|By Nan (Nanook) on Tuesday, October 23, 2001 - 06:52 pm:|
Multispore Mycelium Agar Culturing
This is a preliminary draft that I concocted from my ever-dwindling memory. It will be reinforced with more factual detail when I get a chance to reference TMC on spots that I may have missed/screwed up, however it seems to be in somewhat good shape for those of you who are interested in the basic technique and aren't really worried about 100% accurate information, as I said I'll go through and fix it up when I have some free time, it's got all the basics.
The name may sound scary but the concept is simple.
I wanted to make this little guide so people could get into agar culturing. A lot of people seem to want to try it and they are interested in it, but they also seem scared of it at the same time. Fear no more! This is a general procedure that is adaptable to your resources, time and effort that you want to put into it.
All of this information and lots lots more is available in Paul Stamets book "The Mushroom Cultivator". This is a great book that I suggest everyone and anyone who is serious (or curious) about mushroom growing or mycology in general to check out.
Table Of Contents:
2. Preparation and Sterility
3. Agar Preparation
4. Petri Dish / Test Tube Preparation #Petri Dish / Test Tube Preparation
5. Inoculation and Incubation
6. Contamination, Strain Isolation and Slants
6b. Strain Isolation
12 Petri Dishes or 1/2 pt canning jars (Wide-Mouth preferred)
You probably won't use them all but it's a good idea to prepare a bunch at the same time.
Spores (Spore Syringe or Spore Print)
An Inoculation Loop (if using a Spore Print, not needed if you use a syringe)
Scalpel Or Knife
Plastic Wrap or Parafilm
Alcohol Lamp (Butane Lighters are OK)
2 Test tubes or other air-tight cylindrical containers
* Formula for 44g of MYA+ for 1 liter water (As given in Growing Medicinal and Gourmet Mushrooms by Paul Stamets)
20g Fungi Perfecti Agar Agar
20g Light tan high-quality malt sugar
2g nutritional yeast
2g Gold Medal Softasilk superfine flour
Preparation and Sterility
First, prepare a sterile area. This involves find an enclosed area (Small room or closet is great). The area should be draft free and at a constant temperature. Sterilize the room by washing everything down with a 10% bleach/water solution and letting the solution sit for 15 minutes. Go through the room again to remove the bleach/water solution and then spray a liberal coating on everything. Once this is done don't enter the room again until you are ready working or you will possibly be introducing contaminants into the room. Moving air is a great way to introduce contaminants into your process, when moving in your sterile area use methodical movements to minimize air currents. Make sure all fans are off, windows and vents are closed and when working in your sterile area it's a good idea to place a towel under the door to minimize any possible air currents. When you are ready to work, take a shower, brush your teeth (wear a long sleeve shirt, long pants and a hat) when working (this is to eliminate any contaminants that may fall off of you. It is also a good idea to wear some type of mask to filter your breathing.
Mix all of the ingredients listed above *, then add a liter of water to a container big enough to hold everything and something that can be safely pressure cooked and easily poured (A Quart Canning jar works). Once your mixture is made, place into pressure cooker and cook at 15psi for an hour.
Petri Dish / Test Tube Preparation
You will need to gather your pressure cooker, spores, petri dishes and test tubes before you enter the room. Once you enter the room, spray some Lysol in the air towards the ceiling and let it settle.
Lay out all 12 petri dishes on your work area.
Make sure your pressure cooker is SAFE to open (all pressure is released) BEFORE opening it!
Open your pressure cooker and remove the container that is holding the agar. Using quick yet methodical motions remove the top of each petri dish and fill each about 1/2 way with the agar mixture and replace the covers. A better method is to only slightly open the tops of the petri dishes and pour through the opening that is created on the side. The key is that you want to disturb the air as little as possible, so as to minimize the possibility of contamination. You will also want to fill each test tube about 1/4 - 1/3 full. Next wrap the sides of the petri dishes (tops of test tubes) with parafilm or plastic wrap (either is fine).
Once this is done let the agar filled petri dishes / test rubes cool to room temperature. Remember when entering or exiting the sterile area try to disturb the air as little as possible. Lay the test tubes diagonally on something (slanted) so that when the agar dries it is dried slanted as well.
Inoculation and Incubation Inoculation
Once your petri dishes have cooled to room temperature it is time to inoculate them. If you are using a spore print you will want to re-hydrate the spores for 24 hours before continuing, you can do this by placing the spores in a dish of sterile water for 24 hours with plastic wrap covering it (to minimize contamination) leave the dish in your sterile area. If you are using spore syringes make sure to shake them well before continuing Once your spores are ready flame sterilize your needle and squirt some spore solution through to cool it or flame sterilize your inoculation loop. Open the tops of the petri dishes slightly and inject/scrape some spores into the center of the agar plate. Repeat this (sterilizing your needle/loop between dishes) until you have 3 completed petri dishes.
It is not necessary to use a lot of spores, if you can visibly see the spores in the agar you probably already have too much. Spores are tiny, the smallest speck that you can see is hundreds of spores.
It is now necessary to let the spores incubate. Place the agar filled petri dishes that have been inoculated in a dark, warm (86 F) place for a week. After a week check on them, they should show signs of growth. Over the next week or two you will start to see white mycelium forming and growing out from the center of the dish. During this phase you will probably notice that there are different types of mycelium growing. Some may seem strandy (rhizomorphic) and some may seem cottony (tomentose). The characteristic of different types of mycelium growing on the same dish is called Sectoring, this is normal and expected. Keep an eye on the dishes and look for mycelium that looks especially fast growing and that is very rhizomorphic (strandy, rope like), this is the mycelium that we want. You do not want to let the mycelium reach the sides of the dishes, as the sides of the dishes are prime spots for contaminants to be lurking. Once the dish is 3/4 colonized it is time to go onto the next step.
Contamination Check, Strain Isolation and Slants Contamination
This is the part that a lot of people are interested in. To get the best possible fruiting and yields from our precious mycological wonders we want our mycelium to be the strongest and most virile. You will want to check each dish for anything that is non-white (or a slight off white), be on the lookout for green, blue, pink or brown these are most likely contamination. Avoid sections of the dish that are contaminated (better yet toss/sterilize the dish and start over with it).
Now, go through each dish and since you were keeping an eye on the fastest growing mycelium (right?) you want to look for the spots where mycelium colonized quickly, is thick and prolific and is rhizomorphic. This is where a flow hood or glove box comes in handy, it is useful but not a requirement (reduces contamination). If you do have a glove box of flow hood make sure you do all transfers in this environment. Get a previously prepared petri dish and a colonized petri dish. Sterilize your scalpel or knife until red-hot and allow it to cool. Carefully open the colonized petri dish and cut a slice from the most promising section of the mycelium roughly the size of a dime (rhizomorphic, prolific, fast colonizing, contaminant free), now carefully remove the slice of mycelium and close the petri dish cover. Open the pre-prepared dish and place the slice of mycelium in the center and close. Repeat these steps for all other good cultures. You can save the rest of the mycelium for later use (refrigerate), toss it or create more dishes. With the newly inoculated dishes you will want to follow the basic steps in Inoculation and Incubation however since the spores are already incubated, colonization times will be reduced. You want to continue this process (colonization, selecting promising mycelium, and transferring) until there is no sectoring apparent. Once this is accomplished you now have a pure strain of mycelium that is virile, rhizomorphic and healthy.
You will want to now cut a slice of mycelium from the pure strain you have created and transfer it to the test tubes, then refrigerate. This is called a slant which you can use at a later date to get back to a pure strain if necessary (without repeating all of the steps above). You can then use the remaining mycelium in the petri dish(es) to inoculate your favorite substrate. This will save time as the spores have already been incubated. Growth should be apparent in a couple of days, with full colonization in under 2 weeks.