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Cloning a Mushroom

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Cloning: Excising a piece of pure living flesh from a mushroom, under sterile conditions, and placing in sterile, nutrient rich medium, in order to capture the characteristics of that particular mushroom.

Selecting and Preparing material to clone[edit | edit source]

Choose young, fresh mushrooms, preferably in the button stage. (Be careful that you are certain of the species, as many appear the same in the button stage.) Cut away debris before bringing in front of the transfer unit.

Use a damp paper towel to clean off outside of mushroom. Best areas for taking the tissue:

  • Directly above the gills
  • Directly above the disc of the cap
  • Interior tissue at the base of the stem

Procedure[edit | edit source]

1. Loosen cap of clean tube and lean against frame of filter,

2. Lay the mushroom on a piece of paper towel in front of the sterile transfer unit.

3. Flame-sterilize a sharp scalpel until it is glowing red.

4. Cool the tip by:

  • touching the nutrient agar in the petri, dish or culture tube , or
  • waiting a few seconds, or
  • dipping in alcohol bath and re-flaming to burn off the alcohol

5. Tear the mushroom from the base, up the stem and through the cap, revealing fresh, clean material.

6. Cut out a small section of flesh, (the size of a grain kernel). Cutting out an inverted pyramid shape helps to free the piece of flesh with more ease.

7. Quickly transfer the piece of tissue into the culture tube, which you are holding horizontally. Scraping the piece off using the first inside fold of the tube may help. Screw the lid on tightly and shake the tube gently until the tissue is in contact with the agar. If transferring to a petri dish, place the tissue in the center, in the cut where you cooled the blade. Re-flame the scalpel for each new tissue transfer.

Keep the area being transferred to closest to the filter, in the area that is “upstream”. Downstream from that are the cultivator’s hands, down stream from that is the specimen on the towel.

8. label the tube with:

  • date
  • genus, species (P. ostreatus)
  • your initials

9. Optional: log the above amount and comment on anything that happened that may affect the procedure .

Preparation of Equipment[edit | edit source]

Set up[edit | edit source]

  • Laminar flow hood on the table with a clear workspace in front.
  • Find extension cord and plug in unit.
  • Remove front panel and wedge under squirrel cage to form a little overhang protecting the first few inches of space in front of the filter.

Set out[edit | edit source]

Alcohol burner (denatured alcohol), lighter Scalpel Scalpel rack Gloves Alcohol Paper towels Sterile agar tubes Labels, pen Parafilm or tape

Clean[edit | edit source]

Entire workspace. I.e., area in front of filter, and bottom of “roof” over area, and any implements that you are using in that area (outside of lamp, scalpel handle, tube rack or scalpel holder, pen) using a 10% Clorox /water solution, hydrogen peroxide, or rubbing alcohol. Light the alcohol lamp and place at either side of the work area, inside the flow area. Put on gloves and place a little alcohol in your palm and rub over the surface of the glove. (Be sure to keep your gloved hands away from the flame, if you have recently used the alcohol to clean.)