As a beginner, pouring agar can seem daunting. This is because pouring agar improperly can cause fast, irritating contamination and be very disheartening. For those who don’t know agar is a naturally occurring gelatine like substance extracted from seaweed, once mixed with nutrients it provides a growth medium for all sorts of microbiological applications. In mushroom growing, agar has many uses, it can be used to isolate cultures from contamination, test a cultures viability, expand existing cultures, isolate single spores, and preserve cultures for long periods of time among a myriad of other uses. One of the most common agar recipes used by mushroom growers is MEA.
Malt extract agar is a simple solution of 20:20:1000. 20 parts agar, 20 parts malt extract and 1000 parts water. This is because of its simplicity, ease of procurement and mushrooms general love for malt. I almost exclusively use this recipe since it has always provided me with great results, I have tried many other recipes and the only one I alternate with is PDA, potato dextrose agar.
The liquid agar is normally sterilised in a pressure cooker at 121c for about 15 minutes. Once cooled to around 40c it is poured under sterile conditions, either In-front of a flow hood or in a SAB (still air box), into a sterile receptacle, usually Petri Dishes. Once set, these are then used or sealed up and stored for later use. Improper pouring can cause contamination to show itself very quickly on the uncontested agar. The most common causes of contamination come from pouring in unsterile conditions, into unsterile receptacles or improper storage. The types of contamination vary greatly but more often it appears as a green sporulating bacteria or a white ‘wet spot’ looking bacteria.
The lowest risk agar method is called the ‘No Pour Tek’. This method doesn’t directly expose the agar to a potentially unsterile environment, reducing the risk of contamination. The basic steps are; find a suitable receptacle either glass or polypropylene, fill with liquid agar, seal it up, sterilise it, allow to cool. This method, as you can see, doesn’t require any exposure outside of the sterile environment. I make a slightly different version of the ‘No Pour Tek’ pots which include an injection port. This way I can test my liquid culture without having to fire up my flowhood and clean my lab down. In short, it’s a cheap and fast way to test your cultures.
There are thousands of different jars, pots, and dishes that you can pour the agar into. I use Polypropylene deli pots from Amazon, you can get them here. I use them because they’re cheap, washable and haven’t let me down yet. Whatever you choose, make sure the mouth is wide enough to work in with a scalpel if that is your desired goal. Avoid containers that are too deep as this would make it very tricky to extract your culture later on.
You don’t have to make your lids the same way I do. However, if you’re using plastic and not glass, I would suggest the minimum you do is to include a small breather port. During sterilisation the agar may boil over inside the container, not really a problem if your container is glass with a screw on lid, however, if you’re using plastic it can blow the lid off and leave you with a bit of a mess, adding this small breather prevents that. For mine, I make 2 holes one for an injection port and one for the breather. I start by punching a ¼” hole in the centre for my injection port. Make this smaller than the injection port, that way you can squeeze it through, and the injection port will self-seal around the hole. You can get these ports in my shop, here.
I then make a 3/32” hole somewhere around the edge for the breather port. I use this punch tool for mine but you could easily use a hot needle, paper punch or even a belt buckle punch, just make sure it can punch the hole big enough.
Next, I squeeze the injection ports through, you can see it bulging out, it’s always better to undercut the hole size and squeeze these through.
I then tape over the breather with micropore tape. If you wanted to get a bit fancier, you could secure some Tyvek over the hole with micropore tape, but the tape alone is fine. This hole isn’t for gas exchange for the mycelium, it’s for the cooking process, there is plenty of air inside the container for the mycelium to grow.
Sometimes these deli-pots aren’t the cleanest and can have residual tags from the injection moulding process. Make sure you rip these off as they may cause the lid to seat improperly.
Like I said above, I use MEA at 20:20:1000. 20g Agar, 20g Malt extract and 1000ml of water. You can buy MEA kits in my store, here. Simply blend, in the correct ratios, with hot water until the malt and agar dissolve. With MEA, there will always be bits floating around, these are just crystallised/burnt sugars and nothing to worry about.
I pour these dishes about 5mm – 10mm deep, depending on how steady my hands are, these pots are 100ml and I pour around 20ml in each. Pouring too deep is a waste of agar as it’s unusable by the mycelium, while pouring too shallow will cause the agar to curl up and dry out prematurely.
Place them in your PC and sterilise for 15 – 20 minutes at 15psi. Don’t stack them more than 2 high as the heat and weight of the pots on top may weaken and distort the plastic, If you want to fill your PC with these, you can use a round cake cooling rack like these (just make sure it fits your PC’s diameter), and use a suitable item as a spacer, I use some spare glass jars like these for spacers. I also use those glass jars as the spacer for the bottom of my PC too!
Once sterilised, allow the PC to cool naturally. Force venting will definitely blow the lids off the deli pots even with the breather holes. Make sure the PC is on a level surface, so the agar solidifies level. Allow the PC to completely cool to room temperature before removing. The heating process warps the deli-pots as the plastic softens, as they cool, they reform their shape, however, they also draw in air as they cool so leave them inside the PC where the air has been sterilised. It also helps to reduce the levels of condensation inside the container.
Once cooled run a strip of parafilm or micropore tape around the lids to join them to the bases, this avoids them accidently being popped off or drawing in contamination. Unless you’re going to immediately use them all, you’ll want to store them. I tend to keep 1 or 2 pots in my incubator for a few days to grow out any contamination, if there is any, as a control. The rest I store anywhere really, some say store in a fridge but since they’re sterile and sealed it shouldn’t matter where they’re stored. Use them as you would any agar plate. Grow out your spores, test your LC before putting it on grain, transfer cultures, or store your cultures for a short while, the choice is yours. I hope this little guide helped; any questions just ping me in my email! Cheers!