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underground
welcome to the underground


Registered: 06/23/03
Posts: 191
Loc: colorful colorado
Last seen: 1 year, 1 month
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Strain Isolation for P. mexicana - A
#19381476 - 01/07/14 09:24 AM (10 years, 1 month ago) |
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Hi folks,
I started two rye jars with a multi-spore inoculation, just to get things rolling, but I am also trying to isolate an aggressive, fast-growing strain that will be a good sclerotia producer in subsequent jars. I have done one transfer so far, but I was wondering what characteristics should I be looking for in the mycelia for the next transfers?
Faster growth or denser growth? Is rhizomorphic growth desirable with this species, as with cubensis? What about color - yellowish, light brownish, white?
Thanks,
-underground
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Ganzig
It's for the street cred


Registered: 11/29/06
Posts: 8,206
Loc: Oregon
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Re: Strain Isolation for P. mexicana - A [Re: underground]
#19381484 - 01/07/14 09:27 AM (10 years, 1 month ago) |
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I have a question.
How are you going to separate any traits when the culture is on grain?
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skippydude
Myco-curious



Registered: 09/08/12
Posts: 1,827
Loc: "Alice's Wonderland"
Last seen: 9 years, 2 months
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Re: Strain Isolation for P. mexicana - A [Re: Ganzig]
#19381535 - 01/07/14 09:39 AM (10 years, 1 month ago) |
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Put a piece of your spawn on agar and isolate the strongest fastest sectors
Transfer the strongest fastest sector from this dish to a new dish
Repeat the process until you have isolated an aggressive mono-culture
You only want white mycelium, yellow and brown stained myc is covered in metabolites and goes bacterial very easily on agar.
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Ganzig
It's for the street cred


Registered: 11/29/06
Posts: 8,206
Loc: Oregon
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Re: Strain Isolation for P. mexicana - A [Re: skippydude]
#19381559 - 01/07/14 09:46 AM (10 years, 1 month ago) |
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Quote:
skippydude said: Put a piece of your spawn on agar and isolate the strongest fastest sectors
Transfer the strongest fastest sector from this dish to a new dish
Repeat the process until you have isolated an aggressive mono-culture
You only want white mycelium, yellow and brown stained myc is covered in metabolites and goes bacterial very easily on agar. 
If it is on grain how do you know if you have more than one "strain"? It is most likely a specific genetic make up and you would only be cloning, not isolating.
If one was to use agar and spores it makes more sense to just start from spores. Or else you might just be wasting your time.
Right?
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underground
welcome to the underground


Registered: 06/23/03
Posts: 191
Loc: colorful colorado
Last seen: 1 year, 1 month
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Re: Strain Isolation for P. mexicana - A [Re: Ganzig]
#19381570 - 01/07/14 09:50 AM (10 years, 1 month ago) |
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Ooops! Forgot to mention that aside from the grain jars, I squirted some spores onto agar, and have been trying to isolate that way.
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Ganzig
It's for the street cred


Registered: 11/29/06
Posts: 8,206
Loc: Oregon
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Re: Strain Isolation for P. mexicana - A [Re: underground]
#19381579 - 01/07/14 09:52 AM (10 years, 1 month ago) |
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Quote:
underground said: Ooops! Forgot to mention that aside from the grain jars, I squirted some spores onto agar, and have been trying to isolate that way.

Good luck.
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skippydude
Myco-curious



Registered: 09/08/12
Posts: 1,827
Loc: "Alice's Wonderland"
Last seen: 9 years, 2 months
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Re: Strain Isolation for P. mexicana - A [Re: Ganzig]
#19381585 - 01/07/14 09:55 AM (10 years, 1 month ago) |
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Quote:
Ganzig said:
Quote:
skippydude said: Put a piece of your spawn on agar and isolate the strongest fastest sectors
Transfer the strongest fastest sector from this dish to a new dish
Repeat the process until you have isolated an aggressive mono-culture
You only want white mycelium, yellow and brown stained myc is covered in metabolites and goes bacterial very easily on agar. 
If it is on grain how do you know if you have more than one "strain"? It is most likely a specific genetic make up and you would only be cloning, not isolating.
If one was to use agar and spores it makes more sense to just start from spores. Or else you might just be wasting your time.
Right?
You will know if it is a mono soon after it starts growing, whether it is spores, grain spawn or even cloned primordia
Some of my best mono cultures were isolated from cloned fruits
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FungiJB
smot poker



Registered: 05/29/12
Posts: 174
Last seen: 4 years, 11 months
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Re: Strain Isolation for P. mexicana - A [Re: skippydude]
#19381601 - 01/07/14 10:00 AM (10 years, 1 month ago) |
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Heres how im looking at this. Quick isnt allways good when It comes to stones. The longer they grow, the stronger they are. I dont see much point in isolating something that grows quickly, when the desired end result is not as strong as it would be if you waited. If I were to isolate, I would isolate the biggest fatty ones. Just my .02
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dusttodust


Registered: 11/26/12
Posts: 491
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Re: Strain Isolation for P. mexicana - A [Re: FungiJB]
#19381614 - 01/07/14 10:04 AM (10 years, 1 month ago) |
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Ganzig
It's for the street cred


Registered: 11/29/06
Posts: 8,206
Loc: Oregon
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Re: Strain Isolation for P. mexicana - A [Re: skippydude]
#19381624 - 01/07/14 10:07 AM (10 years, 1 month ago) |
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Quote:
skippydude said:
You will know if it is a mono soon after it starts growing, whether it is spores, grain spawn or even cloned primordia.
If it is primordia you already know it is mono because it is a clone. I get that you would soon know about the grain but one could just end up putting one genetic version in hopes of getting multiple "strains" which would be a waste of a plate and time.
If one starts from spores then they know they are getting variability and can isolate what they want out of it.
Quote:
skippydude said: Some of my best mono cultures were isolated from cloned fruits
Makes sense. It was already a mono and did not need any isolating though. Unless you were isolating it from contams but that is not what we are talking about here.
BTW I love watching your grows skippydude. You are very good at it.
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I must keep reminding myself of this. I must keep reminding myself of this. I must keep reminding myself of this. I must keep reminding myself of this.
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skippydude
Myco-curious



Registered: 09/08/12
Posts: 1,827
Loc: "Alice's Wonderland"
Last seen: 9 years, 2 months
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Re: Strain Isolation for P. mexicana - A [Re: Ganzig]
#19381650 - 01/07/14 10:14 AM (10 years, 1 month ago) |
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Quote:
Ganzig said:
Quote:
skippydude said:
You will know if it is a mono soon after it starts growing, whether it is spores, grain spawn or even cloned primordia.
If it is primordia you already know it is mono because it is a clone. I get that you would soon know about the grain but one could just end up putting one genetic version in hopes of getting multiple "strains" which would be a waste of a plate and time.
If one starts from spores then they know they are getting variability and can isolate what they want out of it.
Quote:
skippydude said: Some of my best mono cultures were isolated from cloned fruits
Makes sense. It was already a mono and did not need any isolating though. Unless you were isolating it from contams but that is not what we are talking about here.
BTW I love watching your grows skippydude. You are very good at it. 
Sorry but your dead wrong about this one A single mushroom primordia(1 cap 1 stem) can consist of 100s or 1000s of individual organisms If you didn't know this I doubt you have you ever cloned a mushroom from a MS grow
Consider what your saying when you fellas are suggesting a weaker slower growing sector As all sectors will produce sclerotia, you should only work with the healthier, faster growing stuff, it's just common sense
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underground
welcome to the underground


Registered: 06/23/03
Posts: 191
Loc: colorful colorado
Last seen: 1 year, 1 month
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Re: Strain Isolation for P. mexicana - A [Re: dusttodust]
#19381679 - 01/07/14 10:22 AM (10 years, 1 month ago) |
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So RR says that brown IS a good characteristic in this strain. Thanks, dusttodust!
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skippydude
Myco-curious



Registered: 09/08/12
Posts: 1,827
Loc: "Alice's Wonderland"
Last seen: 9 years, 2 months
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Re: Strain Isolation for P. mexicana - A [Re: underground]
#19381708 - 01/07/14 10:28 AM (10 years, 1 month ago) |
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Quote:
underground said: So RR says that brown IS a good characteristic in this strain. Thanks, dusttodust!
Brown spawn? or brown mycelium on agar? Y'all got me curious now 
If you really want professional advice on this strain I would call Ben @ Earths Tongue (210)772-3336 He is the one that brought this jewel back from Mexico and did the original isolation work No body knows it better
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StickyIcky Fingers
Free DOOM!! Available Here

Registered: 12/03/13
Posts: 178
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Re: Strain Isolation for P. mexicana - A [Re: skippydude]
#19381769 - 01/07/14 10:44 AM (10 years, 1 month ago) |
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IME ATL and now P. mex have been white on agar. Once they are part way though colonizing the jar then the myc will start turning orange/brown/ yellow and such
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skippydude
Myco-curious



Registered: 09/08/12
Posts: 1,827
Loc: "Alice's Wonderland"
Last seen: 9 years, 2 months
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Quote:
StickyIcky Fingers said: IME ATL and now P. mex have been white on agar. Once they are part way though colonizing the jar then the myc will start turning orange/brown/ yellow and such
Thanks, this is what I figured, had me going for a minute there
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Ganzig
It's for the street cred


Registered: 11/29/06
Posts: 8,206
Loc: Oregon
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Re: Strain Isolation for P. mexicana - A [Re: skippydude]
#19381853 - 01/07/14 11:03 AM (10 years, 1 month ago) |
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Quote:
skippydude said:
Sorry but your dead wrong about this one A single mushroom primordia(1 cap 1 stem) can consist of 100s or 1000s of individual organisms
Will you show me what you are talking about? Link?
Quote:
skippydude said: If you didn't know this I doubt you have you ever cloned a mushroom from a MS grow
i
Are you always this condescending to people?
No reason to be a jerk. Just because you may know something I do not does not mean you need to get all defensive and talk like a child.
Seriously though.
I don't see how what you are saying is possible. A fruit is Dikaryotic I don't get how that makes 100-1000's of genetic outcomes.
Now realize by me not knowing how that works does not mean I am attacking you.
I am just curious about what you know. No need to be defensive. So please don't respond with a snotty remark.
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I must keep reminding myself of this. I must keep reminding myself of this. I must keep reminding myself of this. I must keep reminding myself of this.
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skippydude
Myco-curious



Registered: 09/08/12
Posts: 1,827
Loc: "Alice's Wonderland"
Last seen: 9 years, 2 months
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Re: Strain Isolation for P. mexicana - A [Re: Ganzig]
#19381932 - 01/07/14 11:21 AM (10 years, 1 month ago) |
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Quote:
Ganzig said:
Quote:
skippydude said:
Sorry but your dead wrong about this one A single mushroom primordia(1 cap 1 stem) can consist of 100s or 1000s of individual organisms
Will you show me what you are talking about? Link?
Quote:
skippydude said: If you didn't know this I doubt you have you ever cloned a mushroom from a MS grow
i
Are you always this condescending to people?
No reason to be a jerk. Just because you may know something I do not does not mean you need to get all defensive and talk like a child.
Seriously though.
I don't see how what you are saying is possible. A fruit is Dikaryotic I don't get how that makes 100-1000's of genetic outcomes.
Now realize by me not knowing how that works does not mean I am attacking you.
I am just curious about what you know. No need to be defensive. So please don't respond with a snotty remark.
My apologies no harm intended honestly I get a little bent when I start getting the "But RR says" comments. I have had too many on this site talk to me in this fashion and my excuse is lame so again I sincerely apologize
I was of the same impression that a single mushroom was a single organism After being told by a veteran grower that from MS grows the mushrooms are many different organisms just like the spawn, I still didn't believe
Only after cloning a mushroom myself from a leucistic mushroom Growing it out on agar where it had several sectors Then when the sector I chose no longer carried the leucistic trait, did I believe it
Since then I have cloned quite a few mushrooms Every one cloned from a mono-culture grow stays mono Every mushroom from MS or wild finds produces sectors
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Ganzig
It's for the street cred


Registered: 11/29/06
Posts: 8,206
Loc: Oregon
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Re: Strain Isolation for P. mexicana - A [Re: skippydude]
#19381945 - 01/07/14 11:25 AM (10 years, 1 month ago) |
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Thank you for the apology. I totally understand the "Well RR said so" mantra.
It does get annoying.
Thanks for the explanation.

Very interesting stuff here. It's got my brain hurting.
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bodhisatta 
Smurf real estate agent



Registered: 04/30/13
Posts: 61,890
Loc: Milky way
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Re: Strain Isolation for P. mexicana - A [Re: Ganzig]
#19382127 - 01/07/14 12:07 PM (10 years, 1 month ago) |
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Once your substrate is colonized the whole thing is a single organism, All the mycelium is one organism. Interestingly the mycelium can be made of multiple dykariotic strains so that the whole organism has multiple genetics that are separate. A fruit from MS grows can have 100s of strains, If you're really lucky a fruit can produce a monoculture/isolate on the first transfer but it likely wont.
Quote:
RogerRabbit said:
Quote:
Sci-Fi said: Agree. A clone is also an isolate, single strain, mono-culture. Same thing. You may have derived it via cloning a mushroom, but it's still a isolated strain.
Two or more strains creating one mushroom, together, without joining or exchanging genetic material? That sounds like science fiction.
You are incorrect, as is scatmanrav.
There can be dozens of strains conspiring to form a single fruiting body, without these strains combining into a single organism. This is well known and established in mycology. Paul Stamets reports isolating up to 200 organisms from a single fruit. It's not science fiction, it's science. Picture thousands of individual soldiers in an army going to battle. Each is separate, yet conspire to form the coherent fighting force.
An isolate can either come from a pairing of two compatible hyphae from separate spores, or from mycelium that has later combined via anastomosis into a single organism. There can be monokaryotic pairings with other mononucleate mycelium, dikaryotic pairings with other multinucleate mycelium, or mono-dikaryotic pairings. You know you have an isolate when there is no more sectoring on agar.
To the OP, rhizomorphic mycelium is not the only characteristic we isolate for. The important thing is to get to single sectors and then fruit out each strain. This is the only proved way to find the best performing strains. Many rhizomorphic strains will be poor performers, but some will blow your socks off. The latter are the ones you keep, discarding the former. The only way to know is to fruit each one. It's a long process, but it's fun and how we come up with the best commercial strains. RR
Quote:
RogerRabbit said: A sector is a strain. A sector develops when compatible hyphae join to become a single organism, sharing genetics. Strains can also join with other strains via dikaryotic pairings, known as anastomosis. An isolate is a single strain, meaning you have transfered all the sectors to their own plates.
By taking tiny pieces of mycelium when I make transfers, I can often get to single sector isolates with three to four transfers. The dish in the picture on the left below is the result of two or three transfers. You can clearly see each sector by holding the dish up to the light. Tiny cuts were made in each of these sectors to move that mycelium to new dishes, and each of those became a single sector isolate. The right picture shows a dish that is still sectoring, and on the right is an isolated strain. Note there is no sectoring at all. Each and every isolate should be grown through the fruiting stage to determine the best performers. Prior to doing so, a small amount of mycelium should be carefully labeled and placed into the refrigerator on a petri dish or culture slant. This way, after fruiting, you can go back to an earlier stage in cell division for each future grow, capturing an aggressive strain. RR
Quote:
RogerRabbit said:
Quote:
Wouldnt they just join together? isnt cube mycelium compatible with trich or cobweb?
Cube mycelium will not join with trich or cobweb. Anastomosis is the pairing of dikaryotic mycelium with other dikaryotic mycelium. In other words, combining strains, but not species. For example, Penis Envy could combine with cambodian cubensis via anastomosis, but neither could combine with oyster, shiitake, or mold. RR
Hence why you can take 4 different "variety" of cube syringes squirt them all into one cake and still end up with 1 cube cake that makes fruits that look like cubes.
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Violet



Registered: 12/06/11
Posts: 4,205
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Re: Strain Isolation for P. mexicana - A [Re: bodhisatta]
#19382208 - 01/07/14 12:25 PM (10 years, 1 month ago) |
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Why hasn't anyone mentioned cloning a sclerotia? Did I miss someone say that?
Pluck a nice stone from a still-sterile grain container with sterilized tweezers, put on agar... If you use petris (I don't) and are cloning a stone too large for the petri (I have done this before) just break the stone open and nick off a chunk onto agar with the end of a flamed needle.
Cloning a nice sclerotia this way is a great way to get what is probably a pretty good "fruiting" strain with at least solid colonization growth as well. Many if not most such strains will also fruit, some may even be great fruiters!
For most growers of these, the stones are the major point and goal, so I suggest taking some clones to isolate the best stone performers then finding out which isolate is also the best fruiter and sporolator. That's always done great for me.
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