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Stage 2: Grain Spawn

Once you have established some healthy, vigorous, and competitor-free inoculum, the next step is toward cooked, sterilized grains. As most species will not fruit on pure grains, this is an inter- mediary step for fruit body production. However, it is recommended as grains provide a cheap, nutrient-dense substrate that the mushroom mycelium can rapidly grow on, ultimately producing a granular spawn that can evenly and easily inoculate the substrates used in Stage 3. A variety of grains can be used in Stage 2. 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 contamination 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 in Stage 3.

which provides for more points of inoculation in Stage 3. 8 Preparation of Grains To produce

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Preparation of Grains

To produce the highest quality spawn, grains must be properly prepared prior to sterilization. Regardless of the grain being used, these are some guiding principles to keep in mind during your grain prep:

• The grains should not be too hard. When you bite into a grain it should be a little bit undercooked (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 mycelium 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, or overly wet grains make grains more prone to contamination 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 or later be broken up during Stage 3 spawning. Pre-rinsing grains and adding gypsum helps to reduce this stickiness.

The following basic recipe for grain prep helps address these three concerns.

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 grains.

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

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Grains can be fermented fol- lowing the practices outlined for bulk substrates earlier and used as a substrate for Oyster (Pleurotus) species. However, as these grains are very wet, mushy, sticky, and acidic they are not a viable substrate for most other species.

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 coffee to add additional nitrogen.

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 incorporating 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 (described in Stage 3) to speed cooling.

9. Add 1 tablespoon of gypsum evenly throughout the grains. Gypsum provides min- eral supplementation while also helping to reduce the stickiness of the grains. Some cultivators add 1 teaspoon of hydrated lime to provide magnesium to the mycelium.

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

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

14. Open the PC and remove the jars. Inspect each jar for cracks 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 collec- tor/pasteurizer. Smaller grains, such as millet, do not need to be cooked as soaking provides ade- quate 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 sterilizing. Brown rice, used for many commercial medicinal products, often turns out very sticky and needs extra attention. After soaking and cooking to the al dente state, 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 substrate from Stage 3 can be added to grain jars prior to sterilization to help initiate the enzymatic expression that the mushroom will ultimately require to consume the Stage 3 substrate. For wood-lovers, I tend to add 10% of properly hydrated sawdust to grains before sterilizing.

(Left) Grains can be quickly cooled and drained on a mix- ing screen.

(Right) When cooking grains for mushrooms, cook some mushrooms for yourself. And if you have one, drink a home- brew, too.

for mushrooms, cook some mushrooms for yourself. And if you have one, drink a home- brew,

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Inoculating Grains With Myceliated Agar Once your grains are sterilized and cooled, they can be

Inoculating Grains With Myceliated Agar

Once your grains are sterilized and cooled, they can be inoculated. Traditionally, myceliated agar has been the main inoculum for grains where, in a similar manner to a Plate-to-Plate Transfer, a wedge of myceliated agar is moved to a sterilized jar of grains.

Materials

Cooked, sterilized, and cooled jar of grains

Myceliated petri dish

Scalpel or spatula

Tool sterilization materials

Method

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 with a heat source 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 on to 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 recovers from the shock of being transferred. It also provides the fungus with easy access to the grains.

10. Wrap the plate. Label the jar with species/strain and date and set it to incubate.

If inoculating multiple jars at once, loosen all the jar lids in advance. The mycelium on the agar should be cut into even sections that each contain some amount of leading edge mycelium. Going down the line of jars, quickly inoculate each jar with a piece of myceliated agar. If the transfer tool touches anything between transfers, sterilize it again. Under ideal conditions, one standard plate can inoculate up to 10 quart-sized jars of grain. However, the high inoculation rate obtained from spreading one plate to only 3–6 jars will increase myceliation while also reducing the time frame that competitors have to invade.

will increase myceliation while also reducing the time frame that competitors have to invade. Working With
will increase myceliation while also reducing the time frame that competitors have to invade. Working With
will increase myceliation while also reducing the time frame that competitors have to invade. Working With

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Measuring Moisture Content

While testing grains by biting into them is a great means for checking the state of a cooked grain, the exact water content can be measured for more pre- cision. Properly cooked grains should have a moisture con- tent of 55–65%. To test this, weigh out 100 grams of pre- pared grains and then place them on a baking tray in an oven heated to 350ºF (177ºC). Bake the grains for 20 minutes or until they are completely dry. Weigh the dried grains. The difference in weight will correspond to the moisture content that the cooked grains held. For example, if the grains come out to be 35 grams, then 65 grams of water had been lost in the oven. This means that the method used for pre- paring the grains yielded a 65% moisture content.

Inoculating Grains With Liquid Culture
Inoculating Grains
With Liquid Culture

Inoculating grains with liquid culture is quick and easy when com- pared to the stress and sterility required for agar inoculations. This technique is similar to that used for Liquid-to-Liquid Transfers.

Materials

1 myceliated LC jar

1 sterilized grain jar with airport lid

1 sterile 60 mL syringe with a 16-gauge Luer-Lok needle

Alcohol and sanitizing materials

Method

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

2. Insert the syringe needle into the LC jar and extract roughly 2–10 milliliters 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 gently, 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 liquid culture jar.

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

times I enter the liquid culture jar. 5. Label each inoculated grain jar and set them

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Scaling Up

The above processes can easily be expanded to larger quantities of grains and larger vessels. Grain prep times may need to be adjusted to properly hydrate larger volumes of grains. Pressure cooking times should also be increased accordingly to ensure complete heating throughout the substrate. Half-gallon jars should be cooked for 90 minutes and one-gallon jars and filter patch bags are cooked for 2 hours. A larger amount of inoculum will also be necessary for these larger quantities of grains. One gallon of grains can be (heavily) inoculated with 120 milliliters of liquid culture inoculum or several large pieces of myceliated agar.

Break, Shake, and 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% myceliation. Shaking at 90%+ myceliation often stunts the mycelium’s growth, neg- atively impacting the success of later expansions.

neg- atively impacting the success of later expansions. Grain-to-Grain Transfers Grain spawn can be expanded to

Grain-to-Grain Transfers

Grain spawn can be expanded to more sterilized grains before being moved on to Stage 3. Taking this extra step to expand your mycelial mileage helps reduce the need for constant agar work. However, compared to the open-air ease of inoculating grain with liquid culture, I do not do many Grain-to-Grain Transfers as they require an aseptic transfer space. But your needs may vary.

Materials

1 jar of healthy myceliated grain spawn that is not overgrown

Multiple jars of cooked, sterilized, and cooled grains

Method

jars of cooked, sterilized, and cooled grains Method (Above, top) A bulk bag of grain spawn
jars of cooked, sterilized, and cooled grains Method (Above, top) A bulk bag of grain spawn

(Above, top) A bulk bag of grain spawn being myceliated by an agar wedge.

(Above) Elm Oyster millet grain spawn. This bag was inoculated two weeks earlier with 120 milliliters of liquid culture.

(Left) Resihi grain spawn be- fore and after shaking. The spawn on the left is at an op- timal state for transferring to a fruiting substrate. The grains are fully myceliated but the individual kernels are more or less visible and the mycelial mat hasn’t turned into a hard mass.

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1. Under aseptic conditions in your transfer space, loosen (but don’t fully open) all jar lids and arrange them for easy access during transfer.

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

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8 If left in a container for an ex- tended period, mushroom my- celium often produces
8 If left in a container for an ex- tended period, mushroom my- celium often produces

If left in a container for an ex- tended period, mushroom my- celium often produces a bath of digestive enzymes, antibiot- ics, and waste products.

3. Quickly and carefully open the lid of the first jar just enough to introduce 10–20% of the myceliated 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 and set the jars to incubate.

Grain-to-Grain Transfers may be done twice (creating three generations) for most strains. Pushing for a fourth grain generation is not recommended as the mycelium will likely lose vigor after consuming so much of the same substance. Most growers prefer to only expand once (Gen. 2).

substance. Most growers prefer to only expand once (Gen. 2). Liquid Grain Spawn to Grains Some

Liquid Grain Spawn to Grains

Some cultivators use an autoclavable Erbach blender to pulp grain spawn with water, creating a form of liquid inoculum. Alternately, many standard blender bottoms fit on a canning jar and can be autoclaved to achieve the same effect. This liquefied grain spawn can then be used to inoculate more grains or the substrates used in Stage 3. Compared to the ease of the liquid inoculum pro- duced in Stage 1, this approach is less appealing to me personally. But some cultivators stand by this approach due to the high density of mycelium and nutrient load it affords.

When is Grain spaWn ready for staGe 3?

Learning to spot when grains are ready to move onto another substrate is critical for successful cultivation. If grains are overgrown, the mycelium may become stunted in its growth or begin to initiate fruiting. Overgrown grains also tend to form a very dense mycelial mat, making it difficult, if not impossible, to break them up by shaking. Myceliated grains should be moved to their next substrate as soon as every kernel is covered in a visible coat of mycelium. After shaking the grains at around 30% myceliation, watch the mycelium distribute throughout the jar. Once all the visible grains are myceliated and no bare grain surface is visible, it can be assumed that the grain spawn is ready to move.

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