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InvisibleMycolorado
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Re: Strain Isolations on Agar, Pics and Questions [Re: bodhisatta]
    #23666364 - 09/21/16 08:58 PM (7 years, 4 months ago)

I pretty much just grow oysters...curious how the LI would do on a highly supplemented sawdust block.  I typically sup with 10% bran and spawn with rye at 10-20%.  Maybe a higher sup rate..20% with a plate of LI would produce something...?


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Re: Strain Isolations on Agar, Pics and Questions [Re: Mycolorado]
    #23666369 - 09/21/16 08:59 PM (7 years, 4 months ago)



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InvisibleMycolorado
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Re: Strain Isolations on Agar, Pics and Questions [Re: Inocuole]
    #23666378 - 09/21/16 09:02 PM (7 years, 4 months ago)

Thanks, Inoxious!


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Re: Strain Isolations on Agar, Pics and Questions [Re: Mycolorado]
    #23666508 - 09/21/16 09:43 PM (7 years, 4 months ago)

NP, Mycoloblo.


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Re: Strain Isolations on Agar, Pics and Questions [Re: Mycolorado]
    #23666709 - 09/21/16 10:53 PM (7 years, 4 months ago)

Quote:

Mycolorado said:
I pretty much just grow oysters...curious how the LI would do on a highly supplemented sawdust block.  I typically sup with 10% bran and spawn with rye at 10-20%.  Maybe a higher sup rate..20% with a plate of LI would produce something...?




im kinda confused by your question my friend... LI is what you would use to inoculate your grainspawn, which you would then spawn to the highly supplemented block. in this case, the LI wouldn't really affect the bulk expansion process, but it would probably knock some time off your grain colonization times (unless you are doing g2g). unless im misunderstanding something?

my quart jars of wheat or rye colonize in 4-7 days with LI


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InvisibleMycolorado
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Re: Strain Isolations on Agar, Pics and Questions [Re: c10h12n2o]
    #23667530 - 09/22/16 09:36 AM (7 years, 4 months ago)

Sorry, I should prolly post this over in gourmet, but was just curious as I know you're getting into eddies and from the looks of it, kicking ass!  Anyway, the question, yes you Innocule, could also be applied to any wood lover species.  I was curious if anyone has had success skipping spawn and going straight to inoculation with the LI.  The additional supplementation would make up for the nutrients lost to not spawning. It might take a bit longer but speed isn't really a concern for me.  I know Gourmet Mushrooms, Inc. runs bottles inoculated from agar...it appears they skip the spawn...check their video if you haven't seen it, it's a pretty incredible operation they have.


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Re: Strain Isolations on Agar, Pics and Questions [Re: Mycolorado]
    #23668145 - 09/22/16 01:43 PM (7 years, 4 months ago)

lol yeah a little off topic, but thats definitely an interesting question! i think i have seen a few bottle and invitro teks that you could use LI to inoculate, i am also curious if anyone has any experience with something like this? i imagine the drawbacks might outweigh the pros , but still interesting!

is this the one you are talking about?



OMG wow this video makes me drool, their equipment is so sweet! i love seeing the way people do large scale stuff, when they have money invested and have to turn a profit, the marketplace does some amazing things! those bottles are awesome, gonna have to order a bunch for sure....

all that automation equipment is so cool!!! really makes me want to play around with some CNC controlled machines (or something similar to a 3d printer), my buddy has been mounting lasers on 3-axis CNC controlled machines, and using vacuum actuators to do some really cool stuff therein... i would love to build some bulk automation tools like a ghetto version of that company's stuff, but also interested in small precise automation/machine assistance, maybe like a chamber filled with inert gas to do agar work... now that would be sexy!!


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

C10's Agar Guide + Tips and Tricks | c10's Flow Hood Build Guide


"Partial knowledge is more triumphant than complete knowledge; it takes things to be simpler than they are, and so makes its theory more popular and convincing."

"Convictions are more dangerous enemies of truth than lies"
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Re: Strain Isolations on Agar, Pics and Questions [Re: c10h12n2o]
    #23668732 - 09/22/16 05:03 PM (7 years, 4 months ago)

UPDATE:
ok so i think i might finally be getting close!!!! only after about 1000 more plates than i thought it would take lol....

it is amazing how differently plates can appear, even from the same sector... all of these look WAAAAAY different from earlier in the series, and even from less rhizo plates from the same point in the series

AA+ plate on 14th transfer, oh so sexy:


top is the plate above, bottom 2 are clones that have been further isolated 2 transfers:


And these are plates from the same point in the series as the first, still showing lots of sectors:


definitely making progess, been an insane amount of work though, considering how similar the clone cultures are by 2 transfers lol


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

C10's Agar Guide + Tips and Tricks | c10's Flow Hood Build Guide


"Partial knowledge is more triumphant than complete knowledge; it takes things to be simpler than they are, and so makes its theory more popular and convincing."

"Convictions are more dangerous enemies of truth than lies"
― Friedrich Nietzsche


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InvisibleTuhdoww
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Re: Strain Isolations on Agar, Pics and Questions [Re: Inocuole]
    #23668875 - 09/22/16 05:55 PM (7 years, 4 months ago)

Quote:

Mycolorado said:
Thanks, Inoxious!



Quote:

Inocuole said:
NP, Mycoloblo.




:excuseme:


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InvisibleMycolorado
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Re: Strain Isolations on Agar, Pics and Questions [Re: c10h12n2o]
    #23671054 - 09/23/16 01:09 PM (7 years, 4 months ago)

Yep...those guys know how to crank out some beautiful fruit on a huge scale!  They've probably been featured on the Modern Marvels show for that setup. 
Per topic, I was speaking with my college mycology professor and he said even a true isolate could still exhibit rhizomorphic growth patterns depending on conditions and that you really need to see it under the scope to be certain.  This was in reference to isolating down from MS.  I'm not suggesting his word is definitive but it might save a lot of people a lot of heartache...


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Invisiblec10h12n2o
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Re: Strain Isolations on Agar, Pics and Questions [Re: Mycolorado]
    #23672741 - 09/24/16 12:32 AM (7 years, 4 months ago)

Do you have any pics that could demonstrate what you're talking about? Like what people should look for in the microscope to distinguish a monoculture? I wish I knew a mycology professor!! I hope you saved his contact info, that's a killer resource!


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

C10's Agar Guide + Tips and Tricks | c10's Flow Hood Build Guide


"Partial knowledge is more triumphant than complete knowledge; it takes things to be simpler than they are, and so makes its theory more popular and convincing."

"Convictions are more dangerous enemies of truth than lies"
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Offlinespacechildo
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Re: Strain Isolations on Agar, Pics and Questions [Re: Mycolorado]
    #23673114 - 09/24/16 06:46 AM (7 years, 4 months ago)

Quote:

Mycolorado said:
Per topic, I was speaking with my college mycology professor and he said even a true isolate could still exhibit rhizomorphic growth patterns depending on conditions and that you really need to see it under the scope to be certain.  This was in reference to isolating down from MS.  I'm not suggesting his word is definitive but it might save a lot of people a lot of heartache...




what made you think isolates can't be rhizomorphic? :confused2:

you dont need a microscope to tell whether or not you got an isolate, sectors become pretty clear once you get further down the line,
I'm sure it has been linked here already but checkout stropharis cleaning & isolating on agar :thumbup:


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InvisibleMycolorado
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Re: Strain Isolations on Agar, Pics and Questions [Re: spacechildo]
    #23673380 - 09/24/16 09:33 AM (7 years, 4 months ago)

You know spacechildo, I didn't get that part of the question regarding sectoring posed to him....he gets going off on shit regarding the genetics, etc....I'll follow up on it.  Stro's thread is great...referenced a lot lately.  I wasn't clear above...I was speculating that one could possibly be working with an isolate that does exhibit sectoring but uniformly, when growing rhizomorphically, if that makes sense?  Here's the same plate as above...it definitely has sectors and they may be distinct, but there are also large sections that have a nice uniform pattern that could still be broken down into "sectors" if you will...



Yeah, c10, he's actually a good friend of mine and lives in my neighborhood, so I can bounce shit off him all time.  I'll have to follow up with him on the microscopy but I'm pretty sure he was talking about looking at individual strands of hyphae containing clamp connections.  You might be able to do a hyphal dilution in the same way you make a LI, but on a smaller scale and streak on a plate to isolate that way...might be worth a shot.


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Re: Strain Isolations on Agar, Pics and Questions [Re: Mycolorado]
    #23673465 - 09/24/16 10:12 AM (7 years, 4 months ago)

there's a BUNCH of strains in that pic, so many they are hard to tell apart.
but you can see its far from a perfect circle and how many different fans of growth stick out all around the edge.

zoom in on the pics where stro shows the sectors on the end of the fans sticking out. devil's in the details.


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Offlinespacechildo
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Re: Strain Isolations on Agar, Pics and Questions [Re: spacechildo] * 1
    #23673475 - 09/24/16 10:17 AM (7 years, 4 months ago)

check this out, I drew on your pic, from 3 - 6 o'clock I can count at least 9 different sectors.


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InvisibleMycolorado
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Re: Strain Isolations on Agar, Pics and Questions [Re: spacechildo]
    #23675046 - 09/24/16 07:45 PM (7 years, 4 months ago)

Yeah, agree with that.  I'm gonna transfer a couple from the opposit side and run them on 2% and 4% mea to see how they differ on different nutrient content.  My thinking is, certain media recipes and % nute content may be more favorable for aiding in isolation.


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Re: Strain Isolations on Agar, Pics and Questions [Re: Mycolorado]
    #23690746 - 09/29/16 02:02 PM (7 years, 3 months ago)

great info space, thanks for explaining and illustrating, very helpful

beautiful plate btw mycolorodo! when it starts looking round, rhizo, and organized like that they look SOOOOO much better than the jungle plates do

im really glad space, SLH, and bodhi primed my expectations, because the first dozen or so times i had what i suspected might be an isolate were quite disappointing. sometimes, i will transfer what REALLY looks like a really well organized single rhizo sector to verify that it is an isolate, only to find out that the next plate has 8+ sectors, sometimes NONE rhizo!

its really wild the way strains can pile on top of each other, sometimes distinguishing themselves, sometimes not. we are basically hoping different strains grow in different directions on the agar, to form sectors. but some of these sectors can be made up of many "compatible" strains, which might take several transfers to really start distinguishing themselves

also, late in the process i have started taking several transfers from the same sector and comparing them against each other, sometimes they can respond dramatically differently on subsequent plates

after testing a few variations on my recipe to aid sectoring and rhizo growth, I have definitely stopped using yeast and peptone, as they seem to make the myc grow thick and not wide. I typically make 500ml MEA with 7g light malt extract and 10g agar, since that seems to be the recipe all my best looking plates have been made of


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

C10's Agar Guide + Tips and Tricks | c10's Flow Hood Build Guide


"Partial knowledge is more triumphant than complete knowledge; it takes things to be simpler than they are, and so makes its theory more popular and convincing."

"Convictions are more dangerous enemies of truth than lies"
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Re: Strain Isolations on Agar, Pics and Questions [Re: c10h12n2o]
    #23690998 - 09/29/16 03:29 PM (7 years, 3 months ago)

So OP, have you isolated yet? If not, what's the closest plate you have?


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Re: Strain Isolations on Agar, Pics and Questions [Re: c10h12n2o]
    #23691964 - 09/29/16 08:02 PM (7 years, 3 months ago)

Here's a close-up of the 4% MEA culture.  Lots of sectors here...but cool pic nonetheless.  This came off the above plate.  I'm gonna try to get a piece of one of these ropes and try a water dilution and streak to a few plates to see if I can further isolate down...


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Invisiblec10h12n2o
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Re: Strain Isolations on Agar, Pics and Questions [Re: Mycolorado]
    #23700929 - 10/02/16 04:58 PM (7 years, 3 months ago)

Quote:

TedTheHighlighter said:
So OP, have you isolated yet? If not, what's the closest plate you have?




:rofl: nope...

been through around 2000 plates (working with lots of varieties, clone cultures, etc.), and learned a hell of a lot, been doing an insane amount of work. cannot understate how insane the amount of work is. SLH was mad correct when he said focusing on more than one variety at a time for isolations is a HUGE amount of work and could easily lead to someone being putt off

the original series i was working with are on between their 15th and 19th transfers. they look ENTIRELY different than they did initially, since i have been mostly focusing on rhizo sectors, the cultures i am working with now are VERY rhizo. though i often do at least one transfer from an aggressive or distinctive sector even if it isnt rhizo, since there is no telling what it will look like in subsequent plates

i currently have lots of plates from that series that look like the plates above, and am down to 2-6 sectors per plate, often quite distinctive. ive been taking multiple transfers from some sectors lately, since they can end up looking totally different on subsequent plates

in contrast to my initial series, plates from subsequent varieties look much better sooner, since i have been diluting spores and streaking them, some of them look great within 2-4 transfers (but still far from a monoculture)

another thing: ive been taking larger transfers from earlier plates, since it seems to speed things up a bit and give the myc more of a circumference and hence more opportunity for strains to grow out in different directions (i think this also has something to do with why it usually takes a while for rhizo myc to develop, further out on the plate, since as the circumference expands the rhizo strain group has more chance to distinguish itself). For the same reason, once the jungle has thinned a bit and there is more distinct sectoring, i have been taking tiny transfers trying to target individual strains/strain groups

i improved the lighting in my SAB (installed a fixture) so should have improved toplit photos now. will try to post some images soon, kinda burried under all these plates haha...

 
Quote:

Mycolorado said:
Here's a close-up of the 4% MEA culture.  Lots of sectors here...but cool pic nonetheless.  This came off the above plate.  I'm gonna try to get a piece of one of these ropes and try a water dilution and streak to a few plates to see if I can further isolate down...





nice pic buddy!!:highfive1:

whats really crazy is how strains group up, and how we hope they choose to grow in different directions :/

btw, are you talking about doing a water dilution with spores? or with myc from a plate? as far as i know, people are usually talking about doing dilutions and serial dilutions with spores, and streaking is a kind of dilution or spreading thin in itself

if you are talking about trying to streak a plate with myc rather than spores, i dont think i have heard of that, but it might actually work now that i think about it. a lot of the time, if a transferred bit of tissue touches the agar anywhere other than where i try to stick it, there is often a satellite colony which develops in the location it touched. kinda makes me wonder if a plate streaked with living tissue scraped off with a loop might give the strains present in the sample more opportunity to differentiate themselves than the limited circumference provided by a transfer... anyone else have any thoughts on this?


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

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"Partial knowledge is more triumphant than complete knowledge; it takes things to be simpler than they are, and so makes its theory more popular and convincing."

"Convictions are more dangerous enemies of truth than lies"
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