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Sectoring / Isolating


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#1 Taproot

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Posted 18 May 2019 - 08:25 PM

Hi! I'm new to sectoring and have been reading a lot about genetics isolation and how agressive rhizomorphic mycelium is desirable. I have some pictures of some spores that I originally inoculated rye grains with, then transferred to agar, then transferred to new plates and am wondering which parts I should further isolate. If it matters, these samples are on M.E.A. They were transfered from the original plate 2019/05/14. These pictures were taken today 2019/05/18. Any tips are much appreciated! Thanks everyone.

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#2 wharfrat

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Posted 18 May 2019 - 09:26 PM

welcome to topia..  just grab a small sample from he strongest looking sector, one of the arms that reach out from it should be a good place to start. I usually take a few samples and isolate further. take best sector of each and grow them all out. keep the best of the flush.. do more than one because all might not grow.


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#3 roc

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Posted 18 May 2019 - 09:41 PM

Welcome to 'topia!



#4 Taproot

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Posted 19 May 2019 - 12:05 AM

Thanks for the advice. The thing is I don't know what sections of each culture I should be selecting. Can anyone maybe draw an arrow to the spots in my pictures that I should be selecting? I am really new to this and don't know where the "strongest sections" are. Or how to properly identify them. Other than looking for "ropey" looking concentrations of hypea. Or is that all there is too it?
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#5 Taproot

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Posted 19 May 2019 - 09:51 AM

Is there any recommended posts I can read that will help me understand what the "strongest looking sector" would look like? I'm pretty new to this and am still unsure. I was hoping to get a experienced member to show me what they would do / where they would sector it.

 


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#6 onediadem

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Posted 19 May 2019 - 10:12 AM

Plate A.. One of the outer strands at 10 0clock.

B.. I would toss. It has bacteria all over it.

C.. I would let grow out a bit more

D.. That lone rope at 7 oclock.

E.. The strand touching the R2

 

Those would be my choices. Sterile work, reseal your original plates also. You can always go back in for more.

 

 

edited because, forgot the color


Edited by onediadem, 19 May 2019 - 10:13 AM.

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#7 wharfrat

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Posted 19 May 2019 - 02:00 PM

Plate A.. One of the outer strands at 10 0clock.

B.. I would toss. It has bacteria all over it.

C.. I would let grow out a bit more

D.. That lone rope at 7 oclock.

E.. The strand touching the R2

 

Those would be my choices. Sterile work, reseal your original plates also. You can always go back in for more.

 

 

edited because, forgot the color

 

thank you one for explaining further.. I was in a bit of a hurry.


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#8 coorsmikey

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Posted 19 May 2019 - 02:25 PM

Is there any recommended posts I can read that will help me understand what the "strongest looking sector" would look like? I'm pretty new to this and am still unsure. I was hoping to get a experienced member to show me what they would do / where they would sector it.

https://mycotopia.ne...k-some-agar-30/


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#9 onediadem

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Posted 19 May 2019 - 02:45 PM

Definitely cannot go wrong using Cat's thread. Awesome good info in there and hey, it's got lots of pics too. Good call Mikey. I admit I was much too lazy to dig for that one lol..


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#10 Taproot

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Posted 19 May 2019 - 04:33 PM

Thank you both so much for the support and suggestions. I can't thank you enough!

#11 roc

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Posted 19 May 2019 - 06:25 PM

Hell's Bell's and Cockle Shells!

 

I really appreciate this thread and the input given!

 

I'm excited to work on some agar skills after seeing the suggestions from one and coors!


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#12 onediadem

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Posted 19 May 2019 - 08:00 PM

Agar has always been my favorite part. I love to see people get into it. When you actually clone a 3rd or 4th generation cluster, the results are mind boggling. Each generation should be taken from a cluster, then a bigger cluster ect. It takes some time to get there, I wont lie. Months in fact. The results are worth it, I promise. 


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#13 jkdeth

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Posted 19 May 2019 - 09:51 PM

I agree. I can't say I love the agar work, but I love the results. Bit time consuming but still fastest route to consistent big beautiful canopies.
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#14 Taproot

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Posted 20 May 2019 - 01:33 AM

I have two follow up questions for the pro's. First, how do you go about producing your 3rd or 4th cluster that you then clone? Do you clone/agar/sector/fruit/clone/agar/sector/fruit until your at your fourth cluster? I was told by some that senescence would be an issue if I didn't use spores from said clones after fruiting/cloning/sectoring/fruiting multiple times. So I guess my question is how many times can I do this cycle before I experience senescence and then have to use spores from my clones. Or do I? The second question, is when you sector a piece of desirable mycelium, do you take the individual ropey rhizomorphic piece and put it on another plate or do you cut a, for example 3mm x 3mm chuck of agar with the desired mycelium and others and place that on a fresh plate. I was worried with some of the sectors I took that I took too little mycelium. Sorry if this is too much to ask I'm still figuring out what i can and should be asking and what is overkill or frowned upon. Once again thank you all for the tips and support / energy invested in your posts. It means the world to me. I'll upload pictures of my work tomorrow for you to see what I've done.

#15 onediadem

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Posted 20 May 2019 - 08:15 AM

I never had a problem using 4th generation clones. 



#16 onediadem

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Posted 20 May 2019 - 04:15 PM

Sorry, and yes. Transfer those sections that (Small sections like no bigger than 1/4 inch square) that I explained above. I prefer to just get that one strand. Once your strand grows out on the plate, get it to grain. Then knock up your substrate. When it fruits, I myself do not mess with anything less that a 5 mushroom cluster to clone on the first generation. To me, anything less is not worth the time or agar and plates. There is an end goal here. Repeat that process 3-4 times. I have done both, and rarely have I ever had a problem. The last generation Is the golden egg. I put that to agar, let it grow out and make vials of sterile water water to put that last generation into. 

 

As I said, it is a very long process. But oh so worth it. Figure minimum of two months per generation, if you have no contam issues. I always wait until the second or third flush after a dunk also. I don't know why second flushes are always the best for me, but they are.

 

I dont know if anyone else does this with their agar, but I always pour two sleeves at a time. I use 1 inch rolls cut from saran wrap to seal the plates so they don't dry out. If they contam, they are not even opened. They last me a few months, and I am not repouring, or leaving a sleeve open. I refuse to use plates from an open sleeve once they have been removed from the glovebox.

 

Anyway, hope that helps and sorry for being so chatty today.


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#17 Taproot

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Posted 21 May 2019 - 12:30 PM

That was extremely helpful. I'm going to follow your recommendations.

Two more follow up questions if you're okay with that:

#1. With the last generation stored in sterile water, would I be making tubs out of that for future production? I would only have so many of these saved and wouldn't want to use them up and have to repeat this whole process again, so do I collect spores from the fruits of the last generation to keep everything going and replenish my stored sterile water samples for future production or do i just keep taking clones of mushrooms indefinitely for this purpose? I was under the impression that the further you isolate, the more isolated the spores from that fruit become. Eventually the spores produced from this golden egg sample will be very similar and I wouldn't have to keep cloning fruits as they would all be more or less the same.

 

#2. Can you recommend a good post for the long term storage with sterile water method? I've tried it before with little success (I wasn't doing it properly) and am realizing now the increasingly important need to learn a proper long term storage method.

 

Thank you so much for your time and patience. You're helping me beyond comprehension with the information you're providing. 



#18 Taproot

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Posted 21 May 2019 - 12:41 PM

<p>Also in case anyone wanted to see, this is how my sectoring went. I don't think I did all of them correctly though.</p>
<p>Also, I took pictures of the top and bottom of each plate so you could see the mycelium better.</p>
<p>&nbsp;</p>

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Edited by Taproot, 21 May 2019 - 12:45 PM.


#19 MsBehavin420

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Posted 01 June 2019 - 07:44 PM

What do you guys do with the agar trays after you're done with them? I have one looking like its gonna pin.

#20 crazy1

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Posted 02 June 2019 - 06:05 AM

Good going on the transfers. It looks to me like you peeled the rhizo myc off of the agar, that should be ok.

Usually I sector out a small healthy/active area, as One said, then put the whole piece of agar on the new plate. 

Best of luck to you!

 

Msbehavin420, I have resealed and refrigerated some for later transfers, and tossed some......

And they can pin and actually fruit on agar. 

 

post-11460-13818542176.jpg


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