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OfflineNo_Life_G33k
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Creating an isolate
    #3981113 - 03/28/05 07:23 PM (11 years, 8 months ago)

What would one do if one wanted to improve/refine a strain, when the starting point is a spore print??

Start with multiple agar isolations, and then fruit it??

or fruit it first, and then start doing isolations???

and could someone define "isolate' for me??

how do you determine if there is just one 'isolation' in a sample??
for example: you have a agar dish full of mycelia- when you remove a small slice- is that an 'isolate'?? or do you have several 'isolates' in one small agar slice???


TIA


--------------------
Hot Water Pasteuriztion - A How To Pictorial
http://www.shroomery.org/forums/showflat.php/Cat/0/Number/4527808/an/0/page/0

Make your own shroom chocolates
http://www.shroomery.org/forums/showflat.php/Cat/0/Number/4500682/an/0/page/5

guide with pix.


Make Your Own Magnetic Stirrer! How-To Tek w/ pix
http://www.shroomery.org/forums/showflat.php/Cat/0/Number/5865949/an/0/page/3


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InvisiblePsychoslut
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Re: Creating an isolate [Re: No_Life_G33k]
    #3981166 - 03/28/05 07:35 PM (11 years, 8 months ago)

Do a search, there are only about 100000000000000000000000000 threads about this.


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[quote]KristiMidocean said:
Good now thats clear.WHO FUCKING CARES. If I am fat u all keep pointing it out like its suppose to be a secret.LIke u really have nothing better to do then make fat jokes. If o know its like I do I know yall can come up with NEW AND BETTER SHIT . This shit is old and boring . I left in the first place cause this shit got boring not because of the fat jokes . Fat jokes dont bother me but seriously its old[/quote]


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OfflineShdwstr
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Re: Creating an isolate [Re: No_Life_G33k]
    #3981374 - 03/28/05 08:32 PM (11 years, 8 months ago)

That would take a bit to answer...

Readers Digest Version...
Do both, try everything,
and only settle for the best!


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Invisibletahoe
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Re: Creating an isolate [Re: Shdwstr]
    #3981433 - 03/28/05 08:47 PM (11 years, 8 months ago)

start spores on agar, then isolate the fastest most rhizomorphic mycelium to another petri, do it again, and one more time. Then make that 3rd generation isolate into a liquid culture


--------------------
Stop experimenting half way through your first grow. Grow it to maturity, watch it, learn from it. Do this a few times then experiment with different ideas and figure out what works best for you.


Teh=The

I need to proofread


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Invisibletahoe
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Re: Creating an isolate [Re: tahoe]
    #3981465 - 03/28/05 08:52 PM (11 years, 8 months ago)

to answer your question, each time you cut a small peice of mycelium from one petri and put it to another its an "isolate" You want to make sure you are isolating sectors of the same mycelium, not 1/2 cottony 1/2 rhizomorphic. this is why you isolate an isolate because there will be cottony sectors in your first isolate. Dont go much past 3 generations though because you will start to drcrease the genetic diversity and may get noncompatiable mycelium that wont produce offspring.


--------------------
Stop experimenting half way through your first grow. Grow it to maturity, watch it, learn from it. Do this a few times then experiment with different ideas and figure out what works best for you.


Teh=The

I need to proofread


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OfflineNo_Life_G33k
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Re: Creating an isolate [Re: Psychoslut]
    #3981546 - 03/28/05 09:11 PM (11 years, 8 months ago)

Quote:

Psychoslut said:
Do a search, there are only about 100000000000000000000000000 threads about this.




actually, that's part of the problem. Too much discussion and supposition, and not enough detail.

I'm looking for a short concise answer. I can't afford to drop $50 for the cultivation guides- and besides, it helps a lot when I can (re)phrase my questions and ask them here, instead of trying to decipher text(s).


--------------------
Hot Water Pasteuriztion - A How To Pictorial
http://www.shroomery.org/forums/showflat.php/Cat/0/Number/4527808/an/0/page/0

Make your own shroom chocolates
http://www.shroomery.org/forums/showflat.php/Cat/0/Number/4500682/an/0/page/5

guide with pix.


Make Your Own Magnetic Stirrer! How-To Tek w/ pix
http://www.shroomery.org/forums/showflat.php/Cat/0/Number/5865949/an/0/page/3


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OfflineNo_Life_G33k
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Re: Creating an isolate [Re: tahoe]
    #3981581 - 03/28/05 09:19 PM (11 years, 8 months ago)

Quote:

tahoe said:
start spores on agar, then isolate the fastest most rhizomorphic mycelium to another petri, do it again, and one more time. Then make that 3rd generation isolate into a liquid culture




Ok, I have 'second' isoaltion on agar. it is still sectoring.
So I'll do one more.
But I'm wondering if i have several 'sub-strains' in my 'isolate'.

Am I on the right track here???


edited for spelin


--------------------
Hot Water Pasteuriztion - A How To Pictorial
http://www.shroomery.org/forums/showflat.php/Cat/0/Number/4527808/an/0/page/0

Make your own shroom chocolates
http://www.shroomery.org/forums/showflat.php/Cat/0/Number/4500682/an/0/page/5

guide with pix.


Make Your Own Magnetic Stirrer! How-To Tek w/ pix
http://www.shroomery.org/forums/showflat.php/Cat/0/Number/5865949/an/0/page/3


Edited by No_Life_G33k (03/30/05 06:36 PM)


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Invisibletahoe
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Re: Creating an isolate [Re: No_Life_G33k]
    #3981819 - 03/28/05 10:17 PM (11 years, 8 months ago)

i would call them genetic difference instead of strains. They will be the same strain. You want to get confused now? If you are planning on using a certain substrate it is best to simulate that substrate the best you can in your agar recipes. That way the fastest growing isolation on the agar will grow rapidly on that sub. But this is over kill.


--------------------
Stop experimenting half way through your first grow. Grow it to maturity, watch it, learn from it. Do this a few times then experiment with different ideas and figure out what works best for you.


Teh=The

I need to proofread


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Offlinehyphae
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Re: Creating an isolate [Re: No_Life_G33k]
    #3981926 - 03/28/05 10:41 PM (11 years, 8 months ago)

Yes those are sub-strains. Also isolate again until you have a pure isolate (no sectoring). You can go quite a few transfers actually the myc isn't really consuming any major substrates at this point. GL and Welcome and remember tahoe means well :wink:
BTW changing substrates gives your myc new vigor once senescing has begun to occur (growing old). :wink:


--------------------
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"We all have priorities. I used a closet once setup a nice little lab trouble was all the shit that was in there ended up in the bedroom that pissed off the GF then I ended up dumping her as she was getting in the way of my sterile culture technique! Ya I got priorities too!!!"


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OfflineIGnosticAbhorI
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Re: Creating an isolate [Re: hyphae]
    #3984525 - 03/29/05 10:49 AM (11 years, 8 months ago)

Wow, that was useful and to the point, i know how to isolate a strain and what it means.

Hey tahoe :handth: :cool: :thumbup:

Time to get a pos. glove box and some agar :wink:

-Gnostic


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InvisibleSoopaX
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Re: Creating an isolate [Re: No_Life_G33k]
    #3985781 - 03/29/05 04:15 PM (11 years, 8 months ago)

Take your spore print and make a syringe. When I'm doing isolation work I like to super dilute them, rather than having a Kool-Aid colored solution that I prefer for inoculations. Use about 2mL of sterile water and scrape a few tracks across the spore print and allow the liquid to flush the spores into a sterile mason jar. Slurp it up. Then, using an "S" form, allow some drops of the mycelia to go onto a petri dish. Get your most ropy looking mycelia and place it on another petri dish. Voila, a big petri dish filled with rhizo mycelia.


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


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OfflineRogerRabbitM
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Re: Creating an isolate [Re: SoopaX]
    #3985835 - 03/29/05 04:34 PM (11 years, 8 months ago)

This might help. It's my strain isolation tek. It assumes you already know agar work and have at least a glovebox, although a flowhood makes it much easier.
RR
http://mycotopia.net/discus/messages/5/41777.html


--------------------
Download Let's Grow Mushrooms



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InvisibleEonTan
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Re: Creating an isolate [Re: No_Life_G33k]
    #3991484 - 03/30/05 06:56 PM (11 years, 8 months ago)

You cannot improve a strain without altering the strain. IE adding new genes to the population. You can find the best the strain has to offer in your particular environment.

A strain is made up of a population of substrains. To make the best possible selection from this population would require isolation of single spore germinations and contolled mating of them to determine which two single spore germinations mate to form the best substrain wihtin the populaiton for your specific environmnetal parameters.

Since this is VERY MUCH OVERKILL for what you are trying to accomplish, you should settle for isolating a single substrain, one that does not sector any more on a petri plate filled with nutrient agar. This substrain may or may not be the best one for your particular environmnet, but it is at least pure.

I think the easiest method for isolating a good fruiter in your environmnet is to multispore inoculate a jar of substrate, fruit it the way you would fruit in the future, and clone the best fruit from the casing. This clone will be a pure substrain of known vigor in your particular environmnet. It can be duplicated, and stored for future use.

Example. Inoculate jar of substrate with GT spores. Case the colonized substrate, and fruit it. Clone the best fruit. You now have a single isolate(substrain) fromthe strain GT.

You already inoculated agar, so just keep transfering small amounts of mycelium to new plates until you get non sectoring growth. You now have a substrain(isolate) to test for vigor. It will most likely fruit, but it may not be a great fruiter from the Strain in your environmnet.


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OfflineNo_Life_G33k
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Re: Creating an isolate [Re: RogerRabbit]
    #3991494 - 03/30/05 06:58 PM (11 years, 8 months ago)

next dumb Q:

how many 'sub-strains' would be present on the agar??

Is it just two- rhizo and the other(cottony)?????

or is it like seeds from plants- where there could be different phenotytpes and a wide selection of substrains???

Thanks again y'all- I'll read Mr. Rabbit's thread latter- I DL a work and burn to cd, and then read at home.


--------------------
Hot Water Pasteuriztion - A How To Pictorial
http://www.shroomery.org/forums/showflat.php/Cat/0/Number/4527808/an/0/page/0

Make your own shroom chocolates
http://www.shroomery.org/forums/showflat.php/Cat/0/Number/4500682/an/0/page/5

guide with pix.


Make Your Own Magnetic Stirrer! How-To Tek w/ pix
http://www.shroomery.org/forums/showflat.php/Cat/0/Number/5865949/an/0/page/3


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InvisibleOlgualion
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Re: Creating an isolate [Re: EonTan]
    #3991528 - 03/30/05 07:07 PM (11 years, 8 months ago)

Quote:

I think the easiest method for isolating a good fruiter in your environmnet is to multispore inoculate a jar of substrate, fruit it the way you would fruit in the future, and clone the best fruit from the casing. This clone will be a pure substrain of known vigor in your particular environmnet. It can be duplicated, and stored for future use.




Definitely my preferred method. At least when working with clean prints. But, I am against the whole "dark syringe" thing for inoculation.


--------------------
Study the past...
See the future...


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InvisibleEonTan
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Re: Creating an isolate [Re: No_Life_G33k]
    #3991590 - 03/30/05 07:32 PM (11 years, 8 months ago)

As few as none, and as many as there were viable compatable spores present, and as many that won't combine via anastamosis.

How about somewhere between zero and a thousand.  :wink:


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InvisibleEonTan
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Re: Creating an isolate [Re: Olgualion]
    #3991603 - 03/30/05 07:36 PM (11 years, 8 months ago)

I don't mind if they are dark. At 1/22 ml per drop of solution. :tongue:

Yes spores most be clean to multispore inoculate substrate. They should be clean for agar too.  I hate having to isolate from bacteria, mold, etc.. Thats why you pay the big bucks to Vendors for a clean start. :smirk:

Save the antobitoics for wild prints of something new and exciting. :thumbup:


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OfflineNo_Life_G33k
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Re: Creating an isolate [Re: EonTan]
    #3991641 - 03/30/05 07:47 PM (11 years, 8 months ago)

Quote:

EonTan said:
As few as none, and as many as there were viable compatable spores present, and as many that won't combine via anastamosis.

How about somewhere between zero and a thousand.  :wink:




well. that clears it all up!!!!  :laugh:

actually, it does (partially) answer the Q.

Let me (re)ask this way-

what purpose does fruiting out an isolate serve???

I'm trying to get to what methodology is better:

agar> agar> agar-fruit >/clone or multispore > agar-agar-fruit
Or
fruit >agar >agar>agar > fruit > clone/ multispore >repeat


geez, I may have to change my S/N to Sofa King..... :crazy:


--------------------
Hot Water Pasteuriztion - A How To Pictorial
http://www.shroomery.org/forums/showflat.php/Cat/0/Number/4527808/an/0/page/0

Make your own shroom chocolates
http://www.shroomery.org/forums/showflat.php/Cat/0/Number/4500682/an/0/page/5

guide with pix.


Make Your Own Magnetic Stirrer! How-To Tek w/ pix
http://www.shroomery.org/forums/showflat.php/Cat/0/Number/5865949/an/0/page/3


Edited by No_Life_G33k (03/30/05 07:52 PM)


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InvisibleOlgualion
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Re: Creating an isolate [Re: No_Life_G33k]
    #3991700 - 03/30/05 08:03 PM (11 years, 8 months ago)

This is somewhat advanced stuff. wish people gave out so much info when I started.

What are you trying to accomplish?

If you want an isolate. IMO it is best to go from multispore to fruit. Then clone on agar. You now have your isolate. No need for more transfers. Once you take the clone from the mushy, you have an isolate.

If you go from multispore to agar, and transfer mycelium to new dishes until you have an isolate, you have less chances of getting that killer strain IMO than multi to grain to nice fruit to agar(iso). Some would say transferring rhizo growth from agar will give your best chances of getting a good strain, but I don't entirely agree. I have seen great strains come from cottony mycelium as well!


--------------------
Study the past...
See the future...


Edited by Olgualion (03/30/05 08:05 PM)


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InvisibleEonTan
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Re: Creating an isolate [Re: No_Life_G33k]
    #3992048 - 03/30/05 09:12 PM (11 years, 8 months ago)

No system is better or worse. One is just more controlled and thorough. That's all.

Spore syringe>jar of substrate>casing>fruit. You then clone a piece of this fruit onto agar. When it grows out on agar, you transfer pieces to as many plates of AGAR as you think you might need to use now, and would like to store for the future. This is the easiest way. Every plate will have the same non sectoring substrain, becasue you cloned a fruit.

The other route, gives you clones before you know what they are clones of. Do you understand? If you have not fruited the isolate, you don't know if it is good or not. But since you typically result in several pure isolates from transfering growth from different sectors, you have several to test for a good one.

You decide what is better.

Spore> substrate>casing>fruit>clone to agar is most efficient for most people.


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