Mushroom Cult - spore cleanup video 4
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- Опубликовано: 5 окт 2024
- Welcome to the Mushroom Cult agar series.
This is video 4 of the spore cleanup procedure that includes a full summary.
This video is designed to help you think through materials and methods you might use in the process of cleaning up one mold contaminated plate.
This video is not a prescription for success. Beginners are encouraged to use a still air box as a minimum precaution.
This video contains minor technique errors and does not implement best practices. The reason for not using best practices like using a still air box for example is two fold. First, the box is not used to improve visibility in the video and Second, this is a creative endeavor. The main philosophy behind the procedure in this video is that layers of protection like gloves, still air boxes, and hazmat suits may be added on top of good technical execution but are not substitutes for good technical execution. Additionally, objective evaluation of the technique is entirely dependent on results and has very little to do with how the technique compares to trusted teks, dogmas, or even best practices. The results judge the technique.
Enjoy and please share your criticism. 
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Is this Myco ASMR 😂 man this videos so relaxing ! But thanks for the info, here in denver as well starting to finally try out agar work.
awesome. email me if you want to meet up. also check out the no pour video for a sustainable plate option.
So I honestly think that the environment's ability to be sterile has a lot to do with the container that you yourself are in (house, office, shed, etc) If you're in an older house, theres gonna be a lot more spores and contaminants floating around than an area like a new apartment. I'd guess its a lot to do with the vents and the A/C unit age. You can turn off your A/C a while before, but you can't ever just stop the movement of air unless you're going through a lot of extra steps. and sure, you can use a still air box, but if we could see on a microscopic level, we would notice that you can never get rid of 100% of all contaminants in the air. So I think the most important things would be the Container you're practicing in, and also moving slowly because of the currents we create, and wearing a mask because our breath has a lot of shit in it. Sterilizing your tools and anything the spores will ultimately be in the presence of should be common sense I'm sure. Having a new air filter and Flow Hood can offset the fact that you're AC is spreading around contaminants too. SAB's are key. you just have to be aware of the air your initially trapping inside and how well you're gonna sterilize that also
Good thoughts. The air movement and your physical movement are too of the list for risks. If the air is still it doesn't matter if the room has spores in it but as soon as the air moves you have problems. I did a video called "aseptic in a septic world" that you may want to watch
I made a small flow hood with MDF sheet, 4 x variable speed controller computer fans and a 30cm 26cm Hepa filter. Easy and cheap and lessens contam risk
Possibly. but I currently have 1 sample that is making pink mold which is what I'm looking up for more information and 2 samples that are not. It really could just be what is in the genetics received.
I bomb my SAB with Lysol and let it sit half hour turned open end down before I do anything
You should take from clean new growth that way spores that maybe not germinated yet on origional sample. Also taking smaller samples with needle will help
Using a long needle is a good idea.
Wow that’s was super impressive to go from a mold infestation to a clean transfer. Which shows technique is more important than how many things are sterilized.
Exactly
Lol it took 10 transfers and luck
I would say that another error is that you are “cooling your blade” when you don’t need to. You’re taking an extra unnecessary step and introducing your blade to a clean plate twice instead of just once, which opens the plate up for more contamination. You’re better off leaving the blade hot and sterile and just going into each plate once.
You also left your bottle of isopropyl alcohol directly next to the open flame on your alcohol lamp, just centimeters away from the walls of the container; a fire hazard and recipe for disaster.
Good thoughts. I was trying to avoid the Agar explosion in the mold plate. It is a pretty common practice to cool the tool in a clean plate but there are other options.
But won't the heat kill the mycelieum, I'm always spray my heated needles and knives after I heat, then wipe, but like your saying, extra steps! I figure the heat may kill my spores or lc cultures as I'm filling up syringes.
Yeah, for real, a huge fire hazard with the alcohol. I usually cool my blade with alcohol and let it sit for about 5-10 seconds after sterilizing with the torch.
Very relaxing. Not to mention encouraging.
Glad you enjoyed it!
Frankly, ive never seen so much mold...
I do all my transfers open air, a/c off, barely as sterile as your environment, and I have a 90% clean rate...
My recommendation would be to move to another house that's not bombarded with mold lol
This was a demonstration of cleaning a mold contaminated plate. Starting with 100% contamination and getting to 0%
Its the moldy bathroom🤣🤣
i use the oven technique for flowhood. got 13 agar totally clean but i spray the shit out of everything with isop. seems to work. mist all around gets sterile for good minute. guess imagination and timeframes are key. dont loose track of not sterile surfaces and devices
Dude great video .
BTW! the rubbing alcohol is unbelievably explosive .torch +isopropyl alcohol ÷mental focus = BOOM
Isopropanol is not explosive, it is flammable, yes.
Explosive? LOL. What a moron.
I have a plate I'm trying to clean up, so thanks for sharing this! Just did my first transfer to clean agar and am very interested to see what happens.
Glad it was helpful!
i recommend using a still air box or flow hood
@@mushroomcult Have you ever heard of oven tek?
@@PredatoryPrey yes. It is more difficult than using a still air box
@@mushroomcult Yea it's definitely a way to do it lol. I mean I would say if you don't have an SAB or flow hood it would be useful, but if you can't go to the store and buy a storage bin and cut some holes in it and attach some rubber gloves to it, then you probably shouldn't be cultivating lol.
Thanks so much. Good video. I learned a lot.
Glad you did. Make sure you use a still air box if your work is important
@@mushroomcult I will definitely try it. Thanks again.
How do you store your unused agar plates? Fridge or room temp? And right side up or upside down? Do you put them In bags or tape them? Thanks man!
they store in the fridge best because it slows evaporation. upside down is best because the condensation won't fall down onto the agar. I store them in the original sleeve after heat seeking them. if i was storing a few plates i might tape them. does that answer your question?
I still don’t know how to tell if it’s contaminated lol
Feel free to send me pictures on Instagram messages.
Thanks I found this cause my first try with agar i made 20 dishes and had a spec of not sure what on one edge of one dish so far 3rd day. its yellow 0ne mm round and was wondering what it is and if i could cut it out. I also dropped spores on 4 dishes ( anticipation ) I used a home made still air box and followed directions as best as possible.
cool. so the yellow spot is probably not mushroom tissue. the proper technique to clean up your plate would be to take the cleanest/healthiest section of the organism you want to save and move it to a new clean plate. it is not recommended to remove the contamination but you could try cutting all the agar around the contaminant if you want to.
your spores could take a week or 2 to germinate but check it every day to catch contamination early. don't wait as long as i did in this video.
Why not just cut out the contamination? As long a the contamination isn’t touching the mycelium is should be ok right?
it can work sometimes but best practice is to subculture the cleanest mycelium away to a clean plate. this is especially important when there is mold in the plate. the mold spores will spread rapidly when disturbed.
So why do the plates appear to get MORE contaminated with each transfer? (For the first couple plates)
the fires plate is contaminated so i take a sample of the cleanest mycelium to a new plate. at this point the first plate is trash but i save it just to keep record of it. the next transfer i watch every day to see if any spores from the first plate moved with the subculture. the moment i see growth that is not from my subculture i move the cleanest mycelium to a new plate and the second plate is now trash. this continues until there is nothing except the subculture growing in the final plate. all the plates except the final plate are trash. does this answer your question? if not let me know. thanks
My first work with agar was 2/5/21 made my first batch of agar, two mistakes I made... one needed a bit more water in pressure cooker then I had almost cooked the pressure cooker dry, 2nd mistake not allowing the agar to cool down enough to handle so I rushed myself. 3rd mistake was not waiting for the agar to solidify in the Petrie dishes before wrapping parafilm tape around them. 2/6/21 inoculated two Petrie dishes one with Cream Lex Luther spores and Cambodian spores. 2/7/21 I ended up inoculateing two more dishes with Cream Lex, 2/13/21 inoculated two more with Natal Super Strength. And also experimental inoculation of dehydrated Thai Koh Samui which didn't mature the right way at all but the dish is colonizing. I inoculated all Petrie dishes total of 7 basically same as you have in bathroom as sterile as possible. I obviously expect some contam this weekend making up more agar and Saturday night first transfers to new plates at least the Cream Lex Luther that is.
no better teacher than making errors as long as you learn from them. you are on the right track!
Thanks for posting the video man. I just did my first agar pour about 3 days ago. I don't see any contamination on any plates yet. I'm excited to grow some mycelium on them.
I wonder if you would have had more success if you caught the mold very early and did the transfer right away. It just seems like it was already releasing a bunch of spores inside the dish when you went to transfer the mycelium.
Great work.. I recommend practicing a pretend procedure before doing the real thing. This will help you make sure you have everything in order before you start
@@mushroomcult Thank you, I'll definitely do a practice run before the real deal. Just like anything I'm sure we will all get better with time the more we practice. Even the most simple of tasks have room for improvement alone or in conjunction with others.
@@ShawnTheRazor exactly
Thanks, great work.... and ...Conclusion?
The conclusion is never give up
8:47 that blade fell off because you didn't fully seat it on the handle. They slide all the way on to sit flush and need to be flexed to be removed...they're quite sturdy that way.
Bruh. Get that damn alcohol lamp away from alcohol bottle. You're killing me, man. XD
Why?
The summery of procedure is nice but I can't wait for a wintery of procedure 😜
I'm not sure what you mean
@@mushroomcult just talking a little trash with a dad joke lol, you spelled summary wrong on your slide. Quick question, if I may, do we assume that the culture is not haploid anymore when it goes rhizomorphic? Or is the only way to really tell by looking through a microscope? Just worried about sending a haploid to grain and then not getting it to fruit. Not even sure if thats a problem but haven't found any good answers googling yet. Thanks for the videos!
@@spearcat710 usually a haploid is cottony and not very fast growing but that is not a good indicator. Some species form clamp connections that can be seen under the microscope
Where did you keep those plates after the transfer ？fridge or normal temperature?dark/bright space? From a beginner’s question…
room temperature. avoid direct sun or heat. a stable temp is best
I wonder if I start from spore print, this can be the technique to clean if the print is contaminated?
@@WPHWw-km1tk exactly
I know the video was a while ago but would it be possible to remove the mould instead by cutting out a large section of the agar instead of transferring the sample to a new agar plate? That way then your already working with an established plate.
Best practice is to move the clean tissue to a new plate and leave the contamination. Especially with mold there is a high risk of spreading spores when removing the mold. If you catch it before spores are produced you may get success.
@@mushroomcult Hi, really appreciate your reply and thank you for the information! I’m just starting out into mycology so trying to gain as much knowledge as I can. Cheers 👍🏼.
@@nousername.4831 try everything. Never make the same mistake twice and youll be fine
..is 70% alcohol Flammable ?...
Yes? Tf
Yes
Hey, at minute 2:00 on the bottom right, i have that same thing on every agar dish, many little white dots with that grey thing. Is that contam? Its weird because its on every dish and on every little white dot
that is probably contamination. are they all in the exact same spot and same size?
@@mushroomcult they are clustered but all the same Size mostly. Heard it would be Penicillin but i cant find anything about it!!
@@ThugFightsBmx penicillin is a mold and would be turning blue at some point
Worth watching
thanks! do you have any criticism? i would appreciate it.
what would you say is the maximum amount of time possible for contamination to show up? meaning if you waited a month (for example) and nothing was there - you're in the clear
On average it would be only a few weeks at most. These showed up within a week
Awesome video. Thanks for sharing. Question for you. Why do you take the culture from the previous plate instead of cutting a fresh piece to with new growth?
i take a piece from the cleanest spot in the previous plate in hopes of leaving the contamination behind. does that answer your question?
What I don't understand, why do you move the pieces from the first place, why not cut a fresh piece around it? Shouldn't it be cleaner?
You have to take cuts from mycelium. If he cut fresh agar from around the mycelium, nothing would grow in the next plate because he would have only transferred agar to agar. The idea is to isolate clean mycelium away from the contamination and get it to colonize a plate in full without any contaminants. It’s never a good idea to transfer the contamination away from the mycelium as this dislodges the contamination/spores and shuffles it around the plates and air. So instead, you cut clean mycelium and transfer it away from the contamination and into a clean sterile plate so that it can continue to grow away from the contamination. If you were to just cut out clean agar and transfer it, there wouldn’t be any mycelium to grow once transferred.
At 2:29 for example, are those round fluffy white balls mold or mycelium? I see two of them look very white and fluffy and one looks more grey in the middle. I poured my first plates about a week ago and a few days ago I had a fluffy white ball like one of those pop up directly on top of where I shot my spore syringe, and you can see spores underneath it. I'm just not sure if it's the fluffy kind of mycelium as opposed to rhizomorphic, or if it is some kind of bacteria 🦠🧫 let me know what you think please. It's coming off of the agar quite a bit and kind of looks like a little pile of that magnetic black dust.
Mold organisms also have mycelium just like mushrooms. Most molds start out with white mycelium and then produce spores which give it color gray black blue orange yellow pink. If the mycelium is growing exactly where you put the spores and it is pure white I would leave it alone. If you're getting any amount of color it is possibly a contaminant
Isn't the bathroom literally one of the dirtiest places you could choose to do this?
Depends on your house hygiene. I would guess most people regularly clean there bathroom. My bathroom has no carpet, smooth easy to clean surfaces. I would guess it is the cleanest room in the house. Watch my other video "aseptic in a septic world"
@@mushroomcult sure I guess everyone is different, but according to many studies which I am sure included people who believe they are the exception, the bathroom is definitely one of the dirtiest, kitchen being even worse.
hmmm, HI, nice video. Hey, if I can help you as you helped me with some valuable information... you definetly should use a bunsen burner every time you open thoses plates. You need to create an sterile enviroment.
Thanks. My standard is a flow hood or a still air box.
Sir, How to protect our pure fungi culture on PDA from white molds?
And at 3:05 time the picture contains two species one is fungi and second is? Pls reply
At 3:05 the plate contains mushroom and either bacteria of a yeast. I'm not sure. To protect from white mold you need to identify how it gets into your plate. Did the mold enter the plate from the air, from a tool, on the mushroom sample, from improperly sterilized plates or agar? This will point you to where you need a solution. Some possible solutions might be;. Laminar flow hood, hand washing, sterile tools, modify your material sterilization method, change your inoculation method. Leaving blank plates can help narrow down the problem.
Hey greetings from Paris!
First I am happy that I found your channel after 3 months with this issues about petris I found some cool information.
I have some questions I hope you could answer please.
1- what’s your recipe por your agar plates?
2-And I saw that you pour not a lot of agar in the plate, that helps the colonization?
Thanks a lot for the video and I think I will make the same to have some information back as you 👍
The other recipes I use most frequently are malt extract agar and potato dextrose auger. I try to divide 1 L of agar across 40 plates. The amount of agar in the plate is not very important except to make a flat surface and to hold enough moisture for the growth process. If you are doing long-term storage look into making slants or the pre-poor plates that can have thicker my amounts of agar for longer storage
@@mushroomcult again thanks a lot for your reply 👍!
Just one last question please when you say “to keep enough moisture” you mean that you don’t let the petry plate upside down? Or how do you arrive to keep some moisture on the plate? Thanks for the answer 👍
@@jimerubio2844 the gel itself holds the moisture. You don't really want water on the surface of the gel. If you are storing mycelium long term you will want to have a thicker agar gel so that the container won't dry up over the course of months or years. You can store a pizza plate with agar-gel upside down so that the condensation falls into the lid instead of on the surface of the gel
Would a loop work better than trying to cut a piece of agar? Still learning here, but seems it wouldn't disturb the surface as much
Loops are usually pretty flexible and I find it tonight to make a subculture with one. Try it and tell me how it goes. I like the #11 scalpel
@@mushroomcult found your vids while trying to do my clean up transfers with a SAB. Feel like I just spread the spores more! Thank you for posting this, and I'll keep you posted 🙏
@@christopherpowell967 it is tough when the mold has spores. Slow smooth shots movents. Keep the air in the room still.
Just had my first flush ever. Did all. My sterile work in my bathroom😂
Great success! What is your next step
As i side note as a project or for home use still air box and flame should be enough.. if i was to be develoving a buisness i would personaly be leaning towards a flow hood and some sonic sterile bath between uses and replace the blade between projects. I hate trash is all haha
yah. i prefer using a flow hood. in this case with spores on the mold i would not want air blowing on the plates and spreading spores everywhere. a still air box would be best for this specific project. thanks for your comment. what are you working on right now?
Is it possible to re-use Petrie dishes as in clean then out hand wash then use 70%Isopropyl to sterilize them for use again if not glass or Polypropylene Petrie dishes?
best practice is not to reuse them. you wash them and soak them in bleach or alcohol. you might be able to reuse them if they dry in a sterile environment. expect some contamination. let me know how it goes.
No. Chemicals sanitize, not sterilize. Heat sterilizes. So only use new pre-sterile plates.
Alcohol isn’t sterile. It is sanitary. You can’t sterilize anything using alcohol. There is a difference between sterile and sanitary.
Gah damn 😂 I’d be worried about air quality 1028479%… for yourself bro, like wth is in the air you’re breathing! 8 mf transfers? Something’s going on homie, get a $50 hepa filter for a small room. Best investment you’ll ever make. Drop it in the bathroom/still room 20min before you start, they can clean 99.9% of air in 180 sq feet room within 15min. Then turn it off and wait 2-3 min for the brand new clean air to settle, and then start. I don’t see how it’s that many fails with how you’re going about it. unless all the other ones fell on the floor and you’re lying about them all being like this because i don’t see how tf you failed 8x going about it like this lmao mfs are doing this is toilets and having better success aint no dang way bro.
The starting plate had spore-producing mold in it. That is where the contamination came from. Have you ever cleaned a moldy plate before?
What kind of contaminant is that white stuff? Is it yeast?
I haven't done an ID on it. It is a yeast or a bacteria almost certainly. To identify it a gram stain and microscopy would be needed.
@@mushroomcult ok thanks for the info. I have a microscope but I've never heard of gram stain. So I will have to look that up. I have a similar white/ yellowish spot on one of my lions mane agar plates. Unfortunately the plate is to thick to place under my microscope. I want to identify the contam but I also don't want to have the contam out in the air. So I will have to look up the gram stain thing. The contam I have is identical to yours. The way it looks like there are 3 circles within the circle of what I am assuming is bacterial yeast also. Atleast it's not the mean green. ;)
@@mycomama4209 great. The gram stand will help you narrow down a large category of bacteria. Next you will need to follow a dichotomous key to get to species.
@@mushroomcult ok will do. I have a bunch of Paul Stamets books that go over all the different forms of bacteria and show what they look like under a microscope. So I am familiar with the different varieties under a microscope. But like I said I can't put the plate under the scope. Im assuming I need to put the contaminant on a slide. So yes. I will do the test you recommended and check out the other way of determining which bacteria it is. I only have one small spot. It shouldn't be hard to clean up this culture at all.
@@mycomama4209 great. Yes you need to make a slide.
Open air?
yes. I do my work with a flow hood but i want to demonstrate that it is not necessary if your technique is good. I recommend using a still air box when you have dirty samples or contamination but i didnt want to block the view. the results are what counts. check out my video called aseptic in a septic world. tell me about your cultivation experience or something you are proud of?
Dang! 👍❤️
what do you think?
Melted my auger
To be fair you could be the only one saying it right. :^)
im not concerned with saying it right. i do what i want.
lucky you
Naw, he just has a speech impediment like his mommy and daddy "did"?
I would recommend cutting down your time and editing out the parts you don’t need. It might end upping the number on your subscribers, and people watching. Some people REALLY don’t want to watch 15 min videos when there’s 5 min videos with the same information and more...
thanks! this is good advice. editing is my bottle neck at the moment
Tbh i really appreciated his extra unedited content because i have dozens of questions that other videos just haven't answered yet
I think all ur contamination is coming from no gloves and also working in an open environment. Use a SAB and it will work much better for you! For agar open air isn’t good.
Booom
All I can hear are your blaring S’s. Jesus
do you know how to fix this? im new to this audio stuff. thanks
Someone please shoot me I can't believe I watched it to 6:49
were you injured?
@@mushroomcult scarred for life and the others exposed to it will surely have their moment of clarity when they realize the waste of time you infect them with...
@@buzzzkill9183 you really are a buzz kill