No-Fuss Agar Tek for Mycology

Agar work is the foundation of serious mycology, allowing you to isolate genetics, germinate spores, clone mushrooms, and clean up contaminated cultures.

The Golden Rule: Sterility is Everything.

Your agar is a perfect buffet not just for mushroom mycelium, but for bacteria and mold spores floating in the air. Your number one job is to prevent them from landing on your agar. Always work with clean hands in a clean space.

I. Materials & Equipment

Agar Ingredients (for ~500 ml solution):

This will make approximately 20 standard 90mm petri dishes.

• Distilled or Filtered Water: 500 ml
• Agar-Agar Powder: 10 g
• Light Malt Extract (LME): 8g (This is the primary food for the mycelium)
• Nutritional Yeast: 0.5g (Optional, but highly recommended as a nitrogen source to boost growth)

(See the "Common Recipes" section at the end for alternatives like Potato Dextrose Agar - PDA)

Equipment:

• Pressure Cooker (PC): A stovetop or electric model capable of reaching 15 PSI is essential for proper sterilization. An Instant Pot with a "Canning" or "Sterilize" function can work in a pinch, but it does not guarantee sterilization.
• Media Bottle: A borosilicate glass bottle with a screw-on lid (e.g., Pyrex brand) is ideal. A standard 500ml or 1L mason jar will also work.
• Scale: A digital scale accurate to 0.1 grams.
• Petri Dishes: Sterile, disposable plastic dishes are easiest for beginners. Reusable glass dishes are an option but require more prep.
• Aluminum Foil: To cover your media bottle/jar lid.
• Still Air Box (SAB) or Laminar Flow Hood: This is not optional for consistent success. A SAB is a clear tote bin turned on its side with armholes cut in it. It creates a space with still air, allowing contaminants to settle.
• 70% Isopropyl Alcohol: In a spray bottle.
• Flame Source: An alcohol lamp, butane torch, or even a simple lighter.
• Personal Protective Equipment (PPE): Nitrile gloves and a face mask.
• Parafilm, Grafting Tape, or Micropore Tape: For sealing the finished plates.

II. Step-by-Step Procedure

Step 1: Mixing the Agar

1. Place your media bottle or mason jar on the digital scale and tare it (zero it out).
2. Pour in 500 ml of your distilled water.
3. Add the dry ingredients: 10g Light Malt Extract, 8g Agar-Agar Powder, and 0.5g Nutritional Yeast.
4. Stir vigorously with a whisk until all clumps are gone. The mixture will be cloudy. If using a magnetic stirrer, let it run for a few minutes.
   • Pro-Tip: Adding the powders to cool water and mixing immediately prevents the agar-agar from clumping badly.

Step 2: Sterilization

1. Screw the lid onto your media bottle or mason jar, but leave it one-quarter turn loose. This is critical! It allows pressure to escape during heating and cooling. If the lid is too tight, the glass can shatter.
2. Cover the top of the lid with a small piece of aluminum foil. This prevents water from the PC from dripping down and potentially contaminating the threads.
3. Place a canning rack or a few mason jar rings at the bottom of your pressure cooker. Add about 2-3 inches of water.
4. Place your media bottle/jar inside the PC on the rack.
5. Seal the pressure cooker lid and bring it up to 15 PSI over medium heat.
6. Once it reaches 15 PSI, start a timer for 30 minutes.
7. After 30 minutes, turn off the heat completely.
8. DO NOT attempt to quick-release the pressure. Let the pressure cooker cool down naturally until the pressure lock drops on its own. This can take an hour or more. Rushing this step can cause your agar to boil over violently.

Step 3: Preparing Your Sterile Workspace

1. While the PC is cooling, prepare your workspace. If using a SAB, place it on a clean table.
2. Thoroughly spray the inside of the SAB (or the work surface of your flow hood) with 70% isopropyl alcohol and wipe it down with a clean paper towel.
3. Wipe down your sleeve of petri dishes, your gloves, and anything else you will be placing inside the SAB (your agar bottle).
4. Wait 10-15 minutes for the air inside the SAB to settle and for the alcohol to evaporate.

Step 4: The Pour

1. The agar needs to cool before pouring. The ideal temperature is around 120-130°F (50-55°C). It should be hot enough to still be liquid, but cool enough that you can hold the bottle for 5-10 seconds without being burned.
   • Why? Pouring too hot causes extreme condensation on the petri dish lids, which can lead to contamination.
2. Carefully remove the now-cool pressure cooker lid. Use oven mitts to retrieve your hot media bottle/jar and place it in your SAB. Lightly spray and wipe it down with alcohol.
3. Put on your gloves and mask. Spray your gloved hands with 70% iso-propyl alcohol and let them air dry.
4. Work inside the SAB for all subsequent steps.
5. Gently swirl the bottle to ensure the solution is homogenous. Don't shake it, as this creates bubbles.
6. Arrange your petri dishes in stacks of 5.
7. Unscrew and remove the lid from your media bottle. You can briefly flame the lip of the bottle with your torch/lighter to sterilize it - just be careful if you're using lab bottles with plastic gaskets on them...a flame will obviously melt it ;) .
8. Pick up the bottom dish from a stack. Use one hand to lift the lid just enough to pour, keeping the lid hovering over the bottom half of the plate like a shield.
9. Pour a thin layer of agar into the dish—just enough to cover the bottom (about 1/4 to 1/3 full).
10. Replace the lid immediately and move to the next dish. Work smoothly and deliberately.
11. Continue this process until all your agar is used.

Step 5: Cooling, Sealing, and Storing

1. Leave the freshly poured plates inside the SAB to cool and solidify, which takes about 30-60 minutes. Do not disturb them.
2. Once they are solid, you can stack them and take them out of the SAB. To deal with minor condensation, leave the plates stacked for a day.
3. Quality Control: Keep the plates for 3-5 days before use. If you see any bacterial or mold growth, your sterile technique failed somewhere. Toss the contaminated plates and review your process. If they remain clean, they are ready for mycelium!

III. Common Agar Recipes (for 500 ml water)

• LME (Light Malt Extract Agar): The recipe used in this tek. Excellent all-purpose recipe.
  • 8g LME, 10g Agar, 0.5g Nutritional Yeast.
• PDA (Potato Dextrose Agar): Another classic. You can buy premixed PDA powder or make it from scratch.
  • From Scratch: Boil 150g of thinly sliced, unpeeled potato in 500 ml of water for 30 mins. Strain the water through cheesecloth, discard the potatoes, and add water back to the 500 ml mark. While still hot, dissolve in 10g Dextrose (or table sugar) and 10g Agar. Sterilize as usual.

Happy cultivating! Practice makes perfect with sterile technique.