I must say, it's VERY refreshing to watch a video by someone who is actually teaching the specific reasons behind every step and technique. Sometimes people have the best intentions, im sure, but end up making it appear more complicated with half-explanations, vague (often incorrect) terminology, videos at 3x speed with music playing over parts where they were clearly trying to explain something important (but forgot to edit or add text)... this man however, leaves nothing to the imagination. I'm not left wondering why half of the steps were taken, and actually feel prepared to try it... just pleeeeease post more videos (quality over quantity is just fine). ...and remember "The Six P's" - Prior Planning Prevents Piss-Poor Performance! (I forget where I'd heard it)
thanks so much. i have more videos in the works. i am setting up a new laboratory and will be back at production soon. can i use your comment in my marketing?
@@mushroomcult I was really unsure of the whole mss to agar, people have been doing it but no one really shows you how far the growth must go before transferring to a new plate of agar for isolation growth. So basically I have an mss to agar plate that’s been growing for almost a week now, how much growth should I let the points have to transfer if you don’t mind explaining that to me? 🥹
@@mushroomcult I’m a total newb and I’m starting with agar, usually liquid cultures to agar and now agar to agar transfers. So just anything showing different types of mycelium and the best parts to use for transfers or best spores to isolate and why. Your videos have helped me be able to identify what I’m looking at a lot better but sometimes I still am unsure which areas on some of my plates are the strongest “more ideal” pieces to use for transfers. Anything agar related really! But I’m going through all your videos as I get time so I’m sure I’ll find a lot more helpful info as I go, so thank you!!🙏🙏
Mushroom Cult honestly if I tell you to improve in a way then I would just be giving you ideas of other videos I’ve seen. Yours are unique. I would like to say though, maybe look into getting a better position on your work..? Like a better angle. I’d like to see what you’re doing up close
Nice vid thanks. One thing though that might be confusing to people.. selective rhizo rather than tomentose to transfer has nothing to do with selective isolation. It's only going to help with faster mycelium expansion, but of what, you have no idea. There is no correlation between rhizo myc and good genes in terms of fruits. The only way to selectively isolate and narrow down your genetics is from selecting a fruit, and making a spore print (slight narrowing down) or a clone (maximum narrowing down). Of course after that selecting rhizo over tomentose is going to provid faster colonisation of your next plate. But, again, good rhizo has nothing to do with big/beautiful/strong fruits
Thanks a million! Your description of how you isolate the leading edge of the mycelium to colonize a plate with singular genetics was super informative and the exact piece of info I was looking for when I clocked on this video! Does it matter how the cut agar piece is laid on the receiving plate? mycelium down or sideways? Or agar up? Mush love
If you have 50 different blooms, which should survive if you only need one fruit? Should you isolate the 2-3 rhyzomorphic blooms? The milkiest bloom? The deepest digger? Do you want something that outraces the other myc early on? Or do you want the bloom that reaches the furthest fastest, ignoring how thick and milky it is? Can you tell what will fruit this early or do you need to keep spawning isos until you get a good one?
Thanks for the video! In your experience, how often do you encounter isolations that are zero or very weak producers? Do you find that isolating from spore is a more efficient path to having desirable traits than is doing a small grow and isolating from clones? Thanks again!
If you are referring to cordyceps specifically I haven't run very many isolations. It is necessary to do the breading in some fashion because the strains are short lived. It doesn't need to be don't this way.
@@mushroomcult more so referring to any mushroom in general. Isolating from spore seems attractive because you save the time needed to go to fruit to clone. It sounds like the risk of isolating "dud" genetics is pretty low if you select for healthy mycelium?
@@BobbyEshleman I did not isolate my genetics, and now having terrible time with non producers. Did spores to LC. Mycelium grows all over, wraps around my small shoe boxes, can't get pins... lol
Very helpful. Thank you for the content. I have a few questions if you don't mind. First, can you speak to a method to acquire monokaryotic v. dikaryotic mycelium? Second, why would one want to acquire monokaryotic v. dikaryotic? Finally, any tips on best agar to use specifically for this purpose?
interesting question! if you buy mushroom spawn it will be dikaryotic. if you allow mycelium from 2 compatible spores to mate you will have dikaryotic mycelium. if you isolate a single spore it will produce monokaryotic mycelium that will not produce mushrooms without mating with compatible mycelium. if you have many monokaryotic isolates you can use them in a breeding project to explore the genetic range of your isolates. does that answer your question? i use pda or mea agar normally.
@@mushroomcult Great. That is very helpful. Do you have any suggestions on a method to make sure that I've managed to isolate a single spore? They are so small.
@@crushyourboy There is a technique called "streaking" where you take diluted spore solution, drop it near the edge of a plate, and take an inoculation loop, and drag in a short straight line from that drop along the edge of the plate. Then you drag from the end of that streak, repeating around the edge of the plate. There are better videos that illustrate it, but that's the term you want to look for: "agar streaking." Whether it's bacterial culture or multi-spore solution, the technique is largely the same. Monokaryotic mycelium is nearly impossible to see with the naked eye. Catching one before it finds a mate can be extremely difficult. But no one said mushroom breeding was easy...
Yep this is what I'll be doing, trying to find the best looking white patch lol. How many times do you need to isolate an agar dish? Will one transfer to agar be sufficient to get dominate genes.
It will depend on how many phenotypes you have in the colony. Just transfer until you have even growth in 360 degrees. If you have different sectors carefully transfer a tiny piece from the leading Edge of each sector
when you say "single isolated strains" you're referring to diploid mycelium, right? In theory you want one set of mixed genetics in order to create a mycelial network that'll produce fruiting bodies., but you did comment on not having mixed genetics - can you confirm what you mean, if what i just said is incorrect? also, when you say "zone of inhibition" i'm assuming you're saying that they are diploid mycelium networks running into other haploid/diploid mycelium networks and not 'mating'... thanks for the video!
You can isolate for single monokaryotic colonies or for dikaryotic colonies. If monokaryotic then you will need to combine more than one colony in the same plate for a chance for them to mate. If you isolate dikaryotic colonies you can move to the next step in the grow process. Does that answer your question?
@@mushroomcult it sounds like it does. There are so many conceptual layers to mycelium that its hard for me to track since I haven't gotten into the weeds about it yet (trying....) I think I'll look into the -karyotics and perhaps your response will be more enlightening to me, if not ill come back!
@@mushroomcult oh that was quick. I guess I thought monokaryotic and haploid were the same thing? And diploid was the same as dikaryotic? Can you explain The diffrrence?? 🙏 thanks a lot for responding
@@g-lurk A diploid cell contains a nucleus with two sets of chromosomes. A dikaryotic cell contains two haploid nuclei. Many mushrooms have 2 nuclei after paring. Does this clarify?
@@mushroomcult getting there.. Does a dikaryotic then have two nuclei AND two sets of chromosomes? And a monokaryotic has one haploid nucleus and a haploid has one set of chromosomes? I guess in my head I'm having a hard time understanding if they're all different outcomes of the progression, or if a haploid graduates to a monokaryotic if not mated. Man I need this drawn out visually I think.
i normally use a flow hood but still air boxes are great. iv done lots of open air inoculations. it is totally possible if your technique is good and you prepare properly.
when you get a print on agar the spores will germinate within a few days. keep them in cool room temps like 60's fahrenheit. it is not difficult. you need to isolate the spores very soon after printing because you don't want them to mate. most other spores dont need any special treatment but may take a week or 2 to germinate. does that answer your question?
it isn't a serious problem. you will have multiple genotypes in the same jar fighting for resources and you may get inconsistent results. clones will give you more consistent results. what questions does that bring up?
@@mushroomcult are the results poor enough that it's best just to wait to use the selected genetics. I would like to clone, but I haven't made it to that stage yet, thus why I was wondering if it would be worth innoculating grain for a couple shoe boxes? How many rounds from spore does it typically take to get to desired results?
@@ImAlwaysHungry I've found that I can usually get strong, rhizomorphic mycelium from spores after 3-4 rounds of isolation. I try to grab intersections or junctions between strands of mycelium. If I don't see those, I grab the leading edge.
Not if you want a single spore isolation. The idea would be to have several single spore isolates and cross them purposely so you can have control. If your goal is different your process can be different too.
@@mushroomcult ok true. i see you take a few different isolations onto seperate petris. what next to cross them purposely? do you have a video i can watch of this
@@zacharyswain7034 I don't have a video but you would let each spore grow on its own plate fora few days. Take a small sub culture of 2 different spores and combine them in the same plate about .5inch apart. If the spores are compatible they will mate.
@@Sepo7 The whole point of agar is that it can't be broken down by the mycelium, forcing it to grow only along the surface, so that you can see all of the growth clearly and visibly. The mycelium would break down and colonize a paste made from BRF like any other PF tek cake recipe.
Наука
I must say, it's VERY refreshing to watch a video by someone who is actually teaching the specific reasons behind every step and technique. Sometimes people have the best intentions, im sure, but end up making it appear more complicated with half-explanations, vague (often incorrect) terminology, videos at 3x speed with music playing over parts where they were clearly trying to explain something important (but forgot to edit or add text)... this man however, leaves nothing to the imagination. I'm not left wondering why half of the steps were taken, and actually feel prepared to try it... just pleeeeease post more videos (quality over quantity is just fine).
...and remember "The Six P's" - Prior Planning Prevents Piss-Poor Performance! (I forget where I'd heard it)
thanks so much. i have more videos in the works. i am setting up a new laboratory and will be back at production soon. can i use your comment in my marketing?
Super appreciate the precise and complete explanation without wasting words. Your a quick favorite. Glad I found you
I’ve been looking for this info everywhere, thank you sir! ❤️
Sure! What was the most helpful part? Are you left with more questions?
@@mushroomcult I was really unsure of the whole mss to agar, people have been doing it but no one really shows you how far the growth must go before transferring to a new plate of agar for isolation growth. So basically I have an mss to agar plate that’s been growing for almost a week now, how much growth should I let the points have to transfer if you don’t mind explaining that to me? 🥹
Very nice and concise video. Thanks!
Glad I could help
Soo helpful, really appreciate these type of videos!! 💯🙌🙌🙏
Glad it was helpful! what else can i help with?
@@mushroomcult I’m a total newb and I’m starting with agar, usually liquid cultures to agar and now agar to agar transfers. So just anything showing different types of mycelium and the best parts to use for transfers or best spores to isolate and why. Your videos have helped me be able to identify what I’m looking at a lot better but sometimes I still am unsure which areas on some of my plates are the strongest “more ideal” pieces to use for transfers.
Anything agar related really! But I’m going through all your videos as I get time so I’m sure I’ll find a lot more helpful info as I go, so thank you!!🙏🙏
really your awesome bro 😍 ,because you clarifying doubts and patience given reply in instagram,youtube comment
it's really helpful bro
tqq so much😍
Thank you so much 😀
Love your videos dude! Thank you so much . Mush love 🍄❤️
Glad you like them! what would you like to see? any questions? what would make my content better?
Mushroom Cult honestly if I tell you to improve in a way then I would just be giving you ideas of other videos I’ve seen. Yours are unique. I would like to say though, maybe look into getting a better position on your work..? Like a better angle. I’d like to see what you’re doing up close
I hope the spirits guide you to unbelievable success
thanks
I never had a multispore plate like that with no contamination, especially after 8 days. Maybe I should find your video on that lol
Nice vid thanks.
One thing though that might be confusing to people.. selective rhizo rather than tomentose to transfer has nothing to do with selective isolation. It's only going to help with faster mycelium expansion, but of what, you have no idea.
There is no correlation between rhizo myc and good genes in terms of fruits.
The only way to selectively isolate and narrow down your genetics is from selecting a fruit, and making a spore print (slight narrowing down) or a clone (maximum narrowing down).
Of course after that selecting rhizo over tomentose is going to provid faster colonisation of your next plate.
But, again, good rhizo has nothing to do with big/beautiful/strong fruits
Correct
So many plates! Love it!
How many do you do in a run?
@@mushroomcult I just finished my first agar plates. Everything looks good so far.
@@thinkingmushrooms2943 awesome to hear. What is your next step?
@@mushroomcult Did about 20 ketchup cups. Want to see the rate of contamination with my current method. Will spawn the clean ones to grain.
@@thinkingmushrooms2943 perfect. Don't pass Agar untill the plates are perfect.
Awsome information thx you
Glad it was helpful!
I wish I could’ve seen a follow up video! The results of what you did. I’m brand spanking new to growing and trying to learn :)
I haven't been making videos for a while. I hope to start again soon. Message me if you have questions. Instagram is best
Thanks a million! Your description of how you isolate the leading edge of the mycelium to colonize a plate with singular genetics was super informative and the exact piece of info I was looking for when I clocked on this video!
Does it matter how the cut agar piece is laid on the receiving plate? mycelium down or sideways? Or agar up?
Mush love
Glad it was helpful! the subculture just needs to contact the receiving agar nothing special
If you have 50 different blooms, which should survive if you only need one fruit? Should you isolate the 2-3 rhyzomorphic blooms? The milkiest bloom? The deepest digger? Do you want something that outraces the other myc early on? Or do you want the bloom that reaches the furthest fastest, ignoring how thick and milky it is? Can you tell what will fruit this early or do you need to keep spawning isos until you get a good one?
A thousand times better audio.
thanks
Thanks for the video! In your experience, how often do you encounter isolations that are zero or very weak producers? Do you find that isolating from spore is a more efficient path to having desirable traits than is doing a small grow and isolating from clones?
Thanks again!
If you are referring to cordyceps specifically I haven't run very many isolations. It is necessary to do the breading in some fashion because the strains are short lived. It doesn't need to be don't this way.
@@mushroomcult more so referring to any mushroom in general. Isolating from spore seems attractive because you save the time needed to go to fruit to clone. It sounds like the risk of isolating "dud" genetics is pretty low if you select for healthy mycelium?
@@BobbyEshleman I did not isolate my genetics, and now having terrible time with non producers. Did spores to LC. Mycelium grows all over, wraps around my small shoe boxes, can't get pins... lol
Very helpful. Thank you for the content. I have a few questions if you don't mind. First, can you speak to a method to acquire monokaryotic v. dikaryotic mycelium? Second, why would one want to acquire monokaryotic v. dikaryotic? Finally, any tips on best agar to use specifically for this purpose?
interesting question! if you buy mushroom spawn it will be dikaryotic. if you allow mycelium from 2 compatible spores to mate you will have dikaryotic mycelium. if you isolate a single spore it will produce monokaryotic mycelium that will not produce mushrooms without mating with compatible mycelium. if you have many monokaryotic isolates you can use them in a breeding project to explore the genetic range of your isolates. does that answer your question? i use pda or mea agar normally.
@@mushroomcult Great. That is very helpful. Do you have any suggestions on a method to make sure that I've managed to isolate a single spore? They are so small.
@@crushyourboy There is a technique called "streaking" where you take diluted spore solution, drop it near the edge of a plate, and take an inoculation loop, and drag in a short straight line from that drop along the edge of the plate. Then you drag from the end of that streak, repeating around the edge of the plate. There are better videos that illustrate it, but that's the term you want to look for: "agar streaking." Whether it's bacterial culture or multi-spore solution, the technique is largely the same.
Monokaryotic mycelium is nearly impossible to see with the naked eye. Catching one before it finds a mate can be extremely difficult. But no one said mushroom breeding was easy...
Yep this is what I'll be doing, trying to find the best looking white patch lol. How many times do you need to isolate an agar dish? Will one transfer to agar be sufficient to get dominate genes.
It will depend on how many phenotypes you have in the colony. Just transfer until you have even growth in 360 degrees. If you have different sectors carefully transfer a tiny piece from the leading Edge of each sector
@@mushroomcult ahhhh, okay, yea I always see those perfect 360 agar plates. So that's the course I'll take to get there then!
Did you do these transfers in open air without a flow hood or SAB? How did you not get a bunch of contamination?
This is done in front of the flow hood.
when you say "single isolated strains" you're referring to diploid mycelium, right? In theory you want one set of mixed genetics in order to create a mycelial network that'll produce fruiting bodies., but you did comment on not having mixed genetics - can you confirm what you mean, if what i just said is incorrect? also, when you say "zone of inhibition" i'm assuming you're saying that they are diploid mycelium networks running into other haploid/diploid mycelium networks and not 'mating'...
thanks for the video!
You can isolate for single monokaryotic colonies or for dikaryotic colonies. If monokaryotic then you will need to combine more than one colony in the same plate for a chance for them to mate. If you isolate dikaryotic colonies you can move to the next step in the grow process. Does that answer your question?
@@mushroomcult it sounds like it does. There are so many conceptual layers to mycelium that its hard for me to track since I haven't gotten into the weeds about it yet (trying....)
I think I'll look into the -karyotics and perhaps your response will be more enlightening to me, if not ill come back!
@@mushroomcult oh that was quick. I guess I thought monokaryotic and haploid were the same thing? And diploid was the same as dikaryotic? Can you explain The diffrrence?? 🙏 thanks a lot for responding
@@g-lurk A diploid cell contains a nucleus with two sets of chromosomes. A dikaryotic cell contains two haploid nuclei. Many mushrooms have 2 nuclei after paring. Does this clarify?
@@mushroomcult getting there.. Does a dikaryotic then have two nuclei AND two sets of chromosomes?
And a monokaryotic has one haploid nucleus and a haploid has one set of chromosomes?
I guess in my head I'm having a hard time understanding if they're all different outcomes of the progression, or if a haploid graduates to a monokaryotic if not mated.
Man I need this drawn out visually I think.
did you do this in open air?? or is there a laminar flow hood out of frame?
i normally use a flow hood but still air boxes are great. iv done lots of open air inoculations. it is totally possible if your technique is good and you prepare properly.
@@mushroomcult Nice. Thanks for the response, as well as making this video for us all to learn
H2O2 agar makes open air xfers much easier and way, way less chance of contam taking hold.
After getting spores: How do you germinating them (How long, temperature, time, with or without light?)
when you get a print on agar the spores will germinate within a few days. keep them in cool room temps like 60's fahrenheit. it is not difficult. you need to isolate the spores very soon after printing because you don't want them to mate. most other spores dont need any special treatment but may take a week or 2 to germinate. does that answer your question?
What substance would you use in a plate to isolate entomopathic fungi that for example grow on aphids?
Would there be an issue using the orginal multi spore agar, and innocculating a jar or jars with that? Is this a waste of time, it not?
it isn't a serious problem. you will have multiple genotypes in the same jar fighting for resources and you may get inconsistent results. clones will give you more consistent results. what questions does that bring up?
@@mushroomcult are the results poor enough that it's best just to wait to use the selected genetics. I would like to clone, but I haven't made it to that stage yet, thus why I was wondering if it would be worth innoculating grain for a couple shoe boxes? How many rounds from spore does it typically take to get to desired results?
@@ImAlwaysHungry I've found that I can usually get strong, rhizomorphic mycelium from spores after 3-4 rounds of isolation. I try to grab intersections or junctions between strands of mycelium. If I don't see those, I grab the leading edge.
@@crushyourboy Many thanks for the good info. Is there any way to clean a contaminated tissue sample before putting it to agar?
@@shainemaine1268 that’s one of the reasons to do agar work. Cleaning up and isolation of whatever culture you’re trying to work with.
Is it ideal to get a sector from two colonies that have fused together?
Not if you want a single spore isolation. The idea would be to have several single spore isolates and cross them purposely so you can have control. If your goal is different your process can be different too.
@@mushroomcult ok true. i see you take a few different isolations onto seperate petris. what next to cross them purposely? do you have a video i can watch of this
@@zacharyswain7034 I don't have a video but you would let each spore grow on its own plate fora few days. Take a small sub culture of 2 different spores and combine them in the same plate about .5inch apart. If the spores are compatible they will mate.
@@mushroomcult ty man
Where did u buy ur Petri dishes
I pour the agar plates myself. i recommend trying the no-pour method. i have a video on this too. does that answer your question?
@@mushroomcult yes thank you so much for responding
Can you do a brf paste video please?
i did a recent video on preparing BRF. did you see that?
@@mushroomcult I’ll check and see thank you
@@mushroomcult ok so I checked and you did a brf tek but I’m wondering if you can do a brf paste that can be used as a agar substitute
@@Sepo7 The whole point of agar is that it can't be broken down by the mycelium, forcing it to grow only along the surface, so that you can see all of the growth clearly and visibly. The mycelium would break down and colonize a paste made from BRF like any other PF tek cake recipe.