Introduction: Becoming With Schizophyllum Commune: Create a Sterile Culture From Found Mushrooms
This instructable is focused on explaining how to create a sterile culture of the mushroom Schizophyllum Commune (common name Split Gill mushroom) on a petri dish using found mushrooms. Schizophyllum Commune has been found to have over 28,000 sexes, can be found on all continents except Antarctica, and has the ability (like other "white and brown rot fungi") to degrade endocrine disrupting pollution (plastics, hormone mimicking industrial petrochemicals like the fertilizer Atrazine, the plasticizer Bisphenol-A, or the synthetic hormone Diethylstilbestrol).
I'll go over wild mushroom identification techniques, how to make a nutrient agar petri dish, and how to use your found mushrooms to grow a sterile isolated culture in your petri dish. Other instructables in this series go over techniques such as genetic identification of mushrooms, simple enzymatic assays to test the mushroom's ability to breakdown plastics and hormone mimicking pollution, and how to make cheese with S.Commune.
This project came out of the collaborative work of Mary Maggic, Paula Pin, and myself during a residency at HANGAR in Barcelona Spain fall September 2018. For more info about the project read on ~~ or skip all the Bla Bla and go straight to Step 1 for the tutorial :)
Images are from the Schizophyllum Commune Fanzine ~ files can be downloaded for free here
During the residency our three main goals were to:
1 ~ Research and digest the scientific literature on Schizophyllum Commune and it's various interactions with hormones and endocrine disrupting petrochemical pollutants. S.Commune is one of many "white and brown rot" fungi with the ability to break down hormonal and endocrine disrupting pollution. These fungi feed off of trees by excreting "lignocellulosic" enzymes from their networked bodies or "mycelia" and the enzymes are able to break down the cellulose and lignin that trees are made of. Because of structural similarities between cellulose, lignin, and EDS' / Hormones like Atrazine, BPA, Estradiol, Diethylstilbestrol, and Nonylphenols, the "lignocellulosic" enzymes secreted by the fungi can also break down a wide variety of toxicants. In addition to this, S.Commune produces immunostimulating polysaccharides that are helpful as cancer preventatives and treatments (Approved in Japan for treatment of cervical cancers), can enhance phytoestrogenic properties of certain plants through fermentation, can be used in place of rennet and lactobacilius in cheesemaking processes, and produce thrombin clot dissolving enzymes (useful in treating thrombosis). Strangely, these same "beta-glucans" that can be extracted with boiling water as cancer treatments and immunostimulants, are also used by the petroleum industry to enhance oil yields, tying S.Commune in a bizaar feedback loop to the production of the endocrine disrupting petrochemicals it is able to degrade and remediate.
2 ~ Develop DIY and DIWO (do it with others) protocols that simplify these recipies for working with EDC's and fungi to translate codified scientific knowledge into a language that is more accessible. Use these recipies for education, for questioning, for resistance to molecular colonizations, and creating new narratives about multispecies becomings and our collective mutagenesis.
3 ~ Challenge the dominant cisheteronormative, queerphobic discourse surrounding endocrine disrupting chemicals (EDC's). To quote Malin Ah-King and Eva Hayward's scholarship on the issues, "Many news outlets are reporting these frightening endocrine tales from our backyards. In an effort to foreground these issues—as we will describe in the following—media has gaslighted a Frankenstein metamorphosis that threatens sex and sexuality. Rather than addressing the many other health risks associated with toxic exposure, the most sensational and polemical issues stand in for debate and critical response. It raises questions: Why is sex more central than cancer, auto-immune disease, and even death? What cultural nerves (many of which are globalized), are triggered? And, for those of us with feminist concerns, how do we reorient the debate away from essentialism, sexism, and heteronormativity?" (Toxic Sexes).
Schizophyllum Commune is a unique "white rot fungi" in that it has been found to have over 28,000 genetic sexes. This adaptation, many theorize, has enabled the incredible genetic diversity of this species which exists on every continent around the world except Antarctica. We wanted to celebrate the beautiful sexual multiplicity of this organism as one example of the queerness of biology. The dominant discourse surrounding EDC's in the media, and even from environmental activists focuses heavily on unscientific narratives, that these molecules cause certain sexualities or genders that our society pathologizes, labels non-normative, and often seeks to eliminate or avoid. Not only is the sex-panicked, queer phobic focus of public outcry about EDC's scientifically unrealistic (hormones and endocrine disruptors do not make people queer or trans) but it is ignoring all of the devastatingly real effects that we can link to EDC's: obesity, thyroid disorders, certain cancers, among other health risks.
Step 1: Find Schizophyllum Commune Living in Your Community
Lab grade sterile cultures of Schizophyllum Commune can be purchased for around $350 from a microbiological culture bank such as ATCC... which is why we opted to culture from wild mushrooms for free :)
I had it easy because I had been reading about Schizophyllum Commune and just happened to notice it growing on a tree in front of my apartment one day. If you're not so lucky to accidentally stumble upon S.Commune in your neighborhood, you'll want to go to a wooded area to search for "bracket" mushrooms growing on sickly hardwood trees (trees that look like they're not doing too well) or fallen trees. S.Commune is one of the most common existing fungi around the world and can be found basically anywhere there is hardwood to grow on.
Mushroomobserver.org provides a great detailed description of where and when you're likely to find S.Commune, as well as how to identify it and how to avoid "look alikes" that might lead to misidentification:
" IDENTIFICATION: Schizophyllum commune is easily identified by its split gills, dense hair, and fan-shaped cap. Cap is 1-4.5cm wide and usually a shell or fan shape with a gray to whitish surface. Basidiospores are 5-7.5 × 2-3 micrometers. A similar mushroom known as the “Crimped Gill” Plicaturopsis crispa is not as widely distributed.
HABITAT: Schizophyllum commune grows on hardwood using enzymes to degrade lignin and causes “white rot.” So it is found in most any woody areas. It can be found year round due to its ability to dry out, rehydrate in better conditions, and then continue to sporulate.
This fungus can be parasitic on small diameter trees that are under stress from other factors, such as drought.
LOOK ALIKES: Plicaturopsis crispa or the “Crimped Gill” is similar to the Split Gill. It also has a shell/fan shaped fruiting body. However the cap is a reddish brown or yellow brown, and the underside is very distinctive. It has crimped vein like gills that are much different than the split gills of Schizophyllum commune. The Crimped Gill is less common but distinctive when found."
Step 2: Make Nutrient Agar Plates for S.Commune to Grow On
To isolate a culture of the S.Commune you're found you'll need some petri dishes filled with food for the mushroom. Malt extract Agar is a simple to make food for S.Commune, or "nutrient media" that can be made with easily accessible ingredients. It's also a good food source to use when doing enzymatic assay experiments with S.Commune and other white or brown rot fungi ~~ this process will be covered in another instructable.
SIDE NOTE: If you had trouble finding S.Commune, there are many other fungi in the same family that can be cultured using the same processes and are just as good if not better at degrading endocrine disrupting pollutants. Turkey Tail (Trametes Versicolor) has been found in many studies that looked at hundreds of white and brown rot fungi to be the highest producers of the "lignocellulosic" enzymes that degrade petrochemical pollution. Oyster mushrooms are also very good at this.
I do my microbiological work in a community biology lab called BUGSS (Baltimore Underground Science Space) where I have access to glassware, digital scales, graduated cylindars, an autoclave (for heat and pressure sterilization of the media), etc. Labs like this have sprung up around the world in the past decade, so you might want to check this list to see if there's one near you. If you don't have a great community lab like this in your area, you'll have to invest in some basic equipment for this process such as:
1 ~ A digital scale for measuring your ingredients precisely
2 ~ A pressure cooker to sterilize your media with heat and steam
3 ~ Petri dishes, can be bought online pretty cheaply, reusable glass or disposable plastic will work but re-usable is always better :)
4 ~ Some glassware for mixing your ingredients in, sterilizing in, and storing in.
If you don't want to invest in that equipment you can find pre-poured malt extract agar plates to buy on websites like Amazon ~ it will just be more expensive in the long run if you plan on continuing to do microbiological work.
There are already many great guides and youtube videos on how to make your own nutrient media petri dishes at home. So rather than go into great detail on this, I'll just link to a couple -- and provide the recipe for 2% Malt Extract Agar, which is the nutrient media you'll be using.
2% Malt Extract Agar (MEA) ~~ Recipe for 1 Liter:
20g Malt Extract (common beer making supply, can be purchased online)
15g Agar (solidifying agent - this is used to cook with often so it can be purchased online or at some grocery stores)
1 Litre H20 (I usually use Reverse Osmosis water which can be purchased at most grocery stores in gallon jugs)
This recipe can easily be modified if you want to make smaller quantities. I often make only 250 mL at a time for example, so I only end up using 5 grams of Malt Extract, 3.75 grams of Agar, and 250 mL of water.
Step 3: Innoculate Your Petri Dish With Fungi!
The mushroom you see is called the “fruiting body” it is only a small part of the organism. The majority of it’s body is hidden in the tree (or in the ground), consuming the wood. This part of the mushroom is called the mycelium. To culture Schizophyllum Commune’s white filamentous, tentacular, rhizomatic, mycelial body onto a petri dish, you'll take a sample of the stem, sterilize, and place on your MEA plates. Mycelium will grow from the sterilized mushroom stem core sample and colonize the petri dish, looking like a white cottony mat.
- The Malt Extract Agar (MEA) plates you prepared in the previous step
- 70% Isopropyl alcohol (rubbing alcohol) and a spray bottle
- Paper towels
- Scalpel, or just a stainless steel razor -- box cutter will work
- metal tweezers
- A flame -- I use an alcohaul lamp for sterilization of instruments, NonDisjunction has a great tutorial on how to make one here.
- Sterile surface to cut on (I just used a sterile petri dish for this)
- Parafilm, a paraffin film used to seal petri dishes -- you can also just use masking tape if you don't have this
The main thing you want to keep in mind when trying to use sterile technique is that bacteria and spores are continuously falling in the air, so you want to keep your petri dishes covered as much as possible. When you do lift the lids to pour media, or inoculate, it's always best to hold the lid just above the dish, allowing you to access the inside horizontally, but keeping the lid above the dish to prevent bacteria from falling in. Secondly, you are a giant conglomerate of human, bacterial, fungal, and other organisms ~~ although you cant see it, you are surrounding by a dense cloud of microbes that are diffusing off of your body. Rather than just sitting on the surface of your skin, your micro-biome extends out into the air ~~ sooooooo, don't ever wave your hands over the petri dish, or lean over the open dish to look in. And wear gloves if you have them ;)
SET UP YOUR SEMI-STERILE WORK SPACE
Choose a non-porous work surface clear of debris and junk. Spray the area down with your 70% Isopropyl Alcohol solution and wipe it down with a paper towel. Place your flame in the center of your work area and light it. You'll want to work as close to the flame as possible because it creates a small sterile field around it. Place your MEA plates to one side and your mushroom sample onto a sterile cutting surface on the other side. I normally use a sterile petri dish as my sterile cutting surface as well as a tool rest. Pour some Isopropyl Alcohol into a small glass -- you'll use this to sterilize your blade and tweezers while working.
*¡~ INOCULATE ~!*
Before doing anything, sterilize your blade and tweezers by dipping them into the isopropyl alch and then holding them over the flame for a bit. After sterilization, rest them on your sterile cutting surface if you set them down. Using the tweezers, hold the mushroom stem still and cut a chunk off the end of it. Take this chunk and cut all the sides so that you end up with a small cube of mushroom stem core with all the outer surfaces removed. This helps get rid of all the other microbes that live on the mushroom so they don't contaminate your culture. Then dip the stem core into the isopropyl for about 30 seconds, pull out, and let dry within the sterile field of the flame (don't use the flame to dry it, this will kill the mushroom). Flame sterilize your tweezers again while you wait for the stem core to dry. Then simply lift the lid of one of your MEA plates just enough to slide the tweezers in from the side, and use the sterilized tweezers to place the stem core in the center of the plate. Seal the plate with parafilm, and over the next few weeks, you should see S.Commune mycelium colonizing the plate.
I usually make several plates when working with wild samples so that if I do get contamination, I'm more likely to have at least one clean culture.
We have a be nice policy.
Please be positive and constructive.
It's been a while!
I'm having a bit of trouble with the project because in here it is so cold! I cannot find anything alive! We found some fungi on rotting wood and collected them, but they are hard as chips, and I am not confident they will go back to life. However it would be great to identify them (or just know if I should throw them away). We are trying to develop some project for our science fair, and we are making this DIY PCR
I was going to ask you about the genetic identification part of your project :) ... Just to know if you could give us a hint of what to do once we get finished our PCR machine
I attach the pictures of what we have found so far,
Thanks a lot! and best regards,
Amaya and June-Si (partners on the project)
If you really want a living specimen you could try to find a mushroom farmer locally, or you can order liquid cultures or petri dishes fairly cheaply online. Mycosymbiotics is a great trustworthy grower. They're in Pennsylvania and sell liquid cultures on Etsy for just 10 dollars each ~ they don't have S.commune, but they do have have over 20 other species to choose from. One of the oyster varieties would be great for dye decolorization, I had good success with pearl oyster. From what I've read all the oyster varieties have pretty high lignocellulosic enzyme production/secretion. Here's a link to the Mycosymbiotics Etsy store https://www.etsy.com/listing/234827841/liquid-culture-mycelium-syringe-your?ref=shop_home_active_3
Before doing PCR analysis of fungi, you've gotta extract their DNA. I found this protocol for a simple microwave extraction from mycelium ~ haven't used it yet but it looks promising. My only thought with the dried mushroom is that if it's super tough and dried out the protocol might not work as well as with tender, living mycelial mass. Here's a doc that I put together summarizing protocols for: DNA extraction, PCR amplification of of a DNA region called ITS region from fungi that is unique in different species, and performing a restriction digest for "DNA fingerprinting" with electrophoresis. If you don't have the equipment to create an agarose gel and do electrophoresis then you can skip the restriction digest and look for a local lab that will do DNA sequencing for you. Usually local universities will have labs that offer sequencing services very affordably. In Baltimore I usually outsource sequencing to a lab at Johns Hopkins University and it's only 6 dollars per reaction, so pretty affordable. https://docs.google.com/document/d/1QXOMzBIf8EbQDCFGZvVUKJN_JQeIdprzRgwQyA3J6aA/edit?usp=sharing
Thanks a lot for your answer!
The assays I found are called YES/YAS assays (for Yeast Estrogen or Androgen Screen) and they are produced by Xenometrix. http://www.xenometrix.ch/en/products/details/endoc...
In their websit I cannot see the prices but their official seller in UK (Cambio) sells one kit for like 285 pounds + a yeast strain vial for around 200.(https://www.cambio.co.uk/0/1487/132/products/xenos... That goes way out of my budget!
I think I'll go for the dye. Do you think they will sell it to a school laboratory? I can only think of my Bio teacher to place the order for me... Also, I wanted to ask for about how long does it take for the fungi to eat the dye, and if you think it is possible to control it with a spectrometer in order to get some obective measurement of the change. My school lab has some basic Pasco spectrometers and I think I could borrow them.
Also, did you test the whole hiphae system or did you isolate the enzymes? Some of the studies I've seen have done it in different ways.
Ah, okay. YES screen is good, just so you know - in case you eventually want to go further with this stuff, you can also buy a plasmid to make your own yeast estrogen sensor from addgene: https://www.addgene.org/23062/
But this would require you have the facilities to do a yeast transformation (to genetically modify yeast with the plasmid) in you school's lab. Probably too big an undertaking for what you want to do right now.
As for the Sigma order - I'm not sure if they sell to educators but it's worth a shot. I just did a quick search for "remazol brilliant blue reactive science supply" and looks like fisher scientific also sells it here https://www.fishersci.com/shop/products/remazol-br... I'd search around and check your options. Not all science supply stores require buyers to provide credentials for non-hazardous purchases.
And yes! You definitely could use a spectrometer to measure the dye degradation. You could do this by growing your mycelium in liquid culture rather than on a petri dish. In liquid culture, the action of the enzymes on the dye will be the same. Just when it comes time to load a sample into the spec, you'll probably want to centrifuge a sample and only put the supernatant into the cuvette so that the mycelium does not interfere with the optical reading. I had good results growing liquid cultures in baffled flasks at 25C shaking on an orbital shaker at 100 RPM, but have seen studies suggest 30C as the temperature for optimal growth https://www.ncbi.nlm.nih.gov/pmc/articles/PMC37415...
I didn't isolate the enzymes, this is a more complex task. I simply grew the mushrooms on agar solidified media containing the dye. As the mycelium grew outward from the center of the petri dish, a halo of decolorization grew in those species that were high producers of certain lignocellulosic enzymes.
It's me again! I'm sorry to be insistent. I've done a little bit of research today looking into the "testing whether the fungi does something to the plastic". I've found a couple of test kits but they are quite expensive and I'm not sure of what I need... I also need a good research question on this, like an aim or some application...
Idk, if you have a suggestion I'll be glad to take it!
Hey Amaya ~ what are the test kits that you found? I won't have time to write up the rest of the tutorials in this series for a while, but I can just summarize the lignocellulosic enzyme assay technique I used, and provide some links. The process is fairly simple in concept, the lignocellulosic enzymes that the fungi secretes are what break down endocrine disrupting chemicals and plastics. There are a few petro-derived fabric dyes that these enzymes are able to break down, and when they are broken down, their visible pigmentation disappears. So by adding these colorful pigments to the petri dish, or flask that you are growing the mycelium in, you will have a visual indicator of whether the fungi is producing lignocellulosic enzymes. The dye that I used is called "Remazol Brilliant Blue R," which I purchased from Sigma chemical company https://www.sigmaaldrich.com/catalog/product/sigma/r8001?lang=en®ion=US Sigma is tricky because they will only sell to "approved institutions," not individuals, so I had to get someone who runs a biotech company to place the order for me. Sigma is also generally very expensive, so I would see if you can find some through a different supplier. For the assay, I mixed some of this dye in with 2% malt extract media solidified with agar, and this is what I poured into petri dishes and grew the mushrooms on. I actually ended up seeing more results with Pearl Oyster mushroom mycelium decolorizing this dye than with S.Commune. There are also many other dyes that can be used...
here's a link to a 2% Malt Extract Agar recipe: http://himedialabs.com/TD/M1964.pdf
here's a link to a paper that describes decolorization of remazol brilliant blue by 3 different mushroom species, as well as many other dyes. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3564790/
Hope that helps, good luck!
This project is super cool! In fact I think I'll try to replicate it for a science fair at my school... I was wondering when you could continue the series of posts :)
Thanks a lot for sharing this instructable!