Mycology (the study of Fungi) is rapidly gaining popularity as humanity comes to the realization that fungi can provide several physical, cognitive, economic, environmental, and psychological benefits. Fungi are some of the oldest organisms on the planet, and interestingly shared an ancestor with humans until about 1.5 billion years ago when they split from the “animal” branch of the tree of life. This split occurred when organisms in the animal branch began to encapsulate nutrients in a cellular sack (a stomach) for digestion, whereas Fungi continued to digest at an individual cellular level. Fungi propagate their genetic code through the dispersion of spores, and survive by consuming nutrients from their environment (mostly in the form of detritus and decaying material). Therefore they play a crucial role in the health of any ecosystem, as they are the primary decomposers of both plant debris like cellulose/lignin, and dead/decomposing animal tissue. Furthermore, they also have extraordinary capacity to recycle/decompose toxic material, from petroleum oils, to nuclear waste.

The benefits which Fungi provide are not limited to the ecosystem level, they can also have significant benefit to the individual. Several edible/gourmet species (Lions Mane and Shiitake in particular) promote increased cognitive, cardiovascular, and mental health, while Psychoactive varieties (Psilocybe Cubensis in particular) have shown remarkable results in the treatment of many psychological conditions. These benefits have catalyzed many people to begin growing their own mushrooms, and the greatest information repository known to man (the internet) is awash with forum posts and tutorials. Unfortunately many of these tutorials overlook details and do not explain why certain parts of the process need to be done.

We at Spores Lab designed this manual to give a quick gloss over of our tried and true process, and also try to explain the mechanism behind why our process works.

Before we get into the specifics of cultivation, let’s go over the basics of the mushroom life cycle under natural conditions.

If spores are successfully dispersed in an environment with sufficient nutrients and specific environmental conditions, they will begin to utilize available nutrients to grow and form what is called “mycelium”. Mycelium consists of a mass of branching root-like strands, each strand a single cell thick, called Hyphae. Mycelium can be described as the vegetative portion of a fungus (where all nutrients and energy are put towards growth instead of gene propagation).

Mushroom mycelium will continue to grow and spread as long as nutrients are available, and as long as the environmental conditions are congruent with this “stage” of the mushroom life cycle. This part of the life cycle (where the mycelium is growing but no mushrooms are present) is often called “spawning” or “colonization”.

The next step in the mushroom life cycle happens once the mycelium has “colonized” (utilized most of the nutrients available). At this point (under natural conditions) changes in environmental conditions (like temperature and humidity) will trigger the mycelium to switch from a “spawning” or “colonization” state to a “fruiting” state. It is in this fruiting state that mushrooms grow out of the mycelial mat, and mushrooms will continue to sprout until all available nutrients and moisture in the environment are used or environmental conditions are changed back to conditions congruent with the colonization state.

Explained above is the mushroom life cycle under natural conditions, however when cultivating in an artificial environment certain steps (like working from an isolated culture*, or adding extra nutrients via a fruiting medium before switching to fruiting conditions) can be taken to increase fruiting body size and yield.

*Advanced mycologists can clone mushroom tissue by placing it in an *agar medium. This process (called “isolation”) creates a culture that has a narrow genetic profile, and results in faster ‘colonization’ time, higher yield, and larger fruiting bodies (mushrooms). Further isolation from the initial mushroom tissue sample can be done using agar, by selecting especially strong mycelial strands and propagating these onto another agar petri dish. Another way in which yield can be increased when cultivating indoors is through the addition of a high-nutrient “fruiting medium” when the mycelium has completely colonized its initial medium. When the fruiting medium is combined with the initial grain based medium (that is fully colonized) it is called a “fruiting substrate”.

Agar is a high-nutrient gelatinous medium, the caveat to working with isolations/agar is that a “flow hood” is highly recommended. (a flow hood consists of a HEPA filter enclosed in a box with a fan situated opposite the filter. This allows filtered are to be continually blown over your workspace)

Since this manual is aimed at the “hobby” cultivator, we will cover the process of cultivation using a Spore Syringe. If you are interested in learning more about agar cultures please contact us!

Regardless of whether you will start with an Isolation in an agar medium or a Spore print/syringe, you will need to procure some supplies and equipment. We recommend budding mycologists begin with a “Still Air Box” (instead of a Flow Hood which is quite expensive), a stovetop pressure cooker, and use jars with inoculation port lids for spawning/colonization.

*An inoculation port lid has a rubber self-healing injection port for error-proof inoculation.

*A pressure cooker is used to sterilize the mediums that the mushrooms grow in

*A Still Air Box (SAB) is a container that provides an area with no airflow. One can be built for ~$50. sporeslab.io for a video tutorial on building your own SAB.

It is imperative that we now stress the importance of STERILITY. When you cultivate Mushrooms, you aim to create a perfect environment for fungal growth. Unfortunately this environment is also ideal for bacterial spread, or the spread of other unwanted fungi. Every cubic meter of air (in an unfiltered environment) contains millions of fungal and bacterial spores, and each of these spores can potentially compete with your mushroom culture. You must take extreme care to clean/disinfect all of the surfaces, tools, and body parts that will come into contact, or even come near your mushroom culture. This is also why colonization and fruiting mediums must be sterilized/pasteurized in a pressure cooker, and why a SAB/Flow hood is so important.

STEP 1 — Preparing Colonization Medium

Once you have sourced a sterilizer, sourced your growing mediums, and built a still air box (or acquired a flow hood), the first step is to prepare the colonization medium. There are several mediums that can be used, however in our experience the best results have come using Rye Grain Berries. (they must be organic berries that have not had a fungicide applied to them).

certified organic NON-FUNGICIDE rye grain berries

Begin by placing the grain in a bucket, then fill the bucket with cold water and pour out the water (just the water) several times until the water is pouring “clear”.

Then fill the bucket 6–8” above the grain level and leave the grain to soak for 18–24hrs (depending on the humidity of the environment, if you live in a dry environment soak for 24 hours, if you live in a humid environment soak for closer to 18)

When you return (18–24hrs later) the water level will have dropped significantly, meaning that the grain has absorbed moisture. Pour out the remaining water and fill the bucket 6” above the grain level, this time with HOT water. Let the grain sit in 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 and strain the grain using a colander. Next spread the grain out evenly in a tray/tub/tote and place it in a high-airflow area for about 1 hour to dry the exterior of the grain berries.

Grain drying in a high-airflow area

Ideally you want the grain berry to be as saturated as possible, but little moisture on the exterior surface of the grain berry (the industry term for this level of saturation is “field capacity”). A rough “rule of thumb” you can use to estimate the correct dryness is picking up a small handful of grain then turning your hand upside down. A few berries should stick to your hand.

Now place the grain in your colonization container (we recommend beginners use mason jars), seal the container, and place it in your sterilizer. Cook the grain at 15PSI for 240 minutes (4 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 — then cook for 300 minutes (5 hours).

Also be cognisant of how much grain you put in the jar/bag, keeping in mind that putting more grain will take longer for the grain to colonize. We recommend filling a 1L jar about ¾ and filling a type 3T bag about ½ full of grain.

After the cook, let the sterilizer cool for ~1 hour and then clean the surface of the sterilizer 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 want the area/surface as clean as possible when this happens.

If you are using a mason jar we recommend also using an inoculation port lid, and if you are using a bag we recommend a Type 3T 0.2 micron filter autoclavable bag. If you are using a bag we also recommend cutting a small slit in the corner of the bag prior to sterilization, so the bag does not rupture during sterilization. Re-seal the bag (using an impulse sealer or a “mushroom bag clamp”) immediately after opening the sterilizer.

STEP 2 — Inoculation

After you have removed the jars/bags from the sterilizer, you are now ready to inoculate! Inoculation should be done within 24hr of sterilization. This is because of the finite amount of moisture in the grain, moisture which has to last the entire life cycle. You will not only lose out on potential yield if you wait too long to inoculate, but you will also be giving the mycelium a more difficult (dryer) environment to grow in, and giving potential contaminants a “head start” over the mycelium.

Inoculation should be done in front of a flow hood, or in a SAB. Begin by cleaning, and don’t be afraid to “overkill” for this step, as it’s this point where the risk of contamination is greatest. Wipe down the surface of the syringe, the surface of the colonization container, your arms and all surfaces in the area with disinfectant. Gather your supplies and place them in the still air box, or intelligently position them in front of the flow hood (with regard to the direction of airflow coming from the flow hood). If using a SAB place all supplies in the box, then put the lid on the box, put your hands in the gloves and liberally disinfect the interior of the box with an aerosol disinfectant spray.

Left — Inoculation in front of a flow hood, Right — Inoculation in SAB (lid removed for photo)

Unwrap the sterile 18ga needle (which is usually included in spore syringe kits), remove the plastic tip from the syringe, and attach the needle to the syringe. Sterilize the needle (with either heat or disinfectant) and inject the needle into the inoculation port (if you are using a jar with an inoculation port lid). If you do not have an inoculation port we recommend drilling a small hole in the lid of the jar (if you are using a bag then the needle can be used to poke a hole in the bag). Immediately cover this hole with Micro-pore tape after inoculation.

STEP 2.5 — Colonization

After you inoculate, shake the bag/jar to evenly disperse the liquid spore solution, and then leave the bag/jar in an environment which has the right conditions for colonization. Colonize in an area that is dark, has about 40% ambient humidity, and has steady temperature between 73–78F. It is crucial that the temperature remains below 80F during colonization.

Colonization from a spore syringe will take anywhere from 2–6 weeks depending on how much grain is in your container.

Various stages of colonization

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, quarantine that container from the rest of your operation immediately and dispose of it. It has likely become contaminated and if you do not remove it from the area it will contaminate the containers around it.

Examples of contamination

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 mycelium.

STEP 3 — Building a Fruiting Substrate

Once your colonization medium is fully colonized you are now ready to add a high-nutrient fruiting medium to form what is called a “Fruiting Substrate”. Like with colonization mediums, there are many options for a fruiting medium. We have found the best results using a mix of vermiculite, coconut coir, mypsum, manure, peat moss, and worm castings.

Coconut Coir and Vermiculite, the two main ingredients in a fruiting substrate

Begin by mixing the Vermiculite and Coconut coir at a 50/50 ratio. Next add 10 grams of powdered lime for every 10 litres of dry mix. Then add 1 litre of water for every 10 litres of dry mix, and mix well. Keep mixing and adding small amounts of water until the medium drips slightly when lightly squeezed. The reason we recommend starting with 1L of water for every 10L of mix, then adding water as needed is due to variance in the ingredients from different suppliers.

When “field capacity” water content has been achieved (when the mixture drips slightly when lightly squeezed) place the medium in a Type 14A 0.5 micron filter autoclavable bag and pressure sterilize the medium at 15PSI for 90 minutes. Again, if your cooker cannot reach 15PSI add 1 hour to the cook time for every 1PSI below 15PSI. Also don’t forget to cut a small slit in the corner of the bag so it does not rupture during sterilization. Once you remove the sterilized fruiting medium from the cooker you are now ready to mix it with the colonized grain to create a “Fruiting Substrate”. Reseal the bag immediately after removing from the sterilizer.

This job will be very difficult to perform in a SAB, so we recommend it’s performed in a clean area, and preferably in front of a flow hood. Cleanliness is not AS crucial for this job (compared to inoculation) as by this point the mushroom culture is established and can fight off potential contaminants, however sterility is still very important and overkill doesn’t hurt. Wipe down the surface of the colonization bag/jar, the surfaces of the tray/tub/tote that you will fruit in, your tools, your hands and arms, and the surface of the bag that the fruiting medium was sterilized in. Position your supplies so that you don’t have to reach over the mushroom culture to gram them, and dont position them in between the culture and flow hood (if you are using a flow hood).

Begin by putting on your PPE (gloves, mask, hairnet) and placing the fruiting container (the plastic tub/tote/tray that you will fruit in) inside the black plastic garbage bag. Next disinfect your hands and arms again. Reach into the tub/tray/tote and tamp the garbage bag to the corners of the tray, being careful to touch the bag minimally. Now cut along the top of the fruiting medium bag and pour this into the tub/tray/tote. Repeat this process for the colonized grain bag or jar. We recommend the mixture be roughly 20% colonized medium and 80% fruiting medium.

Left — Mixing substrate, Right — Tamping substrate surface

Mix the two mediums well. You want everything to be as well dispersed as possible to allow optimal colonization of the fruiting substrate in the shortest amount of time. After the substrate is mixed well, tamp the surface lightly with a BBQ flipper so that it is as flat as possible. This is to avoid water pooling during the incubation of the fruiting substrate (colonization/spawning refers to 1 medium being colonized, whereas incubation refers to a substrate, or a mix of mediums being colonized). Now put the lid on the tray/tub/tote and cut the garbage bag about 2” below the lid all the way around the tray, and remove the excess bag. The lid should also have four 2” holes cut at opposing corners, and these holes should be covered with micro pore tape. This allows a small amount of air exchange during incubation.

STEP 3.5 — INCUBATION

After you build your fruiting substrate, leave it in an environment which has the right conditions for incubation. Incubation should take place in an area that is dark, has about 40% ambient humidity, and has steady temperature between 73–78F. It is crucial that the temperature remains below 80F during incubation. You should also check the incubating tray/tub/tote periodically to make sure there is no water pooling on the surface of the fruiting substrate. If water is pooling remove the lid, wipe any water collecting on the lid, put the lid back on, and lower the ambient humidity.

Incubation will take between 7–10 days. If at any point during the incubation period you notice a pungent odour coming from the tub/tray/tote, or notice any discoloration that is NOT white mycelial growth, quarantine that tray/tub/tote from the rest of your operation immediately and dispose of it. It has likely become contaminated and if you do not remove it from the area it will contaminate the trays/tubs/totes around it.

The fruiting substrate is fully incubated when the surface of the substrate is completely white with mycelium.

Fruiting tray fully incubated

STEP 4 — Triggering Fruiting

Once the fruiting substrate is fully incubated you are now ready to trigger fruiting by changing the environmental conditions. The three major changes you will make are the humidity level, the light schedule, and the amount of airflow.

These changes mimic the natural environmental changes that occur when a mycelial culture reaches the “edge” of the medium it is colonizing. A good analogy is a compost pile. When mycelium begins life deep inside the compost pile it is in a dark and low-airflow environment. As it grows towards the edge of the pile it is exposed to light and higher oxygen levels, which trigger pinning. Adding humidity also serves to trigger pinning, and additionally can extend the fruiting period by providing moisture for the culture to absorb and turn into fruiting bodies (which are almost all water).

At this point you should switch from a constantly dark environment to a 12/12 light cycle (12 hours light 12 hours dark). Any light spectrum will work, however slightly “cooler” lighting between 6000 and 7000 Kelvin is ideal.

At this point you will switch the lid of the fruiting tray/tub/tote for a “fruiting dome” to allow more airflow to the mushroom culture. Usually using the same tray/tub/tote as you built the fruiting substrate in, flipped upside down works well. Cut a 2” hole in each corner of the dome and stuff this hole with cotton batting. This serves as a barrier for particulate matter/contaminants but allows airflow. We also use plastic clips to hold the dome to the tray.

Left — Incubation lid, Right — Fruiting Dome

At this point the humidity in your tray/tub/tote environment should also be raised. Accomplish this by misting the surface of the fruiting substrate daily, or whenever there is NO humidity build up on the sides of the fruiting dome. Set your sprayer to create as fine of a mist as possible. You do not want large droplets or pools of water on the surface of the fruiting substrate. How much you will need to mist also depends on the ambient humidity and the amount of ambient airflow in your growing space. You can skip a day of misting if there is excessive humidity buildup on the sides of the fruiting dome or if there is water pooling on the surface of the mycelial mat. Ideally you want to put as much (clean) airflow as possible through the controlled dome environment, but you also want the highest humidity possible in the controlled dome environment.

After approximately 7–10 days in these conditions small Primordia or “pins” will form. These will quickly grow into mature fruiting bodies within 3–5 days. You should cease misting the surface of the fruiting substrate once pins begin to show, but still try and keep the humidity as high as possible in the tray/tub/tote. You can do this by misting the sides of the dome (instead of directly misting the surface of the substrate).

Primordia, or “Pins” beginning to form

STEP 5 — Harvesting/Taking Spore Prints

You are now at the most enjoyable and rewarding part of the cultivation process! Your patience and hard work over the past weeks/months is finally paying off, mushrooms are fruiting out of your substrate. You should aim to harvest these mushrooms as they begin to sporulate (release spores).

Cut the mushrooms cleanly off at the base of the stalk using sharp scissors. Scissors with slightly curved blades work excellently. Also try not to touch the surface of the mycelium when harvesting, wear gloves when harvesting, and try to handle the mushrooms the least amount possible. You should be harvesting a few mushrooms almost daily, as some caps will open before others. When all the mushrooms in a “flush” have been picked you can mist the surface of the fruiting substrate again to keep the humidity as high as possible for the next flush!

Successive flushes will continue to happen until the mushroom culture has used all of the available nutrients and humidity in the fruiting substrate. Typically a home/hobby cultivator should be happy with 1–2 flushes before contamination starts to appear, at which point the culture needs to be disposed of. If you are cultivating in a cleanroom environment you can get up to 5 or 6 flushes.

If you will be taking a spore print (for most species - this does not apply to oyster mushrooms) you will need to let the veil separating the cap and stem fully break and the cap open about ½ way. Then cut the cap off close to where the stem meets the cap, and place the cap on a piece of tin foil. Leave the cap on the tin foil for about 12 hours (this should also be left IN a SAB or in front of a flow hood). A good rule of thumb for knowing when to cut the cap off is when the cap is at its most triangular shape (the cap starts as a concave bulb shape and opens to become convex if you let it go long enough). The triangular cap shape occurs at about the halfway point in this process and is ideal for catching the entirety of the spore print.

When you return remove the cap from the tin foil and the spore print will have been deposited. You can now scrape the spores into a sterile aqueous solution to make a spore syringe!

If you want to see a video tutorial covering the same content as this manual, or are looking to source the necessary equipment, supplies, and genetics for mushroom cultivation check out our website!

Bio - I am a Mycologist living in British Columbia, Canada. Over the past decade I have perfected a method of home-cultivation that works for several Agaricus genus mushroom species. In my spare time I love to write about Psychology, Fungi, and the Psychedelic experience.

Jeff Lebowe