From Agar to Liquid Culture

Part 2 : Unit 2 : Transferring Agar to Liquid Culture

Now that your spores have germinated and you have live, clean mycelium in a petri dish, it’s time to use this mycelium to inoculate some more substantial food, or substrate. While most mushroom cultivators use colonized agar to directly inoculate grain spawn, we take a slightly different route, which we believe has benefits for first time growers. We will use our colonized agar to inoculate Liquid Culture, in order to grow plenty of “liquid mycelium”. This will allow us to use larger quantities of mycelium when inoculating our grain spawn (in the next step), as well to jump start any future inoculations.  In addition, the fact that we use injection ports in our jar lids, means that this extra step doesn’t introduce any additional significant exposure to contamination. Liquid culture can be kept in the fridge for several months and still be viable, so it’s a good storage medium. All in all, Liquid Culture is a tool you should  familiarize yourself with in your mushroom cultivation journey. In this chapter, we dive into the Liquid Culture medium and get our hands….well, clean.

Liquid culture jar with mycelium growing in it
Liquid Culture Jar With Mycelium Growing In It

Table of Contents - From Agar to Liquid Culture

Making Liquid Culture in Mason Jars

Items needed
  • 1 Mason Jar + Modified Lid
  • Pressure Cooker + Rack
  • 1g Light Malt Extract or Honey
  • 600 ml Non Chlorinated Water
  • Weight Scale
  • Jar Stirrer
Notes & Tips
  • Avoid PC'ing Honey and Kero Corn Syrup LCs for more than 25 minutes. This causes them to caramelize.
  • Remember to add a stirrer into the jar before PC'ing, which will allow you to mix the liquid mycelium.
  • LME absorbs water and moisture. Avoid exposing it to either by storing it in an air tight, dry bag.
Liquid culture

Video Tutorial : Making and Sterilizing Liquid Culture in Mason Jars

The video markers above 👆🏼correspond to the the various Steps in this Unit. Click on them to skip to a Step.

Step 1 : Mix the Liquid Culture ingredients in Flask

Clean and disinfect the jar, the lid, the stirrer and the silicone seal, and let them dry out. Once dry, put the silicone seal in the lid. Put the stirrer in the jar. If you don’t have a magnetic stirrer, then a couple of marbles or clean, hard stones will work as well. Measure out one gram of LME powder (or 6 teaspoons Kero / Honey) and put it in the mason jar. Pour about 600 ml of non-chlorinated water into the jar and mix well. Seal the jar with the lid and cover it with aluminium foil to protect the liquid culture.

Step 2 : Pressure Cook for 20 Minutes, then allow to Cool

Fill the pressure cooker with about 1.5 inches of water, and then pressure cook the Liquid Culture jar for about 20-25 minutes. Allow the PC to cool down completely before opening it and removing the jars. Once cool, your Liquid Culture jars are ready to be inoculated. Store the Liquid Culture jars in a cool, dry place until you put them to use.

Pictures : Liquid Culture in Various Colours and Occupancy

Liquid culture close up
Liquid Culture Close Up
Liquid culture jar with mycelium growing in it
Liquid Culture On Grass
Liquid culture
Various Shades Of Liquid Culture

Inoculating Liquid Culture with Colonized Agar

Items needed
  • Colonized Agar Petri Dish
  • Still Air Box
  • 70% Alcohol Spray
  • Sterile Knife or Blades
  • Parafilm
Notes & Tips
  • It helps to think out the process in your head before actually doing it.
  • Try to lift the agar from the petri dish and not stab it. This way it will be easier to drop the agar in the LC jar.
  • Try to choose the healthiest and fastest growing mycelium from the petri dish.
Inoculating agar

Video Tutorial : Inoculating Liquid Culture with Colonized Agar

Step 1 : Prepare SAB and Items for the Mycelium Transfer

Clean your work environment and SAB. Disinfect the Liquid Culture jar, colonized petri dish, blade wrapper and handle, and anything else you will be using inside the SAB. Remove the aluminium wrapper from the Liquid Culture jar, and remove the parafilm from the petri dish. Disinfect the jar and the dish again, as well as your hands. Spray the air inside the SAB with some fine mist alcohol. Loosen (but don’t open) the LC jar lid. 

Step 2 : Transfer Colonized Agar into the Liquid Culture Jar

Carefully open up the sterile knife blade and attach it to the handle. Open the colonized petri dish and cut out a healthy and clean looking triangle of mycelium-covered Agar using the sterile blade. Try to lift the triangle with the blade, rather than poking it. Close the petri dish and open the mason jar lid slightly, just enough to allow you to gently drop the Agar triangle into the jar. Then cover the LC jar and tighten the lid. Take the aluminium cover you unwrapped earlier from the jar, lightly spray it with alcohol and place it back on top of the jar. Make sure you can see the agar floating in the LC jar, and give it a gentle swirl. You want to avoid any liquid from touching the air filter sticker.

Step 3 : Allow Mycelium to Colonize the Liquid Culture

Place the LC jar in a dark and quiet place. Temperatures should be between 22-27 °C or 70-80 °F. Mix the liquid in the jar using the magnetic stirrer once a day. If you didn’t use a magnetic stirrer and placed marbles or stones instead, gently swirl the jar to get the stones moving. These will cause the water to stir, and help break up the mycelium inside the jar. After about 7 days to a week, you should see a significant increase in the amount of mycelium inside the jar. Once at least 1/3 of the jar is full of the cloudy mycelium, you can store the Liquid Culture jar in the refrigerator. 

Previous

Next Unit: Preparing and Inoculating Grain Spawn. Learn how to properly hydrate and sterilize corn kernels as grain spawn. Once sterile, you inoculate the grain with liquid mycelium culture and allow full colonization.

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7 Responses

  1. Just a question here
    Wouldn’t it be safer to inject a bippsy of the age into the lc through the injection port ?
    Like would t theree be a way to suck up some age into the needle and stick it in without opening the jar ?

    Reply

    1. In theory, that would work. However, the agar is very thick and cant always be biopsied in a way that will hold mycelium in the 18g needle. I would think you would need a much larger needle for this, which at this gauge might cause damage to the injection port.

      Reply

  3. Don’t you have to test the Liquid Culture to see if it is contaminated? I heard that you can’t tell unless you put it on agar.

    Reply

    1. You are correct, you can’t really tell if the liquid culture is contaminated or not, other than obvious signs of discoloration or murkiness. To be certain, you could put a sample of your LC on agar to check for healthy growth, and only then spawn to grain. Saying this, if you stick to the process described here, there’s not much risk of contaminating your liquid culture, especially with the needles and injection ports.

      Reply

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