Agar Plates

How To Make Agar Plates for Growing Mushrooms at Home

Anyone who really wants to dig into the hobby of growing mushrooms needs to learn how to deal with nutrient agar. Not only is it a way to experiment and learn to grow mushrooms more effectively, it allows you to save and propagate cultures, allows you to create your own spawn and liquid cultures, and best of all… its fun! I want to show you how the best way to make agar petri dishes for growing mushrooms at home. Scroll to the bottom of the article for a video of the process!

Step 1: Mix up dry ingredients

There are lots of different recipes for agar mix, including PDA (potato dexrose agar), MYA (malt yeast agar) and even DFA (dog food agar), but they all consist of essentially the same idea- adding some sort of food source to agar to create a nutrient rich agar media for mushroom cultures. The mix we use most is Malt Extract Agar.

For 1000 ml of water you’ll need to mix up:

20g agar agar
20g malt extract (we use barley maly extract)
2 g nutrional yeast

Agar might be a little hard to find locally. There are various sources online and some grocery stores will even carry blocks of Agar that can be ground up and used for your mix. Malt extract can usually be found a your local brew store. Nutritional yeast can usually be found at organic grocery stores. Alternatively, you can buy premixed Malt Extract Agar powder that can just be added to water.

Feel free to modify the recipe depending on how much agar you want. 1000 ml will be enough to pour about 35 standard petri dishes. I usually make up 750ml agar mix in order to pour a sleeve of 25 100mm x 15mm plates.

Agar Pre-Mix

Tools for the job, yeast, agar agar and barley malt extract.

Step 2: Add water and sterilize

Mix your dry ingredients in correct proportions with hot tap water. If the water is too cold it will start to set up right away and will be difficult to mix properly. The agar mix will have to be sterilized at 15 psi for 45 minutes to kill all potential contaminants. I like to use a whisky bottle with a modified lid for sterilization and pouring. To do this, simply drill a hole in the lid and pull some through some pillow stuffing. This filter will allow for sterile gas exchange while the agar cools off after sterilization. Cover the lid with tinfoil and place in the pressure cooker.

bottle for sterilizing agar

Whisky bottle with modified lid.

agar pressure sterilizing

Place in pressure cooker. The extra jars allow the bottle to sit in there at an angle.

A common problem when sterilizing agar in a pressure cooker is boil over. This caused when the water outside the bottle cools off faster than the agar inside the bottle, which forces the agar to boil over through the filter, making a mess in your cooker. A way to remedy this is by adding lots of water to your pressure cooker so that the cool off rates will be similar. I usually place the bottle in the pressure cooker at an angle so that most of the agar will be at the same level as the surrounding water. Boil over rarely ever occurs if using this method.

Step 3: Cool down

Once the agar is sterilized, it will need to cool down sufficiently to be able to handle it, but be warm enough so that it remains in its liquid form. This usually takes at least a couple hours. This is a good time to prepare the area where you will pour your plates and washing up for sterile work.

Using agar requires an extremely high level of cleanliness and attention to potential contaminates. Any mold spore or other contaminant in the air, from your skin or fingernails or breath could ruin your project. If a contaminate land on your petri dish at any time, it will likely sprout into action, ruining your potential projects and cultures. Wearing hands and arms, wearing freshly laundered clothes, wearing nitrile gloves and even surgical masks can greatly increase your chances of success.

You will also need to use a “still air box” ( SAB for short) or even better- laminar flow hood. These tools will provide a clean area to pour your plates, and without using either of them, it is almost certain that your plates will contaminate. A SAB can be as simple as a clear tote with a lid, and holes cut for arms. Use alcohol to clean the inside of the SAB and make sure that everything that goes in an out of the both is clean.

If using a flow hood, turn it on a couple hours before intended use to scrub the air. Wipe the surface of the counter with alchohol and set up everything you think you will need.

Pouring Agar Plates

This picture was taken mid pour. Note how the plates are stacked. The plates should be filled starting with the bottom one and working your way up. Also note the open bottle is upstream of everything else.

Step 4: Pour the plates

Start by stacking the plates in front of your flow hood or in your SAB in a sanitary manner. This is where disposable plastic petri dishes work best. They usually come in a pre-sealed and sterilized pack of 25. Clean the outside of the bag with alcohol, cut the bag on the bottom and slide the bag off the plates. Arrange them in about 3 or 4 evenly tall stacks.

Remove the tinfoil from the agar bottle and open the lid. If usuing a laminar flow hood, ensure that the mouth of the bottle remains in the sanitary flow of the hood. Start pouring the dishes in a rythmic manner starting from the bottom plate and working your way up. Add just enough agar so that the bottom of the dish is completely covered. Try to leave the lid of the petri dish open for as little time as possible. Also, ensure no part of your hands touches the inside of the dish.

Once all your dishes are poured, leave them to cool. If using a flow hood, leave the hood on while cooling. This will cool them off faster and prevent contaminants from being sucked into the dish while it cools down.

Pouring Agar Plates

Fill plates with just enough agar to cover the bottom of the plate.

Step 5: Seal and store

It will take about 45 minutes to 1 hour for your plates to cool down and solidify. Some of the plates will have condensation on top. This will eventually dissipate and is not a problem. At this point you can either use them directly for agar work, or seal them for later use. In order to prevent contamination during storage you will need to seal the edges of the petri dish. The best option for this is laboratory parafilm. It provides a breathable but contaminate preventing barrier that easily wraps around your dishes. Simply cut off a slice of about 3-4 inches long, peel off the paper and fold in half. Then wrap around your dish, stretching it enough so that it sticks to the edges of the plate.

Parafilm around the plate

Stretch the parafilm around the edges of the plate.

a sterilized agar plate

A freshly poured and ready to go plate.


Now you have nutrient rich agar media! You can use it to propogate cultures, store cultures for long term use, germinate spores and even clone mushroom fruitbodies.

Feel free to add questions or comments below!

All images and words by Tony Shields, FreshCap Mushrooms

Here are some products that you might find useful for agar work. We especially recommend parafilm!

Spread the spores!

Comments 8

  1. Hi, How can i order plates you are using? Also the ready mixture of fresh cap 🙂
    can i also buy spawn of yellow, blue and pink oyster?

    Thanks for the reply.

    1. Post
      Author

      Hi Cherish! Thanks for reaching out. We will soon be selling spawn of Blue and Pink Oyster, and a few others. Unfortunately we won’t have yellow oyster just yet- but may have that as an option in the future! Sign up to the mailing list, and we will notify everybody when the spawn is available. Or just check the site periodically for updates. We do not have plates or the ready mix available yet, but again, may soon have it in the future. I will let you know! All the best.

    1. Post
      Author

      Hi there! Thanks for reaching out! That is an excellent question. I have always had good results storing the new plates at near or below room temperature (around 18 degC), and have not been concerned about the amount of light. The plates can stay good for a long time. If there are large temperature swings, you will have condensate build up inside the plate, but it should not be too much of a concern.

  2. Hi Tony,

    Thanks for the great write up and video and for the whole Learn section!
    I was just wondering about the Parafilm stuff when you wrap petris with nutrient supplemented agar media before they are inoculated – since there is no mycelium/spores/tissue/pins inside yet, is gas exchange really needed (if we have developed a really sterile technique, at this stage there should be no life in the pertis whatsoever, hence no need to import oxygen or export exhaust CO2, right?). I mean can’t we go cheap and wrap “empty” agar plates with saran tape or any other cheap non-breathing media for the storage period (before inoculation), at the same time ensuring an even better contamination barrier?

    Thanks!

    1. Post
      Author

      Hey G, glad you like the site. And ya, you are correct, there is no need to wrap the petri’s in a breathable material like parafilm when there is nothing on the dish. I just use it because I have a bunch, and it works great. But you can use whatever works for you!

  3. Tony,

    I am interested in making petri dishes. You explain it very well in the video. However, what tek do you use to inoculate virgin petri dishes? Spore prints, syringes. I am talking about starting from scratch not having a culture already colonized from one petri dish to colonize another petri dish. What would be the easiest method. Can you perhaps make a video of you doing this? I have also read about using an inoculation loop and using a zig zag pattern. Can you elaborate on all this a little. Thank you.

    Michael

    1. Post
      Author

      Hey Mike!

      Thanks, I am glad you like the video! You can use either spore prints, liquid syringes or even a piece of tissue from another mushroom. For spore prints, you simply heat up a scalpel, cool it in the dish and scrape the spores onto the dish. For a liquid syringe (spore syringe) simply squirt about 1 mm onto the dish. For a tissue culture, cut off a small piece of tissue from inside the mushroom, underneath the gills and transfer it onto the dish. This produces a clone rather than a new strain.

      For all of these methods, you may need to do a number of “transfers” before getting a single clean culture.

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