MUSHROOM LAB 101

Rod Stafford August 2014

WHAT CAN YOU GROW AT HOME?

• Start with something easy ★ any oyster variety (white, grey, blue, yellow, pink,

brown) ★ shiitake (albinos) ★ reishi (antlers and conks) ★ black poplar (exotic?) ★ turkey tail (rosette forms) ★ Panellus stipticus (bioluminescent) ★ hen of the woods, chicken of the woods,

lion’s mane, bear’s head take a little more time

• Inoculate dowels for log cultivation

GOALS

1. General Cultivation Overview 2. Making a Glove Box (“Old-Tech”) 3. Working with Agar 4. Producing Grain Spawn

NOT COVERED

• Spawn Techniques (other than grain)

• Commercial, Large-Scale or Bulk Growing

• H2O2 Techniques

• Laminar Flow Technique

• Strain Isolation

• Anything Illegal

CHEATING MOTHER NATURE

For mushrooms to grow free from

competition, provide an

environment as sterile as a

semi-conductor manufacturing

factory.

Even though a mushroom may produce millions of spores, complex interactions among hundreds of other fungi, bacteria, etc. in nature maintain an ecological equilibrium.

Source: Growing Gourmet and Medicinal Mushrooms, Stamets

1. Spores 2. Mycelium

A. Buy i. Syringe ii. Agar Plate

B. Library i. Agar Slant/Plate ii. Cardboard Pellets

3. Living Tissue

SOURCE MATERIAL

PART’S LIST TO MAKE ROD’S GLOVE BOX

• A transparent storage bin (Walmart, Target, etc) with a lid, 24”L x 16”W x 12”H

• 2 fluorescent light fixtures 120V (IKEA, etc.) • velcro tape (Hancock Fabrics, Michaels, etc) • 254 nm uv germicidal bulb, 8 Watt G8T5 (11.3”)

(uvclamps.com, atlantalightbulbs.com, etc) ($6.39) • Hole saw (Harbor Freight, etc)

• or Drill bit and hack saw (any hardware store) • Disposable Tyvek suit (Amazon, Lowes, etc.)

Total cost about $25 - $75

SAFETY ISSUES• Never expose bare skin under a germicidal lamp • Do not look at a germicidal lamp without protection • Do not use an open flame inside a glove box

ANOTHER OPTIONmakezine.com/projects/home-mycology-lab/

GLOVE BOX WORK ACCESSORIES• Disposable Gloves • Isopropyl alcohol • Disinfectant spray/wipes • Parafilm*

ITEMS DOWN THE ROAD• Scalpel (handle and blades) • Inoculant Loop • Filter Disks for mason jars • Micropore Tape • Self-Healing Injection Ports • Empty syringes (BD Luer-Lok) w/ needles (16G)

PARTS LIST

Working with Agar

Agar is an extract from a seaweed used to solidify media

• Functions as a scaffolding, it has no nutritive value • Allows you to observe growth in two dimensions

A petri dish is a shallow cylindrical glass or plastic lidded dish that biologists use to culture cells

Petri Dishes

• Disposable plastic • 100 x 15 mm • Gamma sterilized • Solid vs segmented

My preferred specs for mushroom culturing:

WORKING WITH AGARMake your own

Pros • Make as many as you need • Cheaper per plate

Purchase pre-made

Cons • Requires sterile environment • More prone to contamination

Pros • Convenience • Uniform, sterile plates

Cons • More expensive • Usually in batches of 10-20

WORKING WITH AGAR

Agar Sources (there are many more…) • mycosupply.com • Amazon • EBay • fungiperfecti.com • sciencebobstore.com/products/Nutrient-Agar-Powder.html

WORKING WITH AGARTypes of Agar Formulations

General Purpose Fungi • Potato Dextrose (PDA) • Malt Extract (MEA) • Cornmeal • 1% Peptone Agar • Dog Food/Rabbit Food/Ovaltine….etc.

Yeasts and Molds• Brain Heart Infusion Agar (BHIA) with 5% Sheep Blood

• Czapek Dox Agar (for Penicillium) • Sabouraud's Dextrose (dermatophytes) • Tryptic Soy Broth (for bacteria, not fungi!) • many others…..

Specialty

WORKING WITH AGARAgar Notes

I use a 500ml glass liquor bottle with a plastic cap, drill a hole in the cap and use a square of Tyvek

★ 25 grams of agar will make ~ 30 plates

★ Buy pre-mixed agar to save time and effort!

★ Sterilize MEA for 35 minutes at 15 psi!

★ Provide gas exchange during sterilization!

WORKING WITH AGARTo make your own plates you will need:A. Sterile Petri Dishes B. Nutrient Agar C. Distilled Water D. Pressure Cooker E. Bottle to sterilize solution F. Graduated Cylinder and/or Scale G. Piece of Tyvek or cut a filter disc H. Aluminum foil I. Glove Box and GB goodies

breathable foam plugs

WORKING WITH AGAR1. Dissolve agar in water at a ratio of 1:20

• 25 g MEA in 500 ml H2O for ~ 20-30 plates • 5 g MEA in 100 ml H2O for ~ 6 plates

2. Cover flask with aluminum foil and sterilize at 15 P.S.I for 15 minutes in pressure cooker (avoid caramelizing sugars)

3. Remove from PC once pressure normalizes 4. Allow to cool to ~ 150°F and place in glove box 5. In glove box, stack plates 3-6/stack 6. Working from bottom to top, pour plates ~ 1/3 full 7. Allow to cool while stacked

GRAIN SPAWNRye Berries, aka winter wheat grain• larger kernel size helps prevent clumping

• commonly available

• low in bacterial endospores

GRAIN SPAWN PREPARATION

• add CaCO3 and Ca2SO4 • soak raw grains overnight - forces

germination of endospores • bring to boil for 10 minutes

GRAIN SPAWN PREPARATION

• drain in colander

• use steam from cooking to dry berries

GRAIN SPAWN PREPARATION

• fill quart canning jars 1/3 to 1/2 full with grain

GRAIN SPAWN PREPARATION

• use filter disks or tyvek squares under lids

• cover with aluminum foil

GRAIN SPAWN PREPARATION

• sterilize for 90 minutes at 15 p.s.i.

GRAIN SPAWN PREPARATION

• allow to cool COMPLETELY before using, store covered until use

AGAR TO GRAIN TRANSFER• Place all the grain jars in your GB along with the agar

culture • Loosen the cap from each jar but don’t remove them • Flame sterilize your scalpel and place it in the glove

box to cool ★ or use a pre-sterilized blade inside the GB

• Open the culture dish and cut a small wedge of agar • Spear the wedge with the scalpel blade, replace the

culture lid, lift a lid from a grain jar and drop the wedge inside without touching anything

• repeat with 1 or 2 more wedges before moving to next jar

SUCCESS

MAKING DOWELS IS EASY

RESOURCES• makezine.com/projects/home-mycology-lab/ • www.mykoweb.com/articles/cultivation.html • shroomery.org

WEBSITES

SPORES/MYCELIUM/SPAWN• ebay (Caveat emptor) • sporeworks.com • fungiperfecti.com • plantpath.psu.edu/facilities/mushroom/cultures-spawn

(very pricey, but they have EVERYTHING)

SUPPLIES• www.mycosupply.com • www.cynmar.com • www.mycopath.com/