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Sectoring / Isolating


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#41 Misfit

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Posted 18 June 2019 - 10:53 AM

Posts like this are pushing me to pick up isolation ASAP. Have most the stuff on the way
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#42 Microbe

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Posted 18 June 2019 - 12:47 PM

After reading through this a second time it seems like the wisest thing to do would to collect spores from each cluster instead of clones to prevent senescence.

As Mycotopiate said, "there is a limit"

Now the big question is, does collecting spores from large clusters each generation yield the same results as cloning clusters from each generation? If so I'm not sure why anyone would do anything but collect the spores since it would be the best way to ensure the healthiest most vigorous young mycelium time after time instead of using a 5th generation clone that's been alive for a year.

If your attempting to isolate and preserve specific phenotypes then collecting spores and germinating them, then your going to have all sorts of diffrent phenotypes being displayed as a result of genetic recombination.

Its good to take prints often so that you have fresh prints to go back to in the event you lose a culture or you notice scenscens setting in and allthough not as rapid as cloning, but over many generations and decades, it will happen with spores.

So the best thing to do is stay on the youngest generation as long as you can by keeping master coppies and expanding from them. Smear a few plates from a slant and then those 2 plates can go on to make 100's more thus staying on the current generation and without the rapid loss in vigor that is associated with cloning from a clone from a clone. I clone from clones up to about 5 times before i see a very noticeable loss in speed. But then i go back to my master slant and i have a much younger culture as far as biological processes it has went through. Obviously based on time, it is the oldest culture i have when i go back to it.

Another thing i want to talk about is sectoring. I know your way past that as it was early on in this thread but none the less good info to have.......sectoring isn's just visible lines you see between the culture(s). Sectoring is any irregular growth. You may not be able to see any visible lines with the naked eye and it may look like a monoculture but for example if one side is growing out slightly faster not giving you uniform radial growth, that is considered sectoring.

When you get that thick ropy rhizo growth on a plate, those are some of the hardest plates to spot the sectors and are often inaccurately determined to be a mono culture.

Anyway beautiful cultures right out of the gate. You excelles in agar very quickly.
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#43 Taproot

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Posted 18 June 2019 - 01:24 PM

Thank you. I started doing agar work for the first time this year in april. Also thanks for clarifying what sectoring is in mycology. I was looking for definitions online and couldn't fine one. I didn't realize each culture on a dish was considered a sector hence "the act of sectoring"

I was also unaware that genetic recombination was in play. You're information has given me even more clarity into the subject.

A lot of tubs have been giving me a carpet of pin sets but there's one that is very young and from spore that just started and is producing a bunch of individual clusters. I looks like a promising place to start cloning. When keeping a master slant or culture, would I be using a 5th gen clone or would I stick to the first gen clone and always work my way forward from there?

You mentioned earlier that you have experienced senescence around the 5th gen mark and if so using that as a master culture in my VERY limited experience seems like I'm setting myself up for failure.

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#44 crazy1

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Posted 19 June 2019 - 05:10 AM

If you're going to make master slants, use the most aggressive myc you have on plates. 

 

Make your slants, noc them up, let the myc run and cover the top of the agar, put in the fridge in a light blocking container.

 

Senecense only happens when you continue using the SAME myc for numerous generations. As mentioned 5 in one persons experience. 

 

I'm thinking you're talking about the flushes as generations in that question. In my experience, you just take the most awesome cluster and clone it, which flush it comes from doesn't really matter. Once on agar that is your first generation. 


Edited by crazy1, 19 June 2019 - 05:11 AM.

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#45 Taproot

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Posted 19 June 2019 - 09:38 AM

If you're going to make master slants, use the most aggressive myc you have on plates. 
 
Make your slants, noc them up, let the myc run and cover the top of the agar, put in the fridge in a light blocking container.
 
Senecense only happens when you continue using the SAME myc for numerous generations. As mentioned 5 in one persons experience. 
 
I'm thinking you're talking about the flushes as generations in that question. In my experience, you just take the most awesome cluster and clone it, which flush it comes from doesn't really matter. Once on agar that is your first generation.


No I really was talking about the 5th generation so clone to agar to grain to clone 5 times.

Also I'm going to have to look up how to properly make a culture slant. Im still unsure of how to properly do that.

#46 CatsAndBats

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Posted 19 June 2019 - 10:05 AM

 



Its good to take prints often so that you have fresh prints to go back to in the event you lose a culture or you notice senescence setting in and although not as rapid as cloning, but over many generations and decades, it will happen with spores.
 

 

^^^This is worth repeating. I put post-it notes or write directly on my FC's so that I remember to print.

 

Speaking of post-it notes, it's also a good idea to label everything along the way, in addition to spawn date (which if you're not doing, please start), birthing/placed into fruiting conditions date, germination date (then you don't have to worry about senescence sneaking up on you). I don't mess around with transfer notes, but I'm sure there's a reason to but I write like a gigantic toddler, so I don't have room.

 

I use these:

rG-57-Columns-Extreme-Notes.jpg?MOD=AJPE

 

They're expensive but work really well inside FC's, if you're on a budget, decent duct tape works well too.

 

Oh and another semi-pro tip that microbe77 and I kick around, is the use of water/sanitizer/dirt resistant markers.

 

China markers are decent, so are other construction markers:

 

MILW-48223100_MED.jpg

 

Cat out.


Edited by CatsAndBats, 19 June 2019 - 11:07 AM.

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#47 Microbe

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Posted 20 June 2019 - 08:59 AM

If you're going to make master slants, use the most aggressive myc you have on plates.

Make your slants, noc them up, let the myc run and cover the top of the agar, put in the fridge in a light blocking container.

Senecense only happens when you continue using the SAME myc for numerous generations. As mentioned 5 in one persons experience.

I'm thinking you're talking about the flushes as generations in that question. In my experience, you just take the most awesome cluster and clone it, which flush it comes from doesn't really matter. Once on agar that is your first generation.

No I really was talking about the 5th generation so clone to agar to grain to clone 5 times.

Also I'm going to have to look up how to properly make a culture slant. Im still unsure of how to properly do that.
You will not see senescence in a 5th generation or probably any generation thereafter in your lifetime. Were talking and this is speculation, and were probably talking a several hundred generations so fruit>print>germinate a few hundred times and you might begin to see it. For it to cease to grow where probably talking 10's of thousands of generations maybe more but it does and will happen even germinating from spore from recent harvest but again you wont ever have to worry about that.

As far as 5th generation, if you found a good phenotype and isolated a mono culture or even if you want to preserve the culture regardless of why, you will have a very young culture with 100% of its genetic potential so slant it if you want.

When i said 5 times, and i dont care if its a 1st generation or 159th generation, i wont clone for then 5 times. And i certainly would never print after a clone that has been taken 5 times. Remember senescence is simply a fancy word for aging which is in fact a mutation of DNA meaning it can be inherited by the parent(s) potentially resulting in a newly germinated spore from the next generation to exhibit early signs of senescence and contradicting genetic recombination.

You want to preserve the lineage and only print from elite cultures to ensure you are optimizing subsequent gens to germinate and be able to perform at its maximum genetic potential.

Here is what i would do exactly. Fruit your culture(s) and run multiple tubs/bags/trays whichever you use and then clone from your best fruit or fruits that display the target phenotypes you are looking for. Speed, potency, flavor, colors, shape, are several examples of phenotypes.

Grow the clone out and transfer at least 2 times monitoring for any sectoring.

Fruit it and if you like what you see make several master slants and slant it. If you maintain your slants and do the required maintenance, and assuming your going to do this with a 5th generation...........you will be able to work with that 5th generation culture indefinitely. No need to clone or subculture anymore because you only get closer to senescence and eventually the culture will cease to grow and die if you continue to clone.

Fungi are fascinating and in the wild they are designed to have infinite growth. Look at the armillaria ostoyae nicknamed honey fungus, and not only is it the largest living organism on our planet, its anywhere between 2,500 and 10,000 years old. I know right thats a wide range but even on the low end its still impressive. By using slants and agar plates, you are essiently allowing for infinite growth.


I attached a short read on Senescence and there are many out there but this is one of my favorites. Attached File  0051-0055.pdf   977.04KB   12 downloads

Edit: i forgot to mention and in a earlier post i said take prints often, always print them from the youngest generation as possible as a best practice. Again assuming you work with a 5th generation and slant it, all your prints you take will be a 6th gen. If you go back to print just know that you will be starting the process of pheno hunting over again.

Edited by Microbe, 20 June 2019 - 09:13 AM.

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#48 crazy1

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Posted 21 June 2019 - 06:48 AM

Great info there Microbe!!!!!

 

I totally agree with what you've written. But senescence can happen much more quickly with agar to agar.

In doing edibles, and keeping close track on the transfers of myc on agar to agar for expansion, I have seen growth slow considerably after bout 40 transfers. 

 

But as you said, if going from print to agar then growing out, I agree we wouldn't see it in our lifetimes. 

 

 

 

To make slants, you want to add a bit more agar to your original mix. I'd suggest a gram more. Slants are nute rich thick pours.

You'll make your agar > pour into test tubes (or container of your choice) > PC as you would normally > remove and somehow make the tubes lean at an angle so you have more surface area for the myc to grow on. Then you would proceed as you would doing plate transfers. Once grown out to cover the slant area of the agar, store as I mentioned above

 

Peace and good vibes 



#49 CatsAndBats

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Posted 21 June 2019 - 07:46 AM

Great info there Microbe!!!!!

 

I totally agree with what you've written. But senescence can happen much more quickly with agar to agar.

In doing edibles, and keeping close track on the transfers of myc on agar to agar for expansion, I have seen growth slow considerably after bout 40 transfers. 

 


Peace and good vibes 

 

40 transfers? Damn. I don't think that I've ever done close to that number. Are you changing agar recipes? I do batches of 15 and I always add something that the myc has never seen before and/or change up the nutrition.


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#50 peacefrog

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Posted 22 June 2019 - 01:17 PM

I have done 100+ transfers before in my day and have never seen senescence from agar to agar transfers as of yet personally.

But I also do as cat asked, I change up my recipes from time to time during isolation.

From the way I understand it, senescence is directly influenced by cell divisions. In a plate that has been inoculated on it’s 50th time from agar to agar, very little cell divisions have occurred. But if one were to G2G transfer 50 times or super spawn 50 times, there would be a shit ton more cell divisions than A2A. Senescence would be more likely to happen in those scenarios IME.

That being said, I have been at this on and off since the 90s and I have never personally seen true senescence.

Might just be me though.

Edited by peacefrog, 22 June 2019 - 01:18 PM.

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#51 onediadem

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Posted 22 June 2019 - 11:36 PM

You can actually G2G from 1 jar into 1000. No worries there.


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#52 crazy1

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Posted 23 June 2019 - 05:55 AM

No I didn't change my agar recipe, that could have been a factor. And after 40 I began to see it slow down and that was enough for me to say no more.

So not true senescence.

Over 100 transfers, wow great job peacefrog! 


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#53 peacefrog

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Posted 23 June 2019 - 07:03 AM

Yes that probably was the issue. If you change it up say every 10-15 transfers, it will keep its vigor from my experience. And 10-15 is probably over kill.

Case in point with my favorite Cope culture. I probably A2A transferd it ~30+ times (I really don’t recall anymore) and I keep it stored in slants from which I take out transfer to a new plate then back to a fresh slant once or twice a year to keep it alive. It is going on 9 or so years old with multiple A2A transfers and it still grows and fruits exactly as it did when I first isolated and tested it. It’s a very fast and prolific fruitier.
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#54 crazy1

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Posted 24 June 2019 - 05:56 AM

Thanks my friend!!!! I'll change up the agar recipe when I try next time. 


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