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Invisiblecaptaincache
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Help with sector identification
    #25591641 - 11/04/18 01:14 PM (6 years, 2 months ago)



This is my first time using agar to try to isolate a PE strain. I grew out one petri from a colonized grain and then did one transfer to this petri on 10/25. This plate is made with 10g agar, 10g ELME and 1g yeast.

I poured more agar plates and am ready to do another transfer.

I need help identifying the sectors that I should transfer. I know at a certain point just pick 6-12 spots and do the transfers but I figured that this would be good practice identifying the sectors.

I think this has a little over 15 sectors but am looking for your help in verifying what I am seeing. I think the 6 to 9 o'clock area looks promising.

What do you see and what would be your next move with a plate like this?

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Re: Help with sector identification [Re: captaincache]
    #25591809 - 11/04/18 02:35 PM (6 years, 2 months ago)

Is that a clone? :confused:


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Invisiblecaptaincache
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Re: Help with sector identification [Re: LotKid]
    #25591837 - 11/04/18 02:49 PM (6 years, 2 months ago)

Oh no why the sad face?!

No it's not. I have a couple of other ones going now that are clones but they are 5 days behind this one. It's the first transfer from a colonized grain that I took from a jar.

This is my first attempt at agar. Read a ton of different teks and guides and must have watched RR's videos a dozen times.

Since this plate, I've made about 80 plates. It's a great learning experience!

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Re: Help with sector identification [Re: captaincache]
    #25591994 - 11/04/18 04:14 PM (6 years, 2 months ago)

Did you put a print directly on an agar dish? Trying to figure out what I'm looking at here.


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Re: Help with sector identification [Re: TreasonX]
    #25592009 - 11/04/18 04:24 PM (6 years, 2 months ago)

Now I have been doing agar work for only a few weeks now and have not had them look like this either, so these are just my observations and attempts to learn..
the black eh? back lighting off maybe? I get the clone guess from the center that looks like a piece of stipe to start, though around that it looks like a calidascope or some such. There is also what seems like tons of sediment in it? idk..
can you post pics of some of them other 25 plates, if I read that right, to see if they all look similar.


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Re: Help with sector identification [Re: ithikuss]
    #25592056 - 11/04/18 04:46 PM (6 years, 2 months ago)

Sorry if that was confusing.

I made the picture an HDR so you could see the different sectors. It doesn't look like that in real life.

I had colonized grain jars directly from MS syringes. Then I did  a few G2G transfers of the best performers. I plucked one of the colonized rye grains out of the jars and put it on agar. I did a few transfers (the picture being one of the first transfers).

Here is the same plate without the light behind it and HDR settings


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Re: Help with sector identification [Re: captaincache]
    #25592131 - 11/04/18 05:16 PM (6 years, 2 months ago)

I was confused as to the where the transfer on this plate came from. It wasn't clear to me by reading your post and the center kind of looked like a tissue sample.

You're a looong way from an isolate. Trying to count sectors at this point isn't important. If that plate is clean you can refidge it for now and use it as a master plate to make work plates. Then you can grow out those work plates and start looking for clones. That plate has enough genetic diversity that if you don't like what comes off the worker plates you can just make more from other sections of the master plate.


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Re: Help with sector identification [Re: LotKid]
    #25592192 - 11/04/18 05:37 PM (6 years, 2 months ago)

Thank you! This is exactly what I needed to hear.

I'll refrigerate this one since I've already taken some sectors out of this and put on some other dishes.

I can post the pics to the 3rd and 4th dishes once those grow out a bit more.

My agar technique has definitely improved and I started using food coloring so you can see the myc better.

Thank you guys for the help and sorry it was so confusing.

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Re: Help with sector identification [Re: captaincache]
    #25592345 - 11/04/18 06:39 PM (6 years, 2 months ago)

I don't always be getting all my thoughts articulated all pretty and shit my damn self.

I actually thought the lighting and effect on your first pic are cool. Kinda like the filters used by the Hubble to photograph a nebula.


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Re: Help with sector identification [Re: LotKid]
    #25592384 - 11/04/18 06:59 PM (6 years, 2 months ago)

Haha. You mean you don't always be talkin' good n' shit?

I'm super excited about this hobby. I just started in March and went straight to grains and a monotub. I think I had some early luck but recently had one tub not produce which motivated me to start isolating strains that produce.

Thanks for the comment on the pic. I saw a few other pics of myc emphasized the sectors and I tried to recreate it.

Building a flow hood next week. Once I get closer to an isolate I'll post more pics.

As always thank you and thank the community for creating such a great area for noobs to ask questions.

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Invisiblestareatclouds
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Re: Help with sector identification [Re: captaincache]
    #25592478 - 11/04/18 07:51 PM (6 years, 2 months ago)

Quote:

I think I had some early luck but recently had one tub not produce which motivated me to start isolating strains that produce.




The fastest way to do that is to clone a tissue sample from the inside of a fruit body. You seem eager to learn with a good attitude. You'll go far with it. Glad to have you on board.

Also keep in mind that mycelium does degrade over time based on number of cell divisions. Agar to grain jar and grain back to agar is fine, but the more growth that happens, and the longer it sits out, the worse off it gets. There's no clear defining rules for when it's shot, but you can't just expand it forever. Perhaps you know this already, but worth pointing out. That's why people fridge stuff and use slants, so they can come back to earlier/younger cell lines and start over again.


Quote:

You're a looong way from an isolate. Trying to count sectors at this point isn't important. If that plate is clean you can refidge it for now and use it as a master plate to make work plates. Then you can grow out those work plates and start looking for clones. That plate has enough genetic diversity that if you don't like what comes off the worker plates you can just make more from other sections of the master plate.




I feel like he's not that far away from an isolate given he didn't swipe spores, but pulled a piece of grain from a colonized jar. Anastomosis should've taken place in the jar so he'll have less strains present than a transfer from a spore streak. That's probably why his shit already shows some preliminary sectoring?

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Re: Help with sector identification [Re: stareatclouds]
    #25592512 - 11/04/18 08:09 PM (6 years, 2 months ago)

Quote:

The fastest way to do that is to clone a tissue sample from the inside of a fruit body. You seem eager to learn with a good attitude. You'll go far with it. Glad to have you on board.




Thank you! Yeah I didn't know that this was a thing until I started reading up on it. I realized that I couldn't ride that sweet sweet G2G transfer train forever so I figured agar was the way to go. I have definitely spent my nights and weekends researching, reading and watching as much information as I can handle.

I'm going to do my first slant after I build my flow hood. I'm going to follow BOD's flow hood tek. Just ordered the HEPA filter and blower and have the wood cut already.

Once I get down to an isolate, do I want to go to other agar dishes or do I want to go LC route? I found so many different opinions but the LC route seems like the updated method.

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Re: Help with sector identification [Re: captaincache]
    #25592561 - 11/04/18 08:34 PM (6 years, 2 months ago)

I used to use LC quite a bit and have cut back significantly. Haven't made any in a long time. Stick with dropping wedges into jars for now. It works really well.

C'mon stare? You don't think that's still a good ways from an isolate? You can break a clone down into individual isolates thru transfers but that still takes time and not all will perform well.  He still took a grain from a jar that was shot up with spores. What's the difference between that and making 5-10 transfers on agar away from spore? It can take hundreds of transfers to get a true isolate and it's not always worth it.

:2cents:


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Re: Help with sector identification [Re: LotKid]
    #25592658 - 11/04/18 09:37 PM (6 years, 2 months ago)

Quote:

captaincache said:
Thank you! Yeah I didn't know that this was a thing until I started reading up on it. I realized that I couldn't ride that sweet sweet G2G transfer train forever so I figured agar was the way to go. I have definitely spent my nights and weekends researching, reading and watching as much information as I can handle.

I'm going to do my first slant after I build my flow hood. I'm going to follow BOD's flow hood tek. Just ordered the HEPA filter and blower and have the wood cut already.

Once I get down to an isolate, do I want to go to other agar dishes or do I want to go LC route? I found so many different opinions but the LC route seems like the updated method.




That's awesome, man. I'm excited to see how far you can take it.

So an isolate by itself is not guaranteed to be shit as it literally just means one strain and is often called a monoculture. So when you're making transfers to find an isolate, you want to take many at a time and fruit them all. So keep that in mind, because you can easily isolate a strain that doesn't even fruit. For a cool example of isolate performance, check this. And for an introduction into how much work it is to transfer to from MS to isolate through agar only, check verum's PE project.

Regarding what to do with it is up to you and your goals. The advantage to LC over agar to grain is speed and expansion. You can only transfer a wedge to so many jars, but using that to make LI/LC can go to dozens of jars. But liquids are a different animal and have lots of drawbacks. The main issue is agar is a 2D surface allowing you to visually inspect cleanliness before going to grain. With liquid, you have no way of inspecting it and guaranteeing it's clean. There's also many added vectors, such as making/prepping broth, transferring, usually shaking it up, and then putting that LC to grain (which often involves syringes). So it's up to you really. If you want to learn everything at once (which is what I did), just do a bunch of shit and keep your expectations reasonable. LC is very easy to fuck up and even experienced/professional growers just go agar > grain and then G2G expansion.

But with this example, I would definitely not go to LC at all. Why go to those lengths to expand an isolate with unknown performance? Once you have isolates, I'd personally put them on maybe 2 plates and 1 slant (optional at this point). I'd fridge the slant and 1 plate, and put the other plate to grain (how many jars depends on your situation/goals). Then I'd fruit each out, observe their performance, and keep what works. Then when you have proven isolates, pull from the slant/plate and put them to whatever your heart desires.


Quote:

LotKid said:
C'mon stare? You don't think that's still a good ways from an isolate? You can break a clone down into individual isolates thru transfers but that still takes time and not all will perform well.




I wasn't saying he's close, just that he's not as far as your post seemed to imply. And agreed on performance. I don't think I suggested that finding an isolate means success as I've always known that to be incorrect.


Quote:

LotKid said:He still took a grain from a jar that was shot up with spores. What's the difference between that and making 5-10 transfers on agar away from spore?




My understanding is the difference being spores on a plate from a syringe drop or loop swipe are tons of individual MS strains, just starting out. And you transfer early on when you see germination. A fully colonized grain jar is left alone and going to have less strains present due to anastomosis. However many, I don't know, maybe not enough to make a difference from a single grain to a single transfer. But anastomosis is why a clone from an MS grow has less strains present than an it started with.

It's my understanding that anastomosis would also occur in a colonized grain jar, just like it would in a fully colonized plate. And I also believe a transfer from the center of a plate will have less strains present than the leading edge, because the ones in the center have fused together and shared genetic information, making them their own strain.

If you agree that a cloned fruit will have significantly less strains than MS, what's the difference between a fully colonized tub and a fully colonized grain jar WRT anastomosis? There could absolutely be one so I'm genuinely curious, but my understanding is that strains growing together is what causes significantly less strains present in fruits. And that's what happens on any substrate you allow to fully colonize, such as a grain jar.

I could definitely be off on this, though. But that's how I understand anastomosis to work. And again, I don't think he's super close, either, just that I do think he might be closer than a spore streak. My initial post I had typed up was about how those aren't true "sectors" in the way he thinks, because when he transfers a tiny piece of one "sector", he's going to see more sectoring.

My comment was definitely rooted in theory of the life cycle, not a recommendation for anything.

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Re: Help with sector identification [Re: stareatclouds]
    #25592696 - 11/04/18 09:55 PM (6 years, 2 months ago)

First of all, you guys are amazing. My only wish is that this kind of information was easier to find and distill. Thank you guys for taking the time to write this up.

I have no problem learning everything at once. I have actually already done a batch of LC from a MS syringe and had some beginners luck. I purchased a cheap magnetic stirrer on ebay and was really cautious about being as clean as possible.

I also think you are right about being realistic about my expectations. I've been keeping a photo and video journal and have been really anal about labeling. If I fail, I go back and try to find the point of failure and improve.

I have two 66 quart tubs pretty dialed in. Just had my first one not fruit and definitely bummed me out to throw out $25 of hpoo and 7 quart jars of spawn. The funny thing is that I have been throwing this in dirt in the back corner of my yard and have harvested 6 oz. dry from spent substrate. Topic for another day.

The isolate performance experiment was fascinating. My main goal was to have a more predictable yield every harvest. It's just too much damn work to have to throw out an entire tub.

So to summarize, I don't have to have a true monoculture to have an even pinset with decent fruits. Not really concerned with HUGE harvests but just a consistent fruit for micro dosing. (and some macro dosing)

I know I said it already but super grateful for you guys.

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Invisiblestareatclouds
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Re: Help with sector identification [Re: stareatclouds]
    #25592710 - 11/04/18 10:10 PM (6 years, 2 months ago)

Here's a cool post from RR that kind of demonstrates what I'm talking about (emphasis mine): 

Quote:

When spores germinate and then pair up with compatable hyphae to form multiple strains that then fuse by anastomosis, the results can be seen in the picture below.  The single sector at the bottom of the growth is steadly becoming more of the total as it assimilates the other strains into its network.  If allowed to grow, it might have consolidated the entire dish into one strain or it may have ran up against an incompatible strain as seen in the pics above and stopped.

RR




I'm not sure if that's a sector to MS, but the concept still applies, I think. No idea of the numbers involved or how much it changes things, but I'm pretty sure the pool of strains present will always diminish if allowed to fully colonize something. Would absolutely love to be corrected on this as the topic is fascinating to me.

captain,

You remind me of when I first started, ha. And I still keep notes on tons of shit. Don't get discouraged. Just keep starting new things and you'll not care when shit goes bad. One thing that helped me early on was to record videos of my sterile work. That's where everything happens so it's best to develop solid habits early on.

This is a personal thing, but another thing that helped me hammer down new methods early on was making my own notes. First I'd find a trusted and current Tek and read the OP, taking notes along the way in a step-by-step process. Then I'd go through EVERY comment because people have already asked questions I'd have or been corrected on problems I'll run into. And these little tips/notes would get worked into my own notes on that specific Tek. When done compiling, I'd write out the entire Tek in a step-by-step process in my own words, and print it out for my binder. It reinforces the important information and helps build solid habits. And soon after, you won't need them anymore.

Also, for the most part, any cube spores you get are from stabilized and domesticated varieties. Combine that with an MS print with so many strains present, you're very likely to always get a decent yield. If you'd like a more guaranteed result, the quickest way is to clone a fruit from a cluster and run that bitch out. That's what I was saying in the original post. When cloning a fruit, you're taking tissue (genetics) from cell lines that you 100% already know will fruit. Read up on that shit and give it a try sometime.

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Re: Help with sector identification [Re: stareatclouds]
    #25594285 - 11/05/18 04:26 PM (6 years, 2 months ago)

Quote:

One thing that helped me early on was to record videos of my sterile work. That's where everything happens so it's best to develop solid habits early on.





Ha. I did this and wondered why i had some grain jars get contaminated. I was working in my SAB and didn't realize that I had touched my face and my phone 3 times while working on 7 jars. I didn't even realize that I did it until I went back and looked at the video.

I definitely go through this forum like crazy! I love reading the comments but there is so much conflicting information. I have an Evernote workbook of posts that I have found useful and teks that I have edited to fit my setup. Sounds like we are cut from the same cloth. Sometimes things are outdated. Sometimes teks leave out one very valuable piece of information. And sometimes you just have to do it until you figure out what works for you. Like how much polyfill is "tightly packed" vs. "loosely packed".

The one common thread is that everyone has varying levels of success with varying levels of effort and equipment. Do I go ghetto or do I pretend to be a professional level mycologist? Definitely trying to error on the side of pro status!

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Re: Help with sector identification [Re: captaincache]
    #25594546 - 11/05/18 06:17 PM (6 years, 2 months ago)

Glad you're recording stuff. My hands get a splash of iso every time they enter the SAB. The exception is flame sterilizing. You should make note of who the solid posters/cultivators are and seek out their posts. That's a good way to avoid shitty or outdated information. Keep it up, you'll be killing it in no time.

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Re: Help with sector identification [Re: stareatclouds]
    #25594689 - 11/05/18 07:24 PM (6 years, 2 months ago)

You have lots of sectors, yes. But I suggest you don't bother isolating what you have. Chances are you're gonna get shit performance from 99% of what you end up with, which will take a long time and a shitload of plates.

Just get it to fruit and take a clone. Isolate from that. You'll have much higher chances of finding something good that way.


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Re: Help with sector identification [Re: 36fuckin5] * 1
    #25597935 - 11/07/18 07:19 AM (6 years, 2 months ago)

Good points stare. I can't argue with you :lol:


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Re: Help with sector identification [Re: LotKid]
    #25634149 - 11/23/18 10:25 AM (6 years, 1 month ago)



Harvested this yesterday (pic taken about 8 hours before harvest) and took 12 clone samples from the biggest clusters and biggest fruits.

Also I am about 70% done building my flow hood. Followed Stro's Build and added a couple of modifications of my own to it. Is it worth posting a build guide on the site or is it too redundant?

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Re: Help with sector identification [Re: captaincache]
    #25634329 - 11/23/18 11:37 AM (6 years, 1 month ago)

Quote:

captaincache said:


Harvested this yesterday (pic taken about 8 hours before harvest) and took 12 clone samples from the biggest clusters and biggest fruits.

Also I am about 70% done building my flow hood. Followed Stro's Build and added a couple of modifications of my own to it. Is it worth posting a build guide on the site or is it too redundant?




Definitely post your flow hood guide. And harvest closer to the base of the fruit. You're leaving tons of weight behind judging from the stump in the back.

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Re: Help with sector identification [Re: stareatclouds]
    #25634344 - 11/23/18 11:49 AM (6 years, 1 month ago)

Quote:

captaincache said:


Harvested this yesterday (pic taken about 8 hours before harvest) and took 12 clone samples from the biggest clusters and biggest fruits.

Also I am about 70% done building my flow hood. Followed Stro's Build and added a couple of modifications of my own to it. Is it worth posting a build guide on the site or is it too redundant?



Flow hood guides are like porn here.


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Re: Help with sector identification [Re: Grimsweeper]
    #25634411 - 11/23/18 12:34 PM (6 years, 1 month ago)

Yes good call on harvesting closer to the base. That was one that I had to get out of there because the veil broke early. I took that one out 2 days ago and took a few clone samples from it. Harvesting might be my least favorite thing to do. I almost feel bad...

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