ArkFab Mushroom Cultivation Protocol

for the Green Phoenix Initiative C.R. Armbruster and L.E.D. Rattray
Objective 1: Isolate a pure culture of an environmental mushroom specimen onto an agar medium. Materials: Lab: Autoclave Incubator (ex: for P. ostreatus mycelia, 25oC at 85-95% relative humidity, in dark, for 7-14 days) Refrigerator at 4oC HEPA filter Laminar flow hood Processing: 50mL Falcon tube or sandwich bag (for sample collection from environment) 70% and 95% EtOH 10% bleach Alcohol wipes Sterile forceps (2) Gloves MEA or PDA agar plates (poured thick; 1L 1.5-2 sleeves) Empty, sterile petri dish (1/mushroom) Parafilm Permanent marker Method: 1.1) Collect a fresh specimen from the environment. Store mushroom in a sterile 50mL Falcon tube (or sandwich bag) at 4oC until ready to culture, no longer than 24 hours. 1.2) Run the HEPA filter in a small lab space or hood for 20-60 minutes. Wipe all surfaces with 10% bleach or 70%EtOH. 1.3) Wipe outside of mushroom sample with an alcohol wipe to reduce the likelihood of contamination. Place the mushroom in a sterile, empty petri dish. 1.4) Using two sterile forceps or clean, gloved hands, cleanly break the cap of the mushroom in half. 1.5) Dip scalpel in 95% EtOH and flame sterilize. Cool the scalpel by stabbing into the center of the petri dish. This will melt a small hole in the agar. 1.6) Cut a ~1.2cm3 piece of flesh from the interior of the cap of the mushroom. To reduce contamination, avoid touching any external surface of the mushroom. Isolate only the interior tissue of the mushroom cap. 1.7) Using flame sterilized forceps, transfer the mushroom tissue sample into the center of the petri dish, on top of the melted agar area. Partially embed the tissue sample into the melted agar. 1.8) Label the plate with isolate ID, date, and initials. Invert the plate and incubate at the appropriate temperature and humidity for the specimen. Isolate multiple tissue cultures per specimen to overcome loss from contamination. Optional: Wrap each plate in one layer of parafilm to reduce contamination. 1.9) Check petri dish periodically for contamination. If there appears to be anything but a pure culture, discard the plate. Optional: To increase likelihood of isolating a pure culture, aseptically remove a sample of the mycelia early in the incubation period and transfer to a new petri dish using same method. Choose section of mycelia that is running well. To create a culture library: Transfer a section of mycelia into a slant of the same agar. After incubation, slants may be stored at 4 oC. Long term storage of mycelia from the mother culture can be achieved in liquid nitrogen and/or paraffin sealing. Last modified: 12 July 2011 by C.R. Armbruster teach you what Liam taught me Page 1 of 5

ArkFab Mushroom Cultivation Protocol

for the Green Phoenix Initiative C.R. Armbruster and L.E.D. Rattray
Objective 2: Produce spawn starter for downstream use to inoculate bulk fruiting substrates, or to make more spawn (G2G transfer). A spawn starter should be inoculated with mycelia cultured from an environmental mushroom sample or a stored slant culture of mycelia from tissue culture, rather than mycelium whose parent was a spore. Each spore is likely to yield a new strain; therefore its performance would be unpredictable. Not all mycelia fruit. Materials: Lab: Autoclave (ex: for rye grain spawn: 30 minutes, 95-120C, ~15 psi, Fast Exhaust) Incubator (ex: for P. ostreatus spawn, 25oC at 85-95% relative humidity, in dark, for 7-14 days) HEPA filter Laminar flow hood RO water system Rye jars: Mason jars (780mL size) 0.3 mircron autoclavable synthetic filter discs, 9mm diameter Dried rye grain (rye berries; 250mL/ jar) RO water (500mL/ jar) Calcium carbonate (lime; 2g/ jar) Calcium sulfate (gypsum; 1g/ jar) Hot plate Drill Processing: 70% and 95% EtOH 10% bleach Alcohol wipes Sterile forceps (2) Sterile Hemostats or Fork Gloves Permanent Marker Method: 2.1) Begin preparing rye grain jars at least 12 hours before inoculating with mycelia. Combine dried rye grain and RO water 1:2 volume. Heat over a hot plate until boiling for 5 minutes. Do not overcook. The grain should remain intact (i.e. not split). 2.2) Drain and rinse the cooked rye grain with RO water. Resuspend rinsed rye grain in boiling RO water 1:2 volume. Allow the rye grain to soak overnight. Note: Cooking and soaking overnight is not required, since autoclaving will cook the grain. However, cooking and soaking in advance may induce any heat-resistant endospores notoriously present in rye grain to germinate before being exposed to heat in the autoclave. 2.3) Modify mason jars for use as spawn jars. 2.3.1) Drill a 2cm diameter hole in the center of the mason jars metal lid. The hole will allow gas exchange to occur during incubation. The hole may also be used if a syringe inoculation technique is employed. 2.3.2) Place the metal lid onto the mason jar. 2.3.3) Place a synthetic filter disk on top of the metal lid. Last modified: 12 July 2011 by C.R. Armbruster teach you what Liam taught me Page 2 of 5

ArkFab Mushroom Cultivation Protocol

for the Green Phoenix Initiative C.R. Armbruster and L.E.D. Rattray
2.3.4) Tighten the metal ring around the disk and metal lid. 2.4) After approximately 8-12 hours of soaking, fill mason jars with approximately 500-700mL of rye grain. 2.5) To each jar, add 2g of calcium carbonate and 1g of gypsum. Calcium carbonate (lime) will help buffer the pH. As the mycelia grow, they secrete metabolic products that reduce the pH of jar. Calcium sulfate (gypsum) reduces clumping of the rye grains. 2.6) Shake the jars to distribute the lime and gypsum. 2.7) Close the jars as described in steps 2.3.2 2.3.4. 2.8) Cover the top half of each jar in aluminum foil to separate the outer surface of the jar near the opening from environmental contaminants after autoclaving. Put a piece of autoclave tape on the aluminum foil on the top of each jar. 2.9) Autoclave the jars for 30 - 60 minutes at 95-120C and ~15 psi, with fast exhaust. Allow the jars to cool completely (~2 hours) before inoclulating. To inoculate the rye jar with mycelia isolated from a petri dish (Alternative: Syringe method, not described) 2.10) In a clean environment as described in step 1.2, remove the aluminum foil and loosen the lid of an autoclaved rye grain jar. Do not remove the lid. 2.11) Remove any parafilm from a petri dish that is fully covered with a pure culture of the mushroom specimens mycelia. 2.12) Using a sterile scalpel, divide the agar and mycelia into 6-8 sections by cutting a grid. 2.13) Using sterile technique, transfer 3-4 sections of mycelia into each rye grain jar with sterile forceps. Flame sterilize the forceps between jars. 2.14) Shake the jars to distribute the mycelia sections throughout the rye grain. 2.15) Replace jar lids as described in steps 2.3.2 2.3.4. From this point forward, when opening jars, wipe all glass and metal surfaces of the jar with 70% EtOH, allow EtOH to evaporate, then quickly flame sterilize around the metal ring. When closing jars, quickly flame sterilize around the glass mouth of the jar and replace the lid. 2.16) Allow the inoculated mason jars to incubate at room temperature for ~3-4 weeks. Beginning a few days after mycelia becomes visible, vigorously shake jars every 2-3 days until the entire jar is colonized with white or off-white mycelia. Shaking will distribute the mycelia and help avoid anaerobic fermentation by any potential contaminants. Contamination is anything black, blue, dark grey, green, or slimey. Spawn jars are ready for use inoculating bulk fruiting substrates once the jar is completely white with mycelia. Optional: Grain to Grain transfer (G2G)- Spawn from a fully colonized jar can be used to inoculate a new rye grain jar by aseptically transferring an aliquot of mycelia spawn into the new jar as described in steps 2.13 1.25. G2G should be performed no more than 3 times per mother spawn strain, if at all.

Last modified: 12 July 2011 by C.R. Armbruster teach you what Liam taught me

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ArkFab Mushroom Cultivation Protocol

for the Green Phoenix Initiative C.R. Armbruster and L.E.D. Rattray
Objective 3: Colonization of bulk fruiting substrates to produce edible or medicinal mushrooms. Example: Pleurotus ostreatus (oyster mushrooms). Oyster mushrooms are typically grown on an autoclaved straw substrate/compost that has been inoculated with mycelia from spawn starter jars (Objective 2), at an optimal pH of 5.0 for fruiting. The inoculated fruiting substrate can be combined with a sterilized casing material to retain moisture over time, as the mycelia runs through the substrate and eventually fruits. Vermiculite and peat moss are common casing materials, often used in combination. Vermiculite is extremely efficient at retaining moisture but contains no additional nutrients, whereas peat moss both contains water and offers nutrients for continued growth of the mycelia. The casing and fruiting substrates are layered in the fruiting chamber (or garden bed) as follows: Bottom: a 5cm layer of autoclaved casing material. Middle: The fully colonized spawn starter (rye grain jar) is broken up and mixed with a sterile fruiting substrate, like straw. This mixture is evenly distributed over the surface of the bottom layer. Top: another 5cm layer of autoclaved casing material covering the entire surface. For Liams garden bed Pleurotus ostreatus fruiting on straw: http://arkfab.org/?p=232 Table 1. Bulk fruiting substrate materials required for oyster mushroom cultivation (Modified from: Oyster mushroom-cultivation technology and management. Cha et al., 1997) Nutrients Materials C-source cellulose humus materials: wood, straw, leaf, etc. Organic hemicellulose " N-source protein " amino nitrogen " K, P, Si, Fe, Mg, etc. Inorganic Table 2. P. ostreatus Environmental Conditions for Fruiting (Source: Growing Gourmet and Medicinal Mushrooms Stamets, 1993) Spawn Run Primordial Formation Fruitbody Development Incubation Temperature (oC) 24 10-15.6 10-21 Relative Humidity (%) 85-95 95-100 85-90 Duration (days) 12-21 3-5 4-7 CO2 (ppm) 5,000-20,000 <1000 <1000 Fresh Air Exchanges (per hour) 1 4-8 4-8 Light Requirements (lux) n/a 100-1,500 1000-1500

Cropping Cycle 3-4 crops, 7-14 days apart, over 45-55 days.

(Source: Growing Gourmet and Medicinal Mushrooms. Stamets, 1993)

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Last modified: 12 July 2011 by C.R. Armbruster teach you what Liam taught me

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ArkFab Mushroom Cultivation Protocol

for the Green Phoenix Initiative C.R. Armbruster and L.E.D. Rattray
Appendix 1 Agar Recipes

Dog Food Agar (500mL 1 sleeve of plates, poured thick) 10g dry dog food, ground 10g agar 50mL RO water Autoclave: 95oC, ~15 psi, for 30 minutes. Slow exhaust.

LC Media (for Liquid Culture) 500mL 5g Malt Extract 500mL tap water or RO 1 glass bead Autoclave: 95oC, ~15 psi, for 30 minutes. Slow exhaust.

Malt Extract (Yeast) Agar (ME(Y)A) (500mL 1 sleeve of plates, poured thick) 10g agar 1g yeast (MEYA) 10g malt extract 500mL RO water Autoclave: 95oC, ~15 psi, for 30 minutes. Slow exhaust.

Potato Dextrose (Yeast-extract) Agar (PD(Y)A) (500mL 1 sleeve of plates, poured thick) Potato broth* or 5g instant potato flakes 9g agar 7g dextrose (or 10mL honey/corn syrup) 1g brewers yeast (PDYA) 500mL RO water Potato broth: Boil 150g sliced potatoes in 500mL RO for 30min Autoclave: 95oC, ~15 psi, for 30 minutes. Slow exhaust.

Last modified: 12 July 2011 by C.R. Armbruster teach you what Liam taught me

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