Agar for Mushroom Cultivation

Mushroom cultivators use agar to observe the development of mycelium. The agar media is created by combining water, nutrients, and a dried substance called agar-agar (deriving from red algae). The mixture is dissolved in hot water, then pressure sterilized and finally poured into petri dishes. As the agar cools, it will solidify into a jello-like substance. Spore germination and mycelial growth are easily observed on the surface of the agar. Unwanted guests such as bacteria or other spores thrive on agar and will likely overtake the vulnerable mycelium. To prevent contamination, you must maintain a strict sterilization protocol whenever working with agar.

To save time and resources, I highly recommend growing mycelium on agar before going to grain spawn. Even if an agar plate begins to show signs of contamination, the healthy mycelium is still salvageable by conducting an agar to agar transfer. However, contaminated grain spawn should be thrown out, wasting time and money. Below you'll find various recipes for agar, sterilization protocol, transfer methods, and more.

Agar Recipes

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  Light Malt Extract Agar

  Light Malt Extract Agar or LMEA is a standard but highly effective recipe to create agar.

  LMEA Recipe
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  Black Agar

  Black agar is a nutrient culture dyed with activated charcoal powder. Cultivators use black agar to easily observe mycelial growth.

  Black Agar Recipe

Agar Products on Amazon

Sterilization Protocol

Below are the rules I follow while working with agar. You'll want to begin sterilization protocol before removing the agar media from the pressure cooker. Every step you follow will increase your success rate.

Hygiene: There's likely a host of contaminates on your person. Clean your fingernails, take a shower and change into clean clothes before working with agar.

Flow Hood / Still-Air-Box: Pour the agar in front of a laminar flow hood or within a still-air-box (SAB). Pouring in the open air will likely result in contamination.

Air Flow: Air is full of particles that could contaminate the agar, so you'll want the air in your work area to be as still as possible. To achieve this, turn off the ventilation in your home and close any door or windows about an hour before pouring agar dish.

Air Particle: Before opening the agar media, mist the air in your work area with disinfectant, isopropyl alcohol, or even water. The droplets will cling to any suspended particles floating in the air and drag them to the ground. If using a SAB, mist the inside and wipe the surface.

Gloves: Wear nitrile gloves when handling agar. You'll want to clean them with disinfectant or isopropyl alcohol anytime they leave the SAB or flow hood.

Face Mask: Remember, we want the air to be clean and as still as possible. Breathing could aid in contamination, so wear a face mask.

Wipe Down Everything: Disinfect any surfaces or items near your work area.

Sterilizing Tools: Flame sterilize any metal instruments before and after they come in contact with anything. Wipe down anything else with isopropyl alcohol or disinfectant.

Pouring Agar Plates

Petri dishes come pre-sterilized packaged in a plastic sleeve. Never open the sleeve until the bag is disinfected and contained within your work area. Carefully inspect the Petri dishes for cracks and damage. Remove any damaged plates once you are ready to open the sleeve.

The air particles mentioned above could settle on the surface of your work area, causing a contamination hazard. To combat it, conduct agar work on a raised surface such as a wire rack or even an empty Petri dish.

Open the sleeve, ensuring the plates are right-side-up; the lid will encompass the narrower bottom half. From here, you'll need to choose a method for pouring the plates:

• Pour the plates while they are stacked, starting from the bottom. You'll pick up the stack using the bottom lid and pour, working your way up the stack. You may find this method difficult when using a SAB, but it is very efficient.
• Pour each dish individually and stack them once completed. A bit more time consuming, but less awkward.

Whichever method you choose, conduct a few practice runs to become comfortable with the motions. Always ensure the lid hovers over the top of the base and never turn the lid upside down. Your goal is to reduce the time the inside of the plate is exposed to the elements.

Open the lid of the agar media and begin pouring. Only fill the dish halfway or 2/3 full. There's typically a line indicating the half waypoint. Remember to hover the lid above the plate while pouring.

When completed, allow the stack of agar dishes to cool and solidify. They should be ready in about an hour. Keeping them stacked will help reduce condensation. To prevent contamination from entering the SAB, I'll cover the armholes with freshly sterilized monotub lids.

Finally, you'll want to tape the sides of the Petri dishes until you are ready to innoculate them. Ideally, you'll want to use parafilm, but micropore tape or even plastic wrap will work.

Wrapping Petri Dishes with Parafilm

With your one hand, pinch the middle of the dish with your middle finger and thumb. Pin down the end of the parafilm to the side of the dish with your index finger. With your other hand, place your pointer and middle finger on top of the parafilm, as close as possible to your index finger that is holding it down. Pinch the parafilm with your right thumb, then pull and stretch, wrapping the film around the dish until you begin to feel tension. Readjust your hands and repeat the process until wrapping the entire plate.

Storing Blank Agar Dishes

Store blank plates in a refrigerated environment. They are good for around 4-6 weeks. Many of you may cringe, but I store my blank plates in the refrigerator. I seal them within a shoebox tub for added protection. Many precautions are taken to reduce contamination, and I've been successful so far.

Types of Agar Transfers

Below you'll find a few ways to utilize agar plates in your next mycology project.

Spore Print to Agar

There are a few techniques for transferring a spore print to agar. The definitive way is to scrape a spore print using a flame sterilized inoculation loop and drag the loop across the agar in a zig-zag pattern.

Spore Syringe to Agar

Placing a single drop from a well-shaken and flame sterilized spore syringe in the middle of an agar plate is all that is needed to begin germination. This technique extends the life of your spore syringes and limits the risk of wasting grain spawn if the syringe is contaminated.

The typical spore syringe contains 10cc of spore solution. Each jar of grain spawn needs 2cc of the inoculation solution, leaving you with only five spawn jars. There's also a factor of contamination if you've purchased the spores from a vendor.

Grain to Agar

Transfering colonized grain spawn to agar is a simple and effective way to grow mycelium. Using flame sterilized tweezers, pluck a single grain from a colonized jar and place it in the middle of an agar plate.

Tissue Sample to Agar (Cloning)

Tightly packed hyphae comprise a mushroom fruiting body. A tissue sample from a mushroom will continue to grow mycelium when placed on agar. This process is also referred to as cloning. In a sterile work area, carefully rip the specimen in half. Cutting the mushroom with a knife could pull contamination from the outside to the clean inner flesh. With a flame sterilized tool, cut or scrape a tissue sample from the newly exposed flesh and place it in the middle of an agar plate.

Agar to Agar

It is a common method to transfer an agar culture to a blank plate. The technique will extend the life of the mycelium and can help to isolate genetics. If a section of a dish is suffering from contamination, the healthy mycelium could be salvaged by an agar to agar transfer.

Air Quality Test

As a more practical use of agar. Place an open agar dish in rooms to determine the number of spores and bacteria in the air. This test works well around your work area. If the contamination is high, you may want to take some steps to clean better.

In the photo, you'll see agar placed in my lab [left] vs. agar placed in my bedroom [right].

How to Incubate Agar Cultures

After inoculating agar dishes, you'll want to store them upside-down in optimal growing conditions. For incubating most mycelium, the ideal temperatures are between 68-77°F (20-25°C). Spores may take a few days to germinate. Before long, you'll begin to notice hyphae branching out from the inoculation area.