Agar Culture:
Agar is a polysaccharide derived from marine red algae. Agar is unique because it has the ability to remain liquid until cooling below 36 degrees C. The advantage here is that nutrients and other growth media can be mixed together before the Agar solidifies. The agar will remain solid at room temperature. Once the solution is mixed together it can be poured into individual Petri dishes for use in inoculating with one of the following: mushroom spores, a liquid culture, an agar wedge from a colonized Petri dish or live tissue from the mushroom itself. Using live tissue is a method for cloning a desired species and capturing its unique characteristics.
There are 5 different types of media and their name refers to their functionality.
•??General Purpose Medium: This is designed to grow a broad spectrum of microorganisms. Nutrient Agar is a general purpose media.
•??Enriched Medium: This media is enriched with some kind of special growth factor such as blood, serum, hemoglobin etc.
•??Selective Medium: This contains a chemical that inhibits the growth of certain organisms while promoting the growth of others.
•??Differential Medium: This can grow several types of microorganisms, but can distinguish among them due to different appearances on the medium; color of the colonies or color of the medium where the organisms are growing.
•??Fermentation Medium: This determines whether a microorganism can ferment a particular carbohydrate. Fermentation is an anaerobic process that often generates acids. If the carbohydrate is fermented, acid is produced, and this can be detected by a PH indicator dye.
For our purposes in growing mushrooms we will be using a Selective Medium. We will be adding various nutrients, minerals, etc. to promote the growth of our mycelium. We can even add antibiotics to hinder the growth of some bacteria.
There are many Agar recipes that can be used but two of the most common are Potato Dextrose Agar (PDA) and Malt Extract Agar (MEA).
Malt Extract Agar (MEA)
10 grams light malt extract
9 grams agar agar
500 ml potable or distilled water
Potato Dextrose Agar (PDA)
Broth from boiling 150 grams sliced potatoes
in 500ml water for 30 minutes (add water to 500ml)
9 g agar agar
7 grams dextrose
1 gram brewers yeast or yeast-extract (optional)
Amaranth Soy Agar
20 grams amaranth flour
20 grams soy flour
9 grams agar agar
500 ml potable or distilled water
Cornmeal Dextrose Agar
25 grams yellow cornmeal
3 grams dextrose
9 grams agar agar
500 ml potable or distilled water
Dog Food Agar DFA
20 grams dry dog food
20 grams agar agar
1000 ml distilled water
One thing to note when preparing Agar recipes is that it is definitely a circumstance when less is more. If too many nutrients are added to the Agar it becomes hypertonic. What this means is there will be a higher concentration of particles in the nutrient solution than there are in the dividing cells of our mycelium. Nature prefers balance. When mycelium is placed into this hypertonic solution, equilibrium will try to assert itself. Because the cell membranes of the mycelium are selectively permeable; only allowing certain things across the membrane. The water inside the cell will cross the membrane by osmosis to the outside and the cells will dehydrate; halting growth. This is why sugar, an energy source for bacteria, can be used as a preservative in jams and jellies. The jam becomes hypertonic and inhibits growth.
When preparing Agar recipes, you will need a container to sterilize the mixture in. A quart size jar works perfectly for this. Drill a 3/8” inch hole in the metal lid and fit the inside with a tyvek filter disc. Mix the contents thoroughly inside the quart jar. Place the lid loosely on the jar and load into your pressure cooker (optional: you can preheat your water to boiling before adding the quart jar. This will prevent caramelization of the ingredients). Fill the pressure cooker with water until an approximately 1 “ water level is on the side of the jar. Sterilize the jar for 30-40 minutes at 15 p.s.i
After the sterilization time, allow the cooker to cool to zero pressure before attempting to open. The pressure cooker should be opened in front of a flowhood or a sterile glovebox should be used when pouring agar plates. Each recipe above will fill 20 agar plates.
When the jar is cool to touch but the contents are still liquefied, pour enough MEA or PDA to fill the bottom of the Petri dish. The longer the lids are left open to the air the greater the chance for airborne contaminates to enter the culture medium. Allow the agar plates to cool to room temperature and solidify. Optional: You may seal the edges with parafilm to help stave off bacteria contaminations.
When inoculating agar plates, again, it’s important to use a sterile glovebox or flowhood to decrease the chances of contamination. Each agar plate can be inoculated with agar wedges from a colonized plate, spores from a mature mushroom or a prepared liquid culture.
If you have purchased our pre-sterilized medium please follow these directions for reheating it.
Directions for Using our Agar Culture Medium.
Place your quart jar in a pot of water and fill the pot with water until it covers half of the quart jar. Leave the lid in place to prevent contaminates from entering. Raise the water temperature to boiling in order to liquify the medium. Agar will remain liquid until it falls below 96.8 degree F.
When the Medium has liquified it can be poured into waiting agar plates. It is important to pour your agar plates under sterile conditions. We highly recommend using a Flowhood but a glovebox might work. We make no guarantees if a Flowhood is not used. Bacteria are present everywhere including the air. It only takes a few particles to land on your medium to contaminate it. When the agar has solidified again you can seal the edges of the agar plates with parafilm or other sealing device.
