Getting Started: The Colonization Stage

Arguably the most difficult part of mushroom cultivation is the “colonization stage” which includes the preparation of the colonization medium, the sterilization of the colonization medium, the inoculation of the colonization medium with mushroom genetics, and the colonization of the spawn. There are several tasks that one must perform during these steps, paying close attention to detail and sterility, as there are several avenues for potential user error that may result in contamination. This guide aims to provide a clear and concise explanation of this tricky stage so that the home/hobby mushroom cultivator can refer to it should he/she have any questions.

STEP 1 - Preparing the colonization medium

For this step, you will require the following materials:

  • Rye grain berries (or another colonization medium)

  • A large bowl

  • A colander

  • A tray/tub/tote

  • A fan (optional but recommended)

First, select your colonization medium. Here at Spores Lab, we have experimented with several colonization mediums but we have had the best results using rye grain berries. Other colonization mediums that you could use include but are not limited to white wheat, corn, oats, millet, and birdseed, though note that using any of these mediums will typically result in less overall yield of mushroom compared to rye grain berries. Whichever colonization medium you decide to use, ensure that the medium is organic and that it has NOT had a fungicide applied.

Organic, non-fungicide rye grain berries.

Once you have your rye grain berries, place however much grain you will require (keeping in mind that the grain will expand in volume once it is hydrated), in the large bowl and fill the bowl with cold water (tap water is fine). Pour out the water and repeat this several times until the water is pouring noticeably clearer. Now fill the bowl with cold water and cover the grain. The water level should be 6-8 inches above the grain because the water level will drop as the grain absorbs the water. Allow the grain to soak for 18-24 hours (the amount of time is dependent on the humidity of your environment - if you live in a dry environment, soak for 24 hours, if you live in a humid environment, soak for closer to 18 hours).

When you return to your grain, the water level will have dropped significantly. This is normal and simply means your grain was able to absorb the moisture.

Pour out any remaining water in the bowl and then fill the bowl with HOT water (NOT BOILING WATER - JUST TAP WATER AT MAXIMUM HEAT), and let the grain sit in the hot water for 15 minutes. This heat differential allows the grain to fully “plump” and absorb the maximum amount of moisture possible. After 15 minutes, pour out the hot water, strain the grain using a colander, and wait until the colander stops dripping.

Now spread the grain out evenly in the tray/tub/tote and place it in a high-airflow area for about 1 hour to dry the exterior of the grain. If you have a fan, you could use it to dry the grain more efficiently.

Grain being dried by a fan on a drying rack.

Ideally you want the grain to be as saturated as possible, but with little moisture on the exterior surface of the grains (the industry term for this level of saturation is “field capacity”). A rough rule of thumb that you can use to estimate the correct dryness of your grain is to pick up a small handful of grain and then turn your hand upside down. If a few grains stick to your hand, your grain is at the correct dryness. If no grains stick to your hand, the grain is probably too dry, and if too many grains stick to your hand, the grain is probably still too wet.

A handful of grain at field capacity.

Step 2 - Sterilizing the colonization medium

For this step you will require the following materials:

The All-American 75X Electric Sterilizer

Prepare your pressure cooker (PC) or electric sterilizer by filling it to the notched line with water (this notched line is usually found on the inside of the pressure chamber). If your PC does not have this notch, fill the cooker so the water level is approximately 3” above the bottom “plate” of the cooker (see image below). Also ensure that you have a proper weighted release valve so that your sterilizer/PC can reach the proper operating pressure (15PSI). If you are using an Instapot to sterilize, follow the manual instructions for the “pressure cook” or “sterilize” button (most models will have one of these two options).

Add your prepared grain to the jar or container that you wish to colonize in and screw the lid of the jar or container on (we recommend using a Spores Lab leak-proof injection/filter port lid for easy inoculation). If you do not have an injection/filter port lid, drill a ¼ inch hole in the lid of the jar or container that you will be using, and cover the hole with a piece of micropore tape.

Place the sealed jar in your PC or electric sterilizer and cook at 15PSI for 150 minutes (2.5 hours). If your sterilizer does not have the capacity to pressurize to 15PSI, add 1 hour to the cook time for every 1PSI below 15PSI. (for example if your cooker only pressurizes to 13.7PSI - which is common - cook for 210 minutes (3.5 hours).

Some other tutorials and online forums suggest sterilizing for 90 minutes (1.5 hours), but we recommend a longer sterilization time. The reason why we recommend a longer sterilization time is simply that sterilizing for longer does not negatively affect the spawning medium or container in any way (provided that the container is autoclavable) but does ensure that all bacterial and fungal spores present in the medium will be killed. Just ensure that there is enough water in your PC or electric sterilizer to last throughout the whole cook, as if all the water evaporates before sterilization is complete, this could cause the container or lid to melt and could also damage your PC or sterilizer.

After the cook, let the sterilizer cool for approximately 1 hour and then clean the outside surfaces of the sterilizer with disinfectant BEFORE opening it. When you open the sterilizer there is a brief pressure differential that sucks some air into the sterilizer as the pressurized air inside the sterilizer escapes, and you will want the surface of the sterilizer to be as clean as possible when this happens.

Step 3: Inoculation with mushroom genetics

For this step, you will require the following materials:

A Spores Lab spore syringe

Once you have removed your jar or container from the sterilizer, you are ready to inoculate! We recommend inoculating your grain within 24 hours of sterilization. This is because there is a finite amount of moisture in the sterilized grain that must last for the entire mushroom life cycle. The longer you wait to inoculate, the more the grain dries out and the more difficult of an environment it will be for the mushroom mycelium to grow and thrive in.

Inoculation is a sensitive step so don’t be afraid to go overboard on cleanliness during this step - doing so will serve to reduce the chances of your mushroom culture becoming contaminated.

Start by putting on your personal protective equipment (gloves and mask), and then wipe down the syringe, the surface and lid of your colonization container, and your gloves and arms with disinfectant or alcohol swabs. Place your supplies (syringe, container with sterilized grain, and disinfectant) inside your Still Air Box (SAB) or in front of your flow hood. If using a SAB, put the lid on the SAB and liberally disinfect the interior of the SAB (including the gloves) with an aerosol disinfectant spray.

If you are inoculating with a spore syringe, smack the syringe against the palm of your hand to dislodge and break up clumps of mushroom spores. If you are inoculating with a liquid culture syringe, shake the syringe vigorously to ensure the contents are mixed well.

Now unwrap the sterile 18ga needle (this comes with each Spores Lab spore syringe and liquid culture syringe), remove the plastic Luer Lock tip from the syringe, and attach the needle to the syringe by inserting and twisting the needle. Sterilize the needle with more disinfectant spray, and then insert the needle into the self-healing injection port on the lid and push the plunger of the syringe to add the desired amount of syringe fluid into the jar. If you are not using an injection/filter port lid, insert the needle into the ¼ inch hole you drilled into the lid prior to sterilization, inject the desired amount of spore syringe or liquid culture syringe fluid and then immediately cover the hole with a new piece of micropore tape.

Inoculating in front of a laminar flow hood (left) and in a still air box (right).

We recommend injecting 5mL (cc’s) of syringe fluid for a 1L jar. Each Spores Lab spore syringe and liquid culture syringe contains 10mL (cc’s) of syringe fluid, which is enough to inoculate 2x 1L jars. You could use more or less syringe fluid, if desired, but note that your colonization time will decrease or increase, respectively.

Step 4: Colonization of the Spawn

Once you have inoculated your sterilized grain with mushroom genetics, shake the jar to evenly disperse the spore syringe or liquid culture syringe fluid and then simply leave the jar in an environment that has the proper environmental conditions for colonization, namely:

  • A steady temperature between 75F-77F

  • An ambient humidity of about 45%

  • Near or total darkness

Note that it is crucial that the temperature in the environment you are colonizing in does not exceed 80F at any point during colonization. At temperatures close to 80F, your grain will begin to “sweat” due to the excess heat and this invites contamination.

If at any point during the colonization period you notice a pungent odour coming from the container, or notice any coloration that is NOT white mycelial growth (ie. green, black, or grey growth), quarantine that container from the rest of your containers immediately and dispose of it. It has likely become contaminated and if you do not remove it from the area it will contaminate the other containers around it.

Examples of Contamination

Colonization of spawn from inoculation via a spore syringe will take anywhere from 2-6 weeks depending on how much grain is in your container, how much spore solution you inoculated with, and what the environmental conditions of the room you are colonizing in are. Inoculation with a liquid culture syringe will take 2-3 weeks. The mycelium is “fully” colonized when you are barely able to see grain, and the majority of the jar/bag is a solid block of white mycelium.

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