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OfflineNo_Life_G33k
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Registered: 03/08/05
Posts: 356
Last seen: 14 years, 1 month
Creating an isolate
    #3981113 - 03/28/05 05:23 PM (19 years, 1 month 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 05:35 PM (19 years, 1 month ago)

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


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



[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 06:32 PM (19 years, 1 month 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 06:47 PM (19 years, 1 month 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.


My Legacy
https://www.shroomery.org/forums/showflat.php/Number/22140987#22140987

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 06:52 PM (19 years, 1 month 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.


My Legacy
https://www.shroomery.org/forums/showflat.php/Number/22140987#22140987

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 07:11 PM (19 years, 1 month 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 07:19 PM (19 years, 1 month 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 04:36 PM)

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Invisibletahoe
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Re: Creating an isolate [Re: No_Life_G33k]
    #3981819 - 03/28/05 08:17 PM (19 years, 1 month 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.


My Legacy
https://www.shroomery.org/forums/showflat.php/Number/22140987#22140987

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 08:41 PM (19 years, 1 month 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:


--------------------
Getting the most out of your casings!, A pinning strategy.
Oyster Shell "Flour" $2 for 1lb. a hell of a deal :wink:
Not what is overlay but rather what overlay is
Gas Exchange vs. FAE

"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 08:49 AM (19 years, 1 month 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 02:15 PM (19 years, 1 month 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.


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


Jackie Treehorn treats objects like women, man

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OfflineRogerRabbitM
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Re: Creating an isolate [Re: SoopaX]
    #3985835 - 03/29/05 02:34 PM (19 years, 1 month 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



semper in excretia sumus solim profundum variat

"I've never had a failed experiment.  I've only discovered 10,000 methods which do not work."
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InvisibleEonTan
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Re: Creating an isolate [Re: No_Life_G33k]
    #3991484 - 03/30/05 04:56 PM (19 years, 1 month 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 04:58 PM (19 years, 1 month 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 05:07 PM (19 years, 1 month 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 05:32 PM (19 years, 1 month 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 05:36 PM (19 years, 1 month 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 05:47 PM (19 years, 1 month 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 05:52 PM)

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InvisibleOlgualion
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Re: Creating an isolate [Re: No_Life_G33k]
    #3991700 - 03/30/05 06:03 PM (19 years, 1 month 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 06:05 PM)

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InvisibleEonTan
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Re: Creating an isolate [Re: No_Life_G33k]
    #3992048 - 03/30/05 07:12 PM (19 years, 1 month 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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InvisibleCorporal Kielbasa

Registered: 05/29/04
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Re: Creating an isolate [Re: No_Life_G33k]
    #3992261 - 03/30/05 08:00 PM (19 years, 1 month ago)

Check out the local library or go to borders and sit there with a note pad if you cant afford the book just read it and leave it.

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OfflineKaptKid
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Re: Creating an isolate [Re: EonTan]
    #3992268 - 03/30/05 08:03 PM (19 years, 1 month ago)

Sorry for the neb?

The clone has gone from agar to bulk and has fruited. Does the spores of this isolate, represent this isolate or does it take it back to "in general"???


--------------------
Child of the 60's, Tripping ever since.


:sun:

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Offlinedebianlinux
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Re: Creating an isolate [Re: KaptKid]
    #3992283 - 03/30/05 08:07 PM (19 years, 1 month ago)

Quote:

KaptKid said:
The clone has gone from agar to bulk and has fruited. Does the spores of this isolate, represent this isolate or does it take it back to "in general"???




the latter. a section of fruitbody mycelia would represent a modified/evolved version of the isolate. only another piece of the original isolate would truly represent the original isolate. the spores are definitely more in the direction of "in general".

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OfflineKaptKid
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Re: Creating an isolate [Re: debianlinux]
    #3992295 - 03/30/05 08:10 PM (19 years, 1 month ago)

Thanks



:sun:


--------------------
Child of the 60's, Tripping ever since.


:sun:

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InvisibleNecroMyce
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Posts: 747
Re: Creating an isolate [Re: EonTan]
    #18385536 - 06/07/13 10:14 PM (10 years, 11 months ago)

Quote:

EonTan said:
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.

you have several to test for a good one.




I understand this method and why it is used. but just a quick question i have yet to find in servery posts ive been searching through. You say you have several to test for a good one, if i did the said method above of cloning/isolating... when i pick the fruits, i dont necessarily know if they're potent or not, and if they're worth it or out. so id obviously have to eat them and try them out.

Would i just have to pick a few fruits and cut a piece of the fruit off and drop it in agar to start colonizing it, and then try the fruits separately and decide which fruits i liked better? then from there i can transfer the agar and get a master culture?

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Invisiblewildernessjunkie
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Re: Creating an isolate [Re: NecroMyce]
    #18386549 - 06/08/13 05:09 AM (10 years, 11 months ago)

:megacrankey:

Dammit! I read all the way to the end of this before I realized it was an 8 year old thread.

The best resource for you right here is this link.

www.mushroomvideos.com

Spend the 9-10 bucks to download it. Its worth it, and will answer a whole slew of questions you're bound to have.

Edited by wildernessjunkie (06/08/13 05:10 AM)

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InvisiblePanzerCubed
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Re: Creating an isolate [Re: wildernessjunkie]
    #18387042 - 06/08/13 09:33 AM (10 years, 11 months ago)

Good thread but a tad old, thought this might be relevant.



--------------------
Trade List - updated 16/10/15/

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InvisibleNecroMyce
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Registered: 05/12/13
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Re: Creating an isolate [Re: wildernessjunkie]
    #18388145 - 06/08/13 02:48 PM (10 years, 11 months ago)

Quote:

wildernessjunkie said:
:megacrankey:

Dammit! I read all the way to the end of this before I realized it was an 8 year old thread.

The best resource for you right here is this link.

www.mushroomvideos.com

Spend the 9-10 bucks to download it. Its worth it, and will answer a whole slew of questions you're bound to have.





those videos are a waste, unless theres like 3 sections of videos to buy? cuz i bought it for 8.99$ and it only came with PF tek, Spore prints/syringes and a few other videos, didnt cover half the things i needed it to cover.

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Offlineclueless
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Re: Creating an isolate [Re: NecroMyce]
    #18388411 - 06/08/13 04:03 PM (10 years, 11 months ago)

I have the 3rd edition on dvd.
I didn't download it so I'm not sure what you might be missing.

Disk 1 has
Brown rice flour
Grain Prep
Grain to Grain
Rye Grass seed
Manure Sub
Casing Prep
Straw Pasteurization
Laundry Basket- Straw Log

Disk 2 has
Agar and Petri dish work
Isolation of strains
Cloning
Culture Slants
Agar Inoculation
Sawdust-Woodchips
Inoculating Sawdust
Spore printing and Syringes

Not sure what else you need.
This seems like a good start point to me.


--------------------
I'm a rhinestone tiger in a leisure suit.

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InvisibleNecroMyce
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Re: Creating an isolate [Re: clueless]
    #18388445 - 06/08/13 04:18 PM (10 years, 11 months ago)

Well then i only have disk 1, how come i didnt get disk two with the two dvd package i bought? wtf

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Offlineclueless
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Re: Creating an isolate [Re: NecroMyce]
    #18388454 - 06/08/13 04:21 PM (10 years, 11 months ago)

You said you downloaded it for $8.99.
Did you do that or buy a box that should have 2 disks?
I am confused now.

I took very detailed notes from disk 1 in my journal.
Feel free to read through it, and pm for help.


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I'm a rhinestone tiger in a leisure suit.

Edited by clueless (06/08/13 04:26 PM)

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InvisibleNecroMyce
Mostly Ghostly
I'm a teapot


Registered: 05/12/13
Posts: 747
Re: Creating an isolate [Re: clueless]
    #18388466 - 06/08/13 04:24 PM (10 years, 11 months ago)

Let's Grow Mushrooms Download, High Resolution $8.99!



You can now download the full release of Let's Grow Mushrooms in high resolution in two popular formats. The FLV version uses Flash video and plays great using Adobe Flash, Real Player, Quicktime, and many other players available free online. The MP4 version plays well on computers, but is also the standard for portable devices such as iPads, Androids, etc. Download both for less than ten dollars!
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Thats the one i downloaded... Or is it the 49$ one that has both disks?

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Offlineclueless
justnew
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Folding@home Statistics
Registered: 07/21/03
Posts: 783
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Last seen: 4 months, 3 days
Re: Creating an isolate [Re: NecroMyce]
    #18388510 - 06/08/13 04:38 PM (10 years, 11 months ago)

The sample shows all the same chapters, you should have the same in your download.
Contact them if you didn't get all chapters.

ps I have 3rd but the 4th edition appears to have more on it.


--------------------
I'm a rhinestone tiger in a leisure suit.

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InvisibleNecroMyce
Mostly Ghostly
I'm a teapot


Registered: 05/12/13
Posts: 747
Re: Creating an isolate [Re: clueless]
    #18390691 - 06/09/13 12:31 AM (10 years, 11 months ago)

sure nuff he hooked it up :laugh:

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