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^ Permaculture Principles The Five Fungal Needs



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A Brief History of Mushroom Cultivation

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Why Grow Your Own Mushrooms?


The World of Fungi

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The Saprotrophic Basidiomycete Lifecycle






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o m m o n

C o n t a m i n a n t s


P e s t s








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Aseptic Technique

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Designing Work Spaces


Supply List

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Notes on Select Equipment

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Scheduling and Planning


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Treating Substrates

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M u s h r o o m

N u t r i t i o n

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S u b s t r a t e

F o r m u l a t i o n

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Stage 1: Agar Work

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Stage 1: Liquid Culture Work

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Culture Storage


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Stage 2: Grain Spawn Work


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Stage 3: Fruiting Substrates

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Pinning and Fruiting


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Making Spore Prints and Syringes

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o w - Te c h

C u l t i v a t i o n


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Mycorrhizal Fungi

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Cultivating AM Fungi

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Recommended Reading

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Suggested Projects


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Commonly Cultivated Species


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Tracking Forms


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P k r m a c t t l t u r e




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Consider applying Permaculture's ethics (Earth Care, People Care, Fair Share) and principles to your



1. Observe and interact - By taking time to engage with nature we can design solutions that suit our particular situation.

2. Catch and store energy - By developing systems that collect resources at peak abundance, we








3. Obtain a yield — Ensure that you are getting truly useful rewards as part of the work that you are


4. Apply self-regulation and accept feedback - We need to discourage inappropriate activity to ensure that systems can continue to function well.

















to reduce our consumptive behavior and dependence on non-renewable resources.

6. Produce no waste - By valuing and making use of all the resources that are available to us, noth ing goes to waste.

7. Design from patterns to details - By stepping back, we can observe patterns in nature and

society. These can form the backbone of our designs, with the details filled in as we go.

8. Integrate rather than segregate - By putting the right things in the right place, relationships develop between those things so that they work together to support each other.

9. Use small and slow solutions - Small and slow systems are easier to maintain than big ones, making better use of local resources and producing more sustainable outcomes.

10. Use and value diversity - Diversity reduces vulnerability to a variety of threats and takes advan-

tage of the unique nature of the environment in which it resides.

11. Use edges and value the marginal - The interface between things is where the most interesting events take place. These are often the most valuable, diverse and productive elements in the system.

12. Creatively use and respond to change - We can have a positive impact on inevitable change by carefully observing, and then intervening at the right time.

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Fungi Need Lots of Good Water. But Not Too Much

The growth of myceliiam is powered by water. So critical is water to successful cultivation that its pres ence should be seen as one of the greatest limiting factors in any operation. As such, substrates should be hydrated to their maximum water-holding state, or Jie/d capacity. This

hydration level feels different for different materials. But for all of them, the goal is to get the material as moist as possible without creating a product that is "muddy" or that pools nutrient-rich water in the bot tom of its container. An anaerobic pool of water is unwanted in any cultivation stage as it simultaneously

prohibits the growth of the oxygen-dependent mushroom mycelium while also serving as a breeding ground for competitors. It is better to err on the side of making substrates too dry rather than too wet. Most experienced cultivators can tell when a substrate is properly hydrated by giving it a quick "feel test." Once the substrate is inoculated, it is placed in a clean container that will minimize dehydration. Water used should be of the highest quality possible. Most of the chemicals that are commonly added to municipal water supplies are harmful to mycelial growth. Water treated with chlorine gas can be left

standing in open containers for 24 hours to allow the dissolved gas to evaporate. Vitamin C can be added

in equal molar concentrations to neutralize more stable chlorine compounds. Springs, clean ponds, and

artesian wells are all ideal water sources. That said, many cultivators without access to these sources have

good success using tap water.






As with all other Uving organisms, fungi have distinct nutritional requirements that must be properly balanced to ensure their optimal growth. The best diet for a fungus is often one that reflects its natural habitat. Love loving species such as Shiitake, Reishi, and Lion's Mane all fruit best on freshly cut wood- based substrates. Agaricus species and other late-stage decomposers fruit best from thoroughly compost ed materials. In between these two groups we find King Stropharia, Shaggy Manes, and various Psi/ocyhe

species that fruit well on a range of fresh to partially decomposed materials. Finally, the duff-dwelling Blewits, Shaggy Parasols, and Morels have been shown to grow best in soil- and humus-based substrates where microbial interactions are high and nutrients are more dispersed. Curiously, the lines between all of these groups often blur as strains of each species can vary widely in their nutritional requirements and preferred fruiting substrate and many can be acclimated to digest uncommon substrates. Learning to match the picky eating habits of a given species/strain is one of the keys to obtaining consistently high


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At all stages of the cultivation process, oxygen must be provided so the fungus can breathe and metab olize. The level of oxygen provided depends on the stage of cultivation. During stages vegetative growth, low oxygen is called for to mimic the air quality found in the soils and dense wood pieces that fiingi naturally inhabit during their vegetative state. For the cultivator, this means ensuring that any container used for spawn production has holes that allow for passive air exchange, while not being too large in any

given dimension. If a container is too large, the substrate will become too dense for good airflow, leading to anaerobic rotting in the oxygen-deprived core of the material. Airflow should always be provided to some degree as stagnant air encourages the growth of contaminant molds. When mushrooms begin to arise from a mycelial network, oxygen levels are increased, mimicking the fungus' natural exit from their substrate's interior into the oxygen-rich external world.

Fungi Need Warmth

Most mushrooms are mesophilic, meaning that they grow best in about the same temperatures that hu mans prefer. The mushrooms we grow can tolerate a range of temperatures, but most grow best around 70°F (21°C). As temperatures get colder, metabolism and growth rates dramatically decrease, providing an increased window of opportunity for competitors to gain a foothold on a substrate. For most species,

growth rates double with every increase in 10°C. However, temperatures that are above 105°F (40°C) will

kill most mushroom species. Depending on the species, the ambient temperature may need to be raised or dropped to initiate fruiting.

Mushrooms Need a Proper Fruiting Surface

While mycelium can be grown in any shape, high quality mushroom development is dependent on the structure and orientation of the fruiting surface. For example, some species grow best horizontally off of the sides of logs, tree stumps, bags, and buckets while others prefer to fruit vertically from the ground

or top surfaces of substrates. This preference directly influences the choice of container from which a species is fruited indoors and the design of an outdoor installation.


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S t r a i n s

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T i m e - Te s t e d

M a t t e r

T r u i s m s

Every unique combination of two monokaryotic mycelial networks is referred to as a strain. And just as every human, snowflake, or variety of corn has its own appearance and habits, so does every mush room strain differ in its ease of cultivation, medicinal potency, remediative capacity, and flavor profile. For this reason, cultivators cherish strains that are known to grow rapidly and/or produce high quality yields. To fulfill the demand for high quality cultures, commercial culture libraries sell rare or valuable strains at incredibly high prices ($20-$1000!), making these companies the key holders to success in the eyes of many cultivators. This emphasis on commercial cultures has largely overlooked the benefits of working with strains that are found in the cultivator's local environment. For example, imported strains may have finicky fruiting requirements, while local strains are often more tolerant of local weather conditions. Wild harvested strains also tend to be more resilient to stress and local contaminants, reflecting a rugged life history free of sterile environments. In general, the most resilient cultivation strategies work with local or developed strains that prefer the local climate or available substrates, thus avoiding the cost and unknown history




Fungi Are Energy-Conserving and Self-Preserving

Though cultivators often think they are controlling the fungus that they work with, it is really the myce lial networks that determine how successful and long-lived a given project will be. As free and wild crea tures, fungi only perform the work that benefits them and their immediate environment most directly. Of the countless antimicrobial and digestive enzymes that a fungus can release, it only expends its internal resources on producing those compounds that are necessary for its immediate survival. In natural systems, fungi produce an array of substances to defend themselves and their substrates from a dynamic and constantly changing universe of competitors. But in the artificial, sterile conditions of indoor cultivation, the absence of competitive microbes causes a mycelial network to cease produc tion of its defensive compounds. In the short term, this allows the fungus to conserve its energy by

letting down its guard, enabling it to focus on eating and growing. The problem is that the fungus is thereafter much more susceptible to attack by competitors, leading to increased rates of contamination with cultures that have spent a long time under aseptic conditions. Ironically, sterile cultivation creates the need for greater sterility.

When a mycelial network is fed the same substrate for an extended period of time, it may stop produc ing the enzymes required for digesting another substance. If the fungus' diet is constricted for too long,

this lack of variation in its environment can also cause the mycelium to slow in its growth. This slowing of a culture is commonly referred to as senescence and has historically been attributed by a small number

of mycologists to the aging of the mycelium - a theory that is unreflective of the fact that wild mycelial networks can survive for thousands of years. Rather than dying out, most mycologists agree that sterile mycelial cultures senesce because they shut down various metaboUc pathways in reflection of the absence of novelty and external stimuli. In other words, the mycelium gets bored and loses its will to live.

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To minimize the effects of senescence, mycelium should be grown in a variety of environments throughout its life in cultivation. The simplest way to do this is to provide the fungus with a dynamic diet.

Every time mycelium is transferred to a new substrate, the formula of the new media should be different

from the last one used and should ideally contain multiple ingredients. This constant variety of substrates forces the fungus to stay on its toes and constandy change its response to its environment. This range of foodstuffs also keeps the fungus happy. Do you like eating the same thing all of the time? Senescence can potentially be reversed if a mycelial network is sufficientiy stimulated into turning on dormant genes. The introduction of competitor microbes via the use of pasteurized substrates or the in- stallation of the fungus outdoors are two examples of such stimulation. These competitive environments

force the fungus to defend itself or die. If the mycelium has been in sterile culture for too long, it may

not be able to defend itself and could fall victim to competitors. But if the fungus establishes in these more challenging environments, the aggressiveness of the mycelium will increase, allowing a cultivator to reculture its tissue and place it back into aseptic practices where it will grow with renewed vigor. Reinvig- oration does not always work, especially with species that are generally less vigorous to begin with, but it is worth a try if you are at risk of losing a strain to senescence.

Fungi Adapt to Their Environment

Along with the introduction of competitor microbes to elicit metabolite production, cultivators can guide fungi toward greater resilience by helping the fungus acclimate to a new substrate or environment. Upon contact with a novel substrate, fungi often go into a period of stasis in which their growth halts as the fungus scans its DNA to determine which genes will produce the proper enzyme(s) needed to digest the new food source. It is akin to the fungus finding the right key on a key ring to unlock a treasure box, and then mass-producing that key to open all the treasure boxes in the area. This learning process is of ten seen in petri dishes when a mushroom mycelial network encounters a mold or bacteria, stalls in its

growth, and a few days later begins producing a liquid exudate of antibiotics as it starts to grow over the competitor. Constant use of this adaptive capacity is how the fungi have been able to proliferate around the world with such phenomenal success. Advanced cultivation strategies account for this ability by ac-

climating fungi to particular microbes for the production of novel antibiotics, or to toxic chemicals to produce strains that can readily degrade a particular pollutant.

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Though fungi can tolerate significant shifts in their environment, most species can only handle one or two major changes at a time. If too many factors change at once, the fungus may become overwhelmed,

halting mycelial growth. Though the fungus may recuperate, during this stagnancy period its resources

are focused so heavily on recovering from the shock that it has little energy for defense, leaving it highly

vulnerable to attack by competitors. There are three main changes that a fungus can tolerate: substrates, new competitors, and habitat disturbances (such as the breaking of a mycelial network during transfers). While most species can handle one or two of these shifts at a time, the combined stress of aU three influences tends to result in the cessation of the fungus' growth.









Though most mushrooms can defend themselves against competitive microbes, for practical purpos es, the indoor cultivator should work to minimize this stressor. The less energy the fungus expends on

defending itself, the more it has to put toward producing large flushes of fruit bodies. For this reason,

substrates are pasteurized, sterilized, or otherwise cleaned, while transfers are done under aseptic condi- > j


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The majority of the fungus' growth, digestion, environmental assessment, and absorption occurs at the tips of each hypha. At the leading edge of the culture, growth is the most explosive. As a network

ages, the interior mycelium eventually gets cut off from the nutrient flow of the system, resulting in min imal metabolic activity in this inner tissue. To harness a fungus' metabolic maximum, mycelial transfers should include a section of a culture's edge, if possible.

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H o l o g r a m

If a mycelial network is placed in a dynamic environment without physical boundaries and unlimited food supplies, it will grow indefinitely. If the fungus ever runs out of substrate or encounters a physical barrier, its vegetative growth will cease and its energy will shift toward fruit body production. For the cul tivator, this means that if a fungus overgrows its container, or if conditions are otherwise not conductive to fruiting, it wiU become stunted in its growth and less vigorous in subsequent stages of cultivation. To avoid mycelial constriction and promote its endless regeneration, it is best to always perform transfers as soon as the mycelium has consumed its substrate and/or before it hits a growth boundary. When to do this depends on the substrate being used.

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The limits of appropriately applied mushroom cultivation are unknown. Where its history has for so

long been constricted, the cultivators of today should seek to push the current boundaries of fungal culti vation into new fields of research. I cannot overstate the importance of trying new things when working with fiingi, nor can I summarize the great value of learning from mistakes and experimenting. Many of the techniques and insights presented in the following pages are based on the direct experiences of many cultivators who intentionally did things that were not supposed to work. Every time knowledge is gained on how fungi respond to novel conditions or ecologically reflective designs, the skill of the cultivator and the depth of dialogue surrounding cultivation advances one step forward. Without experimentation, we will never fully understand what the possibilities are for working with fiingi. Indeed, many of the greatest advances in science have arisen due to accident and/or intuition. If we only repeat what others tell us to believe about the fungi, we deny our ability to form our own relationships with them. It is by slowing down and paying attention to the responses of fungi that we learn most directly from them, enabling the chance to uncover an understanding of their ways that no book could ever teach.

Mushroom Cultivation is Simple and Scalable

The entire process of mushroom cultivation reflects the holographic nature of mycelial growth, from the microcosm of gestation to the macrocosm of expansion and movement. The cultivation process it self is nothing more than an exponential proliferation of a mycelial network from one container to many and from one substrate to another. Each of the practices outlined below can be done in a smaU kitchen or in a large warehouse. The scale of a project is solely dependent on supplying the tools and infrastructure that will adequately address the underlying principles of the processes involved. Mushroom cultivation is

not a very difficult skill to master once a sound understanding of the rationale behind its methodologies

is fully grasped.


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C u l t i v a t i o n

Mushroom cultivation seems to have originated in China around 600 CE, when Auricularia polytricha

was first cultivated by So. Some accounts attribute the development of Shiitake cultivation practices to the Japanese around 200 CE. Others say it was the Chinese who pioneered the practice around 1000

CE. According to writings by Zhang Shou-Cheng, Wu San Kwung developed Shiitake cultivation during the Chinese Sung Dynasty (960- 1127 CE) in the Lung-Shyr Village of the mountainous Lung-Chyuan

County in Zhejiang Province.

In the West, it wasn't until the late 1600s and early 1700s that French farmers developed rudimentary methods for cultivating Agaricus species on compost and horse manure. By the mid 1700s, this process was moved to abandoned limestone caves where farmers could control the temperature and humidity

levels year round.

In 1917, researchers in Pennsylvania developed aseptic (sterile) techniques for producing grain spawn,

a now central element in the entire cultivation process. This pivotal moment began to offer mushroom farmers greater consistency over the cultivation process, setting the stage for the advancements made

throughout the 20th century. Around this time, techniques were also developed for cultivating molds and yeasts in large fermentation tanks, so as to harvest the industrially and medically important compounds these micro fungi produce. This fermentation process would go on to form the foundation of the liquid

cultivation techniques that are on the leading edge of the cultivation field today. During the first half

of the 20th century, mushroom cultivation research primarily focused on increasing the profitability of

Agaricus production, largely while utilizing intensely clean and costly practices. When psychoactive Psilocybe species reached the counter culture in the 1960s, industrial cultivation

practices were finally distilled into booklets written for the home and dorm room grower. Unable to af ford or build the costly cultivation workspaces designed earlier in the century, iUicit growers devised and

continuously refined a variety of lower-tech and lower-cost protocols for growing mushrooms on a small to medium scale under a range of space and budget constraints. These innovations were soon successfully applied to a wider range of mushroom species, thus ex

panding the number of commonly cultivated species available to farmers. The anonymous community

of psychoactive mushroom growers pushed the world of low-tech cultivation forward in the second half of the century and, in the recent years of the internet, has continued to spread cultivation knowledge and innovation through various online platforms.

W h y

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M u s h r o o m s ?

• Mushrooms are a relatively cheap/near-free, year-round source of healthy whole food and potent natural medicine that can be grown on numerous agricultural and urban "waste" streams. By grow ing food on what is otherwise discarded, we can reduce carbon loss, retaining fertility onsite, and

increasing abundance through a family or community.

• Mushroom cultivation provides many applications for developing local jobs, increasing community

food security while managing resources for ecological renewal.

• The following techniques can be used to grow locally-adapted species and strains that are not com

mercially available.

• Cultivating fungi leads to soil building, increasing nutrient availability, reducing fertilizer inputs,

enhancing water retention, and supporting microbial communities.

• Some fungi can remediate polluted soil and water and regenerate disturbed environments.

• It's sciencey & fun!

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F u n g i

The Fungal Kingdom is vast, hosting an estimated 1.5-6.1 million species worldwide, yet less that 5% of these species are formally described. While many micro fungi (e.g. molds and yeasts) are cultivated

for foods such as miso, tempeh, beer, and bread, in this course we mainly focus on cultivating the larger, fleshier fungi—the mushrooms. All edible mushrooms can be categori2ed based on two concepts: their

spore production strategy and their ecological niche.

Spore Production Strategy: Ascomycete or Basidiomycete?

Ascomycotan Fungi (~65,000 spp. named) include yeasts, molds, and many cup-shaped mushrooms

(among other shapes). The spores of many cup mushrooms form in long tubes {asa) and are ejected out

the top of the tube at maturity. This group includes the morels and truffles. A good book on learning

to cultivate truffles is Taming the Trujfle by Gordon Brown. Morel cultivation is not a refined practice at

this point. A good resource for learning about growing these species is the facebook group **Morchella



Basidiomycotan Fungi (~30,000 spp. described) host the majority of the common edible, medicinal,

and remediative mushrooms, as well as most cultivated mushrooms and wild harvested species.


Mycorrhizal fungi form symbiotic relationships with plant roots and other microorganisms in the

soil. They extend the root system of plants 10-1,000 fold to channel water and nutrients to the plants as well as provide protection from infection. Some of the most popular wild harvested mushrooms are my corrhizal (e.g. Chanterelles, Boletes, and Matsutake). These species have complex mating strategies that

are not well understood and therefore most of these mushrooms are not yet mass cultivated.





Saprotrophic Fungi are the decomposers. They digest organic matter (animal and plant tissues) via

enzymes and solubilize minerals via acids. These mushrooms are the easiest to cultivate as, much like an animal, they only require ait, water, food, shelter in order to grow. Most of the mushrooms we will focus on in this course are saprophytic Basidiomycete mushrooms. Cultivated species include:

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B a s i d i o m y c e t e

L i f e c y c l e

We begin with the spore. Spores develop on a layer of fertile tissue - known as the hymenium -cov

ering the mushroom's gills, teeth, or tube interiors (spore-bearing structures vary between species). A mature mushroom may produce millions or billions of spores in a single day, and thereafter eject them

with a high force. If a spore lands in a moist habitat, it will germinate, producing a network of primary

mycelium comprised of single-cell filaments - hyphae (sing, hypha) — that grow through their substrate

(food source) in search of a genetic mate.

When two compatable networks encounter each other, they fuse to form a network of secondary mycelium that is now capable of forming mushrooms. As this network grows, its thread-like structure densely branches in all directions as it seeks food and water. As mycelium's tips encounter organic and in organic matter they exude a mixture of complex enzymes and acids, converting these substances into sol uble substances the fungus can use as food and/or provide to other organisms (e.g. mycorrhizal plants). As the fungus produces these digestive compounds, it also releases various metabolites to protect itself from surrounding competitors. Being only one cell thick, mycelium has no outer barrier to infection and has evolved to defend itself from other organisms by releasing antibiotic and antifungal compounds that






If the fungus runs out of resources or environmental conditions change signficiantly, the mycelium will be triggered to produce mushrooms. Parts of the network wiU start to form pinheads, or primordia, that quickly mature into fruiting bodies (a.k.a. mushrooms) after a few days. The mature mushroom then

goes on to drop spores by the millions, continuing the life cycle anew.

One can think of spores as analogous to the sperm in mammals in that each contains only half of the genetic information of its parent. However, unlike sperm, spores are not limited to simply seeking a sin- ^ gle "opposite sex." Instead, some species have tens of thousands of combination possibilities due to the

way the genetic information is shuffled when the spores are formed on the hymenium. What this implies for the cultivator is that fungi have an exceptional abilit}^ at adapting to a wide variety of environments and food sources due to such a large number of possible genetic expressions. This comes into play for the cultivator as it provides for the possibility of developing strains that are accustomed to digesting a particular substrate.








As the common name of a mushroom can change by region and country, the scientific or Latin names

of fungi are preferred for more accurate descriptions due to their global recognition. These binomial names use the genus and species titles of the mushroom. A genus is like the last name of a human, while the species name is like a first name. For example, common Oyster mushrooms are in the genus Pkuroius, but each species has a different species name (Pearl Oyster [Pleurotm ostreatus]. Phoenix Oyster [P/euro- tuspulmonarius\ etc.). Cultivators often use the initials of a mushroom's Latin name when labeling their

plates, jars, and other containers (PO, PP, etc.).

C u l t i v a t i o n

P r o c e s s

O v e r v i e w

The process of cultivating fiingi revolves, in essence, around exponentially expanding a mycelial stock.

Through a series of feeding steps, the cultivator expands mycelia until a critical mass is reached that can yield a substantial flush of fruiting bodies. Using moist, sugar-rich substrates for the mushroom,

the cultivator must work in a quick and clean manner to ward off ambient bacteria and fungi that will readily consume the substrate. Once enough competitor-free mycelium has been grown out, the fungus can be fruited indoors or placed outside in various installations. Indoor mushroom production requires a climate-controlled environment that helps signal the fungus into transforming its mycelium into mush


For indoor cultivation, there are four key stages to this process:

1. Spores or a small amount of mycelium is introduced to either a petri dish containing a layer of sterile, nutrified gel or to a jar filled with sterile, nutrified water. Over the course of 7-21 days, a mycelial network will begin to exponentially increase in size inside of the container, covering or filling it. Once this network is large enough for the cultivator to easily interact with, the mycelium

is broken up to become inoculum. 2. A small amount of agar-based or liquid inoculum is transferred to a container filled with cooked

and sterilized grains. Within 2-4 weeks, the mycelium will grow over and through these grains to

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s p a w n .

3. Healthy grain spawn is used to inoculate a final fruiting substrate that will support a large crop. Com mon substrates include wood, straw, coffee grounds, manure, and/or compost-based substrates.

Two weeks to 3 months later, the substrate will be fully myceliated. 4. Once this final substrate is fully consumed by the fungus, the mycelial mass is moved into a humid

environment to support the full development of mushrooms. The first crop, or flush., is often pro duced within 1-2 weeks. With proper care, several subsequent flushes may develop over the pro ceeding weeks and months. Once the mycelium stops producing mushrooms, the "spent" spawn

may be applied in a variety of ways.

In essence, this process has two broad divisions: Stages 1-3 focus on mycelial/vegetative growth (a.k.a.

spawn production), while Stage 4 is dedicated to the reproductive/fruiting aspect of the mushroom life- cycle.

Non-sterile practices also achieve the goal of increasing mycelium to the point of yielding fcuit bodies. However, it is often useful to fall back on aseptic practices when experimental projects fail. While aseptic

practices can incur a greater cost, they are "tried and true" examples of the principles of cultivation and thus foundational to developing novel approaches that can reduce one's dependence on them.

C o m m o n

C o n t a m i n a n t s


P e s t s

Viruses, bacteria, molds, and yeasts are the main competitors in the world of cultivation. In general, anything that looks or smells unlike the mycelium you want grow (typically mushroom mycelium is white) should be considered a contaminant For thorough descriptions of these organisms, visit www.shroomery.

org!5276! What-are-common-contaminants-of-the-mushroom-culture.

• Viruses are very small and impossible to detect apart from the deformations and adverse effects

they cause to once healthy mycelium. These may be responsible for the "aging" that occurs in older strains of mycelium.

• Bacteria presents on agar as slimy, greasy streaks or clusters. They can be translucent, yellowish,

browning, or grayish. On grains they can appear as a "wet spot" of non-myceUated, greasy sub strate and often impart a foul odor {Note: the mycelium of certain species [e.g. Ganoderma lucidum] naturally smells unpleasant). Fruiting bodies can also be infected by bacteria and will present with lesions or


• Molds are one of the most common contaminants as their microscopic spores are easily carried

by air currents during transfers, only to land on your medium. Molds can take many forms though black Mucor^vci molds, blue-green Penicillium, and the notorious olive green Trichoderma species are some of the more common contaminants. Before these molds begin sporulating, however, they can appear white, thus making it important to learn the appearance and growth characteristics of the intended mushroom's mycelium. Essentially anjrthing that doesn't look like the mycelium you introduced, or that is growing where the mushroom mycelium isn't, should be considered a com petitor and disposed of. As molds prefer acidic environments, many of these competitors can be combated with the application of a pH increasing substance, such as baking soda. Other molds can be killed by being exposed to red light or sunlight for a few hours. For spawn bags contaminated with Trichoderma molds, 27% hydrogen peroxide can be injected above the contamination zone with

a syringe and needle to work as an effective fungicide that does not kill the mushroom mycelium.

Many spoilage fiingi can also be inhibited by the directed application of pulsed light and/or elec

tric fields. These techniques may not work for all mold species and if the mold has gone to spore

it might be too late as other areas of the culture may be contaminated. Thus, it is best to learn to

identify contaminants early on and to apply the best preventative aseptic measures.

• Other mushrooms can also take hold of outdoor installations if spawn rates are low or wood

sources are aged. Coprinus (Inky Cap) species are common competitors in chips beds. Turkey Tails and Split gill mushrooms are common competitors on logs.

• Pests such as slugs and flies can become quite a nuisance. The best measures are preventative.

Keep your home clean and your compost pile covered and far from your home. Deer and squirrels are known to eat baby mushrooms so building a small fence around outdoor patches may be nec

essary to protect from these animals. Duck runs can reduce slugs around logs set ups. Fly tape or tree frogs help with insects indoors.

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C o n t a m i n a t i o n

Microorganisms cover the surface of all objects and permeate all porous materials. This fact should be held in mind for all cultivation practices by assuming that microbes are everywhere, regardless of one's cleanliness regimen. This hyper-awareness encourages habits that minimize the movement of microbes from the surrounding environment into the sterile interior of a substrate container, where they are not desired. The following are some of the main causes of contamination, along with suggested practices for minimizing their impacts.

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C u l t i v a t o r

Our body is an ecosystem of microbes that creates a thick coating of bacteria and yeasts on skin and clothing. The cultivator is perhaps the primary vector of competitors in the whole cultivation process. As such, they should ideally be freshly bathed and wearing freshly washed clothing during labwork. Shoes should be removed, dipped in a disinfectant, or covered in a protective sock before entering the transfer

workspace. Some cultivators wear a hairnet, facemask, arm sleeves, and/or latex gloves during transfers.

During aseptic work, the cultivator should not talk without a face mask as contaminants may be sprayed








The Air

To reduce the presence of competitors that naturally fill the air, most work is done in an aseptic transfer space. The air surrounding this transfer space should be cleaned prior to work using one of the following methods:

• Spray the air with 70-80% alcohol, 10% bleach, or a commercial disinfectant 1-3 minutes prior to working. This spray will not only help kill ambient microbes but will also "trap" and pull them down out of the air as the mist sinks. Take care not to inhale these sprays.

• Pump air into the workspace through a High Efficiency Particulate Air (HEPA) filter system. This will create a positive pressure in the work area that pushes dirty air out of the environment.

T h e

E n v i r o n m e n t

Work spaces should lack carpeting, be easy to clean, and cleared of all sources of mold, mildew, rodent infestation, or any other dirty elements. Walls should be painted with an easy to clean latex paint and the

floor made of cement and/or equipped with a floor drain to facilitate cleaning.

Worktables and shelving of non-porous metal or plastic that are easy to sanitize are preferred over wooden infrastructure. In the fruiting space, pockets of stagnant air should be eliminated as this encour

ages mold growth. All walls and surfaces in the workspaces should be regularly cleaned with disinfec tants. Workspaces may also have an anti-chamber where shoes are removed and drafts from the outside environment are reduced. For smaller operations, a small transfer room can be constructed with wooden

framing and plastic sheeting.

T h e

S u b s t r a t e

Most substrates are treated to kill any unwanted microbes living on or within them. These techniques




T h e

I n o c u l u m

The spores or mycelium you work with as inoculum can be a source of outside contamination. Cloned

wild mushrooms often produce bacterial colonies on agar plates due to the presence of beneficial or

benign bacteria living in the mushroom tissue. Sterile mycelium that was transferred and/or stored im properly can also harbor contaminants. Spore prints often harbor competitor spores or bacteria due to

the difficultiy in obtaining 100% pure spore prints.

T h e

To o l s

Despite your best efforts to maintain sterility, all tools and vessels surfaces must be considered covered

in contaminants. Tools should be sterilized along with substrates prior to use as well as in between each transfer by using a heat source.


Fungal gnats and other insects are a major problem in fruiting spaces. These pests not only eat mush

rooms and mycelium, they also spread contaminants between mushrooms and their mycelium.


The use of conscientious, quick, controlled movements during mycelium transfers is essential for

achieving low contamination rates. During every transfer, a keen awareness should be given to the lo cation of one's hands and tools. These should never pass over the opening of a sterile vessel or over

exposed mycelium unnecessarily as microbes could potentially fall off of these objects and into the substrate container, leading to contamination. If a tool touches any surface by accident, it should be res- terilized with a heat source. Containers should be opened for the shortest amount of time and with the smallest opening possible, and mycelium should be transferred quickly. The more time a transfer takes

and the wider a plate or jar lid is opened to the surrounding environment, the higher the risk of contam ination. The beginner, prone to making simple mistakes, is often uncomfortable and shaky with tools

and/or unconscious of the placement of their hands. But, as one gains experience, a refined, graceful

technique becomes second nature. In my experience, a deft and quick mycelial transfer methodology is the most important factor in the success of aseptic work. Should contamination rates remain high and all other sources of contamination have been dealt with, the technique of the cultivator must be thoroughly




Once media has been prepared and cooled for work, the cultivator should spend some time preparing

themself and the work environment. The following guidelines will be referred to through out this manual

by the general term "aseptic conditions." Below are the "best practices" for minimizing contamination. However, many cultivators get away with not taking all of these precautions.



Flow hoods should run for an hour prior to work to clean out the filters. All surfaces should be wiped

down with alcohol. The ak should be sprayed with a disinfectant (Alcohol, 10% bleach, or Lysol). HEPA filters should be tested every few months using blank plates to determine their effectiveness.

T h e

To o l s

Tools should be wrapped in foil and sterilized with the medium. Alcohol flames or Bacti-cinerators should be set out and warmed up.



Freshly bathed and clothed, the cultivator wipes down their arms and hands with alcohol and puts on

a fresh pair of disposable gloves. During work, the cultivator doesn't talk without a mask.

T h e

M o v e m e n t s

With plates unwrapped, jar lids loosened, tools flame-sterilized (and cooled) and the air and surfaces

cleaned, and the cultivator makes their movements with precision and speed. Awareness is paid to any

contact made between surfaces and tools. Should a tool touch an unwanted surface, it is re-sterilized. Tools and jars are wiped down with alcohol and are not actively shaken over sterile media as bacteria or

spores may be shaken off their surface. Mycelium should be easily and quickly transferred. The more time a transfer takes and the wider and longer a lid is opened to the surrounding environment, the higher

the risk of contamination. Tools should be re-sterilized after each transfer. In time, this awareness and




D e s i g n i n g

W o r k

S p a c e s

Designing your working environment is circumstantial to your budget and space availability. Still, for mushroom-to-mushroom cultivation it is often necessary to designate at least a space for doing your transfers cleanly, a place to incubate your mycelium as it grows out, and a humid space for them to de

velop to maturity.

T h e

Tr a n s f e r

S p a c e


S t e r i l e

L a b

The transfer space is where the sensitive work of moving clean mycelium between containers takes place. The space should be as clean as possible, but due to life's limitations, sometimes we have to work with what we have. The standard transfer space for dedicated hobbyists and farms is in front of a laminar flow hood which uses a fan to blow air through a HEPA filter, providing an area of clean, "sterile" air to work in. Flow hoods can be home built using filters and blower bought new or used online, or they can be bought prefabricated. Depending on the materials used, size, and features of a given flow hood, costs

range from $150-2,000 or more. HEPA filters should be able to obtain 99.99% filtration (at least). High quality blowers can be bought from Grainger.com. Many hobbyists start with a well-built glove box made from plastic or painted wood. Boxes should be big enough to allow motility of the user's hands, be well sealed off from outside drafts, allow the user to see clearly what is happening inside, and easy to clean with a disinfectant. Arms enter the box through holes cut in the front or sidewalls of the box. Permanent gloves can be installed in these holes to reduce air currents. A tall (ca. 3-foot [1 m]) box is recommended to accommodate for the height needed to easily inoculate tall containers, such as polypropylene filter patch bags. Prior to working in a glove box, wash all interior surfaces with soap and water and then with a disinfectant. Once the glove box is loaded with materials, close the lid and spray the interior with a disinfectant to "scrub" the air. Caution is advised when using alcohol to spray the glove box interior. If an open flame is present, a fireball can erupt inside



Other low cost clean space options include 1) using the heat in a convection oven that has been set to

high broil for 20-30 minutes then cooled to working temperatures, 2)working inside a clear trash bag that has been sprayed inside with alcohol, and 3) working under a strong flame. The "ideal" clean room is dedicated to mycological work. Entering the space, one would first go

through an anti-chamber where shoes are removed and drafts from the outside are reduced. The space



















not made of exposed wood. There would be no carpeting and the walls would be painted with a white latex plaint, which is regularly be wiped down with a 10% bleach solution. The air passages are equipped with HEPA filters to reduce outside contaminants coming in through airshafts. Work would take place in front of a laminar flow hood (described below) using the best tools available.

O p t i o n a l :

T h e

I n c u b a t o r

Incubators are enclosed spaces that maintain a constant temperature to help increase mycelial growth rates. They can also be used to test contamination on fireshly made agar plates by placing them inside for 24 hours. A simple incubator is a cooler containing hot bottles of water, while a more elaborate one could be a (mini) refrigerator equipped with a thermostat and heat source (e.g. an incandescent light bulb or space heater).







This is the area where all the spawn vegetatively grows. It should ideally be dimly lit / dark and within the temperature range recommended for a given species. For most species, this range is around room

temperature (~70-75°F), so heat-controlled environments are not absolutely necessary. While the myce- lium does not need much oxygen at this stage, the air should be circulated on occasion to provide some

fresh air and to avoid stagnant air, which encourages contaminant growth. While your mycelium will be in a sealed container in the spawn run stage, cleanliness of the environment is still recommended. To tal darkness is not necessary for most species. Note that many bags can release large amounts of C02, which can be harmful with extended exposure.











If you plan to cultivate with much regularity and at a moderate to larger scale, it is recommended to

dedicate a space to substrate preparation. Depending on your needs this might include an area for dry substrate storage; compost preparation; vermiculture; substrate shredding, chopping, mixing, and hydra

tion; vessel storage, loading, cleaning, and drying; and bulk pasteurization and sterilization of substrates. Having all of the tools ready to use greatly increases the efficiency of any cultivation project. If you are cultivating large quantities of mushrooms, having a place to dry them rapidly will ensure

proper storage and help create value-added products. Designs for solar and homemade electric dehydra-

tors can readily be found onHne.

SmaU closets or sheds can be insulated and equipped to serve as a walk-in cooler for spawn and mush

room storage, or to fruit cold temperature species during warmer months. Many farmers use a Cool Bot to trick an air conditioner to run at near-freezing temperatures. The ColdSnap Project has designed an

open-source tool that accomplishes the same goal.







Mushrooms need high humidity and a frequent source of fresh air in order to fully develop, requiring

bags to be opened. As the mycelium and underlying substrate will be more prone to contamination and desiccation than during spawn run.

The grow space should thus provide enough fresh air for the mushroom to breath and to avoid stag nant air, but not so much air that the humidity drops and the mushrooms dry out. Humidity must be maintained at recommended levels or fruit bodies will either not develop or abort before reaching matu

rity. There are several general models of fruiting spaces:

• The Plastic Tent - What once-in-a-while growers use for single sawdust blocks or Oysters-on-

straw kits. It is nothing more than a plastic bag that has some holes or slits cut into it, which is then misted inside and tented over the mycelium bag. For this approach it is important that the plastic

doesn't touch the mushrooms. Likewise, the inside of the bag should be misted at least once a day

to keep humidity high, but avoid spraying the mushrooms direcdy as this can stunt their growth.

• The Perlite Tub - Here, perlite is soaked, run through a colander to drain off excess moisture,

then to fill the bottom five inches of a plastic tote. Bags, etc. are placed on top of the perlite and the lid is closed. The perlite releases moisture, providing humidity. The cultivator must open the lid

multiple times a day (ideally) to exchange the air.

• Shot Gun Fruiting Chamber (SGFC) - An evolution of the perlite tub, the SFGC has holes drilled every 2" in aU directions to provide passive air flow. The inner walls of the tub stiU need to be misted as needed to maintain proper humidity levels.

• Monotubs - These tubs are spawned directly into with pasteuri^d substrates and are fitted with

large holes that are plugged with polyfiU.

• Automated Greenhouse (a.k.a. The Martha) - For the larger hobby si2e operation, any stain less metal shelving unit can be covered in clear plastic and aerated with a humidifer.

• Larger operations — Industrial ultrasonic humidifiers, fogging systems, and filtered air exchange

systems are employed for large warehouses. Outdoor greenhouses also work well if they naturally provide high humidity levels and do not get too hot. Other options include:









• The unused, shady, and vertical spaces of a vegetable greenhouse.

• Buried shipping containers are ideal as they tend to hold ideal fruiting temperatures year-round. A shipping container can also be converted to hold a lab, incubation space, and fruiting envi

ronment in the same space.

• Abandoned civic infrastructure, such as subway tunnels, which often have preexisting ventila

tion and drainage systems.

• Gothic shaped greenhouses or warehouses. Many farms prefer these structures as their shape

helps reduce the collection of heat and moisture in ceiling corners. Better yet, sink the green house in the ground.

• Geodesic domes, which are easy to build and modular.

• Hoop houses or greenhouses. Many farms around the world utilize simple outbuildings for

fruiting mushrooms.

• A plastic-wrapped, wood-framed room-in-a-room built to any size. It is easier to manage hu

midity in several smaller rooms than it is in a large one. If funding is limited for multiple humi- fiers, fans can be used to circulate humid air throughout the fruting space.









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P r e s s u r e

c o o k e r s

a n d

s t e r i u z e r s

A good pressure cooker (PC) is essential for successful cultivation. There are a variety of brands, de

signs, and sizes available. Used ones can be found at thrift stores and garage sales though all parts should be inspected for cracks or excessive wear. A cracked pressure cooker can explode! All American is a popular brand with mycologists as they are well built and use a metal-to-metal contact system (instead a rubber O-ring that can wear out). The difference between the All American pressure cookers and their

pressure sterilizers is, essentially, the pressure control and release system. Instead of using a rocker-top pressure release system used in pressure cookers, the sterilizers use a toggle system to better control the pressure. These toggles can be ordered online and used to replace a rocker top release. Pressure cooker dial gauges should be routinely checked for accuracy at your local county extension office.

If scaling up, an electric, automated pressure cooker or pressure sterilizer such as those made by All

American and Yamato Scientific will help avoid the hassle of monitoring pressure levels and constantly

adjusting a heat source. PID-automated systems can also be home-made. Autoclaves are laboratory de vices that perform the same function as a pressure cooker; they may be small or enormous. Industrial mushroom farms use walk-in autoclaves that are able to sterilize hundreds of pounds of substrate.

Propane tanks can be converted into pressure cookers. 55- or 85-gallon stainless steel drums or 60 gal lon steel wine barrels can be modified to serve as sterilizer units heated by steam that is either generated inside or piped in. In these home-made solutions to the bulk sterilizing question require great caution as

a metal vessel that is cracked and/or under improperly controlled pressure can blow up!




The four main materials for filtering air for spawn jars or bags include:

• Filter discs are a bit old school in my opinion as they are so prone to wicking moisture that they

can easily allow contaminants to pass though them and onto the substrate. Once these filters are infected with mold they are best to be thrown out, adding to their already high cost. Alternately,

they can be soaked in a 10% bleach solution then rinsed and dried between uses.

• Micropore tape is adhesive tape with microscopic holes that allow for gas exchange. Commonly

found in first aid kits, this tape can be purchased online and is not durable enough for repeated





• Tyvek can be purchased onUne from kite supply houses, bought as envelopes from office supply

stores, or gotten from the Post Office in a pinch. This material is breathable and works well for a

couple uses, but is cumbersome. A double layer is preferred.

• Quilt Batting is a synthetic material used to stuff animals, quilts, and pillows. It does not absorb

moisture and due to its malleable nature is versatile for many applications.

" A i r p o r t "

S t y l e

L i d s

"Airport" lids were developed by Hippie3, founder of Mycotopia.org. They are a major innovation in the world liquid culture work. In essence, holes are drilled into canning jar lids and then plugged up with

a glob of high-temp (a.k.a. RTV) silicone / gasket maker to become self-sealing injection sites that enable

needles to aseptically introduce or extract liquids. I prefer the simplicity of a 5/16" holes for the silicone

and a 3/16" hole for an air filter. The fancy "Morelman" lid has a feeder pipe and its a more elaborate


S c h e d u l i n g

a n d

P l a n n i n g

If you wish to cultivate large quantities of spawn or mushrooms, it is helpful to gain a thorough famil

iarity with the time requirements of each stage in the cultivation process. It is easy to get ambitious and spend a late night inoculating a bunch of grain jars only to find yourself overwhelmed two weeks later with mycelium and unprepared for the next stage in the process. Don't make this common mistake! Start small and slow and develop the habit of thinking ahead for all of your projects.

• Agar growth: 5-21 days

• Liquid culture growth: 10-30 days

• Quart jar of grain growth: 7-14 days


O y s t e r

l b .

n u t r i f i e d s t r a w

s a w d u s t

k i t

k i t

i n o c u l a t i o n

t o

f r u


t i

t o n g :

i n o c u l a t i o n

f r u i 1 - 4

t i n g :

3 - 1 2

w e e k s

w e e k s

Once you gain a familiarity with your cultivation process and develop a rhythm, it is possible to de

velop a cultivation schedule that keeps your incubation and fruiting spaces full but not overcrowded. The best route to take is to first select your target species and gain familiarity with its growth habits and

then develop a cultivation calendar for that species that matches your storage capacity, climate, substrate

availability, weather, and budget.

Other work scheduling factors to account for include preparing, soaking, and cooking substrates; sourcing, ordering, acquiring, and unloading supplies; building infrastructure; cleaning and maintaining workspaces; and outreach/marketing. All of these processes and energy commitments quickly add up to

the fact that moderate scale mushroom cultivation can be a relatively time-intensive practice. However,

working with others, using the best tools for the job, and acquiring proficiency and familiarity with tech

niques all increase efficiency. Stick with it. It does get easier.

T r e a t i n g

S u b s t r a t e s




Good pressure cooker usage is essential for effective and safe cultivation. This is the first skill that







1. Put enough water in the bottom of the PC so that by the end of the pressure-cooking there is still some water left. For most runs, 0.5 inches (1.25 cm) of water is sufficient. Never run the pressure cooker dry!

2. Place your materials in the PC and securely close the lid. If using a "rocker top" pressure release system, leave the weight off the vent port. If using a toggle pressure release, open the toggle. Turn the heat source to a level that requires 15 minutes to pass before steam begins to flow from the vent. Heating the PC too rapidly can cause jars to break.

3. Allow a steady jet of steam to escape from the pressure release vent for 1-5 minutes and then place the weight on the vent port or close the toggle. This ensures that everything heats evenly. The PC will come up to pressure in 10-20 minutes. Once the desired pressure is reached (typically 15 psi), reduce the heat level on the burner until the pressure stabilizes. It may take a few minutes of ad

justing the heat to get it just right. If using a rocker top, adjust the heat source so that the rocker maintains a slow, steady rocking motion and/or jiggles once a minute or so.

4. Once the pressure is stabilized, start your timer. Unless you are using an electric PC, you will need to sit with the PC during the entire run to make sure the pressure remains constant and to adjust the heat level as needed. Cook your materials for the specified run time. If you are above 2000 feet (6000 m) in elevation you will need to add 5% to the cooking time for every 1000 feet (300 m) (i.e. at 3,000 feet [900 m] add 5%, 4,000 feet [1,200 m] at 10%, etc).

5. When the run time is over turn off the heat source and walk away to let the PC cool and de-pres surize on its own. I do much of my PC work at night so that everything is cooled by the following morning.

Fractional Sterilization / Tydaluzation

If you do not own a pressure cooker, the process of fractional stmli^tion or tyndalli^tion is a slow, old school means of sterilizing substrates. Here, sealed vessels are heated in a steam bath for 30 minutes, three days in a row. Between steam treatments, the jars are kept warm (85-98°F [30- 37°C] is ideal, but room temperature will work). This heating and incubating process germinates heat-resistant endospore bacterium, making them vulnerable to latter heating stages.

1. Place vessels in a cold, shallow (2-4 inches [5-12 cm]) water bath inside of a pot with a heavy lid. Glass jars should be resting on a small towel or on canning jar rings to keep them off the bottom of the pot.

2. Bring the water to a boil to heat and steam the vessels at 212°F (100°C) for 30 minutes. Living bacteria and fungal spores are destroyed in this treatment but endospores survive.

3. Incubate the vessels overnight to germinate dormant endospores.

4. The next day, heat and steam at 100°C for 30 minutes to kill the germinated endospores.

5. Incubate the vessels for a second night to germinate the remaining endospores.

6. The next day, heat and steam at 100°C for 60 minutes to kill the remaining endospores.


t t s h r o o m



t r


t i o n

a n d


u b s t r a t e

F o r m u l a t i o n

Fungi, like animals, do not produce their own energy by photosynthesis and must find it externally.

Unlike animals, fungi do not digest their food internally, but only absorb simple compounds from their environment. Most of the food cultivated mushrooms consume is composed of complex and insoluble

organic compounds, (e.g. cellulose, lignin, pectin, and starch). In order for this food to be utilized by the fungus, it must break these substances down into simpler soluble molecules that can be transported

through the fungus' cell walls. As a cultivator we must ensure that the substrates we feed the fungi have a properly balanced nutri

tional profile. This process of balancing nutrients is called substrateformulation and is critical to successfully

producing high yields. Below are short explanations of what nutrients fungi need to live.

• Oxygen — Mushrooms are aerobic, meaning they require oxygen for their pimary metabolism.

Oxygen often comes in the form of carbohydrates, alcohols, and amino acids.

• Carbon — Carbon containing compounds are generally the "energy source" of organisms. Fun

gi use the carbon sugars and amino acids for energy and to build their cellular structure. In plant waste, cellulose, hemicellulose, and lignin are the main sources of carbon for the fungi.

• Nitrogen — Nitrogen forms the backbone of the numerous enzymes that fungi produce to de

fend, digest, and metabolize their substrates. Enz5Tnes are a type of protein that accelerate and/ or catalyze chemical reactions; they are responsible for facilitating the vast majority of the chemi cal-based functions of fungal growth and digestion. Without nitrogen, fungi cannot perform these

functions or form chitin. The amount of available nitrogen in a substrate is thus a major limiting factor in the cultivation of mushrooms: when the substrate runs out of nitrogen, the fungus stops

growing. To increase yields, cultivators intentionally add extra nitrogen to their substrates, often in the form of wheat bran or manure, depending on the species being worked with. Too much nitro gen can be counterproductive however as it can lead to abnormal growths, cause contaminants to proliferate, or enable the mycelium to grow so fast that it overheats in its container and kills itself. A concentration of 1-2% nitrogen is generally recommend for most substrate formulas. Organic forms of nitrogen, such as proteins and amino acids, are preferred. Urea should only be used to feed nitrogen to hot compost piles (don't pee on your mushrooms).

• Sulfur — Fungi need trace amounts of sulfur to make the animo acids cysteine and methionine.

This can be provided in the form of biotin (vitamin B7) and thiamine (Bl). Many fungi cannot

synthesize these compounds, so it needs to be supplemented in some form.

• Phosphorus — Used in ATP, nuclei acids, and cell membranes.

• Potassium — Critical to certain enzymatic process.

• Magnesium — Critical to certain enzymatic process, including ATP.

• Vitamins — Fungi cannot produce as many vitamins as plants and need some supplementation, most notably with several B vitamins. The addition of yeast in media is helpful in this regard, and

is only needed in low concentrations.

• Trace minerals — Zinc, Copper, Manganese, Iron, and Molybdenum are all needed in trace


This workbook contains several good starting points for substrate recipes. However, at the farm level, cultivators should design trials comparing various substrate recipes using available resources to determine the most cost effective means to achieving maximum yields from a given species and strain of mush-













• The quality of soil the substrate was grown on — Soil devoid of trace minerals will produce

plants (and plant "wastes") that are devoid of these important elements.

• How THE PLANT WAS GROWN — The quality of your substrates translate to the quality of your mushrooms. If possible, organic ingredients are encouraged to discourage the accumulation of

heavy metals or chemical residues in or on the mushrooms.

• The method of processing the substrates — Many food processing methods and substrate

preparation methods leach sugars or other nutrients out of substrates, requiring the cultivator to later add these missing nutrients back in the form of a co-substrate.

• The actual plants or substances used — Some plants, on average, produce and/or retain high er levels of certain compounds than others.

The measurement of acidity/alkalinity (pH) of a substrate is also important to mushroom growth. The pH scale goes from 0 (very acidic) to 7 (neutral) to 14 (very basic). Most mushrooms grow in the 4-8 range, with an average around 5.5-6.5. Some species are more tolerant of growing in a range of pH levels. Some are less tolerant and grow best in a narrow range. PH can be measured using a variety of test

papers and meters. While the tried-and-true substrate recipes presented in this work book do not generally require pH measurement, the pH of novel substrate formulas should be tested using test papers or meters and ad justed as needed. Generally a substrate is too acidic and needs to have its pH raised with the addition of an alkalinizing supplement, such as hydrated lime. The digestive products of fungal digestion tend to acidify substrates. If the fungus makes their substrate too acidic, it will be no longer be able to grow. For this reason, cultivators may add a pH "buffer" that helps stabilize a substrate's pH regardless of the

acidifying effects of the fungus.

Measuring a Formula's Success With Biological Efficiency

The success of a given substrate formulation is measured by the quality and weight of the mushrooms it produces, relative to the weight of dry materials used to make up the substrate. This comparison of fresh mushroom to dry material weight is known as measuring the "biological efficiency" (BE) of the substrate. For example, if 5 pounds of dry materials produce 4 pounds of fresh mushrooms, the sub strate/strain combination is said to have an 80% BE rate. Six pounds of mush- rooms produced from 5 pounds of dry substrate reflects a 120% BE. Some substrates and strains may produce a 200% BE,

though 100% BE is the minimum hoped for from most species/strains.


S t a g e



A g a r

W o r k

Agar is a seaweed-derived, gelatin-like substance. For cultivation work it is mixed with water and other

nutrients, sterilized, and then cooled inside of small containers to form a semi-solid horizontal platform

on which a mycelial network will grow two-dimensionally. Commonly referred to as plates^ petri dishes filled with nutrified agar are inoculated with spores or a piece of mushroom tissue. Once a contami

nant-free mycelial network is established (usually after a week or two), pieces of the myceliated agar are

moved from the plate to another plate or to a different substrate.

Working with agar is one of the most cumbersome and contaminant-prone stages of the cultivation

process and, personally, my least favorite. However, it does have several distinct applications that make learning the skill of working with agar indispensible to the experimental cultivator. These include the ability to:

• Remove competitor microorganisms from cloned mushroom tissue.

• Begin multi-spore inoculations to isolate and develop individual strains for experimental purposes.

• View, compare, and facilitate the various responses of mycelium to chemicals and other organisms.

A g a r

M e d i a

F o r m u l a t i o n

Agar is nutrient-poor and must be supplemented with carbohydrates, proteins, vitamins, and minerals to ensure healthy mycelial growth. Common sources of these additives include:

• Carbon: Commonly provided by dextrose (corn sugar), light malt extract (from beer-brewing

supply companies), cereal flours, oatmeal water, or the broth made from boiling potatoes. Some

cultivators prefer to mix carbon sources for more robust recipes.

• Proteins: Cereal flour, soy peptone, and potato water are most often used to provide different

proteins and amino acids.

• B Vitamins: Supplied by baker's yeast or nutritional yeast.

• Minerals: I typically add a pinch of azomite dust. A few drops of a liquid trace mineral con centrate is an optional, experimental additive. Just be sure the concentrate does not include silver, which is antifungal.

For most agar formulas, it is recommended to add 3-5 grams of the grain and/or fruiting substrate that will later be used to grow the fungus. Introducing these substrates early on stimulates the mycelium into producing the digestive enzymes it will later need to effectively break down the substrates, leading to quicker myceliation and firuiting times. When cloning wild harvested mushroom tissue, an optional additive is an antibiotic. Gentamycin (at 20 milligrams per liter) and streptomycin are two commonly used antibiotics that can withstand autoclaving and thus can be added to the agar at mixing. Food grade 3% hydrogen peroxide is a cheaper antibiotic option, but it does not withstand the high temperature of

the pressure cooker. Hydrogen peroxide (H2O2) must be added to the agar once it has cooled to below 140°F (60°C) at a rate of 6-10 milliliters per liter of agar. This temperature can be measured with a clean

thermometer or an infrared thermometer. Some cultivators add activated charcoal to their agar at a rate

of 10 grams per liter as a bacterial suppressant and spore germination promoter. It is recommended to

change agar formulas at each transfer of the mycelium to prevent senescence and maintain mycelial vigor. Five hundred milliliters of agar medium will fill around 20 standard petri dishes.

T h e

A g a r

Ve s s e l

Agar should be cooked in a glass vessel that can withstand the high temperature and pressure environ ment of the pressure cooker. Consideration should be given to how the hot liquid and container will be handled after coming out of the PC and easily and quickly poured into the petri dishes without making a mess. Messy plates provide a route for outside contaminants to follow and enter your plates. Pyrex flasks and or long neck liquor bottles can be used. These should be stopped up with polyfil fiber and covered with aluminum foil if they do not have a screw on cap.

P l a t e

O p t i o n s

Pyrex petri dishes are reusable and can sometimes be bought for a deal on eBay. These should be

washed, dried, wrapped in aluminum foil and then PC'd with the agar between each use. Plastic one-time-

use dishes can also be used though are less sustainable. Baby food jars are used by some. As long as the vessel can withstand the pressure cookery and can be easily worked with to extract samples of mycelium, the choice is yours for where the mycelium grows. If you Hke, pre-poured agar plates can be purchased online. The cost is increased over making your own but this may offset the time and frustration spent on plates with high contamination rates. Pre- poured plates are often made with only Malt Extract Agar.

C o o k i n g

a n d

P o u r i n g

P l a t e s

1. Mix and dissolve the ingredients on the stove at a low heat. The liquid should then be PC'd at 15

psi for 20-30 minutes.

2. If using a rattle style pressure release system, do not allow this to rattle too much. This can lead to boil over or caramelization of the sugars, which is toxic to the mycelium.

3. Once the PC is turned off, allow it to sit for 2 hours so the can agar cool a bit.

4. At that point the agar should be poured into your plates inside of a clean glove box or in front of a flow hood as quickly and cleanly as possible.

5. Stack the plates as they are poured to reduce condensation on their lids.

6. Cover this stack in the plastic sleeve the plates came in and allow them to cool for an hour or so.

Optionally, place a cup of hot water on top of the top plate to save the top plate from excess con




7. Once cooled, plates can be wrapped with plastic food storage wrap and refrigerated for long-term

storage or inoculated and then sealed with a single layer of Parafilm and placed in the incubator /




8. You may also opt to place the plates in an incubator for 24 hours to see if contaminants appear.

Inoculating Plates

Once your contaminant-free plates are cooled you are now able to begin growing mycelium! You now have three main options for how to inoculate agar:

1. Obtain a commercial culture of a tested strain in the form of a petri dish or slant.

2. Clone a wild-caught or store-bought mushroom (or a retail piece of grain or sawdust spawn).

3. Use a spore syringe or spore print to start a multi-spore culture of various strains.



Commercial cultxires are an easy starting place for new cultivators to begin cultivating for a few rea


• The strain has been tested and is known to be a heavy fruiter or otherwise valuable.

• No time to wait for spores to germinate and then to isolate a strain from.

• Less risk of contamination compared to cloning.

The disadvantages are:

• Based on the species, strain, and company, a given culture costs anywhere from $20-1,000 or more!

• Depending on its age under sterile conditions, the strain may have senesced to a degree.

• The strain may not be well-suited to your local environment or climate.

It issuggested to copy and back up any commerical culture before expanding it, so as to preserve the

state of vigor it was obtained at. The steps to doing this are the same as a Plate-To-Plafe Tranter, described


C l o n i n g


M u s h r o o m

Under aseptic conditions, a small piece of mushroom tissue can be excised from the interior of a wild or store bought mushroom and then placed on a petri dish. The mushroom tissue will then revert back to a vegetative mode and myceliate on the plate.


• A fresh or slighlty dried mushroom

• A scalpel and set of tweezers

• A clean petri dish

• P a r a f i l m

• Bacti-cinerator and sterilizing materials

• Optionally, a shallow dish of 3% hydrogen peroxide


1. Following aseptic practices, unwrap your petri dish.

2. Wipe the mushroom cap down with alcohol.

3. Flame-sterilize your knife and make a slight cut in to the center of the mushroom cap.

4. Set the knife down and open the mushroom like a book, exposing the sterile tissue inside. Keep

your hands under the mushroom and do not pass your hand over the inner tissue of the mush

room. Alternately, tear open the mushroom if that is easier. Sterilize your scalpel or tweezers and








5. Excise a small piece of mushroom tissue from either just above the giUs (but not the gills them selves), the cap interior, or the stem interior. These are areas where cells are rapidly elongating and

growth is active. With some species it may be difficult to pull tissue from anywhere on the mush room. Do your best.

6. Optionally, once the tissue is excised, dip it in a small dish of 3% food grade hydrogen peroxide

for 5—10 seconds to clean its surface. Many wild harvested mushrooms contain bacteria that in

terfere with current cultivation protocols. In reality, these microbes are likely somewhat beneficial

to the fungus, hence their presence. This cleaning process is recommended for wild, woody, and/

or thin-fleshed mushrooms and/or their mycelium cloned from wood chips, cardboard, or other



7. Quickly open the petri dish and place the mushroom tissue on the agar. Mycelium tends to stick to tools. A good technique to easily remove tissue from a tool is to cut through the mycelium and into the agar, then slide the tool back and through the agar, leaving the mycelium behind.

8. Optionally, repeat steps 5-8 two more times on the same plate to ensure that you obtain at least one clone that will regenerate without contamination. I prefer to use smaller petri dishes for clon ing, so as to conserve agar.

9. Wrap the plate once, label it with species, strain (or harvest location), and date, and place it in an incubator or in a warm space to encourage rapid myceliation. Regrowth should be visible within







Working with spores enables the cultivator to create fresh lineages of mycelium. These novel com binations of genetic information provide for the possibility of creating more vigorous strains that may grow faster, yield higher and taste better than the parent mushroom they were liberated from. However, the selection process for finding a good strain can be time and labor intensive.

M a t e r i a l s

f o r

s p o r e




n t

w o r k :

• 1 clean petri dish

• 1 clean spore print

• Inoculation loop

• Plate wrapping material

• Tool sterilizing materials








1. Clean the transfer area and prepare to work under aseptic conditions.

2. If the petri dish was in cold storage, allow it warm to room temperature in the transfer space.

3. Unwrap the plate.

4. Open the spore print and sterilize the inoculation loop.

5. Open the petri dish like a clamshell and stick the hot inoculation loop tip into the agar to cool it off and to make it sticky with agar. CarefuUy pull the loop out of the plate without touching it to anything. Close the petri dish.

6. Wipe the loop across the spore mass to deposit spores on to the tool. If spores are visible, thou sands of spores are likely on the tool.

7. Open the petri dish as minimally as possible and wipe the spores across the surface of the agar in

a zig-zag pattern. Spread the spores.






Close and wrap the plate with a single layer of wrapping material.

10. Label the plate with species and date and place it in an incubator or spawn run space to initiate

spore germination. Return the inoculum to storage.

M a t e r i a l s

f o r

s p o r e

s y r i n g e

w o r k :

• A clean petri dish

• A spore syringe that has been in cold storage and not exposed to excessive heat

• P a r a f i l m

• Alcohol flame and sterilizing materials

W o r k i n g

w i t h


s p o r e

s y r i n g e

o n

a g a r :


Unwrap your plate. Expose the spore syringe's needle and sterilize the needle tip using a heat




Squirt a small amount of liquid out to cool the needle. A hot needle can destroy spores. Flick the

syringe with your finger to disperse the spores inside of the syringe.


Use the clamshell method to open the plate and insert the needle tip without touch- ing any of the plate's surfaces. Deposit 1—3 drops of spore water on to the surface of the agar. Remove the needle without touching anything and close the lid.










Swirl the plate around to spread the spores.Label and place the plate in your incubator or in a warm area and the syringe in the fridge. In several days visible areas of mycelium should appear. Various sectors will develop delineating the different genetic combinations that have occurred.

S e l e c t i n g


s t r a i n

f r o m


m u l t i - s p o r e

p l a t e

When using a spore syringe or print on agar, numerous combinations of genetic material arise that will each have their own characteristics and performance patterns. Termed "strains," each of these pair

ings should be compared based on a variety of factors to determine which one is to be worked with. Typical factors for selecting the strains include:

Recovery time from transfers or shaking.

Quality of mycelium.


Adaptability to substrates.

Dependence on microflora.

Time from inoculation to fruiting.




Temperature and cold shock requirements.

Number of primoria formed and percentage that mature.

Medicinal quality of mycelium and mushrooms.

Ability to degrade toxins.


Antimicrobial activity.

Appearance, flavor, texture, aroma, nutritional profile, and shelf life of mushrooms.

Deaung with Competitors

Whether you are working with spores or tissue, you will soon see pure mycelium growing for the site of inoculation 3—21 days after inoculation. However, it is also possible that you might see bacterial or

fungal contaminants arising as well, especially if an antibiotic was not incorporated into the agar. Luckily there are several options for dealing with these inevitable moments:

1. Cut out a piece of clean myceuum and transfer it to a new plate: This process is known as suhculturing or subbing. This second plate might also develop the contaminant, as the transferred

piece may be "dirty." A third (and hopefully) final sub might be needed to obtain a clean culture.

2. If possible, cut out the contaminant: Most molds produce a white mycelium during their

initial growth. If the cultivator learns to recognize these colonies as competitors, they can be cut out and removed from the plate. If the mold has gone to spore (i.e. the mycelium has taken on a

distinct color), attempting to remove the mold might shake more mold spores around the plate.

Subbing is preferred in this case.


Cover the mycelium and contaminant in hot agar: Just like it sounds. The mycelium will like

ly climb up through this new layer faster than the mold. The mushroom can then be easily subbed. Alternately, when you clone a mushroom, flip over a piece of the plate's agar to cover the clone from the start. This can help leave behind contaminants that may appear.

4. Use antibiotic agar to kill off bacteria: Place a fresh piece of antibiotic agar from another plate on top of the contaminant in a manner similar to a Plate-to-Plate Tranter. Under this antibiotic agar, the microbe will die and the mushroom mycelium wiU continue to grow.

5. Do nothing: Often, the microbial war that develops on the plate between fungus and foe will result in the mushroom mycelium effectively outcompeting the competitor. Watching this process unfold is in itself a great learning opportunity and glimpse into the habits of the mushroom. If the mushroom overtakes the contaminant, the mycelium should not be used as an inoculum for aseptic















You can learn a lot about the quality of your transfer technique by observing where contaminants

appear on the plate. If a contaminant shows up on the edge of the plate, it may have entered during the tissue transfer, during pouring and wrapping, or during storage. If the competitor shows up on or near where the mycelium was transferred, your mycelium or tool may have been dirty.



Like a petri dish without the agar, sugar and nutrient rich liquid broth can be used as a 3-dimensionai media for mycelium to growth through. This process is commonly done industrially using large sterile fermentation tanks. For the smaller grower, canning jars with airport lids are an incredibly effective and

inexpensive means to work with a liquid culture.

T h e

P r o s

Compared to agar, LC has a number of advantages as an inoculum:

• Liquid culture is cheap: No agar is needed, simple ingredients are used. The jars, lids, and s}^-

ringes used in the process are all reusable. Liquid culture broths can be sterilized in a hot water bath, avoiding the need for a pressure cooker.

• Liquid culture is time efficient: There is no need to cook and cool agar for hours and there's

non of the stress and mess associated with pouring plates. Once a healthy mother jar of LC is es

tablished, it can serve as inoculum for many months, essentially eliminating the need for constandy

starting mycelial lineages with agar plates.

• Liquid culture increases myceliation rates: Mycelium in a liquid suspension grows three-di-

mensionally, increasing growth rates when compared to the two-dimensional surface of an agar

plate. When LC is aerated, the mycelial network breaks into a constellation of fragments, each with its own array of active hyphal tips. This is significant as, compared to an agar plate where the

mycelium is only active at the edge of a single colony, liquid culture inoculum is almost entirely

comprised of leading edge mycelium. • Liquid culture inoculation rates are high: Liquid culture is sprayed onto substrates with a

syringe. As the medium percolates through the substrate, the individual mycelial clusters distribute throughout the material to explode with growth at each point of contact. The result is a much

more even distribution of inoculum than that achieved by a piece or two of myceliated agar.

• Liquid culture work can be done anywhere: Using a syringe and airport lid, one can transfer

mycelium to grain jars outside of an aseptic transfer space. Whole mushrooms can also be cloned in the open air using this method. This reduces the need for constantly preparing and maintain

ing a dedicated transfer space. This is perhaps the greatest benefit of this method over the many

annoyances and costs associated with agar work. The ability to grow grain spawn in the absence

of a clean transfer space using liquid culture inoculum quickly translates to any home mycologist becoming a mushroom farmer. With this challenging step overcome, time and energy increases in

supply, allowing for creativity to spur new applications for cultivation beyond the practices of food and medicine production. Liquid culture allows us to work, think, and grow outside the glove box. How will you work with its many benefits?



LC is my preferred inoculum for everyday use. However, it has some drawbacks compared to agar













If an LC is contaminated, it is very difficult - or near impossible - to clean it up.

Not easy to acclimate strains to novel compounds.

Not possible to isolate strains from multiple spore inoculations. Using spores in LC is also not

recommended due to a high chance of contamination.

P r e p a r i n g

L i q u i d

M e d i a

1. Mix and dissolve ingredients in a pot over low-medium heat.

2. Once dissolved, fill a clean jar (or multiple jars) half-full with the solution.

3. Place a couple of marbles, a piece of broken glass, a crystal, a magnetic nail with its head cut off, or a magnetic stir bar in the jar.

4. Place an airport lid on each jar and cover them with aluminum foil.

5. Pressure cook the jar(s) at 15 psi for 15-20 minutes.

6. Once the pressure has reached 0 psi, the jars can be removed and allowed to cool. Alternately, allow the jars to cool inside the pressure cooker overnight.

If you do not own a pressure cooker, boiling is an alternative means for sterilizing liquid media - just

place the LC jars in a pot of cold water, submerging them half way. Cover the pot with a lid and bring the water to a boil. Boil the jars for 30 minutes then allow them to cool completely before inoculating. Be sure to run a blank jar with this method to confirm that you are effectively sterilizing the media.





The most important points to consider are that the correct types of sugar are used and that the con centration of sugar is no more than 4% (4 grams of sugar per 96 mL water). This is barely sweet to the human tongue but is plenty sweet for the fungus. Any more can be toxic to the mycelium. Household sugar (sucrose) is typically not preferred by most species. Light malt extract and honey can be used alone, dextrose is not ideal as a sole carbon source. Additional nutrients can be added such as peptone and various flours, but they cloud the broth, making it difficult to determine the health of the mycelium and whether there are competitors present.

I n o c u l a t i n g

L i q u i d

M e d i a

Inoculating liquid media must be done with great care as a single bacterial cell or mold spore can ruin a whole jar of broth. Once a clear jar is established, working with the culture is much easier thereafter.

A g a r

t o


This process injects sterile water into a myceliated agar plate with a syringe, scrapes a small amount of mycelium off the agar with the needle and then sucks back up this mycelium water into the syringe






• 1 jar with distilled water & an airport Ud

• 1 jar with Hquid culture medium

• 1 myceliated, clean petri dish

• 1 syringe with 16 ga Luer-Lok needle

• Flame

A l u m i n u m

f o







1. Wrap the syringe and needle in aluminum foil. Cover both jar lids in foil. The foil prevents the filter from getting wet in the pressure cooker, which can lead to contamination problems later. Pressure cook both jars and the syringe at 15 psi for 15-20 minutes. Allow to cool.

2. During the cooking process oxygen was depleted from the liquid media and now needs to be rein- troduced. This can be done using the methods described in the section The Growth and Aeration of a Liquid Culture, below.

3. Wipe both injection sites with an alcohol wipe or alcohol-sprayed cotton ball. Clean the sites well

but avoid excessive force as this may dislodge the silicone, enabling contaminants to enter the jar.

Spray both silicone sites with alcohol.

4. Unwrap the syringe and draw up 5 milliliters of the sterilized water. Alternately, fill the syringes with water, wrap them with aluminum foil, and sterilize them with the LC.

5. In your transfer space, unwrap and open the myceliated agar plate. Quickly, and without touching

any petri dish surfaces with the needle, point the opening of the needle down and inject 1 milliliter of sterile water onto a small portion of the leading edge of the mycelial mat.

6. Using the needle tip, scratch some of the mycelium off the agar, suspending it in the sterile water.

Quickly and cleanly suck up as much mycelium and water as possible.

7. Spray the silicone port on the liquid media jar with alcohol and inject the mycelium water into the media jar.

8. Label and date the jar and place it in the incubation space.




In this approach we will be taking mushroom tissue directly from a fresh mushroom. If the mushroom














• 1 jar with distilled water & an airport Ud

• 1 jar with liquid culture medium


f r e s h

m u s h r o o m

• 1 syringe with 16ga Luer-Lok needle

• Flame

A l u m i n u m

f o




• Sterilization materials & alcohol wipe


1. Follow steps 1-4 for Inoculating A^ar to LC.

2. Wipe the mushroom stem or cap thoroughly with alcohol, spray the mushroom with alcohol and then insert the needle through the stem or cap. Or just tear it open and stab a clean piece of inner


3. Remove




check to


if there

is tissue



needle shaft. If there

is not visible

tissue, stab the mushroom again. Repeat until tissue remains in the needle shaft once it is removed from the mushroom. Try to be quick and avoid breathing on the needle.

4. Spray the LC jar port with alcohol again and insert the needle into the LC jar. Inject the piece of


5. Label and date the jar and place it in the incubation space.

sterilizing and inoculating multiple jars at once. This will help ensure greater success in the event

that the one of the jars becomes contaminated.

G r o w t h

a n d

A e r a t i o n

Once the liquid medium is inoculated, I tend to let the jar sit undisturbed for 1-2 days, during which time the mycelium begins to recover from the shock of being transferred and reverts to vegetative growth. At this point the mycelium should begin to appear as a small cloud of tissue. As this network continues to grow it will begin to consume the dissolved oxygen in the liquid. If this oxygen is not re plenished, the mycelium may suffocate and rise to the surface of the liquid in search of air. To avoid this stress on the mycelium, LC jars should be oxygenated frequently. To dissolve oxygen in the liquid, swirl the liquid by hand or with the aid of the stir plate and stir bars. Take care to not get the lid's filter wet as this can enable contaminants to enter the jar. This agitation also breaks up the mycelium as it grows,

thereby increasing growth rates and enabling easy extraction.


I n o c u l u m

E x p a n s i o n

Plates and liquid cultures can be exponentially expanded to increase the mileage of your labors before

shifting to grain or sawdust spawn. This saves the cultivator the time and risk of constantly pulling from



P l a t e - t o - P l a t e

T r a n s f e r s

An old method for inoculum expansion it to take a small piece of myceliated agar from a healthy plate and transfer it to a clean pre-poured plate. Ideally the new plate would have a different nutrient base than




• 1 myceliated Plate

• 1 (or more) clean, pre-pored plate(s)

• Scalpel or metallic spatula






1. Under aseptic conditions in the transfer space, unwrap both plates.

2. Flame sterile your tool and allow to cool.

3. Open the myceliated plate and cut out a wedge of myceliated agar from the leading edge of the


4. Quickly open the new plate and transfer this wedge to its center.

5. Close the plate and wrap both with a single layer of Parafilm.

6. Repeat with more plates as desired.

Depending on the size of wedge you cut, one plate can be expanded to 10 or more new plates.

Liouid-to-Liquid Transfers

Like a plate, LC jars can be expanded exponentially, though this can be done outside of the transfer

space. Again, changing the recipe of the medium is preferred.


• 1 myceliated LC jar

• 1 clean, sterilized, non-myceliated liquid media jar

• Sterile syringe with large gauge needle (16 ga)

• Flame





1. Select a healthly LC jar.

2. Wipe and spray the silicone ports on both jars with alcohol.

3. Flame sterile your needle and allow to cool.

4. Insert the needle into the myceliated jar, drawing out several CCs of mycelium rich LC.

5. Quickly insert the needle into the new jar of media and inject the LC.

6. Repeat with more jars as desired.

C u l t u r e

S h o r t

Te r m

S t o r a g e

S t o r a g e

o f

P e t r i

D i s h e s

a n d

L C s

For short-term storage of plate cultures, place them in the refrigerator double wrapped in Parafilm. Plates tend to dry out within in a few months, so this method is not preferred for long term preservation



Liquid culture jars can be stored in a fridge for 6-8 months (or longer). Some add a little H2O2 (approx. l-3cc) once to help prevent contamination.

Long Term Storage Strategies

A proper long-term storage strategy of fungal cultures is critical for ensuring projects and growing calendars are not interrupted by unforseen contaminant outbreaks of workplace disasters. Balancing be tween space efficiency for the cultivator and nutrient density for the fungus has lead to several approaches for long term storage.


A common approach is to use test tubes filled with agar. These tubes have been cooled at an angle to

provide a slanted surface for the myceHum to grow on (maximizing surface area in a concentrated vol ume). This works fine for 6 months or so, but many strains start losing viability when kept on a single nutrient for an extended period. Twice a year these cultures need to be moved to another slant with a different nutrient base. Some strains of Agaricus appear to start the dying process anyway, as though agar is not the media they prefer. Every 6 months or so the culture should be brought out of storage and allowed to sit out for 24-48 hours until signs of growth are apparent. A new plate is then inoculated with the slant and then a new slant from this plate, ideally each with a different nutrient base. Optionally overlaying the slants with sterile mineral oil keeps the sample from drying out and acts as an oxygen barrier. Inserting a small wood stick in the slant before PCing will provide a long term reserve for the mycelium in case it dries on the





Following the practice of making nutrified sawdust (described later), small baby-food jars can be filled 2/3 full with this blend, sterilized, and then inoculated under aseptic conditions. Once fully myceliated, the jars are refrigerated. A piece of this sawdust spawn can later be transferred to an agar plate to start a new mycelial lineage. Due to the substrate's densitty, this method can preserve cultures for more than a


D i s t



l e d

W a t e r

Sterile and pour sterile distilled water agar plates (SDWA)(10g agar / SOOmL distilled water, 30 min. at 15 psi). At the same time sterilize small containers filled with distilled water (20 mL scintillation tubes

work well).

1. Transfer leading edge mycelium from a healthy nutrified plate to the SDWA plate using a Plate-to-



2. Once the mycelium has touched down and grown across the SDWA plate a little ways transfer a

piece of just the mycelium and SDWA (avoid the nutrient-rich piece that was transferred) to the

container of sterile distilled water using a Plate-to-Plate Transfer.


Seal the container tightly and label it

4. Store the vials at room temperature away from direct sunlight.

5. In the distilled water environment, the mycelium enters a dormant state and remains in stasis.

6. To reanimate, simply dip a sterile tool into the sterile water and place a piece of the mycelium onto a nutrified agar plate.






I keep the following on hand at aU times: small nutrified agar plates sealed with plastic wrap, larger

nutrified agar plates, sterile distilled water jars, distilled water agar plates, small nutrified sawdust jars, and slants that contain a coffee stir stick. I make up small liquid culture jars as needed as they require the

greatest amount of storage space. To create multiple forms of backups, I first clone a mushroom or mycelium to a small petri dish. This

helps to conserve agar in the event of contamination during cloning. I also clone to cardboard in case the mushroom doesn't regenerate on the agar. Once a pure culture is obtained (perhaps after several Plate-to- Plate Transfers), I then make six backups from this young culture before moving on to spawn production. First, I inoculate a mother liquid culture jar, as this is the most sensitive backup, as well as a larger LC jar to be used as inoculum as soon as possible. Then I transfer pieces of leading edge mycelium into a nu trified sawdust jar, a distilled water agar plate, and three or four slants. If there is extra mycleliated agar I will also inoculate sterilized grains. Later, when the distilled water agar plate is barely myceliated, I transfer

pieces of this plate's leading edge mycelium into small jars of sterile distilled water. All of these backups

are kept in a dedicated refrigerator except for the distilled water jars. Replicates of slants are stored off site in the event that the main back-ups are damaged.

S t a g e

2 :

G r a i n

S p a w n

W o r k

Once you have amassed some mycelium on a petri dish or in a liquid culture just, the time has come to transfer the mycelium to a jar of sterili2ed grains. These grains wiU provide a large amount of nutrients and minerals to feed the mycelium and, once myceliated, will provide a jar full of individual "seeds" to feed the next stage in the process.

S e l e c t i o n

o f

G r a i n s

Rye berries, wheat berries, millet, and sorghum (milo) are common choices in commercial mushroom cultivation operations. Home cultivators also have success with spelt, popcorn, and whole birdseed. All of these grains are preferred due to their low levels of nitrogen and ease of preparation. Many other

grains are too high in nitrogen, which can lead to overheating during mycelial growth or high contamina tion rates. Milo is preferred by some growers as it hosts over 30 types of vitamins and minerals as well as a small size, which provides for more points of inoculation for fruiting substrates.

P r e p a r a t i o n

o f

G r a i n s

Grains must be properly cooked and sterilized so that the mycelium can readily penetrate and myceliate them. There are many approaches to preparing grains for cultivation. Some people will simply mix water and grains in their jars and pressure-cook them immediately as such. Others prefer to soak their grains for 12-24 hours beforehand, as pre-soaking may help germinate dormant endospores of bacteria, making them more susceptible to the heat of the pressure cooker. Others rinse their grains several times to rid them of any dirt and debris that may make the grains stick together while also harboring contaminants. I myself have setded on a combination of these methods as it works consistendy for me. Depending on

your altitude, environment, water and grain source, though, you may opt to adopt a modified version of this approach. However you choose to do it, these factors must be your guiding principles:

• The grains should not be too hard. When you bite into a grain it should be a littie bit under

cooked (al dente) for normal human consumption. There should be no hard center in the grain. In this state, the grain is fully saturated and supple enough for the myceUum to penetrate and digest it.

• The grains should not be too soft. There should be a minimum of sprouted or burst kernels

after sterilizing. Overcooked, burst, and/or overly wet grains make grains more prone to contami nation due to their protective outer layer being broken.

• The grains should be easy to break up. Dirty or overcooked grains can stick together, making

it difficult for the mycelium to grow and/or later be broken up for spawning. Pre-rinsing grains and

adding gypsum helps to reduce stickiness.

The following is a generic grain preparation process for 10 quart jars:

1. Measure out 10-11 cups of dry grains into a large pot.

2. Fill the pot with water and stir the grains to suspend any dirt and debris that is present on the


3. Pour off this dirty water and continue rinsing the grains until the water runs clear.

4. Cover the grains with high quality water.

5. Cover the pot and let it sit for 12-24 hours. Some cultivators soak their grains in 50% strength






6. Place the pot on the stove and bring the grains to a boil for 5-10 minutes or until they are cooked

to the right consistency.

7. Drain the grains through a colander. This nutrient-rich water can be saved and used for incorpo

rating into agar and liquid media recipes. 8. Toss the hot grains around until they have cooled and the excess moisture has steamed off of them. If you are cooking a large amount of grains, spread them on your substrate prep screen to

speed cooling. 9. Add 1 tablespoon of g)rpsum evenly throughout the grains. Gypsum provides mineral supplemen tation while also helping to reduce the stickiness of the grains.

10. Fill each jar one-half to two-thirds full with the grains. A 16-ounce (475 mL) measuring cup and

canning jar funnel significandy help to facilitate this process.

11. Seal each jar with an airport lid and cover the lid with an aluminum foil cap.

12. Pressure cook the jars for 60-75 minutes at 15 psi. Grains can also be tyndallized in the absence of a pressure cooker, but be sure to run a blank.

13. Turn off the stove and let the PC cool overnight.

14. Open the PC and remove the jars. Inspect each jar for cracks and/or an excessive number of burst kernels. Discard cracked jars.

Notes on Grain Prep

Soaking grains for 12-24 hours helps to germinate the dormant endospores of bacteria inside grains, making the endospores more susceptible to the heat of the pressure cooker. The amount of water used

to soak and cook the grains should be the minimum necessary for achieving properly cooked grains. If

too much water is used while boiling, beneficial nutrients will leach out of the grains and be lost. Trial and error will help determine the proper amount of water needed for your grains. Fuel costs can be saved in this process if the grains are cooked to their proper state using a solar collector/pasteurizer. Smaller grains, such as millet, do not need to be cooked as soaking provides adequate hydration. These uncooked grains will be very wet and sticky on the outside and need to be dried off on a clean towel prior to ster ilizing. Brown rice, used for many commercial medicinal products, often turns out very sticky and needs extra attention. After soaking and cooking, spread the rice onto a clean towel and stir it occasionally with a

spoon as it cools. Then load the rice into jars as gently and loosely as possible. After pressure cooking, lay the jars on their side to cool, occasionally turning and shaking the jars to minimize clumping in the rice. A small amount (5-10% by volume) of the fruiting substrate can be added to grain jars prior to ster ilization to help initiate the enzymatic expression that the mushroom will ultimately require to consume



Te s t i n g

m o i s t u r e

c o n t e n t

If you are wishing to attempt a different method of grain preparation, consider measuring the mois ture content of your grain after preparation. To do this simply measure out 100 grams of prepared grains and them place them on a baking tray in a 350°F oven for 20 minutes or until bone dry. Weigh out the

grains a second time. The difference in weight will correspond to the moisture content.

G r a i n

I n o c u l a t i o n s

Once your grains are prepped, it is now time to introduce some mycelium that can grow on them.


Much like a Plate-to-Plate transfer, this technique moves a wedge of myceliated agar to a cooked and cooled grain jar.


Myceliated petri dish (the mycelium should not be to the edge of the plate to avoid contamination)

Cooked, sterilized, and cooled jar of grains





Scalpel or spatula


1. Prepare your transfer space for aseptic work.

2. Shake the jar to loosen the grains. Shake the grains so that they are sloping in the jar.

3. Unwrap the myceliated plate and loosen (but don't open) the lid on the grain jar.

4. Sterilize your scalpel or spatula and allow it to cool.

5. Clamshell the petri dish open then cut out and extract a wedge of agar with your tool. Ensure that the piece has some amount of leading edge mycelium.

6. Close the petri dish lid.

7. Open the loosened grain jar lid as minimally as possible to drop the agar wedge in to the jar onto the lower end of the sloping grains. Do not place your hand over the opening of the jar. Try to not touch the tool to the jar.

8. Close the jar lid and tighten it down.

9. Gently shake the grains over the agar wedge so that they cover the mycelium. This move helps

keep the mycelium from drying out as it recover from the shock of being transferred while also providing the fungus with easy access to the grains. 10. Wrap the plate. Label the jar with species and date and set to incubate.

After inoculation, grain jars should be left alone for several days. Soon, the introduced mycelium will

begin visibly growing on and through the grains. Roughly 3-8 days later, when 25—35% of the grains are myceliated, the jars should then be shaken for 20—30 seconds to break up the developing mycelial network and distribute the myceliated kernels throughout the jar. This helps increase myceliation rates. While I prefer to only shake once, some growers shake their jars a second time at around 70% mycelia tion. Shaking at 90%+ myceliation often stunts the mycelium's growth, negatively impacting the success of later expansions.



Using an airport lid, you can inoculate grains outside of the transfer space, lowering the cost of cul- turing while increasing the fun!


Myceliated LC jar Sterile syringe with needle Sterilized grain jar(s) with airport lid(s)






1. Clean the silicone ports on both jars with an alcohol wipe or alcohol dampened cotton ball then

spray both ports with alcohol.

2. Insert the syringe needle into the LC jar and extract roughly 2-10 CCs of the liquid culture per

quart jar of grains. Be sure you are drawing out mycelium and not just sugar water.

3. Withdraw the needle and insert it into the grain jar. Swirling the needle around gendy, spray the

mycelium across the grains.

4. Repeat with each jar. If the transfers are taking a long time, spray the top of the uninoculated grain

jars with alcohol again before inoculating. I prefer to use a large syringe for this process to reduce the number of times I enter the sensitive liquid culture jar.

5. Label each inoculated grain jar and set them to incubate.


Grain spawn can be expanded to more grains before being moved on to final substrates. Whether

spawning to more quart jars, Vz gallon jars, or gallon jars, an inoculation rate of 10-20% is recommended.

This process is very simple and may be done twice with relative ease for a given mycelial lineage, however pushing for a 3"^ or 4'^ grain generation is not recommended.


• Jar of colonized grain spawn

• Multiple jars of cooked, sterilized, and cooled grain spawn

• P a r a f i l m


1. Under aseptic conditions in your transfer space, loosen all jar lids and arrange them for easy access during transfer.

2. Open the myceliated jar on its side to reduce ambient competitors from entering the jar.

3. Quickly and carefully open the lid of the first jar just enough to introduce 1/10-1/5 of the myce

liated jar's grains. Rotating the jar as you pour helps in this process.

4. Close the lid on the freshly inoculated jar.

5. Repeat for each remaining new jar.

6. Tighten all lids. Label with date and species, shake to distribute the myceliated grains, and set the

jars to incubate.

S t a g e

3 :

F r



t i n g

S u b s t r a t e s

Depending on the species you are working with, myceliated grain spawn is transferred to a variable final substrate from which it can fruit. There are five main types of fruiting substrates:



• Plain, pasteurized sawdust

• Supplemented sawdust (with or without wood chips)

Straw (with or without additives)

Miscellaneous organic wastes

Compost- and manure-based substrates

S u b s t r a t e

A d d i t i v e s

Along with these primary ingredients, other additives can used to balance nutrient needs and increase yeilds. These additives are not always needed and can be overused, so it's important to understand what they are and why they help.

• Gypsum (Calcium sulfate [CaSO]) — A flocculant, gypsum inceases fluffiness in substrates and reduces stickiness. It also provides sulfur and calcium, which help increase yield, speed, and myce lial health. It is typically dded at 2-10% by dry substrate weight.

• Vermicuute or Coconut Coir — These are often added to compost and manure based

substrates to increase texture, aeration, and moisture retention.

• Coffee — A fairly versatile additive, fresh coffee grounds can be added to most substates as a minor additive. Oysters do particularly well on this substrate alone. Weak coffee is used by some to

hydrate substrates, such as grains.

• Spent Malt — Distillery and brewery waste can be used as substrates. Brewery grains are

often acidic and need to have their pH raised. The addition of dry vermiculite or newspaper may be an easy way to offset excess moisture levels. Lightly roasted grains are preferred for their sugar profile.

• Agricultural Residues — Hundreds of byproducts from food and feed industries have been shown to be viable substrate options.

• Worm Castings — Mostly used for compost and manuring loving species, castings add

nitrogen, phosphorus, potash, and other nutrients. They are added at 10-15% by volume.

• Chicken Manure — Mostly used for compost and manuring loving species, chicken manure

is a rich nitrogen source and is only used at around 1-2% by volume.

Substrate formulation is required when working with these alternative substrates. A quick search

through journal databases can easily provide studies that utilized these and many other substrates for



The following additives should be used in limited amounts:

• Thiamin — One tablespoon per gallon of water. Adding too much B1 can throw off nitrogen


• Humic/Fulvic Acid — Aids mushrooms digestion and stimulates growth. Use 1 tablespoon per

gallon of water.

• Kelp — One-half to one teaspoon per gallon of pasteurization water. Adding too much will

promote molds.

• Seaweed Extract / Seaweed — Adds minerals. Use up to 10% (by volume).

• Pollen (not bee pollen) — Aids liquid inoculum growth.

• Soybean / Sesame Meal — A long-term protein source and antioxidant with some anti-mold

properties. Use at 2-4% of dry substrate weight.

• Soybean / Sesame Oil — Adds calcium, antioxidants, fats, and is possibly anti-mold. Only add

small amounts (around 1 tablespoon per gallon of substrate).

• Vegetable/Canola Oil — Contain Upids and nutrients for mycelial growth. Use 1-2 teaspoons

per gallon of substrate.

A l k a l i n i z i n g

A g e n t s

The various types of alkalizing agents all have different chemical properties. Care should be exercised with aU of the products as most are caustic and a skin and eye irritant. Carefully read and follow all man ufacturer directions exactly.

• Wood Ash —Just as it sounds. A cheap way to increase pH. Avoid questionable impurities from

glossy papers, paint, adhesives, etc.

• Horticultural/Hydrated/Slaked Lime (Ca[OH] )2 — Produced by adding water to CaO. Causes rapid pH shifts, but is not long-lasting. When heated above 1077°F (580°C) it dehydrates,

forming the oxide. Reacts with carbon dioxide to form calcium carbonate.

• Pickling Lime — A food grade form of calcium hydroxide with no additives or preservatives.

• Calcium Carbonate (CaCO )3 — Helps buffer pH for an extended time. It comes in the follow

ing six forms:

• Chalk — Soft in texture, chalk holds water well. Using a variety of piece sizes - from 1-inch

thick to dust - helps improve casing structure and provides the longest lasting buffering action.

• Marl — Dredged from dry lake bottoms, marl is a soft lime similar to chalk but has the consis

tency of clay. It is a composite of clay and calcium carbonate with good water holding capacity.

• Oyster Shell — Mainly calcium carbonate along with other minor ingredients. Ground oys

ter shell is similar to limestone grit in its buffering action and its structural contributions to

casings. Oyster shell should not be used as the sole buffering agent because of its low solubility



• Limestone — A sediment mineral composed mainly of calcium carbonate. It is similar to

oyster shells.

• Ground Limestone — Generally, ground limestone is weaker than hydrated Ume, needing

about 30% more to raise the pH by the same amount. Being cheap, it is the most widely used

buffering agent for US Agaricus growers.

• Limestone Grit — Produced in a fashion similar to ground limestone, limestone grit is rated

according to particle size after being screened through varying meshes. Limestone grit is an

excellent structural additive but has low buffering abilities. A number 9 grit is recommended.

W o o d - B a s e d

S a w d u s t

( S p a w n ]

S u b s t r a t e s

From grain spawn the cultivator moves many of the commonly cultivated species on to either plain sawdust or to nutrified sawdust, depending on the course of action to be pursued. Five- to six-pound

bags of this sawdust spawn are standard products from commercial farms. Two common options for making sawdust kits are:

• Spawn grains to pasteurised plain sawdust that, once myceUated, is be used to lay outdoor beds,

plug logs, or make bulk naturali2ed spawn. Some mushrooms can fruit directly from plain sawdust though yields are often not high.

• Spawn grains to supplemented and sterilized sawdust (with or without wood chips added) which, once myceliated, is fruited from directiy. This route results in greater indoor yields compared to

fruiting from non-nutrified sawdust, but is more sensitive to contamination.

Pasteurized Plain Sawdust Kits

If you do not plan to add supplements to your sawdust, you may simply pasteurize the sawdust, saving fuel costs and enabling inoculation to occur outside the transfer space. Pasteurized sawdust should be

given a higher inoculation rate (closer to 20%) to compensate for the lack of readily available nutrients (especially nitrogen) in the wood. Once mycelaited, pasteurized sawdust spawn is not used in sterile work, but can be applied outdoors.

Pasteurizing means that your substrate is only heated to 140-170°F for an hour, as opposed to the high (250°F) sterilizing temperatures obtained by pressure cooking. Pasteurization kills the mesophilic organisms that normally Uve in the 68-113°F temperature range. Pasteurization kills many immediate competitors that may either overwhelm your mycelium or slow down their growth while leaving aUve beneficial mi croorganisms (mainly bacteria) that help guard the substrate against other contaminants, such as molds. Some bacteria and molds still survive however, hence the high inoculation rate to ensure rapid mycelia- tion. Further, pasteurized substrates should be inoculated as quickly as possible to avoid "spoilage."

P a s t e u r i z i n g

S a w d u s t




Appropriate sawdust type







Heat tolerant bags


P o t

o f

w a t e r



• T h e r m o m e t e r


1. Set up your wire mesh unit and lay a 2-6" layer of sawdust on top of it.

2. Alternating between sprinkling water on the sawdust and mixing, obtain a rough field capacity


m o i s t u r e


c o n t e n t


f o r



t h e to

s a w d u s t .





4. If the sawdust feels too dry at this point, add most water. If too wet, add more dry sawdust.

5. Once the proper moisture content is achieved, load the sawdust into heat tolerant bags and wipe

down the top of the bag's interior with a clean cloth. Roll down and tie off the tops of the bags.

6. Place the bags in a pot of water with a weight on top and achieve an internal temperature of 140- 160°F for 1 hour. This is best done by turning off the water once the temp reaches 120°F. It will continue to climb and should stabilize in the correct range.

7. After an hour, remove the bags and allow then to cool in a clean environment.

8. Spawn away!



For larger quantities of bags, a steaming unit can be used to treat larger volumes. One may also set up a

small electric steam filled chamber to achieve pasteurization. The benefits of steam pasteurizing over hot



















Also, this setup can easily be automated with a timer. Simple designs rig up power steamers (such as the 705 Model Wagner Power Steamer) through heat tolerant PVC (CPVC) or copper pipes into a cooler, repurposed deep freezer, or insulated tote. A remote thermometer allows for monitoring of substrate temperatures without opening the unit.




Pasteurized sawdust can be inoculated with myceliated grains outside of the transfer space. This is be

cause the sawdust is not sterile and thus not a blank slate. Plain sawdust also does not provide the readily

available nitrogen that competitors need as the nitrogen is locked up in the lignin of the wood and the