Mushroom Mycelium Tissue Culturing in Liquid and Agar Media
I'm gonna be taking a look inside of my, this is the spawn incubation slash culture media room, and I'm just gonna select out some cultures for today and take a look at some of my spawn. So yeah, I have some liquid cultures that I've been working on. Here we have some lion's mane and a couple oyster species. These have already been used to create spawn, of course. Yeah, examining some of the spawn that I have here, and this is one of our blue oyster strains.
This was inoculated just ten days ago, and it's looking amazing. Then we have some lion's mane that was just an ocular tat ten days ago as well, so these will be getting in to the lab and being used for to inoculate our production blogs. Shadow to Ryan Paul Gates from Terrestrial Fungi. I got some new cordyceps strains in here that I'm actually experimenting with. I haven't grown cordyceps yet, but I'm really excited to start growing it.
So yeah, I have seven different strains that I've played it out here from some liquid cultures. Today, I will be taking little pieces of tissue from these plates and dropping them into nutrient media broth to basically expand the mycelium and inoculate some substrates you assume to get cordyceps mushrooms. So I'm just going to be looking at each strain that I have. Everything's labeled on the back to actual species or genetic lineage to the strain, so I have a couple plates for each one. Pink oyster mushroom Petri plates.
This one is a tropical species, so I don't keep this plate in the fridge. Some constantly happen to do further tissue transfers to keep it actively growing and aggressive. So grab one of these and grab sake. This is a shiitake petri dish, so gonna do some transfers for that today too. That's about it for now.
Right now, I keep my facility at about 70 degrees throughout the summer time. I got to pay a little bit more for cooling down the facility and keeping the mushrooms at an optimum incubation temperature. I like to let the mycelium colonize at temperatures around 70 to 72 degrees, so we get the good optional mushrooms that we have. So get the flow hoods running. So we're in our lab.
We're gonna do some tissue transfers from some of the petri dishes I've selected and transfer them onto these fresh plates that I've poured out, and then we have our liquid media. We're also going to transfer some cordyceps mycelium into each of these jars and let the mycelium grow throughout the broth. We can further use that to inoculate brown rice substrate that's been supplemented with proper nutrients and stuff like that to sustain cordyceps fruit body development, so it would be really cool. Really, really excited to try that out. I'm just gonna be prepping right now, getting all my stuff ready for for my for my clean work to commence.
This is a back tyson orator or just tool transfer tool sterilization unit. Use it to sterilize my scalpels and syringes, anything that's going to come into contact with a sterile culture. So now I'm going to just be wiping down stuff with some isopropyl alcohol, cleaning my my work top to cleaning any of my surfaces. So yeah, this is just basically like a simple room, but it's like an 8 by 12 room that I've converted into my laboratory. At one point, I was just utilizing like a small walk-in closet at home that you can convert into a lab.
A lot of people can convert like pantries or small spaces to basically make an efficient lab space to do all your clean work, transfers, spawn production, you name it. So through the years, I've kind of just added an additional flow hood as my my workload grows, so now I have basically ten feet of this clean clean workspace to do transfers in front of, and it really allows me to to do a lot at once, which I really enjoy, and also it maintains a completely sterile room with HEPA filters basically pushing. Out 99.99% effective sterile air that's been filtered down to 0.2 microns. So good stuff. Also wipe down each of my plates to ensure that they are clean.
This one's I'll just bury down. I like to pre pour a lot of my plates, maybe like just do like 120 plates at a time, and then over the course of like a week or so or to all coming in and oculi those plates as needed. That way I don't mess myself up trying to prepare this agar media. Basically, this is prepared by mixing a malt extract agar powder into one liter of water, and then sterilizing in an autoclave for about 30 minutes. After that, it's poured while the media still hots into these petri dishes and allowed to cool.
Once it's cools at Gillette solidifies and it forms the kind of gels, and it forms this nice 2 dimensional surface that our mushroom mycelium can grow across, and we can select the healthiest spots of growth to basically get optimum results for our mushrooms. So from this thing takes a few minutes to heat up, so I'll let that heat up. So yeah, basically I have nutrient media broth. This is just like a light malt dextrose with about six to eight hundred million. It's been pressure sterilized, so basically ridded of any contamination or mold spores.
At this point it's completely clean. Will drop pieces of tissue into each of these jars. They're all equipped with a magnetic stir bar, so I can put it on a stirrer blade stir plate, and this will spin and break up any of the mycelium, basically further expanding it through the liquid. And then, once it's expanded to a point that I'm satisfied with, I can pour it directly into my substrates or suck it up into syringes through these self-healing injection ports. These lids are equipped with a self-healing injection port and also a syringe filter to basically give the liquid culture adequate gas exchange as the mycelium grows throughout the liquid.
Then also self-healing an injection port. So you can take syringes or whatnot and suck your liquid out and it'll heal right back up that hole so it's really cool and very useful for propagating mycelium and after I kind of wrapped my jars in a pair of film make sure that they're clean when I don't have my flow that's running sometimes it can be a pain to pull off on things but it's definitely aids in preventing contamination and stuff like that that's why I wrap all of our future dishes with a layer of parafilm around the edges I'm sure that no dust or contaminants can enter your culture so yeah I'm just gonna douse my tools some alcohol this is a scalpel it has a blade that you can change on and off as it becomes dull one thing you can do too is just what I do is kind of loosen my lids before I start doing my transfers that way you're not holding a scalpel with some fresh tissue in it and you're trying to Like unscrew a tight
lid at the same time. Scalpels red-hot at this point. Let's take off, just touch it to the middle to cool the school cooler blade, and at this point, yeah, we just dropped a couple chunks of mycelium tissue of cordyceps to this liquid bra. Set that aside. We'll get some parafilm.
Written protocol
Southwest Mushrooms Genetic Library
The strains themselves, the same cultures used in this footage.
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Though that on the ground, so we're not trying to reach towards the ground while we're doing our lab work. And we'll wrap up our dish that we just did the transfer with, and then also we'll also wrap up the rim of our jar lid. That's just because right now I don't have any gaskets underneath my lid, so I'm just using a pair of film. Take her freshly inoculated jar, we'll just turn on the stir bar, start educating the mycelium. Hopefully little fragments of mycelium fall off and get spread throughout the water and form like a chain of growth that we'll see over the next few days.
We'll keep monitoring our cultures and actually Coming in here and stirring them up for 30 seconds, 15 to 30 seconds per jar every day, until we get a really nice growth of nice mycelium. So we'll let that stir, because that's doing that. I'll restore lies my tool with the transfers are a plate right there, and we'll just continue the process of inoculating our color liquid culture. Alright, so when I'm doing my transfers and whatnot, I try not to talk because you don't want any, they can contaminate your work. It's, it's very likely contamination doesn't really happen too often here, and if it does, it's usually because of human error or, you know, I'm a vector of contamination.
You know, I'm the dirtiest thing in my lab, so before coming in, I make it like a routine to make this work the first start of my day, like after I've come from fresh shower and, you know, making sure that I'm hygienic ly clean, I can come in here and start doing my sterile work, whether it be the strictest of sterile work, where I'm making these cultures and growing out fresh cultures on petri dishes or liquid culture, to inoculating my production blocks in here. I maintain like a strict standard of sterility and also just the practice of sterile work and just movement and the way that transfers are done in a timely, cleanest possible manner. The plate right there so I can identify what it is. Just mix this one the same way. If you don't have flow hoods, a lot of people, or how I even started out, you can just do everything in like a still air box or just, you know, really you use strict standards in a clean working closet or something like that that doesn't have carpet.
I mean, I would say that of every, I mean, yeah, we don't really have contamination here unless there's like a hole in the bag or something broke, and then a contamination was able to escape into our our culture or our production block and a lot of thats thanks to just you know modern-day hepa filtration and a lot of this other school technology we have to grow mushrooms with so yeah cordyceps mycelium you can kind of notice the petri dish has like an orange hue to it and try to grow this coaster out in the complete dark because it's very sensitive to light and I know it can affect the pigments of the mycelium and then here next to it we have our pink oyster you can notice the mycelium is pink every mycelium is gonna be different for each strand so it's pretty cool you're gonna see these own unique characteristics to your mycelium and that once you become you know rather familiar with the cultivation process you can really identify strains just by looking at the mycelium so like this right here is the shiitake mushroom and you can notice this is actually an aged culture this is inoculated of March 9th and it starts to brown which we will see in our production blocks as well As it ages, the mycelium browns.
A lot of new cultivators will get scared and think it might be a contamination, but it's actually really healthy and totally natural. Before I had the sterilization tool like this, I could just take like a torch. You can torch your tools and torch them until they're red hot, and you start transferring, transferring. So yeah, grabbing a torch and or a lighter, some people use an alcohol lamp. You know, I mean, you'll have an open flame on your counter.
I do prefer this. It's a little bit more expensive, but it's nice to just have it on and being able to just go and use it as you need it. So kind of use our our blade as a little spatula. There's a lot of liquid culture recipes that you can use utilize to grow your mushroom mycelium. Right now, we're using like a light malt dextrose, but we often, you know, you can use honey.
One tablespoon for 500 milliliters of water is how you Know, it's always a good, good recipe that works. There's other recipes you can experiment with, like a potato starch recipe and stuff like that, so it's all about fun and experimentation with this. So for each transfer, we're reese terrorizing our tool, making sure that we're not gonna cross, cross contaminate any of our work. So this, this part of the process can be kind of intimidating to some people, but once it kind of started grasping more of the concept, it becomes rather easy and really, really fun. Inside the liquid culture, we're noticing like a war pull like vortex, and it's really works great for one, giving your culture aeration and helping basically spread the mycelium throughout the liquid.
If you don't have a magnetic stirrer, one of the things that can be done is just throw in a couple pieces of like broken glass into your jar and just swirling it up yourself by hand every day. Could just do that, and that works just fine, or you could even throw a marble in there or some old coins. Just rewrapping up these petri dishes or possible later use. You can come back to them if you ever need to, to you ever lose the culture or something, try to try to do some more work on it. I have all those pre poured petri dishes in there that you guys seen from yesterday, and I'll be taking these dishes that I've made and kind of transferring and furthering them two more plates.
If realistically, you can make up to 40 new plates with each plate, but I'll just be making like five or so. I'm actually going to go through and look at each plate and see. I have three plates for each strain. I'm going to look at the best one and then put the remained a remainder in cold storage. Um, basically, I'm deciding which one has taken over the plate fastest.
So like for this strain, we have double Sphinx number six times double Sphinx that were crossed with double Spinks number five, and I'm just gonna select this one. It has more areas to select from, and it's more grown out than this plate, so set this one out. If I ever need to go back to it, I can still utilize it. After pouring up my plates, I just used these sleeves to kind of cover them until I need them. That way, it keeps any dust from landing on your petri dishes or on my petri dishes in this case, and they store for a while.
I can come and use these whenever I need them, really don't have to worry about the hassle of preparing the stuff the day that I need it. So we have six plates. We'll have to do thirty little thirty petri dishes total. I'll do five petri dishes per plate. Just make a stack like that right in front of my flow hood, as close to my filter as possible.
That's where you're gonna, that's where it's going to be the most sterile. And also, I'll unwrap these feature dishes too before I start. These are the pre poured, uh, nanaka lated petri dishes that I'm unwrapping. And one technique for for transferring with your petri dish is called like a clamshell technique. Is where you take the dish and only lift up this much and close it right back up.
That's a good way to you utilize if you're just looking for a quick transfer, and that's what it's the best way to do it, honestly. We've got a lot of varying genetics. The cordyceps is new for me, and I got this, this cordyceps trans came from a breeder named Ryan Paul gates, and you can see the names of the strains are pretty crazy. So see what we could do here. Um, yeah, if you're trying to do Eggar at home, I mean, it is you do need a really dedicated sterile space for this kind of work.
So if you do have like a little flow cabinet, I'd recommend you either make one or you can confine the people That make them, you can buy them in smaller sizes as well. So yeah, we have like 2, 2 feet by 3 feet for these 2 filters, and then 2 feet by 4 feet right there, but they even make like a 2 foot by 1 foot one, so you could just do little little transfers at home, or you can build a still air box, which would just be a clear toe with two holes drilled in the front, and you can actually do this work inside of that still air box, being as so you take the necessary precautions to maintain a sterile technique and also a still air environment, a true environment of no, you know, just still air. You don't want any kind of floating debris or anything in your work. So when doing it at home, put all your materials in your still air box. It can be pretty tight in there, but do all your transfers in there.
Make sure that air conditioning is turned off in your home so you don't have any kind of drafts blowing in. But for people doing it at home, I'd recommend just finding like a, you know, a liquid culture that you can use. You can, you can get liquid cultures from from like a mycologist or another grower and a syringe that's already prepared for you. And with the syringe, you can basically just use a lighter or a flame to sterilize the tip of the syringe, and then you can inoculate a jar of your own liquid culture using like the honey, one tablespoon of honey, 2 500 milliliters of water, and then pressure sterilized in a little pressure cooker for 20 minutes, and then take a liquid culture. And after the flame, after you flame sterilize the tip and needle, inject it right into your injection port.
You only need a drop, and then from there you can grow out more of your own liquid culture. With that liquid culture, you can use it to inoculate your own green base substrates and to make your own spawn, so that's what I'd recommend for a home cultivator. It's a to really get started. Also, another recommendation for for people wanting to play with Petri plates at home is you can also buy pre poured petri dishes from other people or other growers and just do the transfers yourself. You don't want to pour your own petri dishes, or you just don't have the capabilities of making them at the moment, so that's another recommendation.
But let's say it's a little bit more advanced in the skill, and you know, do some reading and and some marks research before you really go crazy with it, or just go crazy and find out, you know, through trial and error like like I did. Now, the mushroom mycelium and a petri dish will grow flat across a two-dimensional surface, and then in a liquid culture we give it basically a way to grow out three dimensionally through all sides and in every way possible. And also, another thing about local culture is the fact that you can take just a tiny piece of tissue from a petri dish, drop it into a liquid culture jar, and really just expand the mycelium. I like to utilize the liquid culture for expansion. That would, to be honest with you, you can expand much more mycelium.
I mean, you can do it both ways, but liquor culture is a great way to expand fast, and it's really nice to be able to inoculate with liquid culture and just pour it into your spawn. You get much more coverage with the mycelium covering up all the all the grains, or really it shouldn't be only used to inoculate grain spawn, so you get a lot of coverage with the liquid spawn as opposed to just cutting some wet wedges and throwing it in the spawn, and you just get more points of contact with liquid. But both work great. I use both depending on the species, like reishi mushroom, I really don't even do look at culture with because it's just too. Too aggressive.
When I'm doing my transfers, I like to put the wedge kind of face down onto the plate. That way, the mycelium just grows directly into the agar and just giving it that little boost. If you can't get access to this parafilm, I've seen other people online will use like a saran wrap. I have never had to use that, but that's just an idea if you whoever in that situation. I would say, after every certain amount of every, like, 10 or 15 movements, do, like, I like to Reese terrorize my hands or Reese Anna ties my hands with some isopropyl.
This is our pink oyster. This plate needs to be transferred to some new plates because it's overgrown, so want to make sure that it's still viable and and ready to produce some mushrooms for us. All my pink oysters are basically just one specific strain that I'm growing currently. I've switched it up though over the past, but currently, I'm working with just one strain of pink oyster. Pink Boise is not a strain that I like, I mean it's one, I don't, it's really, it's kind of tedious.
You just can't refrigerate it, so it means that it's gonna grow out really quick, and it's gonna need a transfer here sooner or later. So like with the pink oyster, yeah, like every two weeks, every four weeks, I'm in here transferring it into new plates so it doesn't experience sentences sinesses where it basically stops growing and before it becomes a weak. Pink oyster mushrooms are really known to fruit aggressively. A lot of the times, you'll see the mushrooms growing right out of the petri dish, so it's a great strain for beginners as well. Because it's so aggressive, they can oftentimes, you find yourself fruiting it within ten days of inoculation to substrate.
Being here in Phoenix, Arizona, it's a great strain for me in the summertime. So during the wintertime, it's not so great. It doesn't do good. Like I said, if it gets freezing, then it basically dies. You can't put it in the fridge.
My grow rooms are kind of reliance on like a lot of the the wintertime and or just outdoor conditions. I, you know, it gets cold in my girl room during the wintertime, oftentimes between 50 to 60 degrees, and the pink oyster just does not like it. So it's summertime now, so here's the time to whip out this strain and bring it out. It has a fantastic color and dazzles plates, and people love it. From farmers markets to chefs, it's definitely an attention grabber for sure.
The talk is a classic strain that's probably the most cultivated gourmet mushroom in the world. It's also very medicinal as well, really helps boost your overall main function, and it's really good for you. So I enjoy growing shiitake. It's actually one of my favorite mushrooms to grow, and it definite definitely is one of the most labor-intensive mushrooms to grow also. Compared to most mushrooms, it requires more time for harvesting and more care, but I definitely enjoy the reward that it gives you.
As far as if you want to get into growing shiitake, it's it takes a lot longer to grow than let's say an oyster mushroom. I usually let my shiitakes incubate for two months. Then I can put them into fruit them. The fruiting it also likes a mostly an oak hardwood base substrate with not too much supplementation. If you if you give it too much, like let's say wheat bran, then you're likely to get mutated fruits, but once you get it down, I'd say like 15 to 20 percent of a supplementation to your sawdust recipe, then you will get really nice fruiting zuv top-quality shiitake.
It's a, it's one that likes, you know, it definitely likes attentive care. It likes, you know, a humid environment upwards to 85 to 90 percent at the beginning, and then towards the end before harvest, I kind of lower it to get a nice durable cap and to help preserve the shelf life and the Fridge, and also likes temperatures. It's a wide range strain, wide range mushroom strain, that I can grow all year round because it likes temperatures anywhere from 55 to all the way up to 75, 80 degrees. So I can grow this mushroom all year round. It's a great mushroom for me, and everybody loves shiitake mushrooms. It's definitely one that everybody's familiar with.