3 Agar Media Recipes For Culturing Mushroom Mycelium
There are a number of different agar medias that work well to culture mushroom mycelium. We're going to cover 3 common recipes that are made with ingredients easily accessible for most hobbyists, or people who might not have access to laboratory suppliers. Each recipe can be scaled up or down in volume depending on your requirements.
Two of the three recipes will be for potato dextrose agar (PDA) with different ratios of dextrose and agar. We recommend trying both formulas to see which works best for your mycelium. Both of the PDA recipes will use the broth from boiled potatoes instead of dried potato infusion powder. The reason for this is that dried potato infusion expires and therefore cannot be stored for long periods of time. Most times the minimum quantity purchased can be enough to make 100 litres which no hobbyist would require before it expired. Potatoes don't always store well either but you can purchase them in small quantities as needed. There are recipes across the internet that use instant mashed potato powder but we advise against this for various reasons, unless you're in outer space.
The other recipe will be a malt extract agar. It is crucial to use the lightest malt possible as the sugars in darker malts are very caramelized and this is detrimental to mycelial growth.
It's recommended that you alternate the media you grow your mycelium on in order to veer away from nutritional selectivity as you isolate your cultures. You want your mycelium to be able to grow and digest a variety of substrates. What happens is that the digestive enzymes the mycelium creates actually become selective. This is not the same as scenesence from age or a bad isolation. Alternating the media keeps the mycelium's enzyme development system versatile. Depending where you are in the world, you never know when a substrate you like to use for spawn or beds will become unavailable, therefore the less selective your cultures are, the better. Trial and error is your best bet if you're unable to find published data regarding your strain.
Distilled water is strongly suggested for each recipe. Tap water can be used and it likely won't harm the mycelium but since it varies from region to region, distilled is best. It's important to note that the pH of the media typically doesn't become an issue, however, for some species, and for germination of certain spores, the pH can be important. If after conducting research on the variety you're cultivating you find that it prefers a specific pH, it can be adjusted prior to sterilization by using hydrochloric acid (HCl) to reduce the pH, or Sodium Hydroxide (NaOH) to increase the pH. Using an electronic pH pen is acceptable but cleaning it of agar could be a chore and it can clog the membrane, so we recommend pH strips for this.
It is very important that you prepare your sterile environment prior to starting any work. This means having your flow hood moving or your glove box ready, your petri dishes already sterilized and placed in your sterile environment, or having a package of sterile disposable ones handy.
Conduct all prep work outside of your sterile environment until you are ready to open your pressure cooker/autoclave.
For both of the PDA recipes it is possible to use 4g of dry potato infusion to 1000ml of water in place of making potato broth.
Optional for both PDA recipes below is 2 grams of powdered nutritional yeast.
Potato Dextrose Agar (PDA) (Formula 1)
300 grams sliced potatoes (russet works well)
1000ml distilled water + additional to replace what evaporates during boiling
10 grams dextrose
20 grams agar powder
Potato Dextrose Agar (PDA) (Formula 2)
300 grams sliced potatoes
1000ml distilled water + additional to replace what evaporates during boiling
20 grams dextrose
15 grams agar powder
dextrose (corn sugar)
Step 1: Thoroughly wash and slice your potatoes
Tip: Keeping the skin of the potato intact all the way around the slices will help keep them whole while boiling. This will make filtering easier and faster by reducing the amount of potato fragments in the broth.
Step 2: Place 300g sliced potatoes in 1000ml of boiling water and boil gently for 1 hour. You can cover your pan with a lid to reduce evaporation but allow it to vent a little bit. Top up with distilled water as required.
Step 3: While your potatoes are boiling combine the dextrose and agar powder and set aside.
Step 4: Carefully filter your potatoes through a fine mesh sieve, reserving the broth. If your potatoes didn't break apart too much this is usually sufficient. If you have clearly visible potato fragments in your filtrate you must pass it through a finer sieve or filter paper. Permanent coffee filters also work well.
Note: You can discard the overcooked potatoes or give them to your laboratory's dog for lunch. Just kidding, you shouldn't have a dog in the lab! ;)
Step 4: A lot of water will have evaporated during boiling so it's important at this stage to top up it up to 1 litre with distilled water.
Step 5: Add your powdered mix of agar and dextrose to the potato broth by slowly and steadily sprinkling it over top of the water until all of it has been added. This will help prevent clumping which can remain even after you've sterilized your media.
Step 6: Mix the ingredients thoroughly and pour it into the vessel you will be using to sterilize and pour from. Narrow necked flasks are the most effective and safest for processing. Any vessel that is autoclavable and pours well without running or dripping is acceptable. Polypropylene vessels also hold up to sterilization at 15 PSI (121.2°C/250°F) and are often more affordable than glass. If you're using a polypropylene (PP) vessel rather than glass for sterilization, you must not exceed 24PSI or 130°C/266°F. This wont be an issue here since 15 PSI is the maximum temperature recommended for sterilizing media containing sugars like malt and dextrose.
NEVER FILL A VESSEL OVER 50% ITS VOLUME WITH LIQUID MEDIA DURING STERILIZATION OR IT CAN BOIL OVER
Tip: You do not want to tightly close your vessel or it will form a seal as it cools making it difficult to open. Plugging with cotton or capping with foil is an acceptable practice.
Step 7: Sterilize your media at 15 PSI for 30 minutes, 121.2°C/250°F. Do not sterilize at higher temperatures as you do not want the sugars to caramelize.
Malt Extract Agar (MEA)
1000ml or 1000g Distilled Water
20 grams light malt extract
2 grams powdered nutritional yeast
20 grams agar
This is a very simple blend and is made by combining the dry ingredients and then sprinkling them over 1000ml of distilled water as you would have in step 5 of the PDA recipes. Once mixed simply pour into your vessel and sterilize at 15 PSI for 30 minutes.
ultra light malt extract
Once your media has been sterilized you can pour it into your sterile petri dishes. Pouring hot media can be a difficult task and requires a swift and steady hand for pouring. You do not want to have drops of media leaving deposits on the rims and edges of the dishes.
You can allow the media to cool a bit before pouring but do not let the temperature drop too much otherwise as the volume decreases you will find the media begins to solidify while pouring due to it losing heat more rapidly than when you started. Typically 85°C/185°F is a good temperature to start pouring at. The lowest you would want to pour at is 55°C/155°F.
Tip: Setting up your petri dishes in stacks 3-4 high lined up and pouring starting from the bottom dish first by lifting all dishes on top of the lowest petri dish to be filled in each stack is ideal and allows for swift pouring. Once the stack is filled, slide it back and out of the way, then slide in your next stack. Ensure you have enough room, fumbling over where to place things mid-procedure will only cause issues.
Once your dishes are completely cooled you can use them to conduct tissue culture from fresh mushrooms, or grow and isolate your own cultures from spore.
There you have it. 3 different formulas of agar media that are well suited for growing a variety of edible mushroom cultures including Agaricus, Lentinus, Pleurotus, Stropharia, Hericium, and many other exciting species.