How to use Agar in Mushroom Cultivation

Working with spore prints or spore syringes that are not sourced from, or made in, clean environments can invariably result in contamination. They can be a source of frustration for the beginner and expert mushroom cultivator alike. When reading about mushroom cultivation, chatting in fungi forums, or talking with mushroom cultivators about mycelium or how to solve contamination problems, often the discussion will come around to agar—making “cultures” or trading “plates”. 

This article is a brief introduction to the sometimes intimidating and mysterious art of agar.  Working with agar does require a little dedication, determination, and blind faith, but is well worth the investment. It allows you to take your mushroom growing to a new level, from germinating spores via your spore prints or syringes, and making tissue cultures, to carefully cleaning mycelium from that dirty spore print of a rare magic mushroom.

Read: How to Take Shrooms

You might have been exposed to the use of agar in Petri dishes (“agar plates”) if you did biology in high school or college. Agar allows microbiologists to grow and examine pure cultures of otherwise unseen microbes, with various recipes that can help grow specific species. The most commonly used agar recipes are Potato Dextrose Agar (PDA), and Malt Extract Agar (MEA), but these can be modified to include many novel kitchen ingredients, from Vegemite to Milo.

On the path to agar wizardry, it is guaranteed that you are going to end up with some funky agar plates—and not the James Brown kind of funky: Your housemates may complain about those weird things in the fridge, but it’s all part of the fun. Let’s begin.

What is Agar

Agar is made from red algae and is used as a setting agent in deserts throughout Asia.  It has become popular as a vegetarian alternative to gelatin. It can also be used as a thickener for soups, fruit preserves, and oddly enough, ice cream. In addition to its culinary uses, agar is a fundamental tool in microbiology labs across the planet. Petri dishes containing agar create a perfect environment to grow a variety of fungi or bacteria. 

Overall, the term “agar” is used to refer to a “culture media”—a substrate that contains all the vitamins and nutrients necessary to grow a variety of microorganisms. The first culture media was boiled potato slices, then gelatin in the late 19th century, and then agar-based media became the primary means to grow microbes for study. Due to its higher melting temperature (jelly melts around 95 degrees Fahrenheit), agar plates can be kept at higher temperatures without the media turning to liquid.

What You’ll Need in order to Make Agar Plates

Agar—often sold as “agar-agar”—is easy to obtain, available from most Asian groceries or health food stores, and sold in powder form. Some agar brands are more powdery than others, affecting how well the agar dissolves, but the final product is generally the same. The addition of sugars and nutrients is necessary for healthy mycelium: In high concentrations, these can promote contamination. Not much needs to be added, so tins of Malt Extract syrup can last you a while! When there is a high risk of contamination, you can make agar plates using only agar and water.

One of the oldest and easiest recipes is Potato Dextrose Agar (PDA). When boiling potatoes, they break down somewhat to leave a cloudy broth which is full of nutrients. The addition of Dextrose (dextrose is a simple sugar made from corn, which is chemically identical to glucose) supplies the necessary sugars. You can buy dextrose powder in health food stores.

Molasses, malt extract, malt powder, or malt-based drinks can also be used to make agar plates—malt extract agar (MEA) or Milo agar being familiar recipes. They also contain nutrients so that they can be used on their own. Some recipes require the addition of extra nutrients, with yeast extract being a common additive. More technical recipes call for nutrients in an inorganic powdered form that are then dissolved with specific concentrations.

You will also need a pressure cooker, a glass jar or two, a thermometer (I suggest a milk thermometer), cling film (the tube cut to 45 mm lengths), and clean takeaway sauce containers to use as Petri dishes. A Still Air Box is essential. While in other situations (with PF Tek or Spiderman Tek) you may have been comfortable not using a Still Air Box (SAB) or “glove box,” in this case, it is essential if you want to be successful. 

Shopping list:

• Pressure cooker
• Glass jar or two
• Thermometer
• Cling film (the tube cut into 45mm lengths)
• “Plates” (clean, unused take-away sauce containers make reasonable substitutes for glass or plastic Petri dishes)
• Approx 350 ml jar, a beaker, or Erlenmeyer flask (the jar will be used to heat, and then pressure cook the agar)
• Spores, either as spore print, spore syringe, or swab
• Agar powder
• Potatoes and Dextrose powder for Potato Dextrose Agar (PDA)
• Malt extract for Malt Extract Agar (MEA), or molasses as a substitute
• Concentrated yeast extract (if you cannot find Vegemite or Marmite!)
• Scalpel, or sharp knife
• 70% alcohol, 70% isopropyl alcohol, or methylated spirits diluted to 70%
• Thin glass rod, hooked end of a metal straw cleaner, or dissection probe or seeker
• Still Air Box (SAB) (see below for brief instructions)
• Hydrogen Peroxide (H2O2) at 3% concentration (the type available at supermarkets)

Note: Obtaining lab-grade Petri dishes—“plates”—can be expensive if not bought in bulk, but you can be creative. The plastic sauce containers (or similar) from your grocer or homewares store or catering supplies will work great.

You can also get really guerrilla and use various household products—from powdered malt drinks such as honey, milo, miso, yeast, vegemite (marmite), and even dog food! There are a variety of further modifications you can make to the agar recipes for specific purposes, such as food coloring, hydrogen peroxide, salt, or antibiotics.

A lot of people currently use blue or red food colouring to color their agar. Doing this adds no nutritional value to the agar, and is often used to observe the mycelium as it grows (mainly single spore germination). Plates can also be backlit with a mobile phone light; the technique not only helps to highlight the mycelium you are growing, but also the mycelium of contaminants.

You may have successfully grown mushrooms using the 101 course or followed our how-to guides, and are familiar with creating mostly sterile environments. If you haven’t, though, that’s still okay—but to grow spores on agar (at this point at risk of sounding like a broken record!), you have to be super clean, making sure that your workspace and tools have been well sanitized with 70% alcohol or methylated spirits beforehand.

As an experiment for you to try at some point: Make ten agar plates, and leave them out on your bench. Create a time series exposing the agar to the air, closing the lids and sealing at one minute, five minutes, ten minutes, then at ten-minute intervals. Close and seal the last two plates after one hour and two hours respectively. Place the agar plates somewhere safe and watch for a week or two. Spores from the air will land on the agar and germinate: the longer the time exposed, the more spores. This is a microbiology 101 experiment used to demonstrate how dirty the air in your workspace may be.  

Still Air Box

*A Still Air Box (SAB), or “glovebox,” is a sealed container that some people use to help limit air movement and keep contamination out of their grows. You can buy one or make one using our guide below. Some have gloves physically connected to them so once sealed the contents remain sterile, but with caution and good technique this is not altogether necessary.

What you’ll need for your still air box:

●  Plastic storage box  (53 quart is ok, but bigger boxes will provide more room)

●  Medium-sized Peach tin, or tomato tin (800g to 1kg)

Most SAB’s are made from large clear plastic storage boxes (although my first SAB was a cardboard box with a cling wrap window!) with two holes cut for armholes. The plastic in most plastic storage tubs is brittle, so rather than trying to cut holes, heat a medium-sized tin can (a medium-sized 800g/1kg peach tin or tomato tin) till it is hot and then being careful not to burn yourself, cut two holes in the plastic with a placement where you think your arms would feel most comfortable. The hot tin should cut the plastic effortlessly—best to do this outside. Consider the placement of the armholes before you cut; in time you may find yourself working longer and longer hours, so comfort is essential.

Basic Agar Recipes

The most basic Agar recipe is just plain water and agar powder. A lot of the recipes you will see online tend to work with 1-Litre volumes (that’s a lot of plates!), but whether you use 90mm Petri dishes or takeaway sauce containers, 200ml of liquid is a good amount to make about 10 to 15 agar plates.

Water Agar

• 200 ml of water
• 5 grams of agar powder (approx heaped 1 tsp.)

Potato Dextrose Agar (PDA)

• Boil 100 g of sliced unpeeled potatoes in 500 ml of water for 30 minutes
• Filter the broth through a clean cloth
• Use 200 ml of this potato broth
• 5 grams of dextrose (approx heaped 1 tsp.)
• 5 grams of agar powder (approx heaped 1 tsp.)

Malt Extract Agar (MEA)

• 200 ml of water
• 1 gram malt extract syrup (approx ¼ tsp.)
• 5 grams of agar powder (approx heaped 1 tsp.)

Honey Agar

• 200 ml of water
• 1 gram honey (approx ¼ tsp.)
• 5 grams of agar powder (approx heaped 1 tsp.)

Malt Extract Vegemite Agar (MEA)

• 200 ml of water
• 1 gram malt extract syrup (approx ¼ tsp.)
• 0.1 gram Vegemite (or equivalent US concentrated yeast extract) (approx 1/16 tsp.)
• 5 grams of agar powder (approx heaped 1 tsp.)

You can substitute the malt extract syrup for molasses (1 gram, approx ¼ tsp.), malt powder (2 grams, approx 1/2 tsp.), or milo powder (2 grams, approx 1/2 tsp.)

Measure out 200 ml of liquid (water or potato broth) and pour into your glass jar. You could use a beaker or Erlenmeyer flask, but a 350 ml glass jar (old peanut butter jar) is perfectly suitable. Using a saucepan of water as a water bath, place the jar in the saucepan to help boil the liquid. Be careful of thermal shock (hot and cold often don’t mix, and the glass can shatter), so start the heating process with everything at the same temperature. Measure the temperature of the liquid with a thermometer, and when it reaches 80 degrees, add the ingredients leaving the agar powder until last. Make sure everything is well dissolved.  The agar powder can tend to stick, so my advice is to add it slowly, stirring as you do; otherwise, the powder will form gelatinous chunks. Once the agar is dissolved, cover the jar with a piece of aluminum foil and place in your pressure cooker to cook for 20 minutes at 15 psi to sterilize.

Agar (derived from red algae) can be used as a thickener for soups, fruit preserves, and oddly enough, ice cream. In addition to its culinary uses, agar is a fundamental tool in microbiology labs across the planet.

When the agar has been sterilized, and the pressure cooker depressurized, remove the agar and move quickly to your Still Air Box. Agar readily begins to set below 45 degrees C, so while you are waiting, sterilize the inside of your Still Air Box and your tools. Set up your “plates” in your glovebox by laying them out in a way that is going to be easy for you to pour the agar; loosen but do not remove the lids if you are using takeaway sauce containers. This is going to require a little practice until you are entirely comfortable. Pouring agar can be an art form, so if you find yourself making a mess, don’t worry.  

You will probably get condensation forming on the surface of the plate; pouring when the agar has cooled but not starting to set will help, but another useful method is to stack the poured plates on top of each other after you have poured them. Condensation forms by the hot steam from the agar condensing on the cool lids of the containers, and this can lead to rapid bacterial contamination if bacteria are introduced to the culture media.

Agar: Want to Start Simply? 

Of course, given the internet of all the things, you can skip the whole process of mixing the agar and pouring the plates yourself and, instead, seek out a variety of types of pre-poured agar plates (from PDA to MEA through to Black Carbon Agar). Depending on where you are, you can search Amazon, eBay, or keep an eye on local bulk mycology suppliers.  

Germinating Spores

Spores come either as prints, spore solution in a syringe, or in a form that is gaining popularity—collected on swabs. You can also gently scrape the gills of a mushroom to collect spores, but this is not recommended given the likelihood of contamination, unless the mushroom were grown in a sterile environment.

The best way to grow spores is by adding a small amount of the spores to the agar and carefully spreading them across the surface in a way that makes it easy for you to observe germination. If you spread the spores across the whole surface of the agar, germination becomes harder to track. You can squeeze a drop or two of your spore syringe onto the agar, and spread it across the surface of the agar, or you can scrape up a little of the spores from the print, and as with the solution, then spread them across the agar. With swabs, you gently skim the swab across the surface.

Start with a single point near the edge of the plate, and from that point, create three or four  lines radiating closely from that point. Turn the plate 90 degrees then make three or four more close lines that overlap the first lines, then turn 90 degrees again and create another three or four lines. With the swab, this will spread spores across all those lines. With the solution, with each set of overlapping lines, the solution is diluted, making it more likely that the spores that germinate along the third set of lines will be your desired spores.

You will need to keep the Agar sterile and clean. After adding spores or tissue culture to the surface of the agar, seal the Petri dish well by using the cling wrap to seal it tight.  Laboratories use Parafilm, but cling film wrapped around the Petri dish a couple of times is less expensive and adequate for household situations. Sealing the cultures keeps out dust and more importantly, mites and insects, which will readily introduce contamination. Storing in self-seal bags also adds an extra layer of protection.

Store your plates at room temperature, 18 to 26 degrees C. In a container on top of your fridge or hot water cylinder will work well.  Lower temperatures will only slow the mycelium down, whereas warmer temperatures can promote contamination – Trichoderma for example loves temperatures over 40 degrees C! Otherwise you can use a horticultural or reptile heating mat to keep them warm.

Label each Petri dish with the species name or your code name (something meaningful to you), the date, the media used, and any other significant notes. If you start to develop an extensive culture collection, it is easy to lose track of the details. If you have housemates and are using takeaway sauce containers, the labels help signal that the master cultures in your fridge are not a food product.

Working around contamination

The hardest part of working with agar is working around contamination. Keep an eye on the plates: In time, the spores will germinate (seven to 14 days to be visibly growing), and contaminants will likely be present as well. You should be ready to cut and move mycelium at any point if you notice contamination occurring. 

Mycelium of most mushroom growing species are white and thread-like in texture, sometimes growing evenly almost powdery, or at other times as long thick mycelium referred to as “rhizomorphic.” Even on the same plate the one species can vary in how the mycelium grows – if in doubt, select the stronger rhizomorphic mycelium. Admittedly, it can be confusing telling them apart from contaminants! The mycelium you are watching for will begin as fine clear hyphae, that in time will appear as white tufts that then begin to expand – these should appear consistently along the streaks, enough so that you should observe the differences if another kind of mycelium appears – thick white spots that turn green, grey cobweb like tufts, yellow dust, or little grey hairs forming black balls.

Contaminants become easy to spot in time (as was discussed in the mouldy mushrooms article), they germinate quickly—often more quickly than the spores you are working with—and mature quickly (change color and texture as the spores mature), so you need to be ready to transfer mycelium if and when this happens. You can use the coloured agar method, or backlight the agar plate to observe the fine hyphae as they grow and develop. It’s a good idea to have some peroxide agar plates on hand to move the mycelium onto a clean culture medium (we will learn about peroxide agar next). 

On the path to agar wizardry, it is guaranteed that you are going to end up with some funky agar plates—and not the James Brown kind of funky: Your housemates may complain about those weird things in the fridge, but it’s all part of the fun.

If you are working with spore solutions and they consistently contaminate on agar, use the Dilution Tek and then streak well across the surface of the plate. The Dilution Tek is to add 1ml of your spore solution to 9 ml of sterile water and try on agar. You can dilute multiple times: With each dilution you increase the probability of spores germinating without contaminants. Keep in mind that you also need enough spores so that the full fungi life cycle can continue, as single spores cannot form mushrooms and must join with compatible hyphae in order to form mushrooms and reproduce.

If you end up with no contaminants, well done! Otherwise, keep an eye on good healthy mycelium developing, and transfer them to new plates (it always helps to keep a few for spares) before the contaminants spread too far. If the mycelium are combined (the contaminants and the mycelium you are cultivating are mixed), cut away the leading edge furthest from the contaminants and move to a new plate.

One thing to be cautious about, is that until you fruit your mycelium it can be hard to know for certain (unless you sequence its DNA, or perform in-depth microscopy) that it is the species (or strain) you think it is. Contamination, mislabelled spore prints or spore syringes!

Tissue cultures

Mushrooms are made up of specialised hyphae, and given the opportunity, these hyphae will return to their exploratory form, seeking nutrients and negotiating their environment. By finding the cleanest hyphae within a mushroom, you can take a small sample, place it on agar, and it will start growing. This is referred to as tissue culture: using the mycelium from fresh mushrooms to create new viable cultures.  

The overall quality of the mushroom (how fresh it is), and from where inside the mushroom the mycelium comes from, will contribute to your success. Working with a specimen you have grown in a clean environment is very easy and may result in minimal or no contamination. With foraged specimens, this can become a lesson in frustration and perseverance—where you will have to become creative with your agar technique.

One of the best agar Teks is the Peroxide Agar Tek, invented by Dr Rush Wayne. Hydrogen Peroxide (H2O2), is a powerful oxidizing agent often used as a bleach or, when very diluted, used to clean cuts and wounds. It can also be added to Agar to kill spores and slow down bacteria, i.e. any spores, so this Tek cannot be used to germinate spores. The addition of Hydrogen Peroxide to agar as it cools makes it “functionally sterile”. Because hydrogen peroxide is produced within cells as part of their metabolism in minuscule amounts, mycelium (and some bacteria) can adapt to the larger concentrations. You will notice the mycelium gets “shocked” if you transfer it to peroxide agar and take a few days to recover.

Read: Mold on Mushrooms: What Every Shroom Grower Should Know about Contamination

You can use any agar recipe with a 3% H2O2 solution, adding 1% of the volume to make the water or agar functionally sterile. For the 200ml recipe, you would add 2 ml of hydrogen peroxide (syringes are perfect for this). Heat destroys hydrogen peroxide so use a thermometer as you watch the agar cool and when it gets to between 45 to 55 degrees, add the H2O2 and mix in well and pour quickly.

To dissect the mushroom, use a jet flame lighter to sterilize a knife or scalpel. Cut the mushroom in half, and take a small sample from the fleshy triangle above where the cap and stem of the mushroom join. Place the sample onto the peroxide agar. In time you will observe the tissue sample becoming hairy and then reach out and start growing across the agar’s surface. Keep an eye out for contaminants, and be ready to resample if you observe unwanted contaminants. Try to take as many tissue samples as possible in case of problems. Especially in the case of foraged specimens, you may lose a few or a lot, but even if only one out of ten survives, it’s okay, as you now have a tissue culture. 

Beyond selecting and moving mycelium, there are various tricks, especially if you are dealing with bacteria. Working with unwanted fungal contaminants can get tricky! Novel methods of working with agar include pouring warm agar over your tissue culture, or agar layering, as mycelium can grow through the agar not just on the surface of the agar.

You don’t have to use the peroxide agar tek to do tissue samples; you can place them on standard agar under sterile conditions being very clean as you go, just as you would with spores on agar. The power in the peroxide tek is that as long as you keep things clean, you can do tissue cultures outside a Still Air Box (SAB), or even outside in the field or the forest (spores that land on the peroxide agar are killed by the peroxide). If the mushroom itself is contaminated, this is going to cause other problems. Fungi such as Cobweb Mold are pathogenic on many other fungi and will spread very quickly.

As a side note: If you are working with wood-loving mushrooms, oyster mushrooms, or Psilocybe azurescens, for example, you can start the process on cardboard, by taking a small tissue sample and placing it on a piece of cardboard you have boiled for 20 minutes.  Many contaminants do not grow on the cardboard itself, but keep in mind as with peroxide agar some contaminants do grow on the mycelium and may take a while to become visible.

From Mycelium to Infinity

In time the mycelium will cover the entire surface of the agar. You can keep your fungi cultures alive indefinitely by taking sections of the leading edge of the mycelium and moving them to new agar plates. The leading edge is often the strongest and most healthy, so cut small triangles or squares from along the edge to transfer to new agar plates. Then when the growth is going well with no visible contaminants, you can store it in the fridge. Transfers can help identify the purity of the mycelium as you will often see the contaminants run from the transfer reasonably quickly if they are present.

It is advised that for each transfer you use a different media, e.g. MEA to PDA, then back again, to keep the mycelium active and able to adapt to new substrates and nutrients in various forms. From agar cultures, you can then make liquid cultures, or you can use the agar to create what is called spawn by adding small triangles of the agar to rye grain, corn seed, or wild bird seed.

Over time, if you keep performing transfers, you will notice the mycelium becoming weaker.  A good practice is to have a master culture. One that you know is pure, kept in cold storage, and from which you can take small samples to then re-plate for inoculating your liquid cultures or grain spawn.

Working with agar can be a challenge, but well worth the persistence. Best of luck and may the spores be with you.