Liquid Culture Recipe

Stir in other ingredients until fully dissolved.Using a jar funnel and a measured ladle or pitcher, fill jars not more than halfway.Drop a stir bar or other agitator into each jar.Wipe up any drips from the rim of the jars. Put on airport lids and screw down rings until snug. Cover jar lids with foil to keep filters dry, and load into PC. Include any syringes or other tools in need of sterilization, wrapped in foil.Sterilize for 15-20 minutes at 15 psi. Longer cooking will caramelize and toxify the sugars and melt the syringes.

Tissue Culturing to Liquid Culture

This biopsy technique works well with most fleshy mushrooms, though tough or tiny mushrooms can be nearly impossible to catch This way. Waterlogged mushrooms should be allowed to dry out a little in the fridge first. Dry mushrooms can be soaked in 3% hydrogen peroxide to rehydrate. For improved chances, repeat process with multiple jars of media.

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You’ll need: a mushroom, a sterile syringe fitted with a 16-gauge needle, a sterilized jar of water with an airport lid, a jar of sterilized LC medium with an airport lid, alcohol in spray bottle, alcohol prep pads, cotton balls, tweezers or knife (optional), permanent marker.

1. Gather all tools and supplies on a clean surface.
2. Spritz hands with alcohol and rub together until dry.
3. Unwrap syringe, but don’t uncap yet.
4. Remove foil from jars, spray injection ports of both lids with alcohol and wipe clean with cotton ball.
5. Give a final spritz of alcohol to the SHIP of the water jar. Uncap syringe and quickly penetrate injection port.
6. Tip jar until needle tip is in water, taking care to not wet the filter. Draw up a few milliliters of water.
7. Spritz outside of mushroom with alcohol and wipe with prep pad to sterilize the surface. If very fleshy, use tweezers or knife to start a split and peel away exterior tissue on two opposing sides, without touching tool or fingers to interior tissue. Spritz again.
8. Remove needle from water jar and quickly but carefully stab through the mushroom, avoiding highly textured or spore-bearing tissues (i.e., gills).
9. Look inside the needle to see if you got some tissue inside. If not, try again.
10. If so, give a spritz of alcohol to the LC jar’s SHIP, and penetrate with the needle.
11. Push down the plunger, squirting the tissue sample and water into the culture media.
12. Aerate, label with species, strain, date, TC (tissue culture), and medium, and set to incubate.
13. Wait to aerate again until visible growth from tissue sample is observed, then aerate daily until myceliated.

Making a Spore Syringe

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Once a spore syringe is transferred to LC, grain, or substrate, the result will be multispore germination, which leads to multiple genets growing together. Choice fruits from this lineage can be tissue-cultured to isolate a good strain.

You’ll need: a spore print in a bag (see “Spore Prints,” above), a sterilized water jar with airport lid, cotton balls, a sterile syringe, alcohol spray, and an alcohol flame or substitute.

1. Gather supplies on a clean work surface and sanitize hands.
2. Unwrap syringe, but don’t uncap yet.
3. Remove foil from jar, spritz SHIP with alcohol, and wipe clean with cotton ball.
4. Give a final spritz of alcohol to the SHIP of the water jar. Uncap syringe and quickly penetrate injection port.
5. Tip jar until needle tip is in water, taking care to not wet the filter. Draw up a syringeful of water. Leave needle in port for now.
6. Sanitize outside of spore print bag and remove the needle from the jar. With bag on work surface, stab bag (careful not to go through both layers) and inject water into bag.
7. Use your hand to mix spores into water, then draw water back into syringe.
8. Flame-sterilize needle and cool by squirting out a few drops of spore solution and recap; or, if you want to make multiple syringes, spritz water jar’s SHIP with alcohol and inject spore solution in there.
9. Label and wait 24 hours for the spores to hydrate before using. Spore syringes can stay viable for months to years. Store in a cool, dark place.

LC or Spore Syringe to LC Transfer

You’ll need: spore/LC syringe and alcohol flame or extra sterile needle, OR sterile syringe and LC jar; sterile LC media jar; alcohol spray; and cotton balls. If using pre-made spore or LC syringe, skip steps 5 and 6 and do step 7.

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Gather all tools and supplies on a clean surface.

1. Unwrap syringe, but don’t uncap yet.
2. Aerate both jars vigorously for about 30 seconds, breaking up the mycelium in the LC.
3. Remove foil from LC media jar, spray injection ports of both lids with alcohol, and wipe clean with cotton ball.
4. Give a final spritz of alcohol to the SHIP of the LC jar. Uncap syringe and quickly penetrate injection port.
5. Tip jar until needle tip is in LC, taking care to not wet the filter. This can be done in midair or on the work surface. Draw up 1-10 ml LC, making sure you get some mycelium and not just broth. If the needle clogs, push a little out to clear it and continue. Set the jar upright.
6. If using a pre-made LC or spore syringe, either swap its needle for a sterile one or flame-sterilize needle until red-hot. Allow 3-5 seconds for the needle to cool.
7. Spritz the media jar’s SHIP with alcohol and quickly insert the needle into the media jar. Squirt contents of syringe into jar.
8. Label with species, strain, date, media, source jar and generation, and set to incubate.

Agar Plate or Slant to LC Transfer

You’ll need: an aseptic transfer space (i.e., still air box), a sterile syringe and needle, a sterilized jar of water fitted with an airport lid, a mycelial culture on agar (in either a Petri dish or a culture tube [slant]), a jar of sterilized LC medium, alcohol spray, cotton balls, plate wrapping material, and a permanent marker.

1. Load all supplies into clean SA B or other aseptic transfer space. Mist inside box and allow air to settle.
2. Unwrap syringe, but don’t uncap yet.

More from DIY Mushroom Cultivation:

• How to Build a Home Fruiting Chamber
• What is a Mushroom?

Excerpted from DIY Mushroom Cultivation, by Willoughby Arvalo. Published by New Society Publishers © 2019 by Willoughby Arvalo. All rights reserved.

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