How to Create Liquid Culture for Mushroom Cultivation

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Introduction

Interested in growing your own mushrooms but unsure how to get started? One of the fundamental techniques in mushroom cultivation is making liquid culture (LC). In this article, we will guide you through the process of creating liquid culture in a simple and effective way.

The Recipe

Most liquid culture recipes contain a sugar content of 4 percent or less, as exceeding this amount can be toxic to the mycelium. However, you can add small amounts of other ingredients like protein, lipids, starches, minerals, and vitamins to enhance the culture. It’s important to note that certain ingredients may cause cloudiness, which can hide contamination. For general purposes, regular-mouth pint (500 ml) Mason jars are ideal. Feel free to modify the recipe as you expand your cultures to keep things interesting.

Equipment and Ingredients

Before you begin, gather the following items:

  • A gram scale or measuring spoons
  • Measuring cup
  • Jars
  • Airport lids and rings
  • Agitators (marbles, broken glass, crystals, or magnetic stir bars)
  • A big pot
  • Jar funnel
  • Ladle
  • Paper towel or clean cloth
  • Big spoon or whisk
  • Aluminum foil
  • A pressure canner (PC) or alternative sterilization method

Step-by-Step Guide

  1. Measure out all the ingredients and calculate the total volume based on the number of jars you need to fit in your pressure canner (PC). Leave room for a plain water jar as well.
  2. Heat water in a pot until warm, but not boiling.
  3. Stir in the other ingredients until fully dissolved.
  4. Using a jar funnel and a measured ladle or pitcher, fill the jars no more than halfway.
  5. Drop an agitator (stir bar or other option) into each jar.
  6. Wipe any drips from the rim of the jars.
  7. Put on the airport lids and screw down the rings until snug. Cover the jar lids with foil to keep the filters dry.
  8. Load the jars into the pressure canner, along with any other tools that need sterilization, wrapped in foil.
  9. Sterilize the jars for 15-20 minutes at 15 psi. Be careful not to overcook, as this can caramelize the sugars and melt the syringes.
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Tissue Culturing to Liquid Culture

Tissue culturing is a useful technique for most fleshy mushrooms, although catching tough or tiny mushrooms can be challenging. If the mushrooms are waterlogged, it’s best to allow them to dry in the fridge before using them. Dry mushrooms can be rehydrated by soaking them in a 3% hydrogen peroxide solution. For better results, repeat the process with multiple jars of media.

Equipment and Ingredients

Before starting, gather the following items:

  • A mushroom
  • A sterile syringe with a 16-gauge needle
  • A jar of sterilized water with an airport lid
  • A jar of sterilized LC medium with an airport lid
  • Alcohol in a spray bottle
  • Alcohol prep pads
  • Cotton balls
  • Tweezers or knife (optional)
  • Permanent marker

Step-by-Step Guide

  1. Gather all the tools and supplies on a clean surface.
  2. Spritz your hands with alcohol and rub them together until dry.
  3. Unwrap the syringe, but leave it capped for now.
  4. Remove the foil from the jars and spray the injection ports of both lids with alcohol. Wipe them clean with a cotton ball.
  5. Give a final spritz of alcohol to the self-healing injection port (SHIP) of the water jar. Uncap the syringe and quickly penetrate the injection port.
  6. Tip the jar until the needle tip is in the water, making sure not to wet the filter. Draw up a few milliliters of water.
  7. Spritz the outside of the mushroom with alcohol and wipe it with a prep pad to sterilize the surface. If the mushroom is very fleshy, you can use tweezers or a knife to split it and remove the exterior tissue on two opposing sides, without touching the interior tissue. Spritz again.
  8. Remove the needle from the water jar and quickly but carefully stab it through the mushroom, avoiding highly textured or spore-bearing tissues (such as gills).
  9. Check inside the needle to see if you have collected some tissue. If not, try again.
  10. If you have collected tissue, give a spritz of alcohol to the SHIP of the LC jar and insert the needle.
  11. Push down the plunger, squirting the tissue sample and water into the culture media.
  12. Aerate the jar, label it with the species, strain, date, TC (tissue culture), and medium, and set it aside to incubate.
  13. Wait to aerate the jar until you observe visible growth from the tissue sample. Once you see growth, aerate the jar daily until the mycelium has fully colonized the medium.
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Making a Spore Syringe

A spore syringe allows for multispore germination, which results in multiple genets growing together. Fruits produced from this lineage can be tissue-cultured to isolate a desirable strain.

Equipment and Ingredients

Before you begin, gather the following items:

  • A spore print in a bag
  • A sterilized water jar with an airport lid
  • Cotton balls
  • A sterile syringe
  • Alcohol spray
  • Alcohol flame or substitute

Step-by-Step Guide

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

LC or Spore Syringe to LC Transfer

If you already have a spore or LC syringe, you can transfer its contents to a jar of liquid culture. This allows for further cultivation and expansion of your desired strain.

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Equipment and Ingredients

Gather the following items:

  • Spore/LC syringe and alcohol flame or extra sterile needle, OR
  • Sterile syringe and LC jar
  • Sterile LC media jar
  • Alcohol spray
  • Cotton balls

Step-by-Step Guide

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

Agar Plate or Slant to LC Transfer

Transferring mycelium from an agar plate or slant to liquid culture is another commonly used technique in mushroom cultivation.

Equipment and Ingredients

Before starting, gather the following items:

  • An aseptic transfer space (such as a still air box)
  • Sterile syringe and needle
  • Sterilized jar of water fitted with an airport lid
  • Mycelial culture on agar (in a Petri dish or a culture tube [slant])
  • Jar of sterilized LC medium
  • Alcohol spray
  • Cotton balls
  • Plate wrapping material
  • Permanent marker

Step-by-Step Guide

  1. Load all the supplies into a clean still air box or other aseptic transfer space. Mist the inside of the box and allow the air to settle.
  2. Unwrap the syringe, but leave it capped for now.

What’s Next?

To learn more about mushroom cultivation, check out the following articles:

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