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InvisibleMad Season
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Re: Strain Isolations on Agar, Pics and Questions [Re: spacechildo]
    #23660870 - 09/20/16 07:21 AM (7 years, 5 months ago)

Quote:

spacechildo said:
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:



:whathesaid: I was just curious if this was the thread weetsie made his promise to prove people wrong lol....

I was hoping for an update. I mean if he isn't able to do it, then just say you can't. Leave your pride at the door, and report your results. Scientists fail all the time. I won't have any less respect for someone who can tell people they done fucked up. If anything, it's more respect.


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InvisibleBoogieman47
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Re: Strain Isolations on Agar, Pics and Questions [Re: Mad Season]
    #23661242 - 09/20/16 10:25 AM (7 years, 5 months ago)

Very true thats how you make out the men from the boys you would think on here you would see more of it but i guess even on here like real life people have problems admitting they were wrong


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Invisiblec10h12n2o
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Re: Strain Isolations on Agar, Pics and Questions [Re: Mad Season]
    #23661717 - 09/20/16 01:14 PM (7 years, 5 months ago)

Quote:

bodhisatta said:
I live in Milwaukee

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




not lucky enough to be milwaukee oregon i suspect, haha... i dont make it out to Wisconsin very often, but i would be more than happy to edit and host any footage you get together

lol i learned that the hard way, trying to cool the blade in agar that was too thin, burned straight through the bottom of one plate and the top of another haha...


Quote:

Piezo said:
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...




what works best, per RR, is an ultrasonic jewelry cleaner. works perfectly

great point piezo, i had several plates that didnt do anything at all, i thought maybe i had diluted the spores too much but i think clumping is the more likely culprit

Quote:

spacechildo said:
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:




gotcha. im glad he did it in here, if he keeps updating it will be a very helpful demonstration regardless of the result. I know i damn sure havent been able to get an isolate in 20 transfers, much less 5 lmao... but i would love to learn from those who can

Quote:

Mad Season said:

:whathesaid: I was just curious if this was the thread weetsie made his promise to prove people wrong lol....

I was hoping for an update. I mean if he isn't able to do it, then just say you can't. Leave your pride at the door, and report your results. Scientists fail all the time. I won't have any less respect for someone who can tell people they done fucked up. If anything, it's more respect.




it was certainly a bold claim. I replicated the experiment somewhat, with lots of variations, and have not even come close. if someone does enough digging, its easy to find both experts and naive beginners claiming they can get isolates in such few transfers, so i think the discussion, experiments, results, etc. are very informative and helpful to anyone who is trying to make sense of all the conflicting info that can be found

here is some of what weetsie referenced, to underscore how easy it is for a someone to find confusing info that warranted discussion:

Quote:

weetsie said:

RR lets grow mushrooms - strain isolation:

"Chances are once you transfer from this dish (referencing transfer #3) your next transfer you'll have a mono culture"

The complete guide to cultivating mushrooms - Strain Isolation:

"When starting from spores you may have to transfer your strain to a new dish three or four times before you're able to isolate a single strain"

I really don't agree it takes more than 5 transfers or is in any way that difficult. Dilute your spores, take tiny wedges :shrug:




btw mad i completely agree with you about failures. personally, i learn FAR more from my failures than from my successes, and try to learn from other peoples' experiences as well. it is a shame that people get so emotionally invested in projects/ideas/arguments that they lose sight of how informative a failure can be, for themselves and for readers


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

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."

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InvisibleBoogieman47
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Re: Strain Isolations on Agar, Pics and Questions [Re: c10h12n2o]
    #23661749 - 09/20/16 01:23 PM (7 years, 5 months ago)

Ya thats real shit if we dont fail we dont learn man because you can always back track what happened... and reading other peoples issues have helped me with certain things i know it sucks and makea you feel like ahit when you fail especially investing all the time into this part of life


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InvisibleMycolorado
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Re: Strain Isolations on Agar, Pics and Questions [Re: Boogieman47]
    #23662249 - 09/20/16 04:06 PM (7 years, 5 months ago)

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




I'm going to continue to transfer this sucker down...it'll be interesting to see if it shapes up or shows itself to be multi as you've observed...fun stuff though.

I just got 5 sleeves of falcons for $40 shipped in 2 days!

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Invisiblec10h12n2o
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Re: Strain Isolations on Agar, Pics and Questions [Re: Mycolorado]
    #23662470 - 09/20/16 05:36 PM (7 years, 5 months ago)

@Mycolorodo: 40$ for 5 sleeves is actually pretty expensive! I would recommend buying by the case, it is SOOOOOO much cheaper. for 65-80$ you can get a case of 500 with free shipping, from most lab suppliers. for the longest time i was buying 3 sleeves (of 20) locally for 21$ every single day lol... since i started buying by the case it saves so much time and money. good luck buddy! we will get there one day haha...

BTW, here are the promised UPDATE pics, nothing to write home about, just a bunch of shots of plates at different parts of different series, in different lighting. a few clones in there too



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"Convictions are more dangerous enemies of truth than lies"
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InvisiblebodhisattaMDiscordReddit
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Re: Strain Isolations on Agar, Pics and Questions [Re: c10h12n2o]
    #23662486 - 09/20/16 05:43 PM (7 years, 5 months ago)

$5 for a sleeve of 20 100x15 dishes is expensive. that's the kind of price you should be paying if you're only buying 1-10 at a time

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InvisibleMycolorado
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Re: Strain Isolations on Agar, Pics and Questions [Re: bodhisatta]
    #23662904 - 09/20/16 07:50 PM (7 years, 5 months ago)

And I thought I was getting a deal!  I'll keep my eye out for bulk deals.  I like the Falcons.  Just got ripped off by some cheap ass plates from pulmolabs.  Those guys SUCK!

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OfflineKenetic
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Re: Strain Isolations on Agar, Pics and Questions [Re: Mycolorado]
    #23663096 - 09/20/16 08:40 PM (7 years, 5 months ago)

What's wrong with them?


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InvisibleMycolorado
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Re: Strain Isolations on Agar, Pics and Questions [Re: Kenetic]
    #23664288 - 09/21/16 07:30 AM (7 years, 5 months ago)

I orders a $4 sleeve of plates and wasn't paying attention and paid for 2 day air past the cuttof.  This was on a Wednesday...they processed it on Thursday even though they knew it wouldn't b delivered on in 2 days.  They showed up on Monday and when I reached out to them they told me they did everything they were supposed to and that everyone knows ups doesn't deliver on Saturdays without an additional fee.  The plates themselves are shit and poorly packaged.  I broke 2 sealing them in parafilm.

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OfflineGreg
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Re: Strain Isolations on Agar, Pics and Questions [Re: Mycolorado]
    #23664611 - 09/21/16 10:22 AM (7 years, 5 months ago)

I buy mine 500 at a time from SEOH via Amazon. ~5 dollars each sleeve of 25 100x15 dishes and free shipping.

The plastic sleeves they come in are quite a bit thicker than the ones other suppliers use, so they're less likely to get ripped in shipping.

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InvisibleBoogieman47
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Re: Strain Isolations on Agar, Pics and Questions [Re: Greg]
    #23664647 - 09/21/16 10:37 AM (7 years, 5 months ago)

I saw some on ebay 500 for 50


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Re: Strain Isolations on Agar, Pics and Questions [Re: Mycolorado]
    #23664752 - 09/21/16 11:25 AM (7 years, 5 months ago)

Quote:

Mycolorado said:
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...




I'm far from an expert so I'm not qualified to accurately judge this. However, if I had to I would say that no, that is not a monoculture.
What do you other guys think?


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Offlinespacechildo
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Re: Strain Isolations on Agar, Pics and Questions [Re: TedTheHighlighter]
    #23664757 - 09/21/16 11:28 AM (7 years, 5 months ago)

yeah that's still pretty far from an isolate.

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Invisiblec10h12n2o
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Re: Strain Isolations on Agar, Pics and Questions [Re: TedTheHighlighter]
    #23665115 - 09/21/16 01:45 PM (7 years, 5 months ago)

Quote:

The plates themselves are shit and poorly packaged.  I broke 2 sealing them in parafilm.





lol yeah i think that certainly qualifies as shit

Quote:

noob47 said:
I saw some on ebay 500 for 50




that is a good price, cheapest ive seen was 45$ for a case of 500, but they seemed flimsy. the SEOH plates on Amazon that Greg mentioned are great, i always get them if im impatient and cant wait on an order from a real supplier

lab tech suppliers are the best, since you can get EXACTLY the size and configuration you want. my current favorite is Lake Charles Manufacturing Company, they are THE BOMB, great prices on everything. Here is my last order from them (i also grabbed some of their beaker sets that are made for teaching classes, really great value):

Quote:

Code Item Qty Price Grand Total
206F1 Plastic Petri Dishes, 90x15mm, 3 Vents, Sterile (Case 500)
1 $52.15 $52.15
215G2 Set of 5 Plastic Beakers with 50ml, 100ml, 250ml, 500ml, and 1000ml (Each)
1 $3.42 $3.42
212T2 Tongue Depressor, No Splinter, Birch, Non-Sterile, 6" (Pack 100)
1 $1.65 $1.65
213L14 500ml Round Bottom Florence Boiling Flasks (Single)
1 $3.20 $3.20
402C2 12x75mm, Borosilicate Glass, Round Btm, 5ml Vol, Screw Top, With Cap, Black (Pack 10)
2 $2.41 $4.82

Subtotal: $65.24
Tax: $0.00
Shipping Cost: $23.26
Grand Total: $88.50




pretty damn sweet

Quote:

TedTheHighlighter said:
Quote:

Mycolorado said:
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...




I'm far from an expert so I'm not qualified to accurately judge this. However, if I had to I would say that no, that is not a monoculture.
What do you other guys think?




i highly doubt its a monoculture, definitely a sexy culture though, good job buddy :highfive1:

I saw a post recently that said often many compatible strains will be very well organized (they suggested that anotomosis was involved; im still not sure about even the basics of the dynamics involved in strain interactions/organization though, so i dont know), and in future plates they will often separate. that made a lot of sense to me because it meshed with my observations, where i thought i might have a monoculture and then after a few more transfers started seeing many, many new sectors. its fascinating stuff in any case

if it were me personally, i'd let it grow out a few more days, then use a backlight to try to find some sectors or myc with different characteristics, transfer each of those to 1-3 additional plates (each, to compare transfers from the same "sector" against each other), and then i would use the rest of the plate to make a liquid inoculant, make some spawn, and start fruiting it! that way you can play with some clones while you continue to isolate , and even play with further isolating the clones. but that is just what i would do, others with more experience might have better suggestions


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

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 (09/21/16 01:46 PM)

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Re: Strain Isolations on Agar, Pics and Questions [Re: TedTheHighlighter]
    #23665246 - 09/21/16 02:31 PM (7 years, 5 months ago)

Hey TedTH, yeah, prolly like 20 in there.

Thanks, Dead American!  That's the plan, though I don't like LC...I'll transfer a tad to play with on plates and drop the rest on rye to expand.  I'll clone up some good looking fruit to see what they look like in culture...fun shit!  I was also going to plate it to media with varying nute content to see the effects on morphology, growth rate, etc.  I'll have to read up on anotomosis...

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Re: Strain Isolations on Agar, Pics and Questions [Re: Mycolorado]
    #23665302 - 09/21/16 02:52 PM (7 years, 5 months ago)

not LC, LI! :wink:

when i first started growing, i had great success with LCs, i guess i just got lucky (squirted spores in honey water lol). but as i have learned more, i moved away from them, but LIs rock!

it might have been in this thread, somewhere someone told me that LI > LC, which took a little digging for me to figure out what the hell they meant

best i understand, LC is nutritious and LI is not (at least no more than the agar provides), and where you wait for a LC to colonize, you use an LI immediately

Muda's Badass LI Tek

i have 4 of these blender bottles now lol, i ordered the replacement blade assemblies dirt cheap from amazon :laugh:

i also got a cheap blender base on CL and modified it by cutting out the upper frame, so i can use bigger containers

like you said: fun shit :laugh:


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

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 (09/21/16 02:53 PM)

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Re: Strain Isolations on Agar, Pics and Questions [Re: c10h12n2o]
    #23665686 - 09/21/16 04:51 PM (7 years, 5 months ago)

Thanks for the tip...I'll look into it!

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Invisiblec10h12n2o
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Re: Strain Isolations on Agar, Pics and Questions [Re: Mycolorado]
    #23666032 - 09/21/16 07:06 PM (7 years, 5 months ago)

hey bodhi, ive been meaning to ask you: is there anything wrong with pouring agar as thin as humanly possible? not for long term storage or anything, but in series of transfers involving hundreds of plates

other than burning through the bottom when i try to cool the blade lol... just wanted to double check whether that is acceptable practice for this kind of project

ive been stretching 500ml into 40-50 plates lately, and havent really noticed a problem yet. except when the transfer is so thin that the scalpel burns it from not being properly cooled :facepalm:

thanks in advance


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

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: c10h12n2o] * 1
    #23666218 - 09/21/16 08:12 PM (7 years, 5 months ago)

I don't see the problem if it doesn't cause a problem. I hate how quick it dries up and don't like how it won't cool a scalpel as efficiently.

when I use Lin's wild yeast media I do it really thin. it's done as a spread plate. so you pour the agar let it harden. and then pipette a sterile sample sterily onto the top of the plate then use a sterile spreader or glass hockey stick (glass L rod) and spread it around the surface of the plate. incubate upside down like a bacteria plate

when grab the plate out of the incubator pour off any condensation that forms on the lid(bottom) and then you can count the colonies(enumerate) to give a CFU load of these wild yeasts (most brewers yeast won't grow on these plates so you can quantify non brewing yeast contamination)

so these are poured super thin since this media is very expensive and incubation is only 7 days in a closed container so they don't dry out. they're never cut into either.


so there's valid reasons to pour plates really thin, but I still pour mine about 400ml into 20 dishes so 20ml a plate. realistically it's 15-20mL a plate since I always have a little leftover in my bottle after a sleeve.


here's the wild yeast

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