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spacechildo
proletarians rise up


Registered: 01/24/13
Posts: 19,243
Loc: Babylon
Last seen: 6 years, 4 months
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Re: Strain Isolations on Agar, Pics and Questions [Re: bodhisatta]
#23459746 - 07/20/16 02:01 PM (7 years, 6 months ago) |
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just wait for a plate to start pinning and clone that.
why are you so hung up on getting isolates btw?
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c10h12n2o
serial dilutor


Registered: 01/21/15
Posts: 3,200
Loc: the abyss
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Re: Strain Isolations on Agar, Pics and Questions [Re: bodhisatta]
#23459798 - 07/20/16 02:24 PM (7 years, 6 months ago) |
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.... i dont think you read my posts, as that does not answer any of my questions. but i do appreciate the links, reading them (among others) is a how i got this far, and more reading is how i plan to get further. so i very much appreciate your writing the guides.
however, i do have some specific questions though regarding the specifics of these projects, and would appreciate any feedback or guidance or criticism regarding my techniques, plans, resources, and position on the timeline.
if you find time to read my posts id appreciate any guidance/answers, especially concerning my questions
warm regards
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c10h12n2o
serial dilutor


Registered: 01/21/15
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Re: Strain Isolations on Agar, Pics and Questions [Re: spacechildo]
#23459803 - 07/20/16 02:25 PM (7 years, 6 months ago) |
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good idea thanks
i wouldnt say hung up, more like fascinated 
i like trying lots of things and keeping good notes and learning from mistakes, and isolating strains seems to provide fertile ground for those pursuits, i love it
thanks again to everyone for your input
I am about to pour another sleeve of plates to prepare for the next transfers, along with whatever other great ideas yall throw at me
Edited by c10h12n2o (07/20/16 02:33 PM)
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bodhisatta 
Smurf real estate agent


Registered: 04/30/13
Posts: 61,889
Loc: Milky way
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Re: Strain Isolations on Agar, Pics and Questions [Re: c10h12n2o]
#23459813 - 07/20/16 02:31 PM (7 years, 6 months ago) |
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You can press edit so you don't have to post twice in a row.
https://www.shroomery.org/forums/showflat.php/Number/23459704#23459704 These questions? Answered in the links. Read between the lines, there's many ways to do things the right ways and thousands more ways to do it wrong. You're not going to get someone to make your decisions.
You have more ambition than you need right now it's bigger than your work ethic. Obviouly you're not an idiot since you're asking these kinds of questions in the first place. So get out there and do your part in leaning now, practice and development of skills and experiences that you can't be told or read about.
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Boogieman47
Let's boogie


Registered: 03/05/16
Posts: 9,712
Loc: Under your bed
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Re: Strain Isolations on Agar, Pics and Questions [Re: bodhisatta]
#23459882 - 07/20/16 03:04 PM (7 years, 6 months ago) |
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If you take a fully colonized clean plate of agar and drop it into a jar of water you can do two or three tubs worth of grain depending on how much water to use ... and when I do plates I'll take two or three transfers from different parts of myc then I usually will use the plate I transferred for grain or li
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Boogieman47
Let's boogie


Registered: 03/05/16
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Re: Strain Isolations on Agar, Pics and Questions [Re: c10h12n2o]
#23459924 - 07/20/16 03:30 PM (7 years, 6 months ago) |
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You said you were planning on doing bucket/ mini mono did you mean the bucket tek? I know alot of people use it but it's not the best way to pasteurize I'm not knocking it but if I were you I would properly pasteurize in jars or bags in a pot
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c10h12n2o
serial dilutor



Registered: 01/21/15
Posts: 3,200
Loc: the abyss
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Re: Strain Isolations on Agar, Pics and Questions [Re: Boogieman47]
#23460050 - 07/20/16 04:19 PM (7 years, 6 months ago) |
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please read the thread , there are several clear questions that are not dumb questions, and they might very well help someone besides myself in the future. thats why i took the time to take all the pics and make the post in the first place, certainly didnt photograph 100 plates for a lack of work ethic :/
I obviously know how to make agar and pour plates already,and i have obviously been reading quite a bit based on what i am trying to do, ive certainly clocked my share of time reading agar threads (including yours) so i dont know why you are giving me the standard noob treatment of posting your sig links. Your Agar links/guide is where i have done a large part of my learning (and will continue to), my questions are about the application of those techniques and specific aspects of my cultures and plans that i would like clarification on, so telling me to look back at the material that i got the questions from is kinda redundant
I dont expect anyone to do anything for me or make my decisions, but i very much value insight of others, there are some brilliant people in this lovely community and i am grateful for the opportunity to learn from them thats why i ask questions. lol i think you are severely underestimating my work ethic, i only slept every other day for years while working my way through college :p
Questions:
1. How do these plates look, which need more transfers(any isolates)?https://www.shroomery.org/forums/showflat.php/Number/23458285#23458285 (thanks to everyone who has answered and provided feedback, very helpful and informative) (also thanks for encouraging me to play with clones while i am working on isolates, great idea)
2.Quote:
Supalemonhaze said: I now learned the hard way why you should do a shitload of transfers before you start isolation.
when doing these transfers before isolating, do you just go for tiny wedges of leading edges that seems aggressive (and clean obviously)? is the objective to reduce the number of strains in each successive culture? (same goal as diluting the spore solution to begin with, to thin the herd) i think i understand, just double checking my understanding of the method
2b. During those shitload of transfers, how far do you let those generations grow out before conducting the transfer? And does it make sense to do multiple on each plate at this point since it is further from iso than i suspected?
3. I dont have access to any fruits to clone atm, so i want to start a few MS grows to have something to fruit and clone while i am doing my isolations. Since i have lots of clean plates colonized by spore solution and also 1st and 2nd transfers (which i suppose are MS, though less organisms in the herd than the spore inoculated plates). Does it make more sense to make spawn from one of the clean spore inoculated (MS) plates that i made when i started the project, or to use a wedge from the 2nd or 3rd transfer that should have fewer total number of strains present?
4. Here are the wedges i plan to transfer today for the next (4th) series. I have outlined the wedges in red and numbered them for each plate. Does it look like i am understanding correctly, or is there something i should change?https://www.shroomery.org/forums/showflat.php/Number/23459365#23459365
5. 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
i am using yeast malt peptone agar a la paul stamets. It sure has acted different than the MEA i used to begin with, which was more whispy. Several of the cultures on YMPA are VERY thick in the center, even though i only used tiny wedges. Several of them are pressing up against the lid of the petri dish, and others have grown so opaque and thick that light barely passes through them!Any feedback or suggested revisions you had in mind?
6. What distinguishes a liquid culture from a liquid inoculant?
Edit:
Quote:
noob47 said: If you take a fully colonized clean plate of agar and drop it into a jar of water you can do two or three tubs worth of grain depending on how much water to use ... and when I do plates I'll take two or three transfers from different parts of myc then I usually will use the plate I transferred for grain or li
what an great idea i am going to try that today with several from earlier transfers. thanks for the advice! 
Quote:
noob47 said: You said you were planning on doing bucket/ mini mono did you mean the bucket tek? I know alot of people use it but it's not the best way to pasteurize I'm not knocking it but if I were you I would properly pasteurize in jars or bags in a pot
no i meant like this https://www.shroomery.org/forums/showflat.php/Number/6696796/fpart/1/vc/1 , little buckets with shower caps on top. I have a MONSTROUS AA pressure cooker i use for everything 
Thanks Everyone!
warm regards
Edited by c10h12n2o (07/20/16 04:25 PM)
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spacechildo
proletarians rise up


Registered: 01/24/13
Posts: 19,243
Loc: Babylon
Last seen: 6 years, 4 months
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Re: Strain Isolations on Agar, Pics and Questions [Re: mupetmower]
#23460101 - 07/20/16 04:37 PM (7 years, 6 months ago) |
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1; already answered, 20+ transfers needed. 2 are all answered here
Quote:
mupetmower said: Here is a good link. give that a good read and look at the pics of monoculture and sectoring near the bottom of OP.
3 already answered,clone from a plate pin 4 doesnt matter because see 1) 5 all cultures are different. 6 LI = myc agitated/blended on agar with water (instant) LC = nutritious water sterilized and inoculated (wait for colonization)
You're way overthinking stuff. It will all make sense once you start growing.
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weetsie
unlicensed tub surgeon



Registered: 05/08/11
Posts: 572
Loc: United Kingdom
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Re: Strain Isolations on Agar, Pics and Questions [Re: spacechildo]
#23460145 - 07/20/16 04:56 PM (7 years, 6 months ago) |
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10 transfers, 20 transfers?!?!
what are you lot smoking?
spores to single strain isolate, 3-5 transfers.
-------------------- Active grow logs: Oysters on Straw Pellets Trade list
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spacechildo
proletarians rise up


Registered: 01/24/13
Posts: 19,243
Loc: Babylon
Last seen: 6 years, 4 months
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Re: Strain Isolations on Agar, Pics and Questions [Re: weetsie]
#23460161 - 07/20/16 05:04 PM (7 years, 6 months ago) |
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lol
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weetsie
unlicensed tub surgeon



Registered: 05/08/11
Posts: 572
Loc: United Kingdom
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Re: Strain Isolations on Agar, Pics and Questions [Re: weetsie]
#23460197 - 07/20/16 05:22 PM (7 years, 6 months ago) |
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by 20 transfers you're gonna start seeing sectoring again as your strain starts senescing
-------------------- Active grow logs: Oysters on Straw Pellets Trade list
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c10h12n2o
serial dilutor



Registered: 01/21/15
Posts: 3,200
Loc: the abyss
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Re: Strain Isolations on Agar, Pics and Questions [Re: spacechildo]
#23460210 - 07/20/16 05:27 PM (7 years, 6 months ago) |
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thanks again spacechildo, i really appreciate your input
i have grown lots of times, successfully every single time because i have made a point to think things through and do lots of reading even way back then. its all making lots of sense, i just enjoy thinking about this stuff and clarifying it both for myself and anyone else who finds it useful.
im not confused about any of the basics, just clarifying some things that myself and others might find useful, most of my questions have just been asking for confirmation of something to verify something i think i understand.
and judging by the number of people (some quite experienced) on these forums who swear they can get isolates in 3-5 transfers, i dont think my questions were stupid at all
Quote:
weetsie said: by 20 transfers you're gonna start seeing sectoring again as your strain starts senescing 
lol see this is the other end of the info available to people trying to research this stuff. so its not crazy to ask for clarification or advice :/ can you add anything weetsie? much obliged my friend
Edited by c10h12n2o (07/20/16 05:30 PM)
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spacechildo
proletarians rise up


Registered: 01/24/13
Posts: 19,243
Loc: Babylon
Last seen: 6 years, 4 months
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Re: Strain Isolations on Agar, Pics and Questions [Re: weetsie]
#23460228 - 07/20/16 05:34 PM (7 years, 6 months ago) |
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Quote:
weetsie said: by 20 transfers you're gonna start seeing sectoring again as your strain starts senescing 
that doesnt even make sense. do you even know what senescense means dude? 
after 3-5 transfers you still dont see sectors you see a bunch of genetics growing together. what you call "sectoring again" is sectoring for the first time.
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spacechildo
proletarians rise up


Registered: 01/24/13
Posts: 19,243
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Last seen: 6 years, 4 months
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Re: Strain Isolations on Agar, Pics and Questions [Re: c10h12n2o]
#23460237 - 07/20/16 05:38 PM (7 years, 6 months ago) |
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Quote:
c10h12n2o said: and judging by the number of people (some quite experienced) on these forums who swear they can get isolates in 3-5 transfers, i dont think my questions were stupid at all
whoa, got any links?
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weetsie
unlicensed tub surgeon



Registered: 05/08/11
Posts: 572
Loc: United Kingdom
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Re: Strain Isolations on Agar, Pics and Questions [Re: weetsie]
#23460272 - 07/20/16 05:53 PM (7 years, 6 months ago) |
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I base 3-5 transfers on what I have read in mycology texts, my own personal experience, and what logically makes sense to me.
The goal when isolating is to get there in the least number of cell divisions. Obviously you're going to be taking very small transfers, a grain of rice is often mentioned, I recon you can consistently transfer a wedge thats around 2/360 degrees from the outer growth on a plate or 1/180th of the perimeter.
If you repeat on the second plate you have 1/180th of 1/180th or 1/32400th of your initial outer growth from the first plate.
By the 3rd transfer you're at 1/583200th which is where I start seeing what is as far as I'm aware, isolates.
You also have to consider that strains will merge, stronger strains assimilate weaker strains which can only speed up the isolation process. To give an example, if you put 100 cubensis isolate wedges in a grain jar then did a couple of grain to grain transfers, you would have less than 100 strains and several new strains due to isolates merging.
-------------------- Active grow logs: Oysters on Straw Pellets Trade list
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spacechildo
proletarians rise up


Registered: 01/24/13
Posts: 19,243
Loc: Babylon
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Re: Strain Isolations on Agar, Pics and Questions [Re: weetsie]
#23460312 - 07/20/16 06:06 PM (7 years, 6 months ago) |
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I dont care how small transfers you take, spores are microscopical and you've gotta understand how there's more going on than what you can see. And senescense will never be a problem when you're simply transfering on agar.
Post some pics of these isolates of yours and we'll tell you how an isolate should look like.
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Boogieman47
Let's boogie


Registered: 03/05/16
Posts: 9,712
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Re: Strain Isolations on Agar, Pics and Questions [Re: c10h12n2o]
#23460319 - 07/20/16 06:08 PM (7 years, 6 months ago) |
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Oh OK sorry I had never seen that before looks pretty cool I just get those small plastic bins like a 6qt or smaller let colonize then put them into a sgfc like this I usually get about a half ounce but I'm using MS your isolated if you get some good ones you should get way more
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c10h12n2o
serial dilutor



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Posts: 3,200
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Re: Strain Isolations on Agar, Pics and Questions [Re: spacechildo]
#23460328 - 07/20/16 06:12 PM (7 years, 6 months ago) |
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well, besides the guy above you,
here is RR saying he can do it in 3-4 by taking tiny transfers: https://www.shroomery.org/forums/showflat.php/Number/7746074#7746074
another person: https://www.shroomery.org/forums/showflat.php/Number/21847276#21847276
another thread: https://www.shroomery.org/forums/showflat.php/Number/17335289
another "I never fail to make an isolate in less then 2 transfers": https://www.shroomery.org/forums/showflat.php/Number/14917711#14917711
i know i have seen lots more too... do i really read on here that much more than you? 
not that i am saying any of that, i am just asking, specifically about the pictures, for the benefit of myself and anyone else who might be confused by the wide variety of things that can be read
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Supalemonhaze
Spore syringe hater.



Registered: 10/02/15
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Re: Strain Isolations on Agar, Pics and Questions [Re: weetsie]
#23460335 - 07/20/16 06:14 PM (7 years, 6 months ago) |
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Honestly, .
I will however try to help you just one last time.
There are 0 isolates in your current plates. 3 transfers isn't nearly enough. What you should do is grab just 1 plate with the nicest growth and keep only that for isolation. The rest use them for grain MS inoculations to get some clones in the meantime.
Now, grab that 1 plate, and transfer it at least 5-10 more times, each transfer being taken when the growth is about the size of a large-ish coin. Your transfers should be taken as small as possible and from the nicest looking growth. There is no use of us holding your hand through every transfer, just see which looks clean and fast and transfer that. A lot of experienced folks will tell you that how the mycelium looks will have no bearing on how good the culture is.
Do not take multiple transfers until you see definite sectoring. This means that you will only have 1 isolation plate on hand at a time until you do those 5-10 transfers. Once you notice that the sectors are getting quite big and nicely defined, you can separate your 20 sectors into different plates.
Hope this is enough, goodluck. If you still have a ton of questions, there is nothing that will teach you better than experience. Just jump in, try to follow these instructions to the best of your ability. Your first time will be more about learning anyway.
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Supalemonhaze
Spore syringe hater.



Registered: 10/02/15
Posts: 6,725
Loc: 12" down Europe's butthole
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Re: Strain Isolations on Agar, Pics and Questions [Re: c10h12n2o]
#23460348 - 07/20/16 06:17 PM (7 years, 6 months ago) |
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Quote:
c10h12n2o said: well, besides the guy above you,
here is RR saying he can do it in 3-4 by taking tiny transfers: https://www.shroomery.org/forums/showflat.php/Number/7746074#7746074
another person: https://www.shroomery.org/forums/showflat.php/Number/21847276#21847276
another thread: https://www.shroomery.org/forums/showflat.php/Number/17335289
another "I never fail to make an isolate in less then 2 transfers": https://www.shroomery.org/forums/showflat.php/Number/14917711#14917711
i know i have seen lots more too... do i really read on here that much more than you? 
not that i am saying any of that, i am just asking, specifically about the pictures, for the benefit of myself and anyone else who might be confused by the wide variety of things that can be read
Those are either bullshitters or really experienced cultivators which know what they are doing. You are on your first isolation project and have no idea what you are doing. There is absolutely zero chance that you can get an isolate in 3 transfers for now, that part is entirely true.
You will find out soon enough, you think you're getting close but if you keep transferring, it will keep sectoring. I have been isolating for at least 3 months, you should keep that in mind.
You would need to dilute a spore syringe a lot to be able to do it in a handful of transfers, let alone only 2. This is not something an amateur can pull off so you should really forget you ever read that.
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