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Invisiblec10h12n2o
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Strain Isolations on Agar, Pics and Questions * 1
    #23458285 - 07/20/16 01:58 AM (7 years, 8 months ago)

hi everyone, i am currently working on some strain isolations from 3 different races of cubes.

i wanted to get some feedback on a few plates that i am preparing to either transfer again or start making spawn. these are on the 3rd transfer from multispore.

Plate 20A2a:

Plate 33B1b:

Plate 33A1a:

Plate 33A1b:

Plate 33A2b:

Plate 100A1b:

Plate 100A1a:


I am wanting to know which of these look like they might be ready to go, and which need further transfers, and any other feedback you might have

warm regards

Edited by c10h12n2o (07/20/16 01:59 AM)

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OfflineMMG
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Re: Strain Isolations on Agar, Pics and Questions [Re: c10h12n2o]
    #23458291 - 07/20/16 02:02 AM (7 years, 8 months ago)

most of those look very good, great job.
I'd make some last transfers from each (isolating the most vigorous edges of growth) and then proceeding to either let the plate finish colonizing or putting the remaining wedge directly on grain. That way you have back ups and you can test out each isolate.


--------------------
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Re: Strain Isolations on Agar, Pics and Questions [Re: MMG]
    #23458564 - 07/20/16 06:14 AM (7 years, 8 months ago)

Quote:

MMG said:
most of those look very good, great job.
I'd make some last transfers from each (isolating the most vigorous edges of growth) and then proceeding to either let the plate finish colonizing or putting the remaining wedge directly on grain. That way you have back ups and you can test out each isolate.




None of those are isolates. You can still see it growing at different rates.

@OP: 3 transfers from the MS is nothing, it's easy to go through a box of 500 petri dishes during isolation. What you should do is transfer each of those at least 5 more times, or until you start seeing definite sectoring which you can spot easily. After that, start isolating. Don't start taking multiple transfers from multiple sectors early on in the game, it's just a waste of plates.

Once you start getting real close to getting isolates, you can start separating the sectors. That way you only transfer 1 or two cultures at a time until it's time to separate them.

Honestly, your main project should be cloning for now, with isolation being a side project. I have been isolating a single variety for at least 3 months now, only now do I seem to be getting close (although it always seems like you're getting close when you never did it before :lol:). I would also suggest that you only isolate a single variety at a time. If you want to get something worth the effort, you will easily need to get 20 isolates from each variety. Not every isolate will be good, some will be average, some will be crap and some will be great. RR used to recommend that you should get at least 20 isolates if you want a good chance of getting something good.

You say you are doing 3 varieties, if you get 20 isolates from each, that will be 60 cultures to grow side by side and compare. It's a lot of work in very little time. Start slow, one at a time or you will get overwhelmed.

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Re: Strain Isolations on Agar, Pics and Questions [Re: Supalemonhaze]
    #23458571 - 07/20/16 06:17 AM (7 years, 8 months ago)

Usually 10-15 transfers from MS to even see sectors start. Let alone grabbing an isolate. Maybe in 5-10 transfers if you take miniscule transfers and also on the very first plate use extremely little spore solution

Everyone gets the idea they want isolates. All the best growers grow clones and are still looking for great isolates in spare time. Great isolates are a treat you find in time. Spending all your work on just isolates and agar work is going to get old fast.

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Re: Strain Isolations on Agar, Pics and Questions [Re: bodhisatta]
    #23458577 - 07/20/16 06:20 AM (7 years, 8 months ago)

My only regret is that I didn't count the amount of transfers I made so far. If I had to guess, I already went over RR's 500 petri estimation. I fucked up by seperating the cultures early, I now learned the hard way why you should do a shitload of transfers before you start isolation.

Isolation should always be a side project, you really can't rush something like that.

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Re: Strain Isolations on Agar, Pics and Questions [Re: Supalemonhaze]
    #23458694 - 07/20/16 07:37 AM (7 years, 8 months ago)

what recipe are u using?  the wedges in the center are really thick, I get that look when I use peptone or yeast on top of malt.

u can wrap them in para or cling wrap instead of each one getting their own bag to save plastic….not that pouring plates is the best way to save plastic…but figured I'd throw that out there. 

Ur labels are pretty elaborate (numbers and letters) for being an MS plate thats probably going to be tossed after transfer.  I just label variety/# transfer/date (ex. GT, x-3, 7/14) for MS stuff, but whatever works.  sharpie instead of label maker will save time too. for slants, I throw a piece of scotch tape over the sharpie so it doesn't rub off.

I agree with everyone else, make some spawn and keep a few plates running for random isolates if u wanna go that route.


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Invisiblec10h12n2o
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Re: Strain Isolations on Agar, Pics and Questions [Re: blindingleaf]
    #23459130 - 07/20/16 10:22 AM (7 years, 8 months ago)

i really appreciate everyones responses, very helpful. Im certainly not scared of hard work, though of course it makes sense to work smarter not harder, thanks for helping clarify some things for me. I am really enjoying working on agar, the plan is to isolate about 60 strains and fruit them out, keeping good notes on everything. A couple of questions:

1. I definitely saw some clear sectoring in the last transfer (on some). Are you sure 33A1a and 100A1a arent isolates, or close? most of the others (along with the 30 or so plates from this series that i didnt photograph) seem to be showing clear sectors now. Am i missing something?

2.  when you say i should be mainly doing clones now and isolation should be a side project, do you mean i should be making spawn from multispore and then fruiting it and cloning the fruits?

Bodhi: Much obliged my friend. So should i fruit out multispore first? what do you suggest as far as changing my plan, approach, and/or mindset?

Quote:

I fucked up by seperating the cultures early, I now learned the hard way why you should do a shitload of transfers before you start isolation.




i am a little confused about what you mean here, probably the distinction between the verbs "transfer" and "isolate". Obviously, the first MS plate is a jungle, and you transfer the leading edge of vigorous growth. Then the second plate some seem to already show sectors, but after transferring it is clear that there were multiple strains present in each sample of the sectors i thought i saw. Though some that showed sectors in the 2nd transfer look pretty consistant on the 3rd (like 33A1a and 100A1a). At this point, when i am looking for sectors and transferring them to a separate plate, is that not  isolation?

As for recipe: Stamets YMPA, so yes yeast and peptone both. any suggested revisions?

i really appreciate the feedback! :smile:

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Re: Strain Isolations on Agar, Pics and Questions [Re: c10h12n2o]
    #23459141 - 07/20/16 10:26 AM (7 years, 8 months ago)

you arent seeing sectoring, yet. you are just seeing myc growth. you will know when you see sectoring. give me a sec and ill find a pic of sectoring and an isolate.

or rather, someone here prolly has pics of their own, like leaf.


--------------------
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Re: Strain Isolations on Agar, Pics and Questions [Re: c10h12n2o]
    #23459160 - 07/20/16 10:32 AM (7 years, 8 months ago)

Yeah, 3 transfers in you will definitely be able to tell that there are multiple strains but it's still not what we call definite sectoring.

The idea behind what I did wrong is this. This is what I did:

MS inoculation>2 transfers to ensure cleanliness>started isolating(meaning started separating strains until I had 20 plates)

What I should have been doing is this:

MS inoculation> At least 10 transfers to ensure cleanliness AND to "thin the herd"(could take more than 10)>Once definite sectoring is observed and you can tell that there isn't much strains, you separate 20 sectors into different plates> transfer until isolates.

The difference between these is that with my method, I will have ended up transferring 20 plates a shitload of times, which wastes a lot more agar for nothing. The proper method is to first thin out the herd using just 1 plate and separate the sectors once you notice that you are getting close.


I can tell you with 100% certainty that after 3 transfers, you do not have an isolate. This would only be possible with very little spores(ridiculously little) and transfers that are very very very small. What you need to do is take each variety, keep only 1 plate of each and transfer that until you see definite sectoring. Then separate.

Edit:

Keep in mind that this is a proccess that takes months, that is why you should be cloning as a main project. Cloning is a fast and easy way to get good genetics, isolation takes a lot longer but the result is a very stable culture with a possibly good yield. You are correct about cloning, that is what you should be doing, isolation should be something that is being done in the background. Don't expect to be finished with isolation in less than 2 months, I have been going for at least 3.

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Re: Strain Isolations on Agar, Pics and Questions [Re: Supalemonhaze] * 1
    #23459166 - 07/20/16 10:33 AM (7 years, 8 months ago)

Here is a good link. give that a good read and look at the pics of monoculture and sectoring near the bottom of OP.


--------------------
-The wise man never stops seeking knowledge.


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Re: Strain Isolations on Agar, Pics and Questions [Re: c10h12n2o]
    #23459190 - 07/20/16 10:41 AM (7 years, 8 months ago)

Pretty much everything has been covered by others in this thread on sectors/isolating, but  if i was working with those plates i'd look at 33A2b, personally i like the look of that culture
this would be MY personal choice though great looking plates man and you could keep working with any of those plates and probably get good results. :highfive1:

edit: i'm assuming the darker spot on there isn't a contam but just undisolved/mixed agar


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Edited by MysticMoteToter (07/20/16 10:46 AM)

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Invisiblec10h12n2o
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Re: Strain Isolations on Agar, Pics and Questions [Re: MysticMoteToter]
    #23459365 - 07/20/16 11:38 AM (7 years, 8 months ago)

i am so grateful for the replies and input, much obliged my friends :smile:

I will finish reading the rest of stro's thread, thanks for that.

Im learning, and loving it :smile: I totally see why you said it feels like you are getting close at first haha ... :smile:

When you say i should be doing clones right now, does that mean i should make a LC from multispore and then make spawn (like i did before learning about MS vs Iso), or could i make a LC or spawn from some of the multispore plates i have left from my initial transfers?

and yes the black/dark spots are just where i cooled the scalpel in the agar, and to provide a reference point for lining up photos

Ok, so just to make sure ive been doing this right, here are some of the transfers i am about to make. Do these look about right to yall? am i understanding correctly, or is there something i should change?

33A1a-2-cuts:


33A2b-2-cuts:


100A1a-1-cuts:


100A1b-1-cuts


PS: SLH, were you suggesting i revise my agar recipe?

Edited by c10h12n2o (07/20/16 11:39 AM)

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Re: Strain Isolations on Agar, Pics and Questions [Re: c10h12n2o]
    #23459489 - 07/20/16 12:18 PM (7 years, 8 months ago)

This is a contaminated clone transfer of pe luckily I have two others doing good but I wish I could of still used a bit of this one but having so many plates is hard to keep an eye on to catch a contamination early on.. but like you jumping into agar as a noob is fun and exciting and you can get overwhelmed especially trying to do tubs and transfers and look for good cultures listen to these dudes they all have helped me out alot.. don't waste too much time on that yet it gets to be a pain after you have three + types and thirty transfers of each haha you end up throwing shit away or using them on grains and being left with buying 20 mono tubs to keep up and still have too much spawn lmao


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Re: Strain Isolations on Agar, Pics and Questions [Re: Boogieman47]
    #23459506 - 07/20/16 12:25 PM (7 years, 8 months ago)

I agree with noob, isolating 3 varieties at once is overkill and can easily get overwhelming. All that pouring and transferring is a lot of work, especially because you have to do it constantly over a number of months. I would start with one and once I have a bunch of isolates, I would start isolating another while testing my first batch of isolates.

Anyway, you seem to be confused about what a clone is. A clone is when you make a multispore grow and then take the best mushroom in the tub and put a piece of tissue on agar. This will generally give you better yields pretty fast. You should take 2-3 clones from a tub to see which one is best.

Don't ever inoculate an LC with a spore solution, it's unnecessarily risky, especially since you already have agar. If live liquid inoculant is your thing, you should try LI. LI is safer than LC and it's just as fast.

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Re: Strain Isolations on Agar, Pics and Questions [Re: Supalemonhaze]
    #23459534 - 07/20/16 12:32 PM (7 years, 8 months ago)

https://www.shroomery.org/forums/showflat.php/Number/22833471#22833471 here is a link for li.. I did spore to LC and every jar was bad that li link I used and the jars were done almost half the time the myc growth is a bit different but I might have poured a bit too much solution making it more damp


--------------------

Edited by Boogieman47 (07/20/16 12:33 PM)

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Re: Strain Isolations on Agar, Pics and Questions [Re: c10h12n2o]
    #23459637 - 07/20/16 01:11 PM (7 years, 8 months ago)

Quote:

c10h12n2o said:
When you say i should be doing clones right now, does that mean i should make a LC from multispore and then make spawn (like i did before learning about MS vs Iso), or could i make a LC or spawn from some of the multispore plates i have left from my initial transfers?




no, cloning means taking tissue from a mushroom and grow it out. not using spores.
MS to LC is bad practice but also has nothing to do with cloning.

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Invisiblec10h12n2o
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Re: Strain Isolations on Agar, Pics and Questions [Re: Supalemonhaze]
    #23459704 - 07/20/16 01:41 PM (7 years, 8 months ago)

I definitely hear what you are saying about 3 strains being overkill and a lot of work, it certainly has been in the 200 or so plates i have done so far. If i hadnt started yet and already had several weeks and transfers into it, i would probably just pick 1 to run with (your suggested timeline makes WAAAY more sense lol), but since i already have these this far along i plan to try to finish trying to get isolates from them

I plan to fruit out at least 60 bucket/minimonos of isolated strains, and would be happy if i end up with even 1 good strain. I'm not scared of the work or patience required, I just want to do it right, or at least learn a lot in the  process :wink:

If that is the plan, and assuming those plates are on their 3-4 transfers, how do the proposed cuts look (numbered and outlined in red above)? Should I use all the cuts I numbered? Also, should i be doing 1 tissue sample per new plate yet, or should i be doing 2-3 on each new plate at this point?

I understand what a clone is, sorry if i wasnt clear. I was asking what route i should go to get a fruit to clone at this point. I currently dont have any MS grows going to clone a fruit from, but I have lots of clean MS plates, some with wedges taken out,  some further down the isolation chain than others. So do I have to all the way over waiting for spores to germinate, or can i make MS grain jars from some of the more colonized plates i already have? If so, should i use the whole dish of MS colonized agar and put it in a grain spawn jar, then g2g? Or should i take a vigorous looking wedge(s) from a colonized plate and make grain spawn with it? vigorous wedge or whole dish for LI?

I see what you mean about spore syringe to LC being unnecessarily risky. I actually did it with some success in several of my first grows (including a super soaker filled with it in a cow field lol), though it was probably dumb luck. My first 5 grows all used MS LC(LI?) from a syringe. of course i would hope to avoid anything that risky/dumb again though

I thought i had moved beyond my days of of MS when i started culturing isolates, thanks for the reality check :smile:

I will read up on the distinction between LI and LC, because i have been using the two terms interchangeably from my watergun days until now

Edited by c10h12n2o (07/20/16 01:42 PM)

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Re: Strain Isolations on Agar, Pics and Questions [Re: c10h12n2o]
    #23459723 - 07/20/16 01:50 PM (7 years, 8 months ago)

Multispore to LC is a terrible waste of spores and time

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Invisiblec10h12n2o
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Re: Strain Isolations on Agar, Pics and Questions [Re: bodhisatta]
    #23459729 - 07/20/16 01:54 PM (7 years, 8 months ago)

Quote:

blindingleaf said:
what recipe are u using?  the wedges in the center are really thick, I get that look when I use peptone or yeast on top of malt.






yes i am using yeast malt peptone agar a la paul stamets. It sure has acted different than the MEA i used to begin with, which was more whispy. Several of the cultures on YMPA are VERY thick in the center, even though i only used tiny wedges. Several of them are pressing up against the lid of the petri dish, and others have grown so opaque and thick that light barely passes through them!

Any feedback or suggested revisions you had in mind?

Warm Regards
Quote:

Trusted Cultivator said:
Multispore to LC is a terrible waste of spores and time




that makes perfect sense to me :smile:. care to shed light on some of my questions?

Edited by c10h12n2o (07/20/16 02:27 PM)

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Re: Strain Isolations on Agar, Pics and Questions [Re: c10h12n2o]
    #23459734 - 07/20/16 01:56 PM (7 years, 8 months ago)


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