|By Admin (Admin) on Thursday, August 23, 2001 - 12:08 pm:|
Fatguy's Agar Technique
Agar inoculation has been used for cultivation mushrooms as well as bacteria and molds for several years. The key to using an agar solution in cultivating mushrooms is sterility. Keeping the work surface and your hands clean and free of contaminants is the most important thing in using agar. I recommend that a HEPA system be used but since most people that use this forum cannot afford one, a glove box can be successfully used or a homemade HEPA design (what I use) will work.
Choosing an agar solution to work with should be pretty easy. I purchase agar from Fungi Perfeci but there are several different sources of suppliers out there. A person can purchase plates that have the agar poured and is already jelled in a petri dish. If that is the case them you can skip the agar production portion of this document and go to the inoculation step.
Agar production and pouring
I use a Malt Extract Agar (MEA) solution that is shipped to me in a powderform. Mix the agar solution according to the directions:
-Mix 50 grams of MEA (7 Tablespoons) for each liter of water.
-Autoclave (pressure cook/can) at 15psi for 45 minutes.
-Allow the MEA solution to cool to 150 degrees before pouring .
You can use a Mason Jar as a container to mix and autoclave the solution. This is a good container to use because you have a lid that you can use to cover the jar. Just make sure that when you do cover the jar with the dome lid, that the dome lid is upside down (seal up). I use a coffee decanter shaped like a large flask that I got a thrift store. This is good because I can pressure cook it and it has a handle for pouring. I use extra heavy duty aluminum foil To cover the top of the decanter.
Here is an important point: Unless you have a container that will hold about 2 liters of solution for each liter that you want to make, I would suggest that you make 1/2 liter batches of the agar solution. I had some of the solution boil over in the pressure canner and after the canner cooled, I had about 2 gallons of jelled agar solution sitting in the bottom of the pressure canner. 1/2 of a liter will make
about 12 dishes of agar.
When the 45 minutes is up, slowly and carefully lower the heat and the pressure of the pressure canner. This will prevent the excess boilover described.
Here are a few points regarding the pouring of the plates.. First have all the petri dishes unwrapped and in the sterile area where you plan to pour them. I stack the dishes in piles of three. This comes in handy when pouring. While the Agar is cooling is a good time to do this. Also make sure that your hands are washed and sterilized. I use rubbing alcohol for this but a 10% bleach in water solution will work as well.
When you have everything together and are ready to pour, this is how I do it:
-Take a stack of three petri dishes, uncover the bottom dish while holding onto the lid and the two other dishes, carefully pour the MEA solution in the dish to a depth of about 1/8 to 1/4 of an inch. Cover the dish and do the two other dishes the same way.
-When finished pouring wrap the edges of the petri dishes in either Parafilm or plastic wrap. This will prevent contamination while the agar solution is cooling. Personally, I prefer Parafilm but plastic wrap will work as well.
After the agar dishes have cooled and you are ready to use them, you may notice some condensation on the agar. This is normal and I have not had any problems associated with the condensation. There are those who will disagree with me but until I have some serious problems with the water, I won't worry about it.
When you are ready to inoculate, again, sterility is still the most important thing that there is to keep in mind. Washed and sterilized hands and a very clean environment is what is most important Use the glove box or a homemade HEPA system and spray everything with a 10% bleach water solution before doing your inoculations.
There are three different ways of inoculating that are very effective. There is using the spore syringe, inoculating from spore print and cloning tissue. I will describe all three.
The spore syringe:
- Carefully unwrap your petri dishes from the Parafilm or plastic wrap and set them aside in stacks of three.
- With a heat source (alcohol lamp, propane torch, gas stove, Bic lighter) heat the needle of the syringe provided by PF to glowing red hot. Then cool the needle by forcing some of the spore water through the needle.
- Take a stack of three dishes and carefully open the bottom dish just enough that you can place the tip of the needle in the dish while holding the other two dishes in the stack in your hand. Inject a small amount of the spore water on the agar. Close the lid and proceed to the other dishes using the same technique.
NOTE: DO NOT ALLOW THE NEEDLE TO TOUCH ANYTHING OR YOU WILL HAVE TO RE-STERILIZE THE NEEDLE!
Using only one syringe, I have been able to inoculate upto 25 petri dishes of agar.
The spore print:
To inoculate with a spore print you should use an inoculation loop. This is a loop of wire with a handle. however you can make one of these with some wire that you can get at a hardware store. The wire should be able to be heated to red hot.
- Remove the plastic or parafilm from the petri dishes and stack in piles of three.
- Using a heat source, Heat the inoculation loop to red hot.
- Open the agar dish and cool on the agar, then take a swipe of the spore print with the inoculation loop and spread the spores on the agar that you used to cool the inoculation loop. Replace the lid of the petri dish and seal with Parafilm or plastic wrap.
- Re-heat the inoculation loop between every dish that you intend to inoculate. This will prevent cross contamination.
The Tissue Culture (Cloning):
Once you have living cultures you can keep living cultures on hand by doing live tissue cultures to agar plates. All you need is a living culture of fungus and MEA petri dishes. For me this works well because I can cut the cultured plates into 8 sections, sterilize 7 jars of grain and sue the last wedge of agar to culture about 3-5 plates of fresh MEA. I always have some cultures as a backup and I can cultivate as much as I want of a specific culture.
Here is how it is done:
- Carefully unwrap the Parafilm or plastic wrap off the petri dishes and stack off to the side.
- Choose a culture that you wish to use for the transfer.
- Sterilize a scalpel (a exacto knife will work for this) and cool the knife on the plate that you wish to inoculate.
- Carefully cut a sterile piece of the culture that you want to clone and remove the cultured piece with the sterile knife.
- Place the tissue on the agar plate with the knife, and replace the lid. Seal with Parafilm of plastic wrap to prevent contamination.
All of these techniques are sound and have been written about in "The Mushroom Cultivator" by Paul Stamets. However, he does not go into much detail regarding the use of a spore syringe to inoculate plates of agar. All of this can be done in a home lab and should not be prohibitively expensive.