The purpose of this post is to help me put some of the outstanding info here into a single procedure. Hopefully this will help someone out there.
This is 100% not original. After hours of searching, I compiled a procedure that borrows from so many great teks here that incorporates isolation, grain, LC use, and casing methods. I use coir, but if you have poo that's just as well. I just wanted to put together what I've learned from RR, Roadkill, Magash, Hyphae, creamcorn, Agar, Helltick, and everyone else here that has kickass methods into a plan that uses the best practices from all aspects of this hobby. It's not perfect, and I've omitted some details that I think are obvious, and I may have obviously left out important details...so here goes.
And it starts with step 1:
Making a glove box
Tek & pic from Roadkill
Use a large, clear tub. Use a plexiglass lid to enhance visibility. Cut holes large enough to fit forearms halfway through – silicone in plastic plumbing fittings.
Use 91% iso alcohol as it evaporates much faster than 70%. Insert items to work with, wipe down the interior w/iso and spray w/oust and cover before use. Wait 10 minutes for the iso to kill everything. Use paper towels to plug arm holes when not in use.
Creating Agar Plates
Materials:
Coffee grinder Agar Agar (asian aisle or shop, 9oz package should make 1/4 c) Dehydrated potato flakes Honey Long neck glass bottle Petri dishes (you can use jars as Helltick does in his Grocery store agar tek, same procedure) Glove box Pressure cooker
Procedure:
Recipe from Helltick
Grind agar and add ¼ cup to mixing bowl Add 2 tbsp. potato flakes Add 1 cup water and mix thoroughly Add 1 tsp of honey and mix thoroughly
If you prefer to buy powdered PDA or MDA agar, hydrate it per the instructions. Funnel into glass bottle Drill breather hole in bottle cap and place tyvek over mouth of bottle, then replace cap. This prevents the bottle from exploding during the PC and will keep contams out until pouring time. Cover cap in foil to prevent water from dripping on tyvek. Pressure cook at 15psi for 20 min. Allow PC to cool but do not let agar solidify. Blowing off steam from PC will cause agar to overflow. Be sure to keep heat as low as possible @15psi to prevent steam escaping, which will cause agar to boil and possibly overflow. Place everything in sterile glove box and pour into your Petri dishes (plates).
Technique from RR:
First of all, allow the PC to cool for about an hour and a half so that you can handle the bottle of agar with bare(latex gloves) hands without burning yourself.
Get rid of the bulky rubber gloves and simply cut holes in your glovebox big enough to stick your arms through and be snug around your upper wrists. Wear latex gloves. This will give you freedom of movement, and the much cooler agar will prevent condensation on your petri dishes as well as the sides of your glovebox.
Use a whiskey or other liquor bottle to prepare the agar as it's easy to pour from. The longer the neck the better.
Divide your sleeve of 20 dishes into two stacks of ten. On the first pour, lift the top nine dishes and all ten lids with your left hand and pour the bottom dish with your right, assuming you're right handed. Next, lift the top eight dishes and nine lids and pour the second dish, etc., until the stack is poured, and then pour the second stack. Leave them in two stacks of ten until solidified. When you pull your hands out of the holes, have something ready to stuff them with to prevent drafts, or cover with paper taped into place, etc.
When the dishes solidify, place back into the plastic sleeve. RR
Inoculating Agar and isolating rhizomorphic substrains
Materials:
Spore syringe Sterile agar dishes 91% Iso Alcohol in spray bottle and in regular bottle oust air sanitizer Exacto knife Painter’s tape for labeling latex gloves cotton balls Glad cling wrap or parafilm
Procedure:
Flame sterilize needle before placing into glove box – flame inside glove box w/iso alcohol will cause an explosion Place all items in glove box and sterilize w/iso and oust Shake syringe, alcohol wipe needle, and inoculate 1 drop into the center of an agar plate. Wrap plate with cling wrap or parafilm to keep contams out but allow gas exchange Label this plate with date, strain, and “multi innoc” with tape Incubate plate at room temp (75 deg F) until growth appears (3-5 days to germinate). Look for the rhizomorphic shoots and cut the fastest and strongest looking sector with exacto and place in the middle of a fresh agar plate. Cut only a small wedge about the size of a grain of rice. Label this jar with date, strain, and i0 (isolate zero). Incubate until more rhizomorphs appear. Select the fastest and strongest rhizomorphs and transfer each to their own agar jar. Label each with date, strain, A0, B0, C0, etc. Now isolates can be identified by substrain letter and generation.
According to RogerRabbit, this plate has about 10 sectors. Pic courtesy of RR
Once more isolate the single fastest and strongest mycelia from each of the parent sectors into new jars and label A1, B1, C1, etc. Continue to isolate until there are no distinguishable sectors, only radial growth. Do not proceed beyond 3rd gen siblings, A3, B3, etc. Transfer an agar wedge from each suitable sibling into liquid culture and save the rest of the plate in the fridge for reanimation.
Inoculating grain with agar wedge or LC
Materials:
Pressure cooker Wide mouth quart jars Tyvek Rye berries (one cup per jar) Gypsum Large colander Drill & bits Glove box Labels Incubator (double tub w/fish tank heater set to 78 deg)
Grain tek from Magash – RYE bitches RYE!!!!
Procedure:
Prepare jars by adding 1 cup of grain to each jar. Fill jars with water to cover grain completely Soak for 24 hours to hatch bacterial endospores During this time drill a ½ inch hole in each lid and cut tyvek to cover lids Pour out water, refill, and re-drain each jar Combine all jars into a large pot, cover with water and simmer for 45 min. Pour rye into colander and rinse until completely clean Add 1/8 cup gypsum to keeps grains from sticking while shaking Use measuring cups to evenly divide rye back into jars. Place lid, tyvek, and screw on lid ring. Cover lid tightly with foil to keep water off of the tyvek. Pressure cook for 1 hour at 15 psi. and then let cool to room temp. Transfer an agar wedge (or LC injection) from each sibling (A2, B2, etc) to a corresponding jar in glove box. Label with date, strain, sibling, and generation. Incubate at 75-78 deg F until 1/4 colonized. Shake grain and return to incubator. Wait for total colonization, shaking at 1/2 colonization if needed.
Fruiting
Materials:
Fruiting chamber (Rubbermaid clear tubs w/lids) Drill & bits Aluminum oven pans (sized to fit 2 to a tub, as deep as possible) Coco coir (pet store, sold in bricks such as bed-a-beast) Cooler or 5 gal. bucket for mixing Gypsum powder (calcium sulfate) – garden or homebrew shop Vermiculite Peat moss Calcium carbonate (lime powder) – home improvement or garden store Spent coffee grounds – starbucks grounds for gardens program pH meter colonized jars empty quart jars tyvek
Procedure:
Prepare substrate mix and casing mix while jars are colonizing.
Coir (substrate) mix - Hydrate coir bricks in 1 gal hot (160 deg) water per brick in the cooler. 1 brick of Bed-a-beast will expand to 8 quarts. Before adding the hot water, add 3 quarts of spent coffee grounds, 1 cup of lime, and 1 pound (or 1 pint) gypsum. Add water to coir and let sit, with lid on, for 1 hour. Use gloves to break up the brick and mix to uniformity. Check for field capacity saturation – squeezing a handful should only release a few drops of water. Load immediately with gloved hands into clean quart jars and cover with loose lid and foil. Use PC to pasteurize (NOT sterilize) jars.
Casing Mix - Mix 2 jars peat, 2 jars verm, 1 cup lime, and ½ cup gypsum in a bucket or cooler. Check pH – add lime until 7.5-8.0 is reached. Add water to field capacity. Load immediately into jars and loosely cap and cover with foil. Use PC to pasteurize jars.
Pasteurizing jars of substrate/casing in a PC
Tek from RR:
It's very easy to use your PC to steam pasteurize in quart jars. Load your field capacity casing material or compost/manure into quart jars and place in the PC. Fill with COLD water 1/2 to 2/3 of the way up the jars. Place the jars in your PC or other large covered pot with COLD water. Insert a probe thermometer in the centermost jar, placing the probe tip in the dead center of the jar. Cover and turn on the stove. Do not place the weight on your pc. Leave the vent open. Try to get the temperature to above 140F for at least an hour, but no longer than two hours, and try not to let the temperature exceed 170F. Colonizing substrate
Place aluminum oven pan(s) in glove box. Load spawn and coir jars into glove box and sterilize pans and box as usual. Allow iso alcohol to evaporate before opening jars.
In the pans, combine 1 part grain spawn (shake before pouring out) to 2 parts coir mix and mix to uniformity with gloved and sterile hands. The depth of this mixture should be 3-5 inches (thicker is better) but leave 1 inch from the top edge of pan.
Bulk Colonization lid tek and pic from Agar:
Tightly cover the pans with plastic wrap. Burn holes (Agar uses a cigarette) in the plastic and place sterile micropore tape over the holes to allow gas exchange but keep out contams. Cover with loose aluminum foil to keep out dust and allow gas exchange.
Bulk dunk tek from RR
Incubate at room temp for 3 days. Peek in glove box and see how colonized it is. When it is fully colonized, dunk by uncovering tray and placing in the sink or bathtub for 1-2 hours. The water flow should be just enough to spill water over all sides – the motion of the water spilling off will take any contaminant spores with it. Use a clean jar full of water on top of the substrate to keep the substrate from floating. The water temp should be cool to discourage bacterial growth. After dunking, drain off all of the water and proceed with casing.
Casing
After dunking, case the substrate with ½-3/4 inches max of the casing mix and place in the fruiting chamber with a cool white fluorescent light shining on the casing at least 4 hours a day – 12-14 is optimal. Use a timer. Temp should be about 70-78 deg F.
Fruiting chamber should have several 1/4 in. holes for air exchange and should contain a hygrometer to measure humidity. Shoot for 95%. High levels of humidity can be attained using soaked and drained perlite in the bottom of your FC, or auto-humidification with a cool mist or ultrasonic on a timer. There are many ideas here to search through.
Shotgun FC pic courtesy of RR
Mark each pan with sibling strain and date placed into fruiting chamber. After the first flush, select the best sibling based on colonization time, even pinset, yield, and potency. Clone the superior fruit from each first flush by obtaining a tissue sample and make a liquid culture from each. Select the best isolate and save the original agar dish for prosperity. Use the liquid culture process to inoculate more grain jars and enjoy fruiting the best isolate.
Liquid culture
Thanks to creamcorn for this tek.
Materials:
Tissue biopsy from prime specimen Mason jar – size is up to you Sterile Syringe Glove box Honey Glass marble (for agitation) or magnetic stir bar if you have a magnetic stirrer Micropore tape DISTILLED Water
Biopsy Method: Pick fruit and place in glove box with PC’d syringe (15 min @ 15 psi). wipe fruit stem with alcohol swab and poke all the way through stem. Replace syringe cap.
Alternate biopsy method from RR:
In a sterile glove box, simply tear a stem lengthwise into two halves, then use a sterile razor or scalpel to scrape a bit of mycelium from the center of the stem that hasn't ever been exposed to the outside air.
Procedure:
Prepare sterile LC while fruits are growing Remove needle from syringe to make loading and dispensing honey easy, heat water to make mixing easy Microwave honey for 1 minute to make it more fluid
Mix 96/4 ratio of distilled water and honey – for a quart jar, use exactly 3.5 cups of water (828 mL) and 34.5 mL (which happens to be exactly 7 level teaspoons) of honey – measure with syringe, teaspoon, or graduated cylinder. This gives you total volume of about .91 quarts, leaving room at the top for tilting and extraction.
Add marble or stir bar to jar Use a nail to poke two needle sized holes on opposite sides of the lid near the edge, cover in tyvek, screw on ring, and cover jar top in foil. Pressure cook for 30 min at 15 psi and allow pc to cool to room temp before opening Place in glove box Take tissue biopsy syringe (cap still on) and place in glove box. If using the scalpel method, perform the biopsy at this point. Remove foil, lid ring, and tyvek. Remove syringe cap, swab needle with iso alcohol, and inject tissue biopsy into jar via one of your holes. If using the scalpel method, quickly lift the lid and deposit the sample into the jar. Place micropore tape over holes in lid – never allow these to get wet. Agitate regularly to distribute until colonized (cloudy) should take 7-10 days
Pic of colonized LC courtesy of blutjager
Refrigerate upon full colonization. Keep this jar and label as your master culture – only use it to colonize more LC.
Propogation
Use your master LC to inoculate a new LC via the above tek. Use this culture to inoculate more grain.
LC transfers
When transferring LC to another LC or grain jar, use your glove box. PC a capped, water-filled and foil wrapped syringe for 15 minutes at 15 psi. Allow your PC to cool before opening. Prior to placing the syringe in the glove box, squirt out the all of water and flame sterilize the needle. Swipe the micropore tape on the side you will be aspirating from with 91% iso alcohol. Also swab the needle with the iso. Puncture the sterile tape with the needle and tilt the jar towards the needle. Do not allow the LC to come into contact with the lid. The hole on the other side lets air in through the micropore tape so you aren’t creating a vaccum. Once you have your LC (aspirate 1 cc per jar you want to inoculate), withdraw your needle, swipe it with iso, and replace the cap. Remove the tape you punctured and replace with a fresh strip.
You can immediately inoculate another LC or a jar of grain with this syringe, or you can place it in a Ziploc bag in your fridge to store.
If you run low on master LC, make another master with another tissue biopsy, or from the agar plate you saved of your best isolate. When you biopsy a good specimen, you can optionally do a 2nd biopsy of that same specimen while you’re in your glove box and grow it on an agar dish so you’ll have a “backup copy”.
This process should enable you to be self sufficient with good results.
Mandelbrah
Edited by mandelbrah (02/09/08 10:25 AM)
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