MUSHROOM LAB 101. Rod Stafford. August 2014

Transcription

1 MUSHROOM LAB 101 Rod Stafford August 2014

2 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

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

4 NOT COVERED Spawn Techniques (other than grain) Commercial, Large-Scale or Bulk Growing H2O2 Techniques Laminar Flow Technique Strain Isolation Anything Illegal

5 CHEATING MOTHER NATURE 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 For mushrooms to grow free from competition, provide an environment as sterile as a semi-conductor manufacturing factory.

6 SOURCE MATERIAL 1. Spores 2. Mycelium A. Buy i. Syringe ii. Agar Plate B. Library i. Agar Slant/Plate ii. Cardboard Pellets 3. Living Tissue

7 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

8 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

9 ANOTHER OPTION makezine.com/projects/home-mycology-lab/

10 PARTS LIST 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)

11 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

12 Petri Dishes A petri dish is a shallow cylindrical glass or plastic lidded dish that biologists use to culture cells My preferred specs for mushroom culturing: Disposable plastic 100 x 15 mm Gamma sterilized Solid vs segmented

13 WORKING WITH AGAR Make your own Pros Make as many as you need Cheaper per plate Cons Requires sterile environment More prone to contamination Purchase pre-made Pros Convenience Uniform, sterile plates Cons More expensive Usually in batches of 10-20

14 WORKING WITH AGAR Agar Sources (there are many more ) mycosupply.com Amazon EBay fungiperfecti.com sciencebobstore.com/products/nutrient-agar-powder.html

15 WORKING WITH AGAR Types 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 Specialty Czapek Dox Agar (for Penicillium) Sabouraud's Dextrose (dermatophytes) Tryptic Soy Broth (for bacteria, not fungi!) many others..

16 WORKING WITH AGAR Agar Notes 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! I use a 500ml glass liquor bottle with a plastic cap, drill a hole in the cap and use a square of Tyvek

17 WORKING WITH AGAR To 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

18 WORKING WITH AGAR 1. Dissolve agar in water at a ratio of 1:20 25 g MEA in 500 ml H2O for ~ 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

19 GRAIN SPAWN Rye Berries, aka winter wheat grain larger kernel size helps prevent clumping commonly available low in bacterial endospores

20 GRAIN SPAWN PREPARATION add CaCO3 and Ca2SO4 soak raw grains overnight - forces germination of endospores bring to boil for 10 minutes

21 GRAIN SPAWN PREPARATION drain in colander use steam from cooking to dry berries

22 GRAIN SPAWN PREPARATION fill quart canning jars 1/3 to 1/2 full with grain

23 GRAIN SPAWN PREPARATION use filter disks or tyvek squares under lids cover with aluminum foil

24 GRAIN SPAWN PREPARATION sterilize for 90 minutes at 15 p.s.i.

25 GRAIN SPAWN PREPARATION allow to cool COMPLETELY before using, store covered until use

26 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

27 SUCCESS

28 MAKING DOWELS IS EASY

29 RESOURCES WEBSITES makezine.com/projects/home-mycology-lab/ shroomery.org SPORES/MYCELIUM/SPAWN ebay (Caveat emptor) sporeworks.com fungiperfecti.com plantpath.psu.edu/facilities/mushroom/cultures-spawn (very pricey, but they have EVERYTHING) SUPPLIES