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Stipe-n Cap


Registered: 08/04/12
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Why Agar? 18
#28188696 - 02/15/23 01:21 PM (11 months, 6 days ago) |
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Why do we employ agar for mushroom cultivation? At first glance it may appear perfectly reasonable to grow mushrooms straight from spore; plants grow from seeds planted in soil, after all, so why not reduce the complexity of mushroom cultivation by doing essentially the same with spores? Letβs look at what agar is first before we move on to the why and how. Robert Koch initially utilized potato slices to culture bacteria during his investigations into the germ theory of disease, Koch postulated that: 1. The microorganism must be abundant in all organisms suffering from the disease but should not be found in healthy organisms. 2. The microorganism must be isolated from a diseased organism and grown in pure culture. 3. The cultured microorganism should cause disease when introduced into a healthy organism. 4. The microorganism must be re-isolated from the inoculated, diseased experimental host and identified as being identical to the original specific causative agent. To prove these postulates, Koch would require media to both grow and then isolate these organisms for identification. Gelatin was widely available but failed to produce a growth medium that could reliably culture bacteria due to the protein structure of gelatin. Bacteria would often utilize those proteins as an energy source, gelatin failed to remain stiffened at higher incubation temperatures and would often melt when bacteria produced protease enzymes that break down proteins. Agar, on the other hand, is made up of carbohydrates extracted from seaweed. These carbohydrates have a much higher melting temperature than animal proteins which bacteria do not readily catabolize for energy. Koch would eventually graduate from potato slices to agar-stiffened liquid media poured into jars; Julius Richard Petri would further develop Kochβs culture dishes into the Petri dishes we recognize today. How is this germane? When streaking microbes for identification there may be a wide variety of bacterial species present including yeasts and molds; sound familiar? To I.D. the species of microorganisms responsible for disease, there must be a method for isolating a pure culture, the same is true for the cultivation of mushrooms; this is achieved by streaking to agar. Agar-filled Petri dishes provide a sterile environment to germinate and grow all present microorganisms allowimg you to isolate a pure culture of your target organism. Mushroom fruit bodies grown in an open environment like tubs or tents will be exposed to environmental contaminants like molds and bacteria. The removal of caps for printing, swabbing, etc, risks further contamination via handling. Spores are to be considered septic/contaminated by default.   When spore syringes, spore prints and swabs are viewed from this perspective it becomes obvious why agar is necessary: to produce a pure mushroom culture free of contaminant organisms. Failing to produce a pure culture prior to inoculation will result in a very high risk of failure. Pouring /wrapping blank platesStreakingThere are three common methods for streaking: 1. Quadrant method 2. T method 3. Cell spreader The quadrant method of streaking produces 4 zones of growth. The first contains the initial inoculum at full concentration, each subsequent quadrant aims to pull material from the previous by thinning the cell concentration utilizing a zig-zag pattern while either replacing your disposable loop or flaming in-between:    This process thins the concentration of cells allowing for separation of growth, separation facilitates isolation. Without proper separation the cultivator risks growing mushroom mycelium on top of bacteria, yeasts, and molds, making it difficult to isolate a pure culture free from contamination. T method is essentially the same except with 3 zones of growth as opposed to 4:  If using spore prints, just add a drop or two of sterile water to the edge of your plate to facilitate streaking/spreading of dry spore material: Cell spreaders are used to evenly distribute cells across the surface of the agar, cell spreading is the easiest of the three and produces excellent results:  Add a single drop of spore solution to the center of the plate, then gently smear the solution evenly across the entire surface of the agar with the L shaped spreader. The result:     Notice the amount of growth produced from a single drop of spore solution? This is a great example of spores' septic nature and the sheer number of both target species spores and bacteria. Each milky dot in these pictures are bacterial colonies, had I not used the cell spreader all of the bacteria and mycelium would have grown together, infecting the culture. Should I have chosen grains or any other substrate other than agar, well, I think you get the point. Bacterial contaminations may persist even after multiple transfers. Isolating clean mycelium early on will prevent any downstream frustration. When first starting out I advise you resist the urge to buy or use liquid culture for the following reasons. Don't use LC until you have all of the lead up techniques including the ability to produce reliably clean spawn nailed down. Stay as far from LC as possible until then, otherwise you'll be chasing variables and failures forever. Each skill builds upon the next. 1. Spores to agar to produce culture; cleaning to produce pure culture, making transfers, and agar to grain. This step will familiarize you with the fungal life cycle and introduce you to the basics of identification for both contamination and healthy mycelium. During this phase you'll learn how to reliably produce clean cultures and sterilize both grains and media. Once these skills are mastered, move to skillset #2 2. Grain to grain expansion. This step expands upon the previous production phase by introducing how to expand your pure culture exponentially. This is a more advanced stage dealing with inoculation techniques and aseptic techniques which will prepare you for the next step: 3. Liquid inoculant/liquid culture. Liquids will not work for you if you haven't learned to identify pure culture, contamination, or prepare sterile media which was learned way back in step one. Liquids won't work for you if you don't understand/master the aseptic techniques required for expansion. Walk before you run, otherwise there's too many variables to troubleshoot. Each phase carries it's own set of criteria for troubleshooting problems, if you skip them, you're going to be fucked. Making Agar Agar is quite simple when dealing with non-selective media. Standard 2% gelling and nutrient strength are all that's required to produce healthy fungal colonies. Amending with peptones, yeasts, etc, is not necessary but if that's your thing, to each their own; for most of us this will be all you'll ever need: for a sleeve of 25 @ 2% concentration: 600ml distilled h20 12g agar 12g Dry malt extract for a sleeve of 20 @ 2% concentration: 400ml distilled h20 8g agar 8g Dry malt extract A note regarding yeast extract and peptone:Quote:
Yeast extract and peptone provide nitrogen compounds, vitamin B complex and other nutrients for growth. Yeast extract agar allows separate counting of aerobic mesophilic organisms that form visible colonies in this medium after 24 hours incubation at 37Β°C and those that form colonies after three days incubation at 22Β°C. The two methods give different results.
https://www.biotrend.com/en/buy/cat-yeast-extract-agar-non-selective-5494.html
Peptones are specifically used for culturing bacteria, particularly bacteria associated with fermentation, or food spoilage. When used for culturing fungi, it's used to isolate and diagnose pathogenic and non-pathogenic fungi. Peptones were originally made from anamal products, beef in particular. Peptones are partially digested proteins.
Protein = Nitrogen.
When considering gourmet/active culturing techniques, ask yourself whether a nutrient dense media is beneficial. Agar is not only a growth medium, it's an inoculum. When used as growth medium we utilize for isolation from contamination (cleaning), spore germination, pure culture expansion and storage. When germinating spores, spores are often comingled with aggressive contaminant organisms; a nutrient dense, nitrogen rich medium may not be beneficial.
When utilized as inoculum there may be an argument made for rapid expansion, this would likely be the case with large volume liquid culture broth production. Use of peptones with agar, on the other hand, seems like a waste when considering its wide application for germination, cleaning, culturing, and storage. Nitrogen rich media would be counterproductive when used for cleaning or germination due to the probable presence of aggressive contaminant organisms that would benefit from the excess nitrogen.
Malt extract provides ample nutritional resources, further ammendments are not necessary:


https://consteril.com/steam-sterilization-cycles-part-2-liquids/
Adjust sterilization time to the volume of water. I always cycle for 45 minutes, vent the pressure cooker for 15. The longer cycle will ensure that solids have completely dissolved. Most people are running their cycles for 15-20 mins, this is a mistake. Less is not more with regards to sterility assurance. The standard 15- 20 minute cycle falls well below the minimum threshold for the standard volume people are using for agar and LC. Most people are mixing 500-1000ml when prepping agar.
There's no requirement whatsoever to fill the PC/autoclave with water. Place your agar vessel(s) on an elevated trivet, fill water for the cycle up to the trivet:

Allow the pressure cooker guage to reach zero before removing, any attempt to evacuate steam/hasten cool down will result in boil over.
Before and after pc cycle:
 
No boil over; no caramelization; no maillard effect; no crashing; no sediments; no problems. Your light, dry malt extract will produce varying levels of clarity depending upon the Lovibond rating of your malt:

Clarity does not affect growth, simply aesthetically pleasing. 2 Lovibond will produce the greatest clarity, the same is true for distilled h20: a greater concentration of dissolved solids = more yellowing of the end product.
These are the products I use:
 
Wrap plates with Parafilm or cling wrap.
The following plates were poured using the above 2% agar and dry malt extract powder recipe listed above.
  
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Shrimps
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Great writeup p9!
-------------------- I am not crazy, I prefer the term mentally hillarious. Agar - The way to go! Clean spawn checklist Proper Surface Moisture Recognizing and dealing with contamination π
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dowodenum
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Re: Why Agar? [Re: Shrimps] 1
#28188721 - 02/15/23 01:31 PM (11 months, 6 days ago) |
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Posting in a future oft-referenced thread.
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hazyhorse
scoobin



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hell yeah stipe. excellent resource to point people to when explaining agar. thanks for all the banger write ups
-------------------- you're not the first to set foot here, just another =================================== i love glass petris & you can too!! posts i constantly refer back to new to mushroom cultivation?? read this!! ===================================
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LotKid
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props for taking this kind of time and effort.
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Baba Yaga
β₯ coir grower

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Re: Why Agar? [Re: LotKid] 3
#28188888 - 02/15/23 03:31 PM (11 months, 5 days ago) |
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Always good to keep things tight and fresh. Sometimes it feels contradictory when telling someone they are using old information and then referring to a 6 year old post explaining the basics although these are still valid posts.
This will be the agar thread I'm referring to from now on.
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Stipe-n Cap


Registered: 08/04/12
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Re: Why Agar? [Re: LotKid]
#28188895 - 02/15/23 03:34 PM (11 months, 5 days ago) |
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Quote:
LotKid said: props for taking this kind of time and effort.
Keeps me busy.
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johnukguy
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-------------------- How to post pictures to shroomery TEK Shroomery Trusted Cultivator And Member YouTube Channels. βEvey Hammond: Who are you? V: Who? Who is but the form following the function of what and what I am is a man in a mask. Evey Hammond: Well I can see that. V: Of course you can. I'm not questioning your powers of observation I'm merely remarking upon the paradox of asking a masked man who he isβ
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Sikjakass
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Commenting so I can find again lol
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Advil_Liquigels
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Thank you so much for this.
"Don't shoot spores into grain" should be the number one biggest advice to new people. I wasn't aware that spore syringes had contaminants in them, I thought they could be manufactured to be "clean" but now I know and that's honestly been the biggest thing steering me toward agar work.
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meta_mmxxii

Registered: 08/03/23
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Quote:
Advil_Liquigels said: Thank you so much for this.
"Don't shoot spores into grain" should be the number one biggest advice to new people. I wasn't aware that spore syringes had contaminants in them, I thought they could be manufactured to be "clean" but now I know and that's honestly been the biggest thing steering me toward agar work.
Solid advice.
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Stipe-n Cap


Registered: 08/04/12
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Clean syringes can be made, however, they're the exception and not the rule. Many headaches can be avoided by assuming all spores are dirty, doing so will save you in the long run. Even with proven clean spore solution, agar is still the weapon of choice for the reasons mentioned in the OP.
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SupaThaRipper
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Really appreciate this thread Stipe. Iβve been filling my PC up with water to the level of the agar to prevent boil over. Same thing with LCs. Itβs really nice to know itβs unnecessary. Do you have a write up going in depth, like this one, about transferring and isolating. Iβve read about dropping nutrients with each transfer but that seems to be unnecessary. Not really sure and would like clarification.
Also, βcaramelizationβ is something thatβs definitely heavily circulated. Realizing such a thing isnβt something to worry about. Thatβs why people are doing 20 minute cycles
Edited by SupaThaRipper (11/09/23 07:15 AM)
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Pnin
Riz Gukgak


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This one is ten years old, but it is the one I return to for pics and to read. Might be worth a look. https://www.shroomery.org/forums/showflat.php/Number/18430998/fpart/all/vc/1
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Stipe-n Cap


Registered: 08/04/12
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Loc: Canada
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There's plenty of bad or incomplete info in circulation, I do my best to present reliable information; I'm glad you've found it helpful. I don't have any posts that cover culturing, unfortunately.
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SupaThaRipper
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Re: Why Agar? [Re: Pnin]
#28535189 - 11/09/23 07:22 AM (2 months, 18 days ago) |
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Quote:
Pnin said: This one is ten years old, but it is the one I return to for pics and to read. Might be worth a look. https://www.shroomery.org/forums/showflat.php/Number/18430998/fpart/all/vc/1
Thanks pnin! Iβm going to check this out. Pretty old but hopefully reliable lol
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tryptkaloids
Learner



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That link is 100% reliable and the one I link to peeps.
-------------------- "Remember, kids, the difference between science and screwing around is writing it down" -adam savage Flowchart for Recommended plan of action. Learn the tried and true way to grow mushrooms Use the Damn search engine After you know what you're doing, take a break Pick a book, Make some chips! Josex said:Don't take the site seriously bro, ain't worth it.
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c01h
Aspiring Psychonaut



Registered: 11/24/23
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Stipe at it again
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Stipe-n Cap


Registered: 08/04/12
Posts: 7,623
Loc: Canada
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Re: Why Agar? [Re: Pnin]
#28583740 - 12/15/23 10:10 AM (1 month, 13 days ago) |
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Quote:
Pnin said: This one is ten years old, but it is the one I return to for pics and to read. Might be worth a look. https://www.shroomery.org/forums/showflat.php/Number/18430998/fpart/all/vc/1
I think Stro meant well but this must be the source material for some of the ideas circulation about sectoring, strain isolation, and the use of the term "monoculture" to refer to a single strain isolate. None of that information is correct.
Single strains cannot be isolated on agar from clone or multistore uaing a scalpel, "sectors" are not individual strains, "monoculture" isn't a synonym for "isolate" in the sense that it'sa "single strain isolate".
A monoculture is a single propagated species, all plates cultures free of contaminants are a monoculture (pure culture), which should not be mistaken for a monosporic, or single strain isolate.
Quote:
As you can see the cultures are looking healthy and the strains ar showing their individual characteristics in A and B and as suspected C is a true monoculture.
As a learning tool, I will go over one or two things here. When I first posted the photo that were taken seven days after transfer someone was asking me a few questions and I said;
Quote:
Stropharis said: C is now true monoculture, yes. As for A I believe there are two strains but it is hard to tell
Outdated and definitely incorrect.
Quote:
SupaThaRipper said:
Quote:
Pnin said: This one is ten years old, but it is the one I return to for pics and to read. Might be worth a look. https://www.shroomery.org/forums/showflat.php/Number/18430998/fpart/all/vc/1
Thanks pnin! Iβm going to check this out. Pretty old but hopefully reliable lol
Not entirely.
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SupaThaRipper
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Yeah itβs circulated, thatβs for sure
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Way
The


Registered: 01/14/23
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Hey Stipe, I started with an understanding of strains, sectoring, and monocultures from Stro's post. I've since read some more of your explanations on various posts and obviously have come to realize that it's not entirely correct.
So with my new understanding of "monocultures" and "isolates", I still am left wondering about "sectors". You've explained that they aren't individual strains showing in agar, and I can get down with that. Makes complete sense to me.
What exactly are they though? Why does mycelium "sector" out like that and is there any benefit to paying attention to the "sectors"? Besides trying to get a more uniform culture of course.
I don't generally pay any attention to them besides just looking for the fastest, densest, most organized part of the culture, and that has been working great so far.
Just wondering if you can push my understanding of this forward a bit. Thanks for all the information you've provided to everyone, I really appreciate it.
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Stipe-n Cap


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Re: Why Agar? [Re: Way] 5
#28583919 - 12/15/23 12:55 PM (1 month, 13 days ago) |
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Hyphae extend radially from a central point of inoculation. This central point consists of many aggregated strains which have branched and fused dependent upon mating type compatibility.
 
When starting from multispore, individual monokaryotic hypha branch out and immediately fuse with a suitable monokaryon, producing a dikaryotic single strain, until that dikaryotic strain finds further suitable monokaryons, then mate.
  
The dikaryons can anastomos with compatible monokaryons through what as know as "Bullers phenomenon". Dikaryons cannot fuse with other dikaryons, only compatible monokaryons, and monokaryons with other compatible monokaryons. This monokaryotic stage is short lived due to the high compatibility of mating types. This is taking place at microscopic scale, very tight quarters, which is why any attempt to tease out individual strains is impossible without the aid of serial dilution, microscopy, and specialized tools for manipulation.
Quote:
Hyphal aggregation may fulfil migratory or connective roles in the case of rhizomorphs and mycelial cords which, being corporate structures, are often capable of far more rapid extension than individual hyphae, whilst exhibiting parallel patterns of branching and anastomosis"
-Versatility and degeneracy p.25
Sectoring is the result of differences in growth speed, possible mutation, environmental factors, and possible differences in genetic grouping.
By the time your culture is visible to the naked eye it has become completely dikaryotic. Hyphal branching and fusing has jumbled all of strains together into an intricate mycelial matt which is rhizoid in morphology under a microscope:

Quote:
It is also arguable that without the ability to alternate exploratory and exploitative phases, mycelial margins would be unable, as they do, to maintain a constant rate of extension in more than one dimension without becoming progressively more sparse. The latter would follow from the inability of lateral branches to infill between leader hyphae that are already at maximum extension rate.
- The Growing Fungus
Sectors will contain many strains, how many? I really don't know, I'm not sure if anyone does, tbh. The likelihood of seriously distinct lines of genetic heritage being present on one side of the plate or the other, or between neighboring "sectors" seems to be extremely unlikely due to the tiny central location where the entirety of the genetic material originated from.
Further transfers from x plate will not significantly reduce the genetic diversity found on any given plate, perhaps slightly, but not significantly, but most likely not at all, tbh. The branching hyphae have so completely filled the microscopic gaps and fused where possible that most of the plate culture will be more or less identical, but not like an isolate in the sense that there's only one distinct set of genetics, there's just too much going on to separate those differences on agar, at the macroscopic level.
Genetic diversity remains on the plate, and will be expressed in your grows, but cannot be separated without serious intervention. The only way to separate the parental haplonts would be via chemical dedikaryotization, not scalpel transfers.
A simple thought experiment by argumentum ad absurdum: If heterozygosity was significantly reduced via scalpel/plate transfers, it would be possible to reduce any culture to a pure single strain isolate, eventually (which is what has been claimed). If this were true, we would have broken almost every cultivator multiple times over, making cultivation of demosticated lines either impossible, or incredibly difficult without the constant introduction of new gene flow from wild sources.
This is how I understand the literature, there are surely to be gaps in my understanding, but this is less incorrect than the previous understanding regarding sectors, etc.
I hope that helps
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Way
The


Registered: 01/14/23
Posts: 4,336
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Hey thanks for taking the time to go through all of that. I'm very grateful to you.
Quote:
Stipe-n Cap said: -Versatility and degeneracy p.25
Sectoring is the result of differences in growth speed, possible mutation, environmental factors, and possible differences in genetic grouping.
Quote:
Stipe-n Cap said: Further transfers from x plate will not significantly reduce the genetic diversity found on any given plate, perhaps slightly, but not significantly, but most likely not at all, tbh.
I find these points in particular to be very interesting. I suspected both of these but having you confirm it really solidifies my understanding of how it all works. There really is a large misunderstanding around all of this on here.
Thanks again!
--------------------
That's the way she goes, boys. Sometimes she goes, sometimes she doesn't, cause that's the fuckin way she goes.
Edited by Way (12/15/23 01:33 PM)
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Stipe-n Cap


Registered: 08/04/12
Posts: 7,623
Loc: Canada
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Re: Why Agar? [Re: Way] 3
#28583967 - 12/15/23 01:40 PM (1 month, 13 days ago) |
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Fungis is very counterintuitive. There's so much left unknown, and what is "known" will likely change in the future. Science is all about being able to change your mind when new information is presented.
I'll keep digging, I'm sure I'll be changing my mind about the above sooner or later, but it's the best I have for the time being. Maybe someone else with a deeper understanding will help fill in some of the blanks, contributing to the evolution of our understanding.
Time will tell. But until then, this is at least closer to the truth than what was previously available.
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tryptkaloids
Learner



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Stipe, what's your background if you don't mind me asking?
I hope to be as smart as you one day
-------------------- "Remember, kids, the difference between science and screwing around is writing it down" -adam savage Flowchart for Recommended plan of action. Learn the tried and true way to grow mushrooms Use the Damn search engine After you know what you're doing, take a break Pick a book, Make some chips! Josex said:Don't take the site seriously bro, ain't worth it.
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Stipe-n Cap


Registered: 08/04/12
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Loc: Canada
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Just a guy on the internet who enjoys engaging in these kinds of topics.
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Bajazly
Stranger... Than You Think



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Stipe, who are these guys? The gardeners?
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Stipe-n Cap


Registered: 08/04/12
Posts: 7,623
Loc: Canada
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Re: Why Agar? [Re: Bajazly]
#28584267 - 12/15/23 05:48 PM (1 month, 12 days ago) |
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Lol, probs your friendly neighborhood bacteria 
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HappinessStan
Fungivore



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Quote:
Stipe-n Cap said: Just a guy on the internet who enjoys engaging in these kinds of topics.
Never ending learning. Thanks for the detailed explanation. I'm half way there but there's always still so much more to learn. Organisms are just fantastic, such complex lifeforms that never cease to amaze us. What a beautiful world we live in.
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Stipe-n Cap


Registered: 08/04/12
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Limitless curiosity and some critical thinking goes a long way, you can teach yourself anything.
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Shroomish8
Stranger

Registered: 12/25/18
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#28583919
Wow. That could be its own post, but it's just an amazing reply to an amazing post. Great explanation. Thank you for the clear, detailed write-up on agar AND hyphae interactions! Definitely cleared up some misconceptions and confusion I've had for a while.
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c01h
Aspiring Psychonaut



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Quote:
Stipe-n Cap said: Limitless curiosity and some critical thinking goes a long way, you can teach yourself anything.
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Stipe-n Cap


Registered: 08/04/12
Posts: 7,623
Loc: Canada
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Re: Why Agar? [Re: c01h] 1
#28586450 - 12/17/23 07:25 AM (1 month, 11 days ago) |
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The quickest way to reduce heterozygosity in a culture (other than with serial dilution/microscopy/dedikaryotization) is through successive generations of spore printing. Not by plate transfers:
Generation zero - wild specimen heterozygosity 100%
F1 multispore of wild specimen 50% F2 multispore of F1 specimen 25% F3 ....12.5% F4 ....6.25% F5 ....3.12% F6 ....1.56%
and so on.
After the F6 generation there is such little heterozygosity (variability) left that all the mushrooms generated by multispore are virtually identical and the strain is considered stable.
The immediate descendants of the initial parents are the first filial generation (F1), much of the cultivars in circulation are well past f10, some may be close to f100+, which is why I believe some cultivars are beginning to, dare I say, "break", like golden teacher, for instance; Mutations abound.
Any of the successive generations of descent from an original parental generation contain less genetic material than the previous generation, which means one should be very careful when selecting specimens for printing.
Haphazard printing of just any ol fruitbody is reducing genetic variance along a specific trajectory, whether the cultivator realizes it or not (most very new growers probably don't consider it). Some trajectories are better than others, and once genes have been removed from a given culture, they're gone.
When selecting genetic material for your stock, the source of those spores will be a very important variable, because you'll need to trust that the source materials have been carefully handled/preserved intentionally, otherwise you may be receiving or producing less than desirable traits for future generations.
There are far more haphazardly taken prints in circulation than not, syringes are no different as they're the end product of spore production. With the influx of interest and new growers, people are swapping spores like cousins swap spit in Appalachia.
Food for thought.
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Thankful
Seeker

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Stipe - this is an awesome post full of great educational value. Thank you so much!
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HILLBILLY OUTLAW
Above And Beyond!



Registered: 04/21/22
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 Awesome write up. Your knowledge is and ability to share it is a vital part of our community!!!
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π° πΏ TEAM SPREAD THE LOVE! Smellyhobbit said: Embarrassment and bashfulness are leeches on your ability to learn.
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