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InvisibleSupalemonhaze
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Re: Strain Isolations on Agar, Pics and Questions [Re: Mad Season]
    #23658744 - 09/19/16 01:12 PM (7 years, 5 months ago)

Quote:

Mad Season said:
Wasn't this the thread we were supposed to be proven wrong that isolating takes like 5 transfers or some BS? What's up with that shit?




I think he said three but definitely :whathesaid:

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InvisiblebodhisattaMDiscordReddit
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Re: Strain Isolations on Agar, Pics and Questions [Re: Supalemonhaze]
    #23658750 - 09/19/16 01:13 PM (7 years, 5 months ago)

Takes more than three to isolate from a clone that's chimeric

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Offlinespacechildo
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Re: Strain Isolations on Agar, Pics and Questions [Re: bodhisatta]
    #23658910 - 09/19/16 02:02 PM (7 years, 5 months ago)

not even from a clone, from MS to agar :lol:

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Invisiblec10h12n2o
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Re: Strain Isolations on Agar, Pics and Questions [Re: Mad Season]
    #23659375 - 09/19/16 04:54 PM (7 years, 5 months ago)

Quote:

TedTheHighlighter said:
Glad to see you haven't given up, in fact you've done just the opposite. You are definitely proving that it takes many transfers to reach isolation. I really do wonder how RR did it with just a couple transfers.
I would definitely love to hear about your cloning results along with isolation results. I am still very curious about clones vs isolates




haha thanks brother-man :smile: giving up isnt really my style, but i drive myself crazy trying to cram 100hrs of work into 24 hr days lol... i will definitely keep posting updates, and will include the clone stuff as well, since i am isolating lots of them too. lol right!? i am glad SLH and bodhi realistically primed my expectations and told me what to expect, because it was SO disappointing the first 10 or so times i thought i had an isolate

i knew it would be a lot of work, but i WAY underestimated it. thats why i LOVE when someone comes along and says something super helpful (and someimes obvious from another perspective) that saves me TONS of time and effort, like in the beginning of this thread where leaf said:

Quote:

blindingleaf said:
what recipe are u using?  the wedges in the center are really thick, I get that look when I use peptone or yeast on top of malt.

u can wrap them in para or cling wrap instead of each one getting their own bag to save plastic….not that pouring plates is the best way to save plastic…but figured I'd throw that out there. 

Ur labels are pretty elaborate (numbers and letters) for being an MS plate thats probably going to be tossed after transfer.  I just label variety/# transfer/date (ex. GT, x-3, 7/14) for MS stuff, but whatever works.  sharpie instead of label maker will save time too. for slants, I throw a piece of scotch tape over the sharpie so it doesn't rub off.

I agree with everyone else, make some spawn and keep a few plates running for random isolates if u wanna go that route.




that post was solid gold, saved me hundreds of hours, and helped me refine my technique and fix several mistakes, and will probably be useful to anyone reading as well. great advice, right to the core of the issues i was trying to sort out (what to do at that point, etc). thanks again buddy

I was indeed using yeast and peptone, my labels were ridiculously and needlessly complicated, picked up a big roll of parafilm (God's gift to lab techs), and made LIs and spawn to start playing with clones while i work on the isolates

Quote:

Mad Season said:
Wasn't this the thread we were supposed to be proven wrong that isolating takes like 5 transfers or some BS? What's up with that shit?




:rolleyes: that would be a VERY contentious, antagonistic misinterpretation of the thread

this is the thread where i asked a whole bunch of questions about the isolations i am working on, posted a bunch of pics of plates with proposed transfers outlined, and kept updating based on what might useful for other people in my position.

I have thoroughly enjoyed picking the brains of everyone who has been nice enough to answer my questions and share info, you guys rock :rockon: Ive personally learned a LOT through the process and from the posts in the thread, and hope readers/lurkers will find the discussion as helpful and educational as i have

weetsie suggested that the number of transfers people were citing was way to high, here:

Quote:

10 transfers, 20 transfers?!?!

what are you lot smoking?

spores to single strain isolate, 3-5 transfers.





and he proceeded to initiate an experiment to demonstrate. I had no idea (which is why i made a "questions" thread rather than a "tek"), and also began similar experiments in addition to what i was already working on, but i certainly appreciate any experiments, pics, etc, really anything that contributes to the discussion or brings up interesting talking points, and weetsie's experiment DEFINITELY fit the bill. it is relevant and useful info regardless of the result, i appreciate the sharing of notes/experiments

in my limited experience, i certainly have not been able to get isolates in such a small number of transfers, but i want to learn more about the techniques employed by those who are able to do it

I made the thread to ask questions and learn, and share my experience for the benefit of others, not to "prove" anything


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

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

Edited by c10h12n2o (09/19/16 04:55 PM)

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InvisiblebodhisattaMDiscordReddit
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Re: Strain Isolations on Agar, Pics and Questions [Re: c10h12n2o]
    #23659391 - 09/19/16 04:59 PM (7 years, 5 months ago)

if RR was getting 3 transfer isolates it's because he did a couple serial dilutions then streaked it out.



even still I got dikaryotic growth trying to get monokaryotic growth by diluting spores.

but if your first plate was like these ones you might be a lot closer to an isolate than you would putting a drop on a plate

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Offlinespacechildo
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Re: Strain Isolations on Agar, Pics and Questions [Re: bodhisatta]
    #23659432 - 09/19/16 05:16 PM (7 years, 5 months ago)

weetsie claimed small transfers were key :wink:

and c12h0 that's the longest text I've ever seen where a simple yes would suffice :lol:

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InvisibleBoogieman47
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Re: Strain Isolations on Agar, Pics and Questions [Re: spacechildo]
    #23659448 - 09/19/16 05:20 PM (7 years, 5 months ago)

I have a plate that was from my first drop of solution still havent got an isolate i havent tried either but im sure its on its 50th plate by now maybe more ive got plates that looked like an isolate after 3 or 4 transfers but another transfer proved it was not


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Invisiblec10h12n2o
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Re: Strain Isolations on Agar, Pics and Questions [Re: spacechildo]
    #23660024 - 09/19/16 08:55 PM (7 years, 5 months ago)

Quote:

bodhisatta said:
if RR was getting 3 transfer isolates it's because he did a couple serial dilutions then streaked it out.



even still I got dikaryotic growth trying to get monokaryotic growth by diluting spores.

but if your first plate was like these ones you might be a lot closer to an isolate than you would putting a drop on a plate




i think you are right on the money there bodhi. after I figured out how to properly streak plates and started diluting spores, ive been doing all my MS plates this way. but i hadnt yet thought to do multiple stages of dilution, that would probably be a great step in the right direction

I dont have any pics of any of the initial plates i streaked, but this is the way ive been doing it:



it gives me LOTS more to choose from per initial plate, and greatly increased my chances of getting rhizomorphic and/or aggressive myc to work with on later plates. but doing it this way, you have to time your transfers right, because it will become overgrown quickly. i like comparing the different colonies vigor. been doing single stage dilution of the spores, and have countless colonies per streaked plate, similar to yours but many more individual colonies, and in the pattern above

i dont think i have ever seen monokaryotic growth in person, although i look forward to working with some monokaryotic cultures for some experiments at some point. still obviously have a TON to learn about isolations and working with isolates

Quote:

spacechildo said:
weetsie claimed small transfers were key :wink:

and c12h0 that's the longest text I've ever seen where a simple yes would suffice :lol:




i think it was RR who said that small transfers are key, weetsie just echoed it. its probably safe to say that small transfers are helpful when trying to get from spores to isolate in the fewest number of transfers. do you disagree? if so please explain

and a "yes" would not suffice when the answer is "that would be a VERY contentious, antagonistic misinterpretation of the thread"

some people think/type/read faster than others :shrug: i try to be articulate and leave as little room for misinterpretation as possible, for the benefit of anyone reading

Quote:

noob47 said:
I have a plate that was from my first drop of solution still havent got an isolate i havent tried either but im sure its on its 50th plate by now maybe more ive got plates that looked like an isolate after 3 or 4 transfers but another transfer proved it was not




me too brother! i have been working with a TON of different series, with lots of genetic diversity (both race and species), and i have not had a single one turn out to be an isolate yet, over 1000 plates in

like i said, im glad SLH and bodhi gave me some realistic expectations going into it, because it was so disappointing the first 10 or so times i thought i had a nice rhizo isolate, only to have entirely different characteristics and multiple sectors on the next plates :/


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

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

Edited by c10h12n2o (09/19/16 09:01 PM)

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InvisibleBoogieman47
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Re: Strain Isolations on Agar, Pics and Questions [Re: c10h12n2o]
    #23660061 - 09/19/16 09:10 PM (7 years, 5 months ago)

Ya they both know their shit.. i am not too worried about an isolate right now like i said that one plate has over 50 transfers easy i bet using a microscope would help alot


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Invisiblec10h12n2o
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Re: Strain Isolations on Agar, Pics and Questions [Re: Boogieman47]
    #23660118 - 09/19/16 09:39 PM (7 years, 5 months ago)

ive noticed that when i take transfers that are as small as i can manage, 2/3 do not really grow out on the next plate. maybe my scalpel was still too hot, agar too thin, idk, but ive had about 30 plates total that were minuscule transfers which didnt end up doing anything :/ any ideas why this happens?

lately ive been doing about a grain of rice size, which works every time.

like mad said in the beginning of the thread, it seems like if you are just trying to have a good grow, a well-organized culture is easier to obtain and more important than isolates (which makes a ton of sense to me)

the reason i personally am determined to build a library of isolates is because i want to be able to run meaningful experiments and collect meaningful data points on some arduino/computer controlled automated rooms


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

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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InvisiblebodhisattaMDiscordReddit
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Re: Strain Isolations on Agar, Pics and Questions [Re: Boogieman47]
    #23660130 - 09/19/16 09:43 PM (7 years, 5 months ago)

I wish someone could video me doing agar work for mycology purpose. Not that people haven't already done it but...

I have pisspoor recording equipment

And yes you're probably burning it up. Dab the hot tool in the plate a few times Right next to your transfer area

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Re: Strain Isolations on Agar, Pics and Questions [Re: c10h12n2o]
    #23660134 - 09/19/16 09:46 PM (7 years, 5 months ago)

I'm glad there seems to be a lot of chatter as of late pertaining to agar work.  I think a lot more people would find success in cultivation if they would learn to do it.  I feel like on the cultivation board, agar work is viewed as some special/hard/mystical process or at least regarded as non-essential...I started on agar from spore and worked up to fruit so maybe I'm biased.  But, like I said, glad people are posting agar stuff. 
Per topic, I think some are easier to isolate down than others.  Correct me if I'm wrong, but I believe lower nutrient media will promote rhizomorphic growth, which is easier to see sectoring in.  This is pretty close to, if not a mono and it's at 6 transfers.  It was also made from a loop transfer from a print, so there were very few spores (compared to a syringe) in the initial inoculation. It's 3 transfers from the plate in my sig.  Sorry for the long ass ramble...

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Re: Strain Isolations on Agar, Pics and Questions [Re: c10h12n2o]
    #23660138 - 09/19/16 09:48 PM (7 years, 5 months ago)

I have taken very small transfers just from the agar being stiff and the myc comes off it usually grows ... id say your blade is still hot ? Idk i think if the myc died from that it would show a contam from having dead cells i could be wrong that is strange ... im not knocking isolates im just too lazy to get one haha i think its cool you are so into it i cant knock someomes ambition


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InvisiblebodhisattaMDiscordReddit
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Re: Strain Isolations on Agar, Pics and Questions [Re: Boogieman47]
    #23660152 - 09/19/16 09:54 PM (7 years, 5 months ago)

Agar is easy as fuck. Knowing things about shroom biology and having dexterity isn't.

Either way I can teach children to do culture work on agar. Success rate may be under 99% still beats using a syringe on everything

The natural progression is giving up on isolates it seems

Edited by bodhisatta (09/19/16 10:08 PM)

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Invisiblec10h12n2o
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Re: Strain Isolations on Agar, Pics and Questions [Re: bodhisatta]
    #23660178 - 09/19/16 10:09 PM (7 years, 5 months ago)

Quote:

bodhisatta said:
I wish someone could video me doing agar work for mycology purpose. Not that people haven't already done it but...

I have pisspoor recording equipment

And yes you're probably burning it up. Dab the hot tool in the plate a few times Right next to your transfer area




I would be more than happy to video you, edit it, and host it. im in the PNW but i travel a lot, where would you be able to do it?

i have lots of video/ad equipment, greenscreens, lighting, cameras, etc

Let's Do Agar! from RR Video with bodhi and c10 haha.... i can see it already

i must have been burning it. i have started pouring my dishes really thin, and unfortunately there often isnt enough depth to sufficiently cool the blade, and its surprising how long those things can hold heat, especially once they get a carbon layer built up from the flame

Quote:

Mycolorodo said:
I believe lower nutrient media will promote rhizomorphic growth, which is easier to see sectoring in




this certainly seems to be the case, at least most of the time. i have been using half carb agar recipe for these, since myc seems to reach out and hunt nutrients more on it. though when i have tested using less than that, growth has slowed or stalled

btw great work man, and thanks for sharing! nice looking plates :highfive1:

im gonna try 2 stage dilution next


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

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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InvisiblebodhisattaMDiscordReddit
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Re: Strain Isolations on Agar, Pics and Questions [Re: bodhisatta]
    #23660294 - 09/19/16 11:21 PM (7 years, 5 months ago)

I live in Milwaukee

The blade can have enough heat to burn right through the bottom the dish if you're not careful

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OfflinePiezo
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Re: Strain Isolations on Agar, Pics and Questions [Re: bodhisatta]
    #23660487 - 09/20/16 01:12 AM (7 years, 5 months ago)

What do y'all use for declumping spores in solution ? Glycerin ? Maybe that putting a glass tube in the water jet over an ultrasonic mister could help ?

The benefits of two step dilution is small if the spore are still clumped together at the end...

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OfflineKenetic
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Re: Strain Isolations on Agar, Pics and Questions [Re: Piezo] * 1
    #23660492 - 09/20/16 01:16 AM (7 years, 5 months ago)

Shake it like a salt shaker


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Re: Strain Isolations on Agar, Pics and Questions [Re: Kenetic]
    #23660518 - 09/20/16 01:40 AM (7 years, 5 months ago)

What i did ^
Mycology is all about shaking.

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Offlinespacechildo
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Re: Strain Isolations on Agar, Pics and Questions [Re: c10h12n2o]
    #23660865 - 09/20/16 07:17 AM (7 years, 5 months ago)

Quote:

c10h12n2o said:
and a "yes" would not suffice when the answer is "that would be a VERY contentious, antagonistic misinterpretation of the thread"




you misunderstand man, it wasnt your reason for making this thread
but yes it was this thread some dude claimed he'll show MS to isolate in 3-5 transfers :wink:

for some reason he didnt wanna make his own thread about it :shrug:

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