How We Inoculate Mycelium in the Lab | Clean Room Mushroom Cultivation
So I'm just, uh, getting ready for a lab day. I'm gonna go in and do some inoculation of, uh, grain spawn, and then we're gonna do a few different, uh, a few different ways of inoculation. I'll do some inoculation with some liquid culture and some agar dishes, as well as some grain to grain transfers. So some of this spawn was inoculated about a week ago, and we kind of see the mycelium starting to really take over the grains. I mean, at this point, it'd be a good idea to shake the bag or break up the pieces or the colonized pieces to redistribute them throughout the bag, uh, and that way it'll really speed up colonization.
We should see full colonization at this point within the next few days, kind of like this bag right here, but this one was just shaken up the last couple days. Now we have reached full colonization basically, so some of these will be taken and used to expand the mycelium. We have the bags labeled as G1. That means that I've taken, uh, basically grown out the culture on a petri dish and taken agar wedges and transfer them into the sterilized grain, which is soft red winter wheat. Now that it's fully colonized, we want to expand it further and basically expand it about 10 times.
So we'll turn this into 10 more bags, and they'll all be labeled G2, and with those bags, we can utilize them to start inoculating our production or, you know, making, uh, making grill kits or whatever. So just taking a look at which bags I'm going to be utilizing. We've got some Reishi, some oyster mushrooms, actually a white elm oyster, and again, we have some bags that are almost full colonized. This one was inoculated 9 16, so it's been about 8 days, and this is the pink oyster. This was actually inoculated with a liquid.
As we could see, the mycelium is growing throughout the bag pretty evenly, so it's about to finish up. You could shake this, speed it up, or you couldn't. Same with this one. We'll break up this mycelium to speed up the colonization. We have some oyster mushrooms.
So yeah, I'm just going through my spawn, monitoring the growth, and basically shaking as needed or kind of redistributing the mycelium. Here we have some fully colonized G2 king trumpet, so we could just see a really nice healthy mycelium. Also going to get some some petri dishes out and utilize these to inoculate some grain, and this is actually going to be a cordyceps inoculation. Now we'll make our way into the laboratory. I usually like pre-print out my labels for the day, and I'll just kind of have strips of them that I can just slap on bags.
That way I'm not, you know, I found that it took a lot of time when I'm using markers to, you know, you got to pick up the marker, open it up, right on the bag, close the cap, put it down, and that kind of gets repetitive after doing hundreds of bags a day. So I just print out a bunch of labels, and then just keep them next to me, and I just slap them on bags. That way I'm not, you know, using a bunch of markers and wasting, uh, time, just trying to save time really. That's what it's all about when you run a, when you're working on a mushroom farm. Every second of the day, you know, kind of counts, especially when you're, you know, in the lab, and you gotta get get to other places.
So so I'll just put G2 on my label. That way I know it's generation two to go ahead and use that for our production, and I'll just make sure I put the species or the strain, whatever I'm growing, uh, on the label as well, so I know exactly what it is. You don't want to mix anything up, and make sure you get the date on there as well, and looks like I'm gonna be doing quite a bit of these. Guys, today, so I'm gonna just spray down my preachy dishes, just to make sure that they are sanitized before I do start conducting my inoculations. All right, so yeah, we have some bags of soft red winter wheat berries that have been hydrated, and they've also been supplemented with proper nutrition to basically sustain cordyceps mushroom production.
So really going to be cool. Yeah, like keeping one of these wash bottles handy, make sure you can kind of wash your hands with a little bit of rubbing alcohol between transfers and after, you know, after every certain amount of movements, you always want to re-sanitize your hands, wipe down your work surfaces, just to maintain a sterile technique. So we'll heat up our scalpel and our back die cinerater, go ahead and open our bag into that laminar flow. So when I'm conducting my inoculations, I try not to breathe. So, um, breathe directly through my nose, not through my mouth, and also while I'm transferring, I try to hold my breath in between transfers, so that way I'm not contaminating any of my work or breathing any kind of bacteria onto my sterile cultures.
One of the things I like about utilizing the bags for like grain or tissue transfers is that once you drop the agar wedges or whatever it is into your spawn container, you can actually break up the tissue further with your hands or your fingers. That aids in the spread of the mycelium. So we've got a nice inoculation there, and, uh, yeah, so this will be cordyceps mushrooms, if all goes right, growing on soft red winter wheat and supplemented with the proper nutrition and, uh, what not to actually cultivate the cordyceps. So we push our plates right up close to your right up close to the airflow as possible. So the plan with the cordyceps is to basically lay the bags out flat like this, allow the mycelium to fully colonize, and then once, uh, mushroom production starts to happen or I start to see fruit bodies forming inside of this, I will cut it open and probably have it inside of some kind of little little tub or tote to maintain like humidity and whatnot, and we'll see what happens.
So I'm working with three different three different strains of cordyceps today, cordyceps to see kind of which performs the best in my environment and what grows the best with my conditions. Cordyceps requires a unique diet, so the green is hydrated, and then in addition, uh, supplements are added, like nutritional yeast and stuff like that, to really help boost the mycelium and the actual production of necessary compounds that are medicinal within the cordyceps. Just break up those pieces of agar, just opening up my bags, making sure to use the the outside of the bag, never touching the top or the inside of the bag. So usually I'll use one peachy plate to inoculate up to 12 pounds of fresh sterilized. So in this case, I'm using one petri plate to inoculate six pounds of sterilized grain, just to kind of speed up colonization, and since I'm just kind of running some trials at this point, I'm just really looking to test out this strain.
Now I'm going to go ahead and give the bag and I spray down. This is basically just to clean off the bag from any any dust or anything that could have settled on it, even though it was growing in a sealed environment. You always want to make sure that you're better safe than sorry, so, and at that point, we'll break up the grains. In order to pour bits of grain into different bags, you need to break it up first. Now we'll get ready to start our grinder grain transfers.
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Just kind of make a nice little little spout for you to pour your grain out. We kind of just check it for any any leaks. Just grab the bag and kind of put some pressure on it. If we're good to go, grab our label, just kind of slap it right on the bag, shake up the. Bag or break up the grains even more, give that nice even distribution, so each of the, or each little individual kernel that was inoculated in here, will become a a point, or a new point for growth or for leap off from mycelium to start colonizing even more grains and basically expanding out the mycelium.
And when I'm doing my pores, I always want my bag facing the filter, uh, instead of like having it turned towards me or whatnot, minimizing any chance of anything to get into your. So each bag realistically gets about about a point six of a pound, or a half a pound of grain. So we make about ten bags per bag. You could make up to 20 if you really wanted to, but figure it on, uh, better, you know, faster colonization rates. I think that's better from in my opinion.
Yeah, you currently see me really, uh, using a lot of equipment to be able to manufacture or produce this grain spawn, but when I was young and I first started out, I would basically utilize little, uh, like mason jars as my spawn containers, and those enabled me to work without a flow hood, uh, basically, which is these these big filters sitting in front of me. They provide like this stream of sterile air pushing out at a little bit of force, so any kind of contamination is pushed out of the way, so I'm able to do all this sterile work really comfortably. But for years, I basically didn't have any of this equipment. I started growing at 16 years old, and I basically would find things around the house to create a little sterile environment to work in. And I remember my first glove box was kind of just like a cardboard box lined with foil, aluminum foil.
Then it had two holes cut in the front, and it had some household, like those those really long cleaning rubber gloves, those vinyl ones. So I had those taped into the inside, and then I covered the top of the box with like a clear film, like one of those things that when you buy like a like a comforter, you get like those zip up little plastic things. I covered that and was able to do my transfers in there. That was kind of like a first rough glove box, and it worked fine. All inoculations were done into jars, so really didn't need the flow hood at that point.
I wasn't opening any bags, and everything was done with a syringe, so I could flame sterilize my syringe until the needle's actually red hot. Then, while it's red hot, it's stuck into the it's inoculated or solution is stuck into the jar, and basically that's how I would do it. I would get really good results that way and very successful. But as you start to scale up and you want to produce more, you want to use larger containers, the glove box and the steel air box becomes really, uh, just doesn't really work as well, and I don't know, it's just not feasible at this scale. But yeah, if you're doing it at home, one of the main things too before you start inoculations into a glove box is to kind of turn off any ventilation in your home, making sure all air conditioners and all fans are shut off for at least an hour.
This kind of gives all the dust in your home and any airborne spores or contaminants chances to kind of settle on the ground. And when performing work in a steel air box, that's the whole point. It's still air. There's no moving drafts getting into the way of your work. Before conducting inoculations in a still air box, the inside of the stereo air box is sprayed down with either a soapy water solution or even an alcohol solution, and this kind of gets spores stuck to the walls of the steel air box.
You're able to kind of perform your work without, you know, you might get some contamination, but it's definitely, it's very minimal compared to just. Doing things freely, then when you get into working with the flow hoods, it can kind of decrease your chances of contamination, but it really all falls in with the with the cultivator and you're still sterile technique. I mean, you can still have flow hoods and still get contamination if you just don't have the proper technique of just your work sterile technique. That means, you know, breathing during inoculations shouldn't really be done, um, and the way your movements are placed, is your hand in front of your urinal? Is it in front of your open container where, you know, a possible spore or skin cell even can fall into your substrate and wreak havoc actually on your on your grow?
So yeah, for years I just used like mason jars to make spawn and, um, you know, it's a it's a great spawn container, honestly. I really just started working with, uh, using bags as spawn when I just started really scaling up and really just getting more demand. You know, there's pros and cons to using each container. The pros of using like the glass jars is the fact that they are actually reusable. You can reuse them time and time again for for years, actually.
The bags are basically single use, but the thing is, with them, they make it much easier to get, uh, basically bulk both grows going, and the co, the pros to this actually is that you can break up the grain with your hands. With the jar, you'll have to find yourself beating the jar of grain to try to get the clump of grain to actually break up, so that can actually be quite difficult sometimes, depending on how far along with colonization you are. So yeah, this is what I mean by being able to just massage the bag with the grain in it, something that can't be done with a jar. So if you get like a strain or species like an oyster or even a reishi, and you don't get to it within the, you know, first two weeks of colonization, then it's basically going to be impossible to inoculate your substrate. I mean, I found times where I had just let it go for three weeks and, you know, by the time you get it into the inoculation point, you can't break up the spawn inside of the jar and pour it into any of your bags or any of your containers.
So that's one of the things with using jars. You better, you know, be on time when it's time to start inoculating other other than that, it's going to be very difficult to to work with your material. One thing you can do to check the the potency of your spawn is to just give it a nice whiff. I mean, each spawn, you're going to recognize, has its own characteristics. Shiitake mushrooms will smell like fresh, or shiitake mycelium or spawn will smell like actually fresh shiitake, and with oyster, you get that nice fantasy smell, that fresh smell.
So I just kind of check just to make sure that, uh, you know, just I actually like the smell of it. So that's one way to check the health of your spawn, is to to give it a smell. If you smell anything rancid or sweet, then you know it could be a possible contaminant, but it just, it's just a habit at this point. I mean, I like, you know, like just checking on the smell of it and making sure it's good to go. Reishi's gonna have its own unique smell.
Really, it's just to check the potency of the spawn. That's why I do it, and I can't do it before I inoculate, or I don't want to, because I don't want to put my nose over some, you know, sterile spawn that's going to be poured into another substrate. So yeah, right now I'm just checking temperatures. Uh, that's something I usually do when I first come in, just to kind of see what the spawn is at. Typically, it's it's pretty hot in the laboratory.
It's usually Like, between 80 to 90 degrees in here sometimes. When I'm doing a fresh unload, it can get up to, you know, well into the hundreds in here, about 115, 116 degrees sometimes, just because I'm unloading really hot substrate to bring in here to cool down in front of, uh, you know, in front of the laminar flow hood, basically making sure it doesn't suck in any kind of contaminated air. But yeah, the temperature in here by the time we start inoculating usually is around 85 to 90 degrees, so it does stay pretty warm in the lab. During the winter months, it gets, it gets bearable. But all right, so we're getting some reishi inoculated into some secondary G2 bags now.
This strain of reishi is a super fast colonizer. Usually within five to six days, we'll find our substrates fully colonized, whether it be grain spawn or actually a sawdust production block. Somebody asked, can you fruit your mushrooms off of grain spawn? And typically, it depends on what species you're working with. Some species will readily fruit off of the grain spawn, and I found reishi mushrooms fruiting off of grain spawn.
I found oyster mushrooms fruiting off of green spawn, and even lion's mane fruiting off a great spawn, but some of the strains just won't fruit on grandspawn, like maitake, for instance, and shiitake, for instance. They just, they need the hardwood substrate in their diet to actually produce the fruit bodies. You can get a lot of mushrooms to grow on grain spawn, but although that the yields are typically not going to be as good as if you can grow them on the, you know, agriculture waste or the byproduct that you can find them growing on in nature. So, but yeah, there's some methods of growing on grain spawn where you could just take fully colonized grain spawn and actually, like, case it into a tray and, uh, put a casing layer on top of the grain spawn and basically get mushrooms to grow right out of a tray of just grain spawn. That can be done, but, you know, it's not recommended unless you're, like, just looking for a little bit of mushrooms in a hurry.
Strains like lion's mane and oyster mushrooms, you'll find them growing off of your own grain spawn if you don't get them into, if you don't inoculate with them or use them right away. The mushrooms that do grow on grain spawn for those species typically don't get as productive, and they don't look as good as they would if you grew them on their own, you know, natural substrate or agriculture byproduct. So that grain spawns in an important step into the production. Instead of going from, like, a liquid culture or straight from petri edition to, like, a, we would prefer to go grain spawn production block because, one, you get more inoculation points with the grain spawn. Each individual kernel becomes a vehicle to, basically, a vehicle to carry our mycelium into our sterilized substrate.
So with just, like, a small handful of these grains, you get, you know, 100 or so inoculation points. That really speeds up the colonization process, and also the added nutrients coming from the grain spawn, like the, you know, certain nitrogen benefits and starches and sugars that the mycelium actually feeds on and that utilizes, or mycelium will utilize the nutrients to boost yields and to get better production. So without grain spawn, you could, uh, another thing I do is I like to go grain spawn with certain species to sawdust, and then from there, the sawdust will be used as spawn as well. So I can go grain spawn to, let's say, 12 of these blocks, and then 12 of these blocks can get broken down and go to 120. So you can expand it further.
Those 120 can even broken down and turned into 1200, all starting from just one single petri plate. But the further you start expanding, the the weaker the strain gets or the lower your yields get. So I recommend going from grain spawn to sawdust to Sawdust and then fruiting, that's one of the ways that I like to do it, and it works very well, especially with shiitake. You get really good yields that way. I found with shiitake, the, you know, you over, you, you give them too much nutrition, and you don't get the fruit bodies that you could be getting if you just give it that perfect amount, which tends to be about 20 of a supplementation.
I've seen people, uh, you know, pour liquid culture into like a sawdust substrate and get colonization, but then again, the substrate's lacking the nutrients that's going to be carried over with the actual grain. So using grain spawn boosts your yields, increases colonization times, and those are really key points in using it. So yeah, if you're making grain spawn like this at home and you want to get started, best way to do would be just to use some mason jars, and you can do like a soak method, where you soak your grain spawn overnight, and, you know, 12 to 24 hours is sufficient. And then after that, you can kind of rinse your grains and lay them out to dry over a layer of paper towels or whatnot, and basically, after about an hour or so, once the grains are to the point where they're not too wet, they're not sticking together, you can basically like put a little bit of them on a paper towel, and they'll just roll right off, they're ready to be loaded up into your jars and sterilize.
And then, once sterilization is done, you can just take like a liquid culture syringe and inoculate your grain spawn, and then after that you. Can use that to a pasteurized substrate, or even a sterilized substrate, if you're doing it in, like, a glove box. See that? When opening the bags, just kind of opening it in front of that flow hood, or just to make sure I'm only sucking in sterile air, just kind of want to prop it up really nice so I can get a good, uh, opening to pour into. Usually, I just kind of count to three, or just eyeball out my spawn.
After a while, I really don't feel it's necessary to weigh it, or I don't like using any kind of measurement tools or spoons to dig into my spawn, so I'd want to minimize contact with it. So, when performing inoculations, you kind of just want to be quick, and, you know, whenever you open up your bag to Place the culture into your bag. You want it open for as little time as, and you just want to be very quick about, you know, getting everything done in a timely fashion. I would say this is the most important step, and this is, uh, this is the step that a lot of people can, uh, have more trouble with and, uh, other steps because it's, uh, you know, it takes a while to really develop a clean culture or just good spawn, and then once you get it to the point of production, you get to really see how well you did your work because that's the time where you're gonna either, you know, see, get good results or bad results.
So, yeah, without a good culture, without a good spawn, or just good technique, then it can be really rough. You