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InvisibleWall.E
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Why We Put Spores to Agar and How to Win the Genetic Lottery * 12
    #27045046 - 11/18/20 08:17 AM (3 years, 2 months ago)

Hey all, thanks for stopping by. In this journal I'm going to try my best at explaining a few of the reasons why we put spores on agar. With that being said, please also understand that I have no qualifications to make me an authority on any of this. These are just my observations from learning as much as I can about this hobby. If you want to skip my science lesson and instead learn from a true and trusted source, feel free to read through Pasty's write up Basic principles of mushroom cultivation

But, if you're willing to read through mine, let's get started.

A Brief Science Lesson

Like we learned in 8th grade or whenever, plants, animals and fungi are made up of cells. There are a lot of differences between the cells, but we're only interested in the nuclei for the time being. For the sake of brevity, I'm going to refer to animal cells, because well it involves cum and that's more interesting than pollen for demented minds.

Animal cells spend a majority of their life as cells containing 2 sets of chromosomes called "diploid" cells. Diploid is a Latin-based term, "di-" meaning "two" (think dichotomy) and "ploid" meaning "fold" (I guess this refers to the shape of chromosomes? Either way, doesn't matter here). "Haploid" cells are cells that contain just 1 set of chromosomes, "Ha-" meaning "Single".

Haploid cells are the result of a process called "meiosis". Meiosis is basically when a cell decides that it's time for reproduction. This is when jizz, pollen and spores are all made. A sex cell with the 2 sets of chromosomes will randomly pair up genes, split up into 2 different cells and each of those resulting cells will replicate again to create 4 cells with different sets of new chromosomes.

When these haploid cells meet another haploid cell that it's compatible with (sperm and egg), the two fuse together in a process called “fertilization” and the 2 nuclei fuse together to create a new pair of 2 chromosomes which we will call a zygote (think fetus).

Let's talk fungi and how they differ from our animal example above. When spores are released into a suitable environment, they split and create an exploratory hypha. This hypha will continue to replicate until it meets with another hypha of a compatible sex type (there are thousands of sex types, some fungi have a few, some have thousands. For simplicity we just refer to them as "+" and "-"). When these two hyphae meet, they don't immediately fuse their nuclei together and create a new diploid organism like a fetus or seed. Instead, the fertilization process is broken down into 2 different stages. The first stage is a process called “plasmogamy” in which the cells will fuse their cytoplasm together. This process creates a new structure called a "dikaryon", which is a cell that has 2 different nuclei living inside of it. This dikaryon stage of the fungus is a large part of the cultivation process; as at this point we refer to our fungi as “mycelium”. When the fungal body has decided that it is time to reproduce the nuclei fuse together inside of the cell in a process called “karyogamy”. The result of this fusion is a diploid cell that contains 1 nucleus with chromosomes from 2 different hyphae. These genes are randomly mixed and matched, the cell goes through meiosis and creates 4 new spores with 4 new sets of genes.

Right, now that the shitty lesson from some guy you definitely don't want to be your kid's science teacher is over, let’s get back to that dikaryon stage.

The dikaryon stage of the fungal life cycle is what we’re looking at when we are working with agar plates, watching mycelium devour a bag of grain or we’re pulling our hair out watching yet another tray fuckin’ overlay the casing layer. I’m not the one to get into specifics as I don’t have the expertise, experience or knowledge, but the mycelium will act as one body and also individually at times. At any rate, I’m only going to talk about the structures I’m familiar with, and my hopes are that I don’t make any points about which structures are better or what their real purpose is. I only want to draw the line connecting my shitty understanding of the science with our hobby. With that disclaimer, let’s point out the two mycelial structures that we most commonly work with when dealing with psilocybe cubensis.

Tomentose growth is the structure our mycelium will form when it senses nutrition and begins the process of expanding and taking over our agar plates or substrates. Rhizomorhic growth is the tendril-like structures that mycelium forms when it desires to reach out over long distances in search of food or water. This is part of the reason why if you’re trying to obtain a rhizomorphic looking plate or culture, you would use relatively low nutrients in your agar base. There is debate whether or not tomentose or rhizomorphic growth will lead to better flushes, and part of that is what I’m going to highlight below – genetics.

Enough of the boring shit, why do we use agar instead of just putting spores to grain?

As discussed earlier, the reproductive process knowns as “karyogamy” produces 4 spores after meiosis and each spore’s nucleus contains a set of chromosomes from hypha A (the + sex type) and hypha B (the – sex type). When dealing with spores, we are working with really large quantities of genetic variation as the genes are randomly mixed and matched inside every spore. For simplicity’s sake, let’s imagine a scenario that will help to argue why we go to agar first.

Let’s say you were able to guarantee that a single drop/swab of a spore solution contained 100 spores, 50 – and 50 + sex types. But given the unreliability of the environment, it also contains some other mold spores. The drop is placed perfectly into the middle of an agar plate. Over the course of a few days, we will begin to see the molecular action come to life as hyphae and mycelium start to develop. As the spores grow and continue replicating, let’s say it’s perfect and each + meets a – hypha, they fuse and create 50 new dikaryon. As time goes on, we can begin to see the dikaryon form as mycelium and this is when they begin to take on the different structures mentioned earlier, the tomentose and rhizomorphic growth.

Molds are also fungi, so they will have their own characteristic features of their mycelial growth. This leads me to the first reason why we put spores to agar first, rather than blowing a syringe into grain and hoping for the best.

By allowing the cubensis mycelium to continue its replication process, we will start our elimination process as we can reasonably assume that a portion of the 50 dikaryon mentioned earlier are completely unfit for survival, as they’ll lose fights to other microbial organisms, ineffectively digest food or simply not know what to do (mutations like enigmas I would imagine is an example of this). By allowing the plates to grow, we are able to select for certain characteristics that should give us good results for a few reasons.

By allowing the plates to grow, the mycelium will continue growing in a relatively straight path from an epicenter. Over the course of a few days, the growth should be apparent and this is when we are able to take our first transfers. If we are to go back to the 100 spore example, let’s say that only half of our dikaryon were able to make it through the initial swab and competition between the other molds. So we now have 25 dikaryon that have grown from the initial drop to a point we are able to differentiate. If we were to look at the plates and say that 1/4 of the concentric growth (imagine 1/4 of a pie chart) is workable (no mold or the mold has a clear point where it stopped growing in the same direction of our cubensis mycelium), keeping with the trend of keeping it simple, let’s say we have 4 dikaryon in the shape of rhizomorphic strands and 4 dikaryon in tomentose, so 8 potential “isolates”.

Divvying up our pie slice of agar, we take 8 transfers, each transfer being an isolate. When referring to an isolate here, I’m talking about a dikaryon, or a “strain”. It’s what winds up becoming a mushroom and as such, how it gets to become a mushroom is important. Let’s say that our 8 new plates were all done perfectly just like before and a few days go by. It becomes apparent that a few of our plates are doing really well with great uniform growth taking over the agar plate aggressively. Whereas the other plates seem like they’re not doing as hot. This is where the argument comes up again about tomentose vs rhizomorphic growth, and I don’t really have any answer for it. Ask a TC or something if you wanna know what’s better in terms of mushroom production. What we are interested in at this point in the process is how aggressively the mycelium is colonizing.

When working with sterile substrates and the like, we open ourselves up to failure by allowing perfect environments for bacteria and fungi to thrive. And as such, it is important that we get our substrate colonized as quickly as possible. This is why we are pulling transfers and trying to find efficient mycelium. It’s a race against the odds in a sense, we can try to mitigate the contamination vectors, but the best way to approach it is through sterile work and good cultures. By way of deduction and transfers, let’s say we have 4 plates that are showing great and aggressive growth, 2 rhizomorphic and 2 tomentose. Continuing on with the cult process, we inoculate substrate with the cultures and watch ‘em go. Over a few weeks, we notice 2 jars got botched and now we have 1 rhizomorphic jar and 1 tomentose jar, and they both do their thing. After full colonization, we spawn them into their own shoeboxes. After a week and a half, jar 1 is producing really beautiful fruits and some really big clusters. The other shoebox on the other hand is producing just a few mushrooms and is looking pretty disappointing.

”Well, what the fuck? I thought we were supposed to be winning the genetic lottery?”

You’re right, and we still can, but it’s going to take another step in a process called “cloning”. Cloning is exactly what one would think it is. It’s taking a part of the organism and replicating an exact copy of it. Some plants like cannabis can be cloned if a stem is clipped from the plant and provided a suitable environment that allows root formation. With mushrooms, the most common cloning process involves taking live mycelium from inside the fruit body and placing it onto a piece of agar. If we were to do this with the shoebox example and pull healthy tissue from a giant monster in a cluster in that tub, then we have a really strong candidate for a great culture as we know that the genetics of the dikaryon are efficient at competing against other competitors (agar plate), efficient at digesting food (quick colonization of substrate) and the fruit body was exceptionally large. If everything goes to plan, then after confirming the clone was successful and the agar plate is clean, then when the substrate is colonized and the fungus starts to produce primordia, it should do so with vigor and hopefully produce beautiful looking tubs.

“Right, but what about in reality? Where, ya know, we can’t separate 100 spores from the thousands found on a spore print?”

I thought you’d never ask.

I really did try to dumb it down and make it simple for me to help explain what’s going on and why we do what we do. In practice, we are usually dealing with hundreds, if not thousands of spores. When you swab a plate, there are likely 100’s if not thousands of genetic combinations going on. Just like how we can be tall, black, blonde, strong, smart, whatever, the genetic combinations in fungi result in size, color, chemical content, mycelial structure, etc. When we are looking at plates and we see rhizomorphic growth cutting through the fluffy tomentose growth, these are different dikaryotic strains. We’ll cut and transfer away, but only the really dedicated keep transferring and transferring until they find an isolate. Part of the reason why people don’t hunt isolates unless they were pulled from a tub is because of the genetic lottery. Like you could have a really great culture that colonizes an agar plate in 3 days, and a mycoquart in a week, but if the fruits are tiny and not very potent, then what’s the point?

To help mitigate this, a lot of people find it in best practice to look for healthy mycelium isolated away from the mold and bacteria that often ride along on our spore prints. Then after a few successful transfers, we want to find the mycelium that colonizes the quickest and then see where that gets us. From our flushes we can get the fruits that are providing the characteristics we want and take it back to agar, where we can find which part of the fungus is going to again be the most efficient at digesting and converting the substrate into fruit bodies.


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Offlinestarbones
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Re: Why We Put Spores to Agar and How to Win the Genetic Lottery [Re: Wall.E]
    #27045127 - 11/18/20 09:12 AM (3 years, 2 months ago)

This was a good read and really enlightened me on the methodology and why it is done. Thank you!

:yeahgoodadvice:


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Offlinesmalltalk_canceled
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Re: Why We Put Spores to Agar and How to Win the Genetic Lottery [Re: starbones]
    #27050263 - 11/21/20 10:38 AM (3 years, 2 months ago)

Nice read


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InvisibleWall.E
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Re: Why We Put Spores to Agar and How to Win the Genetic Lottery [Re: smalltalk_canceled]
    #27050776 - 11/21/20 05:09 PM (3 years, 2 months ago)

Thanks to both of you! I'm glad somebody got something out of it, that's all I wanted.


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OfflineCapMeh
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Re: Why We Put Spores to Agar and How to Win the Genetic Lottery [Re: Wall.E]
    #27077715 - 12/07/20 07:49 PM (3 years, 2 months ago)

This was very informative and enlightening thank you Myc!


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OfflineFrozen Jedi
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Re: Why We Put Spores to Agar and How to Win the Genetic Lottery [Re: Wall.E] * 2
    #27119476 - 12/31/20 11:32 PM (3 years, 1 month ago)

Nice write-up man! The sex talk is never easy to have, but I think you boiled it down to the essence very well.


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InvisiblemushboyMDiscord
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Re: Why We Put Spores to Agar and How to Win the Genetic Lottery [Re: Frozen Jedi]
    #27196247 - 02/09/21 07:51 PM (3 years, 6 days ago)

solid read dude:takingnotes:


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InvisibleWall.E
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Re: Why We Put Spores to Agar and How to Win the Genetic Lottery [Re: mushboy]
    #27196250 - 02/09/21 07:52 PM (3 years, 6 days ago)

Thanks mang, appreciate it.


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InvisiblePudinLicous
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Re: Why We Put Spores to Agar and How to Win the Genetic Lottery [Re: Wall.E] * 1
    #27251671 - 03/13/21 06:09 PM (2 years, 10 months ago)

Great write up and nice read.

(Also a proud member of Tomentose Gang)


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InvisibleWall.E
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Re: Why We Put Spores to Agar and How to Win the Genetic Lottery [Re: PudinLicous] * 1
    #27251922 - 03/13/21 10:36 PM (2 years, 10 months ago)

Thanks man, tomentose for life


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InvisiblemushboyMDiscord
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Re: Why We Put Spores to Agar and How to Win the Genetic Lottery (moved) [Re: Wall.E] * 2
    #27264956 - 03/22/21 06:29 PM (2 years, 10 months ago)

This thread was moved from the user's journal.

Reason:
:feelsjohngoodman:


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InvisibleWall.E
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Re: Why We Put Spores to Agar and How to Win the Genetic Lottery (moved) [Re: mushboy]
    #27264980 - 03/22/21 07:06 PM (2 years, 10 months ago)

Thanks brah


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OfflineFailboat
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Re: Why We Put Spores to Agar and How to Win the Genetic Lottery (moved) [Re: Wall.E]
    #27264998 - 03/22/21 07:26 PM (2 years, 10 months ago)

Love me some fatty plate fruits.:wink:


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InvisibleBenson
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Re: Why We Put Spores to Agar and How to Win the Genetic Lottery (moved) [Re: Failboat]
    #27265135 - 03/22/21 09:48 PM (2 years, 10 months ago)

:takingnotes: gimmie dat knowledge

:cheers:


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OfflineKnucklehead
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Re: Why We Put Spores to Agar and How to Win the Genetic Lottery (moved) [Re: Benson]
    #27265198 - 03/22/21 10:40 PM (2 years, 10 months ago)

:congrats:


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OfflineZakkery
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Re: Why We Put Spores to Agar and How to Win the Genetic Lottery (moved) [Re: Knucklehead]
    #27265373 - 03/23/21 02:02 AM (2 years, 10 months ago)

:havesomescience:

I dig it. Fun to read to.


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Offlinehomegrownbrews
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Re: Why We Put Spores to Agar and How to Win the Genetic Lottery [Re: Wall.E]
    #27525553 - 10/31/21 05:04 PM (2 years, 3 months ago)

this was a very informative read sir!  now u should explain senescence in the same manner!  I've always wondered exactly how and what to avoid in my process with that, but don't really have a firm grasp on it... like can u transfer agar to agar too much... does going back to spore reset the genetic wheel, even if it's spores from a cloned fruit?

also, any thoughts on cloning fruits and to which flush I do it from?  I have heard that u don't want to take from later flushes but I'm not sure I understand why?  I don't get any large fruits until after 2nd flush but most often 3rd and 4th?

again, amazing write up.. wish u had a whole series:grin:


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InvisibleWall.E
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Re: Why We Put Spores to Agar and How to Win the Genetic Lottery [Re: homegrownbrews]
    #27525755 - 10/31/21 08:09 PM (2 years, 3 months ago)

About senescence, I really have no idea if that’s an issue that will disrupt the normal flow of someone’s cult work. If you have slants that are years old and you’ve been taking transfers and put them back into the fridge then maybe. But I also recall someone doing up to a hundred transfers or something like that just to find out it was still pretty vigorous. If you practice mycology well and get wedges to grain as soon as it’s looking clean then it won’t ever be an issue. I’ve personally ran some to T7 and didn’t have any issues, and I’m also notorious for letting plates and jars just sit for a week or two just because I don’t have enough time in the day.

As for the flushes and clone worthiness I again have no real idea. I’ve taken some from second flushes and they’ve been okay, and then other species that don’t have real defined flushes, like my tamp grows (they’re just pumping out fruits until they’re done) they seem to be hit or miss. I have super limited experience with clones though, so don’t take my word for it. Maybe someone else will chime in


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OfflineJaylude1904
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Re: Why We Put Spores to Agar and How to Win the Genetic Lottery [Re: Wall.E]
    #27525778 - 10/31/21 08:47 PM (2 years, 3 months ago)

Thx for the knowledge.


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OfflineCrackatoa
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Re: Why We Put Spores to Agar and How to Win the Genetic Lottery [Re: Jaylude1904]
    #27525949 - 11/01/21 01:06 AM (2 years, 3 months ago)

Taking transfer's and going from agar won't cause any issue's. You're taking away genetics be them good or bad, not in a massive way though. Going to spore gives you bigger range of range of fruits, size and speed. If that clone is stabilized you'll get those fruits from that print. Plenty of genetics still though.
For cloning most go for first flush fruits because speed is a factor most want, and cluster's are as well. A fast cluster clone will give better chances of fuller flushes, that's what we hope anyway. Plenty have gotten great clones from later flushes, it's mostly trial and error.


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