Multispore and single strains

A lot of people that start growing mushrooms start running into the same questions and problems. After a number of successes growing, a lot of people wonder why mushrooms of the same ‘flush’ don’t grow equally. They all seem to have their own natural rhythm. Also, there are large differences in the way the mushrooms look. Some are big and healthy, but not all mushrooms grow up to adulthood.

The most important reason for this is that most home growers inoculate their substrate with a spore syringe. Inoculation with a syringe is called “multi-spore inoculation.”

Every spore-syringe contains thousands, maybe even millions of spores, from the same spore print. All these spores, in theory, they can all germinate into a unique genotype with their own characteristics. So, when inoculating a substrate with a spore syringe, a large number of different genotypes end up in the substrate. Every unique genotype that grows to be a mushroom has its own characteristics when it comes to speed of growth size, and looks. This explains the unregular flushes and why one mushroom can look much nicer and healthier than the other.

The more experienced grower, therefore, looks for the stronger genotypes and inoculates his substrate with a so-called “single strain” or “pure strain”. To understand this first, you need to know more about how you can separate the strong genotypes in the mycelium from the weak genotypes.

When you’ve inoculated the substrate with a spore-syringe and large parts of the substrate are colonized, often you can distinguish two different kinds of mycelium. One kind looks somewhat fluffy/down-like; This is called Tomentose Mycelium. The other has a wiry-type structure; this is called Rhizomorph Mycelium. This is the kind we are looking for. Rhizomorph Mycelium has a larger potency to grow into nice, healthy mushrooms than Tomentose Mycelium.

To make sure that only rhizomorph mycelium will develop, it’s possible to take a part of rhizomorph mycelium from a colonized substrate. This part of mycelium can serve as the inoculant for another container of a sterilised substrate. If you took a good part with a strong genotype of the mycelium, within the new substrate will mostly grow healthy and strong mycelium.

But when a container is largely colonized, it can be very difficult to select the best mycelium from it. The risk of contamination is very large; so it’s not advisable to try.

Growing the mycelium on agar in Petri dishes first offers a solution for that problem. The agar media is not a fertile soil for growing mushrooms, but the mycelium develops very nicely on it. Therefore mycelium that grows in Petri dishes can only be used to inoculate a substrate where it can develop further.

Before we’ll continue with why and how agar can be used, we’ll show how you can make this cultivation media from agar.

Throughout time it has appeared that there are many different recipes that can all lead to a good result. These agar media recipes can be bought, ready to use, in local stores or through the internet. It is also not that difficult to make these recipes yourself.

Mix the dry components well together and add the water to it. It is best to do this in special ‘laboratory-bottles’, the ones with the long narrow necks. These bottles are easy to use and have a narrow neck which reduces drastically the risk of contamination. Of course, you can also use a different kind of jar or glass.

Cover the bottle well with a piece of tinfoil and mix it again thoroughly. Place the bottle in the pressure-cooker and sterilize the agar media for approximately 40 minutes.

When the pressure has completely left the pot, you can remove the bottle from it. The agar media is close to boiling, so be careful. Boiling agar can cause nasty burns.

It is now important to pour the agar into the dishes at the right moment. If you wait too long, the agar starts to clot. This means it’s not easy pourable anymore. This situation is not very handy.

If the agar is too hot when you pour it into the dishes, this causes another unpleasant situation. It will start to condense within the Petri dish. Condense is harmful to the growth of mycelium and it also prevents you from seeing what is going on inside the dish.

The right temperature for the agar to be poured is about 40-50 degrees Celsius. When you’re able to hold the bottle for about 10-12 seconds without it being painfully hot, then you know the temperature is right

Place the dishes next to each other in piles of about 10 or 20 pieces and remove the tinfoil from the bottle of agar. Take the whole pile and pour the agar in the lowest dish, then continue to work upwards. Let the dishes sit for a while so the agar can clot. When it is completely clotted, the dishes are ready to use.

Agar is very susceptible to contamination. Especially at this stage, it is very important to work very clean and with a steady and efficient hand.

Now you know how you can make agar media, we can continue with explaining the advantages of the usage of this agar media in Petri dishes.

When your Petri dishes with agar media are ready, you can inoculate them with some spores. Take the printout of the zip bag with a sterilized tweezers, open de Petri dish and scrape some spores with the sterile scalpel so they fall on the agar media in several places. Close them quickly again and place them in the incubation room at 28 – 30 C.

After a few days the spores will germinate and the growth of white mycelium will be visible. Because the mycelium on agar media grows in a flat, 2-dimensional way, it’s possible to extract a part of mycelium with a scalpel and place it in a new dish with agar. So cut out one or more of the germinated spores and place each of them in the middle of a new dish. Close the dish quickly.

After a few days, you can already distinguish the stronger and the weaker parts in the structure of the growing mycelium. Look if there are any sectors of mycelium which are rhizomorphic.

If you’ve made the right selection of mycelium, the new mycelium will grow stronger and larger, compared to the first dish you used.

This process can be repeated a number of times. After a while, you will notice that the mycelium doesn’t grow in “sectors” anymore. Now you have your own “pure” or “single” strain. From this single strain, you can inoculate numerous containers substrate or spawn by cutting a small part of mycelium from the petri dish, and putting this in the new substrate/spawn. In these jars, there will be a development of rhizomorph mycelium of 1 specific genotype. All the mushrooms that develop from this strain will all have the same properties in terms of growth and looks.

The procedure of moving parts of mycelium cannot be repeated too often. If you select sectors too often after some time degeneration will set in. The mycelium in the agar dishes will then produce more down-like/fluffy sectors again. Degeneration has to be prevented.

In general, it’s advised to make no more than 3 parts of rhizomorph mycelium from the same mother culture to petri dishes. 5 times is the absolute maximum for most kinds.