimen.

Begin by gently tearing open the stem of the mushroom to expose the sterile inner surface. You will not want to cut your mushroom open with a knife, as external contaminants can be tracked through the inner portion on the blade. Externally, mushrooms can have yeasts or molds growing on them that can cause problems when attempting to attain a clean sequence. Once the inner surface is exposed, tweeze out or cut out a portion of the inner flesh that is about the size of a grain of rice. Place this piece of material into your eppi tube and close the cover. That is all that needs to be done for this sample, except for ensuring that the tube is properly labeled with the collection number. Now you can sterilize your scalpel and tweezers to repeat this process for your next sample.

Sterilizing your instruments (scalpel and tweezers) can be done by wiping them with an alcohol swab and them giving them a short flame with a lighter. No need to get them red hot, just put the flame to the metal for 3-4 seconds.

It is possible to take tissue from any part of the mushroom in order to acquire DNA. We recommend taking tissue from the stem of the mushroom as to preserve as much of the cap as possible. The cap contains many more parts that could be important for future microscopic study. Thus, we want to maintain as much of it as possible intact.  

Some genera (i.e. Marasmius) may have species that are too small in order to effectively harvest tissue from the inner surface of the stem. In this case, you may decide to place a portion of the stem into the eppi tube, or even the entire stem for very small specimens, preserving as much of the cap as possible.

Tissue can be stored in the eppi tube with Nuclei Lysis Solution for many months, if not years, before any further steps are taken to get a sequence.

Saving Tissue Video Protocol - FTA Cards

This video protocol outlines a secondary method of tissue collection that works with fleshy fungi. This protocol is significantly more expensive than our preffered method, but it may be best in some individual circumstances.

Saving the Tissue – FTA Cards

Another common method for DNA preservation of wild mushroom samples are Whatman FTA Plant Cards. These are cards made of a paper that contains DNA stabilizing agents. The process to use the cards is simple.

  1. Place a small sample of mushroom tissue in a circle on the interior of the card. Once again, you want to get a section of tissue that is from the interior flesh of the mushroom. This will prevent contamination during the DNA extraction process.
  2. Fold the cover of the card over the tissue.
  3. Hit the area containing the tissue with a hammer to macerate the tissue and release the interior liquid into the paper of the card.
  4. Turn the card over to ensure there is a spot of moisture from the tissue that has soaked into the paper.
  5. Remove any excess issue from inside the fold or that may be remaining on the cover. Try not to scratch or damage card material during this process.
  6. Label your card with the collection number for this species.
  7. Allow the card to dry.

While this is not recommended as our primary procedure, it will work very well for both getting a sample of DNA from the tissue and preserving it for future analysis. The cards are somewhat expensive at about $4 or $5 each. This will run you about $1.00 per sample for DNA collection. It is possible to put two or more samples into each section of the card, lowering the overall cost per sample. One advantage to these cards is their compact size, allowing for easier transportation. The primary disadvantage is that it is only possible to take samples from fleshy fungi. Whatman cards do not work well for dry or tough specimens (like polypores).

About North American Mycoflora Project

We are working towards a single goal - the development of the first comprehensive mycoflora of North America. This project is a consortium of citizen scientists and professional mycologists performing a biological survey of all the macrofungi that occur in North America.

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