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Invisiblec10h12n2o
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Strain Isolations on Agar, Pics and Questions * 1
    #23458285 - 07/20/16 01:58 AM (7 years, 8 months ago)

hi everyone, i am currently working on some strain isolations from 3 different races of cubes.

i wanted to get some feedback on a few plates that i am preparing to either transfer again or start making spawn. these are on the 3rd transfer from multispore.

Plate 20A2a:

Plate 33B1b:

Plate 33A1a:

Plate 33A1b:

Plate 33A2b:

Plate 100A1b:

Plate 100A1a:


I am wanting to know which of these look like they might be ready to go, and which need further transfers, and any other feedback you might have

warm regards

Edited by c10h12n2o (07/20/16 01:59 AM)

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OfflineMMG
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Re: Strain Isolations on Agar, Pics and Questions [Re: c10h12n2o]
    #23458291 - 07/20/16 02:02 AM (7 years, 8 months ago)

most of those look very good, great job.
I'd make some last transfers from each (isolating the most vigorous edges of growth) and then proceeding to either let the plate finish colonizing or putting the remaining wedge directly on grain. That way you have back ups and you can test out each isolate.


--------------------
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Re: Strain Isolations on Agar, Pics and Questions [Re: MMG]
    #23458564 - 07/20/16 06:14 AM (7 years, 8 months ago)

Quote:

MMG said:
most of those look very good, great job.
I'd make some last transfers from each (isolating the most vigorous edges of growth) and then proceeding to either let the plate finish colonizing or putting the remaining wedge directly on grain. That way you have back ups and you can test out each isolate.




None of those are isolates. You can still see it growing at different rates.

@OP: 3 transfers from the MS is nothing, it's easy to go through a box of 500 petri dishes during isolation. What you should do is transfer each of those at least 5 more times, or until you start seeing definite sectoring which you can spot easily. After that, start isolating. Don't start taking multiple transfers from multiple sectors early on in the game, it's just a waste of plates.

Once you start getting real close to getting isolates, you can start separating the sectors. That way you only transfer 1 or two cultures at a time until it's time to separate them.

Honestly, your main project should be cloning for now, with isolation being a side project. I have been isolating a single variety for at least 3 months now, only now do I seem to be getting close (although it always seems like you're getting close when you never did it before :lol:). I would also suggest that you only isolate a single variety at a time. If you want to get something worth the effort, you will easily need to get 20 isolates from each variety. Not every isolate will be good, some will be average, some will be crap and some will be great. RR used to recommend that you should get at least 20 isolates if you want a good chance of getting something good.

You say you are doing 3 varieties, if you get 20 isolates from each, that will be 60 cultures to grow side by side and compare. It's a lot of work in very little time. Start slow, one at a time or you will get overwhelmed.

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Re: Strain Isolations on Agar, Pics and Questions [Re: Supalemonhaze]
    #23458571 - 07/20/16 06:17 AM (7 years, 8 months ago)

Usually 10-15 transfers from MS to even see sectors start. Let alone grabbing an isolate. Maybe in 5-10 transfers if you take miniscule transfers and also on the very first plate use extremely little spore solution

Everyone gets the idea they want isolates. All the best growers grow clones and are still looking for great isolates in spare time. Great isolates are a treat you find in time. Spending all your work on just isolates and agar work is going to get old fast.

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Re: Strain Isolations on Agar, Pics and Questions [Re: bodhisatta]
    #23458577 - 07/20/16 06:20 AM (7 years, 8 months ago)

My only regret is that I didn't count the amount of transfers I made so far. If I had to guess, I already went over RR's 500 petri estimation. I fucked up by seperating the cultures early, I now learned the hard way why you should do a shitload of transfers before you start isolation.

Isolation should always be a side project, you really can't rush something like that.

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Re: Strain Isolations on Agar, Pics and Questions [Re: Supalemonhaze]
    #23458694 - 07/20/16 07:37 AM (7 years, 8 months ago)

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.


--------------------
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Invisiblec10h12n2o
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Re: Strain Isolations on Agar, Pics and Questions [Re: blindingleaf]
    #23459130 - 07/20/16 10:22 AM (7 years, 8 months ago)

i really appreciate everyones responses, very helpful. Im certainly not scared of hard work, though of course it makes sense to work smarter not harder, thanks for helping clarify some things for me. I am really enjoying working on agar, the plan is to isolate about 60 strains and fruit them out, keeping good notes on everything. A couple of questions:

1. I definitely saw some clear sectoring in the last transfer (on some). Are you sure 33A1a and 100A1a arent isolates, or close? most of the others (along with the 30 or so plates from this series that i didnt photograph) seem to be showing clear sectors now. Am i missing something?

2.  when you say i should be mainly doing clones now and isolation should be a side project, do you mean i should be making spawn from multispore and then fruiting it and cloning the fruits?

Bodhi: Much obliged my friend. So should i fruit out multispore first? what do you suggest as far as changing my plan, approach, and/or mindset?

Quote:

I fucked up by seperating the cultures early, I now learned the hard way why you should do a shitload of transfers before you start isolation.




i am a little confused about what you mean here, probably the distinction between the verbs "transfer" and "isolate". Obviously, the first MS plate is a jungle, and you transfer the leading edge of vigorous growth. Then the second plate some seem to already show sectors, but after transferring it is clear that there were multiple strains present in each sample of the sectors i thought i saw. Though some that showed sectors in the 2nd transfer look pretty consistant on the 3rd (like 33A1a and 100A1a). At this point, when i am looking for sectors and transferring them to a separate plate, is that not  isolation?

As for recipe: Stamets YMPA, so yes yeast and peptone both. any suggested revisions?

i really appreciate the feedback! :smile:

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Re: Strain Isolations on Agar, Pics and Questions [Re: c10h12n2o]
    #23459141 - 07/20/16 10:26 AM (7 years, 8 months ago)

you arent seeing sectoring, yet. you are just seeing myc growth. you will know when you see sectoring. give me a sec and ill find a pic of sectoring and an isolate.

or rather, someone here prolly has pics of their own, like leaf.


--------------------
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Re: Strain Isolations on Agar, Pics and Questions [Re: c10h12n2o]
    #23459160 - 07/20/16 10:32 AM (7 years, 8 months ago)

Yeah, 3 transfers in you will definitely be able to tell that there are multiple strains but it's still not what we call definite sectoring.

The idea behind what I did wrong is this. This is what I did:

MS inoculation>2 transfers to ensure cleanliness>started isolating(meaning started separating strains until I had 20 plates)

What I should have been doing is this:

MS inoculation> At least 10 transfers to ensure cleanliness AND to "thin the herd"(could take more than 10)>Once definite sectoring is observed and you can tell that there isn't much strains, you separate 20 sectors into different plates> transfer until isolates.

The difference between these is that with my method, I will have ended up transferring 20 plates a shitload of times, which wastes a lot more agar for nothing. The proper method is to first thin out the herd using just 1 plate and separate the sectors once you notice that you are getting close.


I can tell you with 100% certainty that after 3 transfers, you do not have an isolate. This would only be possible with very little spores(ridiculously little) and transfers that are very very very small. What you need to do is take each variety, keep only 1 plate of each and transfer that until you see definite sectoring. Then separate.

Edit:

Keep in mind that this is a proccess that takes months, that is why you should be cloning as a main project. Cloning is a fast and easy way to get good genetics, isolation takes a lot longer but the result is a very stable culture with a possibly good yield. You are correct about cloning, that is what you should be doing, isolation should be something that is being done in the background. Don't expect to be finished with isolation in less than 2 months, I have been going for at least 3.

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Re: Strain Isolations on Agar, Pics and Questions [Re: Supalemonhaze] * 1
    #23459166 - 07/20/16 10:33 AM (7 years, 8 months ago)

Here is a good link. give that a good read and look at the pics of monoculture and sectoring near the bottom of OP.


--------------------
-The wise man never stops seeking knowledge.


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Re: Strain Isolations on Agar, Pics and Questions [Re: c10h12n2o]
    #23459190 - 07/20/16 10:41 AM (7 years, 8 months ago)

Pretty much everything has been covered by others in this thread on sectors/isolating, but  if i was working with those plates i'd look at 33A2b, personally i like the look of that culture
this would be MY personal choice though great looking plates man and you could keep working with any of those plates and probably get good results. :highfive1:

edit: i'm assuming the darker spot on there isn't a contam but just undisolved/mixed agar


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

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Edited by MysticMoteToter (07/20/16 10:46 AM)

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Invisiblec10h12n2o
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Re: Strain Isolations on Agar, Pics and Questions [Re: MysticMoteToter]
    #23459365 - 07/20/16 11:38 AM (7 years, 8 months ago)

i am so grateful for the replies and input, much obliged my friends :smile:

I will finish reading the rest of stro's thread, thanks for that.

Im learning, and loving it :smile: I totally see why you said it feels like you are getting close at first haha ... :smile:

When you say i should be doing clones right now, does that mean i should make a LC from multispore and then make spawn (like i did before learning about MS vs Iso), or could i make a LC or spawn from some of the multispore plates i have left from my initial transfers?

and yes the black/dark spots are just where i cooled the scalpel in the agar, and to provide a reference point for lining up photos

Ok, so just to make sure ive been doing this right, here are some of the transfers i am about to make. Do these look about right to yall? am i understanding correctly, or is there something i should change?

33A1a-2-cuts:


33A2b-2-cuts:


100A1a-1-cuts:


100A1b-1-cuts


PS: SLH, were you suggesting i revise my agar recipe?

Edited by c10h12n2o (07/20/16 11:39 AM)

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Re: Strain Isolations on Agar, Pics and Questions [Re: c10h12n2o]
    #23459489 - 07/20/16 12:18 PM (7 years, 8 months ago)

This is a contaminated clone transfer of pe luckily I have two others doing good but I wish I could of still used a bit of this one but having so many plates is hard to keep an eye on to catch a contamination early on.. but like you jumping into agar as a noob is fun and exciting and you can get overwhelmed especially trying to do tubs and transfers and look for good cultures listen to these dudes they all have helped me out alot.. don't waste too much time on that yet it gets to be a pain after you have three + types and thirty transfers of each haha you end up throwing shit away or using them on grains and being left with buying 20 mono tubs to keep up and still have too much spawn lmao


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Re: Strain Isolations on Agar, Pics and Questions [Re: Boogieman47]
    #23459506 - 07/20/16 12:25 PM (7 years, 8 months ago)

I agree with noob, isolating 3 varieties at once is overkill and can easily get overwhelming. All that pouring and transferring is a lot of work, especially because you have to do it constantly over a number of months. I would start with one and once I have a bunch of isolates, I would start isolating another while testing my first batch of isolates.

Anyway, you seem to be confused about what a clone is. A clone is when you make a multispore grow and then take the best mushroom in the tub and put a piece of tissue on agar. This will generally give you better yields pretty fast. You should take 2-3 clones from a tub to see which one is best.

Don't ever inoculate an LC with a spore solution, it's unnecessarily risky, especially since you already have agar. If live liquid inoculant is your thing, you should try LI. LI is safer than LC and it's just as fast.

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Re: Strain Isolations on Agar, Pics and Questions [Re: Supalemonhaze]
    #23459534 - 07/20/16 12:32 PM (7 years, 8 months ago)

https://www.shroomery.org/forums/showflat.php/Number/22833471#22833471 here is a link for li.. I did spore to LC and every jar was bad that li link I used and the jars were done almost half the time the myc growth is a bit different but I might have poured a bit too much solution making it more damp


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

Edited by Boogieman47 (07/20/16 12:33 PM)

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Re: Strain Isolations on Agar, Pics and Questions [Re: c10h12n2o]
    #23459637 - 07/20/16 01:11 PM (7 years, 8 months ago)

Quote:

c10h12n2o said:
When you say i should be doing clones right now, does that mean i should make a LC from multispore and then make spawn (like i did before learning about MS vs Iso), or could i make a LC or spawn from some of the multispore plates i have left from my initial transfers?




no, cloning means taking tissue from a mushroom and grow it out. not using spores.
MS to LC is bad practice but also has nothing to do with cloning.

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Invisiblec10h12n2o
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Re: Strain Isolations on Agar, Pics and Questions [Re: Supalemonhaze]
    #23459704 - 07/20/16 01:41 PM (7 years, 8 months ago)

I definitely hear what you are saying about 3 strains being overkill and a lot of work, it certainly has been in the 200 or so plates i have done so far. If i hadnt started yet and already had several weeks and transfers into it, i would probably just pick 1 to run with (your suggested timeline makes WAAAY more sense lol), but since i already have these this far along i plan to try to finish trying to get isolates from them

I plan to fruit out at least 60 bucket/minimonos of isolated strains, and would be happy if i end up with even 1 good strain. I'm not scared of the work or patience required, I just want to do it right, or at least learn a lot in the  process :wink:

If that is the plan, and assuming those plates are on their 3-4 transfers, how do the proposed cuts look (numbered and outlined in red above)? Should I use all the cuts I numbered? Also, should i be doing 1 tissue sample per new plate yet, or should i be doing 2-3 on each new plate at this point?

I understand what a clone is, sorry if i wasnt clear. I was asking what route i should go to get a fruit to clone at this point. I currently dont have any MS grows going to clone a fruit from, but I have lots of clean MS plates, some with wedges taken out,  some further down the isolation chain than others. So do I have to all the way over waiting for spores to germinate, or can i make MS grain jars from some of the more colonized plates i already have? If so, should i use the whole dish of MS colonized agar and put it in a grain spawn jar, then g2g? Or should i take a vigorous looking wedge(s) from a colonized plate and make grain spawn with it? vigorous wedge or whole dish for LI?

I see what you mean about spore syringe to LC being unnecessarily risky. I actually did it with some success in several of my first grows (including a super soaker filled with it in a cow field lol), though it was probably dumb luck. My first 5 grows all used MS LC(LI?) from a syringe. of course i would hope to avoid anything that risky/dumb again though

I thought i had moved beyond my days of of MS when i started culturing isolates, thanks for the reality check :smile:

I will read up on the distinction between LI and LC, because i have been using the two terms interchangeably from my watergun days until now

Edited by c10h12n2o (07/20/16 01:42 PM)

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Re: Strain Isolations on Agar, Pics and Questions [Re: c10h12n2o]
    #23459723 - 07/20/16 01:50 PM (7 years, 8 months ago)

Multispore to LC is a terrible waste of spores and time

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Invisiblec10h12n2o
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Re: Strain Isolations on Agar, Pics and Questions [Re: bodhisatta]
    #23459729 - 07/20/16 01:54 PM (7 years, 8 months ago)

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.






yes 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?

Warm Regards
Quote:

Trusted Cultivator said:
Multispore to LC is a terrible waste of spores and time




that makes perfect sense to me :smile:. care to shed light on some of my questions?

Edited by c10h12n2o (07/20/16 02:27 PM)

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Re: Strain Isolations on Agar, Pics and Questions [Re: c10h12n2o]
    #23459734 - 07/20/16 01:56 PM (7 years, 8 months ago)


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Re: Strain Isolations on Agar, Pics and Questions [Re: bodhisatta]
    #23459746 - 07/20/16 02:01 PM (7 years, 8 months ago)

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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Re: Strain Isolations on Agar, Pics and Questions [Re: bodhisatta]
    #23459798 - 07/20/16 02:24 PM (7 years, 8 months ago)

.... 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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Re: Strain Isolations on Agar, Pics and Questions [Re: spacechildo]
    #23459803 - 07/20/16 02:25 PM (7 years, 8 months ago)

good idea :smile: thanks

i wouldnt say hung up, more like fascinated :laugh:

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 :smile:

thanks again to everyone for your input  :cheers:

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 :smile:

Edited by c10h12n2o (07/20/16 02:33 PM)

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Re: Strain Isolations on Agar, Pics and Questions [Re: c10h12n2o]
    #23459813 - 07/20/16 02:31 PM (7 years, 8 months ago)

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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Re: Strain Isolations on Agar, Pics and Questions [Re: bodhisatta]
    #23459882 - 07/20/16 03:04 PM (7 years, 8 months ago)

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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Re: Strain Isolations on Agar, Pics and Questions [Re: c10h12n2o]
    #23459924 - 07/20/16 03:30 PM (7 years, 8 months ago)

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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Re: Strain Isolations on Agar, Pics and Questions [Re: Boogieman47]
    #23460050 - 07/20/16 04:19 PM (7 years, 8 months ago)

:rolleyes: 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 :smile: 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 :smile: i am going to try that today with several from earlier transfers. thanks for the advice! :thumbup:

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 :wink:

Thanks Everyone!

warm regards

Edited by c10h12n2o (07/20/16 04:25 PM)

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Re: Strain Isolations on Agar, Pics and Questions [Re: mupetmower]
    #23460101 - 07/20/16 04:37 PM (7 years, 8 months ago)

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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Re: Strain Isolations on Agar, Pics and Questions [Re: spacechildo]
    #23460145 - 07/20/16 04:56 PM (7 years, 8 months ago)

10 transfers, 20 transfers?!?!

what are you lot smoking?

spores to single strain isolate, 3-5 transfers.


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Re: Strain Isolations on Agar, Pics and Questions [Re: weetsie]
    #23460161 - 07/20/16 05:04 PM (7 years, 8 months ago)

lol :justno:

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Re: Strain Isolations on Agar, Pics and Questions [Re: weetsie]
    #23460197 - 07/20/16 05:22 PM (7 years, 8 months ago)

by 20 transfers you're gonna start seeing sectoring again as your strain starts senescing :lol:


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Re: Strain Isolations on Agar, Pics and Questions [Re: spacechildo]
    #23460210 - 07/20/16 05:27 PM (7 years, 8 months ago)

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:




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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Re: Strain Isolations on Agar, Pics and Questions [Re: weetsie]
    #23460228 - 07/20/16 05:34 PM (7 years, 8 months ago)

Quote:

weetsie said:
by 20 transfers you're gonna start seeing sectoring again as your strain starts senescing :lol:




that doesnt even make sense. do you even know what senescense means dude? :lol:

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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Re: Strain Isolations on Agar, Pics and Questions [Re: c10h12n2o]
    #23460237 - 07/20/16 05:38 PM (7 years, 8 months ago)

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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Re: Strain Isolations on Agar, Pics and Questions [Re: weetsie]
    #23460272 - 07/20/16 05:53 PM (7 years, 8 months ago)

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.


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Re: Strain Isolations on Agar, Pics and Questions [Re: weetsie]
    #23460312 - 07/20/16 06:06 PM (7 years, 8 months ago)

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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Re: Strain Isolations on Agar, Pics and Questions [Re: c10h12n2o]
    #23460319 - 07/20/16 06:08 PM (7 years, 8 months ago)

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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Re: Strain Isolations on Agar, Pics and Questions [Re: spacechildo]
    #23460328 - 07/20/16 06:12 PM (7 years, 8 months ago)

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? :tongue:

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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Re: Strain Isolations on Agar, Pics and Questions [Re: weetsie]
    #23460335 - 07/20/16 06:14 PM (7 years, 8 months ago)

Honestly, :tldr:.

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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Re: Strain Isolations on Agar, Pics and Questions [Re: c10h12n2o]
    #23460348 - 07/20/16 06:17 PM (7 years, 8 months ago)

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? :tongue:

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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Re: Strain Isolations on Agar, Pics and Questions [Re: c10h12n2o]
    #23460366 - 07/20/16 06:26 PM (7 years, 8 months ago)

For clarity I doubt you would see rhizomorphs degrade into tomentose mycelium in 20 transfers, at least not with cubensis, I should of used something other than the ":lol:" emoji as I can see it's not clear. That said, 20 transfers would be 6-9 months or so of continuous growth? That seems incredibly wasteful of young cells.

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:


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Re: Strain Isolations on Agar, Pics and Questions [Re: weetsie]
    #23460425 - 07/20/16 07:29 PM (7 years, 8 months ago)

I'm pretty sure OP didn't dilute his spores, and neither did I(neither do others). It takes an awful lot of time and trasfers  to get senescene strictly on agar. 1 single G2G is probably equivalent to hundreds of agar transfers, especially since we do transfers as soon as possible. Bear in mind that you will be slanting your isolates once you get them, effectively halting senescene. Every single grow from there on out will be from that slant.

AFAIK, p values are taken only after an isolate is achieved.

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Re: Strain Isolations on Agar, Pics and Questions [Re: weetsie]
    #23460432 - 07/20/16 07:31 PM (7 years, 8 months ago)

you can quote all you want it aint doing much difference to OP :shrug: when you say cultures "start sectoring again" is the actual sectoring.
you also seem to confuse sectoring with isolates.

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Re: Strain Isolations on Agar, Pics and Questions [Re: weetsie]
    #23460437 - 07/20/16 07:32 PM (7 years, 8 months ago)

Uniform doesn't necessarily mean isolated. It can be un-uniform on a different ratio of a different type of nutrients. Uniform is what I aim for. Isolating is a hassle.


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Re: Strain Isolations on Agar, Pics and Questions [Re: Mad Season]
    #23460445 - 07/20/16 07:34 PM (7 years, 8 months ago)

Quote:

Mad Season said:
Uniform doesn't necessarily mean isolated. It can be un-uniform on a different ratio of a different type of nutrients. Uniform is what I aim for. Isolating is a hassle.




Yeah, sure is. I kinda enjoy it though. I'm kind of excited to see how uniform the flushes are.

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Re: Strain Isolations on Agar, Pics and Questions [Re: Mad Season]
    #23460462 - 07/20/16 07:38 PM (7 years, 8 months ago)

lmao SupaLemonHaze you are the bomb, somehow manage to be extremely helpful without even reading my my post :thumbup: thats quite a talent my friend , thanks again

but again, this is definitely NOT my first grow, not even my 10th grow, just my first time playing with isolates and had some specific questions, questions that might seem less stupid if they are actually read

And i never said i could do it in 3, i asked a question, he asked for links. and i dont think forgetting what i read is the way to learn anything :/ however knowing the context helps (ie, experience, technique)

i am doing my best to keep things in context and clear, for the benefit of anyone who actually wonders about these things

Quote:

weetsie said:


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:



much obliged my friend, i really appreciate the input :smile: that is what i thought, and what my notes say, and what i keep reading. although it could of course take more than that if you use a ton of spores, take huge wedges, etc. thanks for adding some context

Quote:

Mad Season said:
Uniform doesn't necessarily mean isolated. It can be un-uniform on a different ratio of a different type of nutrients. Uniform is what I aim for. Isolating is a hassle.



that makes lots of sense, thanks Mad, honored to have you in my thread :smile: a few of the first MS plates i did looked very even to begin with, even though they were obviously a jungle of strains. they became very uneven after a few days though. What do you do when you start seeing even growth a few transfers into an isolation, but when you suspect multiple strains are present?

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Re: Strain Isolations on Agar, Pics and Questions [Re: c10h12n2o]
    #23460480 - 07/20/16 07:42 PM (7 years, 8 months ago)

What he means by uneven is disorganized (I think at least). Different strains will grow at different speeds but if they are all organized (clean), they can be used as a MS inoculation.

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Re: Strain Isolations on Agar, Pics and Questions [Re: c10h12n2o]
    #23460490 - 07/20/16 07:45 PM (7 years, 8 months ago)

Play with the nutrients and ratios. Lower nutrients to make it more rizmorphic and spread out. Try it on a different media type. All are legit options. If it truly is isolated, it doesn't matter what you do, it'll always be the same growth, and rate.

And by organized I mean this:


It clearly isn't isoalted on any of those, since it's about 3 transfers in, but you can see that the combination of genetics all are growing at the same rates, the cube myc has thick and thin areas that are hard to notice, but still shows it isn't isolated. The panaeolus is almost impossible to tell if it's truly isolated due to the tomentose nature.. My best guess is it isn't isolated. Still extremely organized and ready for inoculating grains tho.


This is disorganized, and still not even something I'd trust until transfered and "isolated" a bit more.


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Re: Strain Isolations on Agar, Pics and Questions [Re: Mad Season]
    #23460578 - 07/20/16 08:11 PM (7 years, 8 months ago)

Quote:

spacechildo said:
you can quote all you want it aint doing much difference to OP :shrug: when you say cultures "start sectoring again" is the actual sectoring.
you also seem to confuse sectoring with isolates.




I don't know how else to put it.. Ive already explained when I brought up senescens it was a joke, sarcasm call it what you want. I was just trying to suggest how absurd it seems to me that it will take 20+ transfers to get isolates.

Single strain isolates take 3-5 transfers, multiple sources have been given to back that up. Even when you don't dilute spores and start with a big black dot  your first plate you can still get isolates in less than 5 transfers.

Starting with less spores is way less important than taking small transfers. Regardless you can achieve a similar effect as streaking by taking some spores on your loop and stabbing your plate multiple times, I like to go in a spiral pattern starting at the outside. I believe I have seen homokaryotic cultures using this methord with old spores.


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Re: Strain Isolations on Agar, Pics and Questions [Re: weetsie]
    #23460596 - 07/20/16 08:16 PM (7 years, 8 months ago)

are you even reading the stuff mad season is posting?

How should I know when you're joking/being sarcastic and when you're serious? :shrug: what does "start sectoring again" mean to you then? or was that another joke I didnt get?

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Re: Strain Isolations on Agar, Pics and Questions [Re: spacechildo]
    #23460669 - 07/20/16 08:36 PM (7 years, 8 months ago)

Rhizomorphic mycelium can become tomentose as the DNA becomes damaged from too many divisions. I was suggesting that by 20 transfers you would of had an isolated culture for so long that it would start to die of old age and you would start seeing genetically different sectors appearing as it fails.

The main take away for me in this thread is that I need to work on my jokes. :sad:


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Re: Strain Isolations on Agar, Pics and Questions [Re: weetsie]
    #23460755 - 07/20/16 09:01 PM (7 years, 8 months ago)

Quote:

weetsie said:
Rhizomorphic mycelium can become tomentose as the DNA becomes damaged from too many divisions.




another joke? or sarcasm? :lol:

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Re: Strain Isolations on Agar, Pics and Questions [Re: spacechildo]
    #23460776 - 07/20/16 09:06 PM (7 years, 8 months ago)

Nope.


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Re: Strain Isolations on Agar, Pics and Questions [Re: weetsie]
    #23461018 - 07/20/16 10:12 PM (7 years, 8 months ago)

Mad, thanks a bunch, that is super helpful info :smile: brilliant idea to try it on different media and compare characteristics , those are wonderful suggestions and i am going to implement them all :thumbup: i really appreciate your examples

lol i thought your joke was quite funny weetsie, and got it the first time. everything you have said makes lots of sense to me :smile:

btw all the plates pictured started from diluted and somewhat old spores (the first plates in the series). I am about to do another set of transfers using Mckenna's MYPA recipe instead of stamet's which i had been using (7g malt, 1g peptone,.5g yeast, rather than 20g malt, 1g yeast, 1g peptone), hopefully that will encourage less vertical growth and more across (will go back to just MEA if not)

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Re: Strain Isolations on Agar, Pics and Questions [Re: spacechildo]
    #23461025 - 07/20/16 10:14 PM (7 years, 8 months ago)

Quote:

spacechildo said:
Quote:

weetsie said:
Rhizomorphic mycelium can become tomentose as the DNA becomes damaged from too many divisions.




another joke? or sarcasm? :lol:



always thought nute content was what played a major role in tomentose v rhizo growth :wink:
maybe i was wrong....learn something new every day :awehigh:


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Re: Strain Isolations on Agar, Pics and Questions [Re: MysticMoteToter]
    #23461060 - 07/20/16 10:26 PM (7 years, 8 months ago)

The two arent mutually exclusive by any means, it could very well be both.

I have never heard of Rhizomorphic mycelium becoming tomentose as a result of too many divisions before, but it is certainly interesting and something i want to look into, seems plausible :smile:

and it seems relatively well accepted that nutrient concentration plays a role in the formation of rhizo vs tomentose growth (and probably in the super dense vertical growth, as in some of my plates which i suspect is a result of too much yeast+peptone+ME)

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Re: Strain Isolations on Agar, Pics and Questions [Re: c10h12n2o]
    #23461191 - 07/20/16 11:25 PM (7 years, 8 months ago)

Quote:

c10h12n2o said:
The two arent mutually exclusive by any means, it could very well be both.

I have never heard of Rhizomorphic mycelium becoming tomentose as a result of too many divisions before, but it is certainly interesting and something i want to look into, seems plausible :smile:




Quote from TMC

"When a mycelium grows old it is said to be senescing. Senescent mycelium, like any aged plant or animal, is far less vigorous and fertile than its counterpart. In general, a change from rhizomorphic to cottony looking mycelium should be a warning that strain degeneration has begun."


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Re: Strain Isolations on Agar, Pics and Questions [Re: weetsie]
    #23461205 - 07/20/16 11:31 PM (7 years, 8 months ago)

What about when it goes from cottony to rhizo? Cause most of my plates when I drop spores is tomentose


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Re: Strain Isolations on Agar, Pics and Questions [Re: weetsie]
    #23461208 - 07/20/16 11:33 PM (7 years, 8 months ago)

Quote:

weetsie said:
Quote:

c10h12n2o said:
The two arent mutually exclusive by any means, it could very well be both.

I have never heard of Rhizomorphic mycelium becoming tomentose as a result of too many divisions before, but it is certainly interesting and something i want to look into, seems plausible :smile:




Quote from TMC

"When a mycelium grows old it is said to be senescing. Senescent mycelium, like any aged plant or animal, is far less vigorous and fertile than its counterpart. In general, a change from rhizomorphic to cottony looking mycelium should be a warning that strain degeneration has begun."



weird, i've got some cubensis Redboy culture that will go tomentose on just about anything with nutrition but can't get it to go rhizo to save my life...however, it's also put out one of my most potent clones....i don't think tomentose growth is indicative of anything other than colonization speed as long as it's not wispy or weak looking :shrug:


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Re: Strain Isolations on Agar, Pics and Questions [Re: weetsie] * 1
    #23461214 - 07/20/16 11:34 PM (7 years, 8 months ago)

Senescence IME gets wispier, not necessarily more tomentose.. Tomentose is still aggressive and thick, it just doesn't need to stretch as much looking for nutrients, so it gets all thick and bunched up.


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Re: Strain Isolations on Agar, Pics and Questions [Re: MysticMoteToter]
    #23461223 - 07/20/16 11:41 PM (7 years, 8 months ago)

Quote:

MysticMoteToter said:
weird, i've got some cubensis Redboy culture that will go tomentose on just about anything with nutrition but can't get it to go rhizo to save my life...however, it's also put out one of my most potent clones....i don't think tomentose growth is indicative of anything other than colonization speed as long as it's not wispy or weak looking :shrug:




I think it's only referring to if it happens with old cultures on the same media.

Quote:

Mad Season said:
Senescence IME gets wispier, not necessarily more tomentose.. Tomentose is still aggressive and thick, it just doesn't need to stretch as much looking for nutrients, so it gets all thick and bunched up.




I always thought tomentose simply meant cottony, regardless of being strong or weak.

Quote:

noob47 said:
What about when it goes from cottony to rhizo? Cause most of my plates when I drop spores is tomentose




I think all spores start like like, never seen any different.


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Re: Strain Isolations on Agar, Pics and Questions [Re: weetsie]
    #23461447 - 07/21/16 02:22 AM (7 years, 8 months ago)

Quote:

weetsie said:
Even when you don't dilute spores and start with a big black dot  your first plate you can still get isolates in less than 5 transfers.





I'd like to see you try and pull this off and document it. Have you ever even isolated a culture? From this sentence I get the idea that you never really tried it. Even with transfers not even a mm wide, it would still take a lot of transfers to get isolates with a normal spore inoculation (without dilution).



That transfer is what my regular isolation transfer looks like. It's about 1mm wide(if that) and as long as the agar's depth. It's basically impossible for me to take transfers smaller than this. It's actually annoying sometime to pick the transfer up and deposit it on the new plate due to being so small. Still, I've been isolating this variety for no less than 3 months.

And for the sake of not confusing anyone, just because a culture goes from rhizo to cottony, it doesn't mean it's senescening. It takes an awful lot of cell divisions and expansion for a culture to senescene. No one really knows how much agar transfers it takes for a culture to grow old but it's safe to say that no one here will ever experience that through agar transfers alone.

@Mad season: So you only inoculate a grain jar with cultures that grow at the same rate? I get that the one you labelled as "disorganized" is not a good candidate for inoculation, especially the one at the bottom but I constantly get sectors that look clean and organized but grow at different speeds. Never thought of that as a problem as long as the strands look organized and flat.

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Re: Strain Isolations on Agar, Pics and Questions [Re: Supalemonhaze]
    #23461552 - 07/21/16 03:27 AM (7 years, 8 months ago)

Most of us, the vast majority, will never even experience senescence in any culture

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Re: Strain Isolations on Agar, Pics and Questions [Re: bodhisatta]
    #23461555 - 07/21/16 03:30 AM (7 years, 8 months ago)

Quote:

Trusted Cultivator said:
Most of us, the vast majority, will never even experience senescence in any culture




:whathesaid:

Most hobbyists will never even experience it, the ones who do normally do so from excess G2G's.

hey bodhi, how much transfers would you say it takes to get a culture to grow old? Close to a thousand maybe? Or not even?

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Re: Strain Isolations on Agar, Pics and Questions [Re: Supalemonhaze]
    #23461560 - 07/21/16 03:34 AM (7 years, 8 months ago)

What is that when the myc gets old and useless?


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Re: Strain Isolations on Agar, Pics and Questions [Re: Boogieman47]
    #23461567 - 07/21/16 03:38 AM (7 years, 8 months ago)

Yes. Excess G2Gs/expanded mycelium is usually the cause of it. Mycelium growing on a 2d surface(agar) is senescening at a much slower rate than mycelium growing in a 3d manner like it would in grain jars and bulk subs. This is entirely the reason why they say not to G2G a grain jar for more than 3-5 times.

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Re: Strain Isolations on Agar, Pics and Questions [Re: Supalemonhaze]
    #23461577 - 07/21/16 03:49 AM (7 years, 8 months ago)

So what if you did ten jars to 100 jars and so on the third to fifth time it will be pretty useless ? Even if you got 1000 jars ?


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Re: Strain Isolations on Agar, Pics and Questions [Re: Boogieman47]
    #23461583 - 07/21/16 03:53 AM (7 years, 8 months ago)

No, you can do g2g safely for a few times but they don't ever recommend going over 5 times. Some won't recommend going over 3. The mycelium will still be young enough to colonize the bulk and give a normal healthy flush though. It takes more G2Gs to get senescene, 3-5 times is being safe rather than sorry.

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Re: Strain Isolations on Agar, Pics and Questions [Re: Supalemonhaze]
    #23461584 - 07/21/16 03:55 AM (7 years, 8 months ago)

Right I feel you man makes since


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Re: Strain Isolations on Agar, Pics and Questions [Re: Boogieman47]
    #23461845 - 07/21/16 07:28 AM (7 years, 8 months ago)

Quote:

noob47 said:
you man makes since




:lol:

TMC is over 30 yrs old dude, gotta read it like a bible and take everything with a grain of salt.

Best way to learn how stuff works is to grow, simple as that.

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Re: Strain Isolations on Agar, Pics and Questions [Re: Supalemonhaze]
    #23462192 - 07/21/16 09:51 AM (7 years, 8 months ago)

Quote:

Supalemonhaze said:
Quote:

weetsie said:
Even when you don't dilute spores and start with a big black dot  your first plate you can still get isolates in less than 5 transfers.





I'd like to see you try and pull this off and document it.




I'll take you up on that.

Watch this space.


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Re: Strain Isolations on Agar, Pics and Questions [Re: weetsie]
    #23462228 - 07/21/16 10:02 AM (7 years, 8 months ago)

:popcorn:


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Re: Strain Isolations on Agar, Pics and Questions [Re: mupetmower]
    #23462292 - 07/21/16 10:27 AM (7 years, 8 months ago)

Quote:

mupetmower said:
:popcorn:



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Re: Strain Isolations on Agar, Pics and Questions [Re: Supalemonhaze]
    #23462433 - 07/21/16 11:25 AM (7 years, 8 months ago)

yeah lets just hope senescense doesnt make your isolates start sectoring again this time! :rofl:

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Re: Strain Isolations on Agar, Pics and Questions [Re: spacechildo]
    #23462517 - 07/21/16 12:11 PM (7 years, 8 months ago)

Pulling up a chair :popcorn:


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Re: Strain Isolations on Agar, Pics and Questions [Re: lukehighwalker710]
    #23462527 - 07/21/16 12:15 PM (7 years, 8 months ago)

If I had a dollar for every time a noob said he'd prove something wrong and even went as far as updating his thread I'd still be a poor son of a bitch!

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Re: Strain Isolations on Agar, Pics and Questions [Re: spacechildo]
    #23462536 - 07/21/16 12:20 PM (7 years, 8 months ago)

Quote:

spacechildo said:
If I had a dollar for every time a noob said he'd prove something wrong and even went as far as updating his thread I'd still be a poor son of a bitch!



:rofl: :rofl2: right? At least update us that it didn't work out don't just dissapear lmao


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Re: Strain Isolations on Agar, Pics and Questions [Re: spacechildo]
    #23462940 - 07/21/16 03:19 PM (7 years, 8 months ago)

Quote:

spacechildo said:
Quote:

noob47 said:
you man makes since




:lol:

TMC is over 30 yrs old dude, gotta read it like a bible and take everything with a grain of salt.

Best way to learn how stuff works is to grow, simple as that.




Out of curiosity, what parts of TMC do you feel are worth taking with a grain of salt?
I own the the newest revised edition as well as GGMM. Besides coir and small scale fruiting chambers, what is it missing or has wrong?

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Re: Strain Isolations on Agar, Pics and Questions [Re: Rooster Cogburn]
    #23463032 - 07/21/16 03:52 PM (7 years, 8 months ago)

oh man... where to begin? everything on lights/darkness, probably most if not all the charts, temps, overlay, basically just remember mush cult is a rather new thing and a lot of what was written in stone has now been disproved.
doubt everything, try everything, and if you got a theory try to disprove it rather than prove it, science style :lol:

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Re: Strain Isolations on Agar, Pics and Questions [Re: spacechildo]
    #23463076 - 07/21/16 04:05 PM (7 years, 8 months ago)

Quote:

spacechildo said:
Quote:

noob47 said:
you man makes since




:lol:

TMC is over 30 yrs old dude, gotta read it like a bible and take everything with a grain of salt.

Best way to learn how stuff works is to grow, simple as that.



Trust me I'm going to buy it but I have grown maybe not as successful as some of you guys on here I've never done that many g2g because I have 6 types of cubes and three clones on agar with like 75 pasty plates I don't know what you're referring to? Space


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Re: Strain Isolations on Agar, Pics and Questions [Re: Boogieman47]
    #23463094 - 07/21/16 04:10 PM (7 years, 8 months ago)

ya little quote fuck up there.. but you did make me :lol:!

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Re: Strain Isolations on Agar, Pics and Questions [Re: spacechildo]
    #23463106 - 07/21/16 04:13 PM (7 years, 8 months ago)

Haha oh shit I didn't even realize that well I was up late as Fuck that makes "since" to me now Lmao


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Re: Strain Isolations on Agar, Pics and Questions [Re: Boogieman47]
    #23463135 - 07/21/16 04:23 PM (7 years, 8 months ago)

:lolsy:

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Re: Strain Isolations on Agar, Pics and Questions [Re: spacechildo]
    #23463259 - 07/21/16 05:02 PM (7 years, 8 months ago)

Ok, I see what you mean. As far as published work go's what do you feel is the most up to date piece of work concerning mushroom cultivation besides TMC or GGMM?

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Re: Strain Isolations on Agar, Pics and Questions [Re: Rooster Cogburn] * 1
    #23463286 - 07/21/16 05:15 PM (7 years, 8 months ago)

shroomery.org :bigyesnod:

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Re: Strain Isolations on Agar, Pics and Questions [Re: Rooster Cogburn]
    #23463384 - 07/21/16 05:40 PM (7 years, 8 months ago)

Quote:

Rooster Cogburn said:
Ok, I see what you mean. As far as published work go's what do you feel is the most up to date piece of work concerning mushroom cultivation besides TMC or GGMM?



:goodnews: https://www.shroomery.org/forums/search.php this is a good way to refine what your looking for, but published work on the cultivation of actives will be very hard to come by due to drug scheduling laws haha


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Re: Strain Isolations on Agar, Pics and Questions [Re: MysticMoteToter]
    #23463469 - 07/21/16 06:05 PM (7 years, 8 months ago)

all the good regulars here arent getting published but their writeups are way way better than most "published work" we get linked here on a weekly basis :shrug:
I bet they're good lab technicians, but they dont know jack shit about mushrooms, growing MS taking notes thinking they found the holy grail/answer to 43.

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Re: Strain Isolations on Agar, Pics and Questions [Re: spacechildo]
    #23463765 - 07/21/16 07:39 PM (7 years, 8 months ago)

I barely g2g, there's other reliable methods to get things done quickly. G2G for me usually ends up with signs of bacteria so I only do it with stuff that's gonna be fruited outside, like oysters.

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Re: Strain Isolations on Agar, Pics and Questions [Re: Supalemonhaze]
    #23464300 - 07/21/16 10:36 PM (7 years, 8 months ago)

How do you think you are getting bacteria during your G2G transfers, are you using jars or bags?. Personally I like going from my master jars straight to bags, use some bags for tubs and take another bag to bunch more bags and so on about 5 generations, usually 8 lb. or 8 quart bags, I have found that to be the fastest method for me so far.
What do you prefer to do other than G2G?

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Re: Strain Isolations on Agar, Pics and Questions [Re: Rooster Cogburn]
    #23464703 - 07/22/16 01:13 AM (7 years, 8 months ago)

If I wanted anything done fast, I would go with LI.

I think the main problem with me G2Ging is that the endospores would have recovered by the time my jars are colonized. G2Ging will just give those endospores grains to grow on. I can have a very healthy looking jar, G2G it and then see definite signs of bacteria. No stalling or anything but I get grains stuck to sides/thick mycelium etc. All spawnable but the point to using agar is to get the cleanest spawn possible, G2G's basically fucks up all that hard work.

For inside grows, I stick to wedges. Speed is not an issue once you start inoculating jars with a routine, you'll always have something to spawn when you start doing that.

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Re: Strain Isolations on Agar, Pics and Questions [Re: Supalemonhaze]
    #23464752 - 07/22/16 01:45 AM (7 years, 8 months ago)

I've noticed that too when I do g2g the spawn sometimes comes back fucked up I just thought my sterile technique was sloppy


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Re: Strain Isolations on Agar, Pics and Questions [Re: Supalemonhaze]
    #23465263 - 07/22/16 08:52 AM (7 years, 8 months ago)

sterilize your master jars longer supa.

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Re: Strain Isolations on Agar, Pics and Questions [Re: spacechildo]
    #23465283 - 07/22/16 08:59 AM (7 years, 8 months ago)

I had a similar problem, but mine was because i sucked at g2g at first and would get mold in the receivers too much, so i switched to master only.

pasty turned me onto pint masters.  i cook them with qt jars, so they get sterilized a while for the amount of grain in them and they colonize pretty quick from wedge.  ill go to 2-3 qts after that.


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Re: Strain Isolations on Agar, Pics and Questions [Re: spacechildo]
    #23465382 - 07/22/16 09:29 AM (7 years, 8 months ago)

Quote:

spacechildo said:
sterilize your master jars longer supa.





I PC for a good amount, or so I think at least. I PC my jars for a total of 2hr15m. Used to do 2hrs before that but a few weeks ago I timed how long it takes for the core to get completely saturated with heat. It took 45 minutes so with 2hr15m the jars spend 1hr30m at ~121F. Isn't going over this overkill? I always thought it was but IDK really.

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Re: Strain Isolations on Agar, Pics and Questions [Re: Supalemonhaze]
    #23465432 - 07/22/16 09:42 AM (7 years, 8 months ago)

must be your sterile tech then :shrug:

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Re: Strain Isolations on Agar, Pics and Questions [Re: spacechildo]
    #23465464 - 07/22/16 09:54 AM (7 years, 8 months ago)

:lolsy:

Edit:

Unless you're serious... In which case... :elderno:  I figure you're joking though.

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Re: Strain Isolations on Agar, Pics and Questions [Re: Supalemonhaze]
    #23465482 - 07/22/16 09:59 AM (7 years, 8 months ago)

well OK you're right g2g doesnt work :lol:

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Re: Strain Isolations on Agar, Pics and Questions [Re: spacechildo]
    #23465491 - 07/22/16 10:02 AM (7 years, 8 months ago)

Oh, I guess you were serious then. Inadequate sterile tek normally results in mold contams, not bacteria. For me at least. I reckon something else is going on, endospores is the only thing I can think of. Or maybe I'm crap at spotting minute bacteria contams. The former makes more sense to me because it's unlikely for me to have invisible bacteria in all of my plates.

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Re: Strain Isolations on Agar, Pics and Questions [Re: Supalemonhaze]
    #23465593 - 07/22/16 10:39 AM (7 years, 8 months ago)

you should never stop striving for better sterile tech IMO. And I know g2g works great so it shouldn't be a problem for you :shrug:

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Re: Strain Isolations on Agar, Pics and Questions [Re: spacechildo]
    #23465650 - 07/22/16 11:11 AM (7 years, 8 months ago)

But how can you say it's the sterile tek when none of the jars are getting molds? Don't get me wrong, my sterile tek is far from bulletproof and even if it was, you're always learning. I just don't think that if I g2ged 9 recievers and I got bacteria in every single one, it's sterile tek related.

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Re: Strain Isolations on Agar, Pics and Questions [Re: Supalemonhaze]
    #23465678 - 07/22/16 11:25 AM (7 years, 8 months ago)

how did the bacteria get in there then? I think its an over-simplification to think you cant get bacteria from poor sterile tech. sure they dont sporulate but they are everywhere.

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Re: Strain Isolations on Agar, Pics and Questions [Re: spacechildo]
    #23465722 - 07/22/16 11:36 AM (7 years, 8 months ago)

Endospores? Physically contaminating 9 jars in the act of a g2g with bacteria exclusively is next to impossible IMO. I mean, if 5 jars went bacterial and 4 jars went moldy, I would bet an organ it was the sterile tek. ---Edit: Although a scenario more likely would be 9 moldy jars with signs of bacteria.-----

Also, these jars don't have the amount of bacteria that would make a jar stall. They still grew out on bulk and produced shrooms. The bacteria contam is slight, but definitely noticeable. There is only 2 explainations I can think of, endospores or invisible bacteria contam in every single plate I do. :shrug:

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Re: Strain Isolations on Agar, Pics and Questions [Re: Supalemonhaze]
    #23465752 - 07/22/16 11:43 AM (7 years, 8 months ago)

try a different grain and see if that helps, giving up on g2g is not the answer IMO!

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Re: Strain Isolations on Agar, Pics and Questions [Re: spacechildo]
    #23465758 - 07/22/16 11:45 AM (7 years, 8 months ago)

At the end of my sterile work like last week, I took a blank agar plate and walked around my room with the lid off while slowly swiping the plate through the air, then put the lid back on and set it up on a shelf. I did this just as an experiment to check the spore load in there,I have done practices on blanks cutting it in a SAB and came back clean this was just open air. so I expected to return to see some mold growth, but lo and behold just bacteria :shrug: didn't touch or cut the agar at all so I would assume (not educated on it enough to say scientifically) that this bacteria can be in the air. However, like I said, didn't get any while working in the SAB and I know your tech is much more practiced than mine. I would assume it's more than sterile technique at play here as well. I'm excited to try Li as you suggested though, because imo it'd be more efficient to turn one plate into 10 jars to start rather than one jar then 10.


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Re: Strain Isolations on Agar, Pics and Questions [Re: spacechildo]
    #23465794 - 07/22/16 11:54 AM (7 years, 8 months ago)

Yeah, bacteria can definitely be in the air but IME mold spores highly outnumber airborne bacteria in terms of quantity.

Quote:

spacechildo said:
try a different grain and see if that helps, giving up on g2g is not the answer IMO!




I still use it on oysters which is where it's needed most for me. Laundry baskets are a bitch to fill. Haven't had a kick ass enough clone or isolate so far, so as long as I'm just messing with MS and testing cultures, wedges work great for me.

I could do LI if I wanted to get shit done as fast (if not faster) than G2G but really, once you start inoculating jars every week, speed is not an issue. There are plenty of ways to skin a cat.

Edit:

You make a good point about the grain, it is definitely worth looking into if I ever find a grain that contends with oats.

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Re: Strain Isolations on Agar, Pics and Questions [Re: Supalemonhaze]
    #23466032 - 07/22/16 01:20 PM (7 years, 8 months ago)

Quote:

Supalemonhaze said:
Yeah, bacteria can definitely be in the air but IME mold spores highly outnumber airborne bacteria in terms of quantity.

Quote:

spacechildo said:
try a different grain and see if that helps, giving up on g2g is not the answer IMO!




I still use it on oysters which is where it's needed most for me. Laundry baskets are a bitch to fill. Haven't had a kick ass enough clone or isolate so far, so as long as I'm just messing with MS and testing cultures, wedges work great for me.

I could do LI if I wanted to get shit done as fast (if not faster) than G2G but really, once you start inoculating jars every week, speed is not an issue. There are plenty of ways to skin a cat.

Edit:

You make a good point about the grain, it is definitely worth looking into if I ever find a grain that contends with oats.




I'm taking it you only use first generation master jars for inoculating subs? I see what you mean about doing that every week being just as quick, but don't you also need need new slants and plates just as often that way? I dont think of G2G as speed but mycelial mileage which is way more valuable.
Haze, also a tip for G2g jars, when I first started I would get some bacteria during transfers and I couldn't figure it out, I started really going to town with Iso under the ring of the master jars before cracking them open, havn't seen bacteria in G2G bags or jars since. Especially working in front of a hood you have to get that master jar clean as fuck.

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Re: Strain Isolations on Agar, Pics and Questions [Re: Rooster Cogburn]
    #23466106 - 07/22/16 01:47 PM (7 years, 8 months ago)

Agar is cheap, I don't think of that as a problem. A single clean agar plate can make a half pint of LI (more if you wanted to) which can inoculate just as many jars as G2G. That said, I still prefer g2gs for their speed so I still use them for my oyster grows.

I don't use 2pc lids with my jars, my lids don't go down the side of the jar so I can clean it easily with an iso soaked paper towel, I still squirt iso in the threads though out of habit but this doesn't really get iso in there with the jars I use. I figure that if the paper towel is dirty, the squirt of iso is helping wash the shit away. The bacteria you were getting probably wasn't from the outside of the jar though, the grains never touch the dirty side of the lip during a g2g. If they did, or if the flow pushed contams from the outside to the inside your jars you would get as much molds as you were getting bacteria anyway.

You are right though, 1st generation is more than enough for my needs, most home growers don't even have the space to fruit 100grain jars worth of spawn.

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Re: Strain Isolations on Agar, Pics and Questions [Re: spacechildo]
    #23466109 - 07/22/16 01:48 PM (7 years, 8 months ago)

Quote:

Rooster Cogburn said:
Ok, I see what you mean. As far as published work go's what do you feel is the most up to date piece of work concerning mushroom cultivation besides TMC or GGMM?




As far as i can tell (and my post count certainly doesnt reflect how much i have read), the newest edition of TMC and GGMM are definitely the most comprehensive published work for our purposes. They are a wealth of excellent info, and a great starting point and reference material alike.

Of course there are some people who think they know more than Paul Stamets, RR, and the other brilliant minds who investigate great subjects and publish their work for our benefit, but that has a lot more to do with their own arrogance than the availability of better published works

Quote:

spacechildo said:
shroomery.org :bigyesnod:




shroomery is without a doubt the most comprehensive mycology forum on the net, and i could spend a lifetime reading and learning from the resources on here. But bear in mind, a thread or tek is a far cry from a vetted, peer-reviewed publication. random-hippie-reviewed is not the same as mycologist reviewed.

With that in mind, there is no better place to ask questions and learn current techniques (especially when RR and co are around) and learn from others' successes and failures.

Edited by c10h12n2o (07/22/16 01:51 PM)

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Re: Strain Isolations on Agar, Pics and Questions [Re: c10h12n2o]
    #23466117 - 07/22/16 01:52 PM (7 years, 8 months ago)

because some of these "random hippies" here, who make the teks, and/or test those said teks definitely havent grown way more psychedelic mushrooms than almost any "real" mycologist out there.... hope you can sense the enormous load of sarcasm.

sure, there are teks made by noobs, that dont work very well.. but there are also up-to-date, tried-and-true teks here, made by those "random hippies".


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Re: Strain Isolations on Agar, Pics and Questions [Re: c10h12n2o]
    #23466124 - 07/22/16 01:53 PM (7 years, 8 months ago)

Quote:

c10h12n2o said:
shroomery is without a doubt the most comprehensive mycology forum on the net, and i could spend a lifetime reading and learning from the resources on here. But bear in mind, a thread or tek is a way from a vetted, peer-reviewed publication. random-hippie-reviewed is not the same as mycologist reviewed.




Is this a joke? I'd love to hear more about how TMC was peer reviewed before release.. And what makes a mycologist a random-hippie once he logs on to the internet?

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Re: Strain Isolations on Agar, Pics and Questions [Re: Supalemonhaze]
    #23466144 - 07/22/16 01:58 PM (7 years, 8 months ago)

Quote:

Supalemonhaze said:
Agar is cheap, I don't think of that as a problem. A single clean agar plate can make a half pint of LI (more if you wanted to) which can inoculate just as many jars as G2G. That said, I still prefer g2gs for their speed so I still use them for my oyster grows.

I don't use 2pc lids with my jars, my lids don't go down the side of the jar so I can clean it easily with an iso soaked paper towel, I still squirt iso in the threads though out of habit but this doesn't really get iso in there with the jars I use. I figure that if the paper towel is dirty, the squirt of iso is helping wash the shit away. The bacteria you were getting probably wasn't from the outside of the jar though, the grains never touch the dirty side of the lip during a g2g. If they did, or if the flow pushed contams from the outside to the inside your jars you would get as much molds as you were getting bacteria anyway.

You are right though, 1st generation is more than enough for my needs, most home growers don't even have the space to fruit 100grain jars worth of spawn.



Incubating space is crazy with jars as well, main reason I started using spawn bags too. I grow gourmet mushrooms too, that's where I have had most of my experience in large scale cultivation. What kind of oysters are you growing haze?

Edited by Rooster Cogburn (07/22/16 02:00 PM)

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Re: Strain Isolations on Agar, Pics and Questions [Re: Rooster Cogburn]
    #23466195 - 07/22/16 02:13 PM (7 years, 8 months ago)

Regular oysters I cloned from the store. I also have pioppino which I grew once but fucked it up. The pioppinos are fucking beautiful when grown in high FAE. I don't have any edibles going atm, I made an oyster laundry basket ~3 weeks ago and had to trash it in less than a week. The tub that it was colonizing in was easily 90F, it was probably more at the core seeing as the laundry basket was filled with about a foot of straw. I had to remove the moldy straw by hand and it was literally warm as piss. It was uncomfortable to touch to be honest.

I'm waiting for summer to pass before I gear up my edible grows again. I really want to give this pioppino culture a good chance this time around.

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Re: Strain Isolations on Agar, Pics and Questions [Re: spacechildo]
    #23466223 - 07/22/16 02:20 PM (7 years, 8 months ago)

Quote:

spacechildo said:
Quote:

c10h12n2o said:
shroomery is without a doubt the most comprehensive mycology forum on the net, and i could spend a lifetime reading and learning from the resources on here. But bear in mind, a thread or tek is a way from a vetted, peer-reviewed publication. random-hippie-reviewed is not the same as mycologist reviewed.




Is this a joke? I'd love to hear more about how TMC was peer reviewed before release.. And what makes a mycologist a random-hippie once he logs on to the internet?



As far as gourmet mushroom cultivation goes Stamets book GGMM is probably the best, the only thing it lacks is specific techniques for cloning, culturing and making slants in which it refers you to TMC for those things, and bear in mind TMC is still used as a text book in mycology classes still, you can rent it as a text book from Amazon, that doesn't really mean much though, I agree it's way too outdated to be used as a text book, but as far as techniques go all of the ones we still use are based off of TMC and GGMM, cloning, agar work, and spawn production, and then for gourmet mushrooms GGMM is golden for sterilized sawdust, straw, and compost, but is definitely not useful for cubes.
Something I found interesting is that on the Aloha Culture Bank website they claim they use RR's videos for training their lab technicians.

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Re: Strain Isolations on Agar, Pics and Questions [Re: Supalemonhaze]
    #23466247 - 07/22/16 02:24 PM (7 years, 8 months ago)

Quote:

Supalemonhaze said:
Regular oysters I cloned from the store. I also have pioppino which I grew once but fucked it up. The pioppinos are fucking beautiful when grown in high FAE. I don't have any edibles going atm, I made an oyster laundry basket ~3 weeks ago and had to trash it in less than a week. The tub that it was colonizing in was easily 90F, it was probably more at the core seeing as the laundry basket was filled with about a foot of straw. I had to remove the moldy straw by hand and it was literally warm as piss. It was uncomfortable to touch to be honest.

I'm waiting for summer to pass before I gear up my edible grows again. I really want to give this pioppino culture a good chance this time around.




Summer fucking sucks, I run Amycel 3014 in the summer, it's a brown oyster that favors warmer temps, but I like cold weather strains better, and just cold weather mushrooms in general. Never done piopino but really want to, I hear they are delicious.
Never done the laundry basket, I just do sawdust because I never got a straw rig together.
BTW sorry, I dont want to jack this thread, Im new here and I don't want to break the rules.

Edited by Rooster Cogburn (07/22/16 02:26 PM)

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Re: Strain Isolations on Agar, Pics and Questions [Re: mupetmower]
    #23466304 - 07/22/16 02:39 PM (7 years, 8 months ago)


Quote:

mupetmower said:
because some of these "random hippies" here, who make the teks, and/or test those said teks definitely havent grown way more psychedelic mushrooms than almost any "real" mycologist out there.... hope you can sense the enormous load of sarcasm.

sure, there are teks made by noobs, that dont work very well.. but there are also up-to-date, tried-and-true teks here, made by those "random hippies".



I agree the info on here is priceless, but all of the same tried and true "teks" are being used by the commercial spawn and mushroom industry right now by real mycologists growing millions of pounds a year in a multi million dollar industry, TMC was originally written to reveal the trade secrets that commercial cultivators held close for so long.
Stamets has grown a shit ton of cubensis, more than we will ever know, approved and funded by grants none the less.

Edited by Rooster Cogburn (07/22/16 02:39 PM)

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Re: Strain Isolations on Agar, Pics and Questions [Re: Rooster Cogburn]
    #23466422 - 07/22/16 03:22 PM (7 years, 8 months ago)

there's absolutely nothing in TMC or GGMM that isnt covered on shroomery.org
and in addition we add info and noticable tendences here on a daily basis, no book can do that.
thats why shroomery > everything else IMO!

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Re: Strain Isolations on Agar, Pics and Questions [Re: spacechildo]
    #23466815 - 07/22/16 05:26 PM (7 years, 8 months ago)

I have more respect for pasty, cron, muda for example than I do of Paul when it comes to psychedelic mushroom cultivation. Specifically cultivation. Maybe Paul can out biology talk us but he certainly can't out grow us when it comes to cubes. He would be reading the new good teks too

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Re: Strain Isolations on Agar, Pics and Questions [Re: bodhisatta] * 1
    #23466964 - 07/22/16 06:08 PM (7 years, 8 months ago)

Quote:

mupetmower said:
because some of these "random hippies" here, who make the teks, and/or test those said teks definitely havent grown way more psychedelic mushrooms than almost any "real" mycologist out there.... hope you can sense the enormous load of sarcasm.

sure, there are teks made by noobs, that dont work very well.. but there are also up-to-date, tried-and-true teks here, made by those "random hippies".




that is exactly why i said "shroomery is without a doubt the most comprehensive mycology forum on the net, and i could spend a lifetime reading and learning from the resources on here." Nothing compares. my point was that anyone can post to the shroomery, whereas not just anyone is Paul Stamets or RR. this is a point about humility and perspective, not saying anything negative about hippies or their randomness

Quote:

spacechildo said:
Quote:

c10h12n2o said:
shroomery is without a doubt the most comprehensive mycology forum on the net, and i could spend a lifetime reading and learning from the resources on here. But bear in mind, a thread or tek is a way from a vetted, peer-reviewed publication. random-hippie-reviewed is not the same as mycologist reviewed.




Is this a joke? I'd love to hear more about how TMC was peer reviewed before release.. And what makes a mycologist a random-hippie once he logs on to the internet?




im starting to think you dont know what a joke is because you ask people that a lot lol :smile:

TMC has absolutely been reviewed, revised, expounded, criticized, republished, etc, all in an academic seting, all of which constitute peer review. i dont know where you got "before release" from but it wasnt me. and mycology is an actual academic field of study, with phds and gov grants who spend their lives researching and publishing peer reviewed studies on mycology.

just because someone has a shroomery account and knows how to grow cubensis doesnt make them a mycologist on the same level with people like Stamets and countless other academic researchers who devote their lives to the topic, thats just silly/small-minded/arrogant

you are really trying hard to find an argument here when there isnt one, at least not without putting words in someones mouth or misinterpreting what they said

Quote:

Trusted Cultivator said:
I have more respect for pasty, cron, muda for example than I do of Paul when it comes to psychedelic mushroom cultivation. Specifically cultivation. Maybe Paul can out biology talk us but he certainly can't out grow us when it comes to cubes. He would be reading the new good teks too




lol im sure Stamets does read on here, he probably takes advantage of every resource at his disposal to learn about mycology, he is smart like that :smile: and i am sure all the people you listed have immense respect for him.

To argue over one personality vs another really misses the point. So does comparing a 30 yr old groundbreaking book that revealed trade secrets to the masses to a modern forum where people discuss and share ideas and modern techniques (plus a whole lot of bs).

The point is that the principles of mycology that stamets and other academics study are quite relevant to what we discuss here on the shroomery: we could all learn a lot from stamets and other academics. Of course if you try to apply his wood techs to cubensis it wont work well but that isnt because he is old, its because you didnt read carefully enough to catch where he explains that not all species like the same substrates (ie wood-lovers vs manure-lovers)

again, i really dont think there is an argument to be had here, at least not without twisting, misinterpreting, or just not reading


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Edited by c10h12n2o (07/22/16 06:12 PM)

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Re: Strain Isolations on Agar, Pics and Questions [Re: c10h12n2o]
    #23466977 - 07/22/16 06:14 PM (7 years, 8 months ago)

Quote:

c10h12n2o said:
just because someone has a shroomery account and knows how to grow cubensis doesnt make them a mycologist on the same level with people like Stamets and countless other academic researchers who devote their lives to the topic, thats just silly/small-minded/arrogant




No, I'm saying that said mycologists have shroomery accounts. Why do they suddenly become a "random hippie" when they log on?
And you must know how few studies are actually done on cubensis or other actives, most stuff we get taught about stuff like vert or mycogone comes from universities that publish stuff on agaricus production.

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Re: Strain Isolations on Agar, Pics and Questions [Re: bodhisatta]
    #23466982 - 07/22/16 06:15 PM (7 years, 8 months ago)

Have you ever seen his grow rooms? They are fucking nuts.

What "teks" do you think he would be looking into or unaware of?

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Re: Strain Isolations on Agar, Pics and Questions [Re: Rooster Cogburn]
    #23466992 - 07/22/16 06:17 PM (7 years, 8 months ago)

just stick around and you'll both see what we're talking about :super:

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Re: Strain Isolations on Agar, Pics and Questions [Re: spacechildo]
    #23467006 - 07/22/16 06:21 PM (7 years, 8 months ago)

Quote:

spacechildo said:
Quote:

c10h12n2o said:
just because someone has a shroomery account and knows how to grow cubensis doesnt make them a mycologist on the same level with people like Stamets and countless other academic researchers who devote their lives to the topic, thats just silly/small-minded/arrogant




No, I'm saying that said mycologists have shroomery accounts. Why do they suddenly become a "random hippie" when they log on?
And you must know how few studies are actually done on cubensis or other actives, most stuff we get taught about stuff like vert or mycogone comes from universities that publish stuff on agaricus production.




Stamets is one of those few people that had been approved and funded to study cubensis.
And I agree Agiricus cultivation is where we have learned so much, If paul wanted to farm cubensis it would look just like his Agiricus cultivation and with his own compost that he spent a good portion of TMC teaching about.

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Re: Strain Isolations on Agar, Pics and Questions [Re: Rooster Cogburn]
    #23467751 - 07/22/16 10:37 PM (7 years, 8 months ago)

TMC is shit compared to the shroomery. There's no arguing that. Notice all the regulars agree it's shit compared to the shroomery. That isn't a coincidence. Shroomery is reviewed by all the members and has an amazing active knowledge base. Something i know stamets would be jealous af to have. When it comes to the cultivation of actives, nobody does it better then this community of OCD fucks.


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Re: Strain Isolations on Agar, Pics and Questions [Re: Mad Season]
    #23467882 - 07/22/16 11:19 PM (7 years, 8 months ago)

Quote:

Mad Season said:
nobody does it better then this community of OCD fucks.




:ilold:


TMC was certainly to go-to book in the past for anyone wanting to cultivate anything but Paul definitely stopped using some of the stuff that's in there, just like we did. As space said, there is some outdated info in there, which stamets himself is said to have taken back since then during his seminars. I mean, you have to remember that this book was originally released in the 80's. Anything that old is bound to have some outdated stuff, especially with a practice that hasn't been around that long.

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InvisiblebodhisattaMDiscordReddit
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Re: Strain Isolations on Agar, Pics and Questions [Re: Rooster Cogburn]
    #23468082 - 07/23/16 12:39 AM (7 years, 8 months ago)

Quote:

Rooster Cogburn said:
Have you ever seen his grow rooms? They are fucking nuts.

What "teks" do you think he would be looking into or unaware of?



He grows edibles... Not cubes
You could be a edible mushroom farmer your whole life and still fuck up a  cube grow your first time, he hasn't grown cubes in decades. Back when TMC was wrote PF cakes and aquariums with drip shields were high tech.


Staments is a great researcher, great grower, but a miserable scientist. He touts mushrooms as a legitimate cure for cancer, and has never cured a single person

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Re: Strain Isolations on Agar, Pics and Questions [Re: bodhisatta]
    #23468109 - 07/23/16 12:55 AM (7 years, 8 months ago)

That's because he has something to gain by selling medicinal capsules to the public. :lol:

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Re: Strain Isolations on Agar, Pics and Questions [Re: Supalemonhaze]
    #23468242 - 07/23/16 02:18 AM (7 years, 8 months ago)

I bet the entire north west was high as kites the entire time Paul was in school. How do you think he paid for that prestigious schooling:wink:
I'm joking, I see what you mean though, as far as gourmet mushroom cultivation is concerned his book GGMM is still golden and relevant for anyone diving into semi commercial or commercial cultivation.

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Re: Strain Isolations on Agar, Pics and Questions [Re: Rooster Cogburn]
    #23468297 - 07/23/16 03:47 AM (7 years, 8 months ago)

Scraped up at least half a square inch of spores and dumped them in a pile on a plate.



Feeling confident :dancing:





EDIT: for anyone that's intrested...


Quote:

weetsie said:
Quote:

Supalemonhaze said:
Quote:

weetsie said:
Even when you don't dilute spores and start with a big black dot for your first plate you can still get isolates in less than 5 transfers.





I'd like to see you try and pull this off and document it.




I'll take you up on that.

Watch this space.



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Re: Strain Isolations on Agar, Pics and Questions [Re: weetsie]
    #23468411 - 07/23/16 06:48 AM (7 years, 8 months ago)

This ought to be interesting. This deserves a new thread. :thumbup:

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Re: Strain Isolations on Agar, Pics and Questions [Re: Supalemonhaze]
    #23468817 - 07/23/16 10:18 AM (7 years, 8 months ago)

Most people get something looks like the PEU plate on the right

Oh man I got an isolate.

Nope keep going more than a few more transfers

You get plates to look like the two on the right(LOI)


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Re: Strain Isolations on Agar, Pics and Questions [Re: bodhisatta]
    #23468829 - 07/23/16 10:22 AM (7 years, 8 months ago)

Wispy cultures are annoying to isolate, I was going with PE my first time. In the end I decided to go with a more rhizomorphic culture.

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Re: Strain Isolations on Agar, Pics and Questions [Re: Supalemonhaze]
    #23468846 - 07/23/16 10:26 AM (7 years, 8 months ago)

Exactly why I picked EQ, very rhizo every time I have grown it.


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Re: Strain Isolations on Agar, Pics and Questions [Re: Mad Season]
    #23468943 - 07/23/16 11:00 AM (7 years, 8 months ago)

Quote:

Mad Season said:
TMC is shit compared to the shroomery. There's no arguing that. Notice all the regulars agree it's shit compared to the shroomery. That isn't a coincidence. Shroomery is reviewed by all the members and has an amazing active knowledge base. Something i know stamets would be jealous af to have. When it comes to the cultivation of actives, nobody does it better then this community of OCD fucks.




I think we are saying the same thing. a 30 yr old book and a modern forum are two VERY different contexts. To say that one is better than the other is ignoring that context. apples and oranges, reishi and cubensis. only by completely missing the point and misconstruing my post (or not reading it) could someone think i said TMC > Shroomery. context is everything, i really dont think there is an argument here

anyone who has read and has the humility and perspective to appreciate the principles covered in TMC will obviously recognize the tremendous value of a public forum that allows people to discuss those principles (and new ones)


BTW thanks to everyone who has taken the time to contribute their knowledge and experience to this thread, especially regarding the questions. and big thanks to everyone who has posted pics and examples (and even experiments!! :grin: )for the benefit of others who might have the same questions. i very much appreciate it, and im sure countless others will as well (even if they dont post)


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

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Re: Strain Isolations on Agar, Pics and Questions [Re: c10h12n2o]
    #23471909 - 07/24/16 10:32 AM (7 years, 8 months ago)

I didn't mean it directly at you. I meant it in general. Though to say these random hippies who have spent their time on 1 species don't know more than a mycologist who has a generalization of all species is just a perspective. Every other week I'm correcting a person who works in a mycology lab or someone's mycology friend. Every species is different, but on this forum there's lots of people of all different types who collectively know more than any mycologist.

Anyways..

@supale might be a bit late but this is a jar of grains literally covered in bacteria:



I know because it's been sitting on my cupboard thing with the rest of my contaminated shit without being PCd for 2 months now. Also the bacteria kept out all other competitors even tho it was prepared in open air, soaked for 12 hours and never saw any heat.

Grains are almost impossible to tell if they have bacteria until the very end when the mycelium shows you what's happening, and even then it's kinda hard to tell. There's over 200,000 bacterium on every mm of our skin. Especially our hands. Grain masters MUST be clean. Both inside and out. Bacteria also travels on dust particles too. Also yes there is endospores to think about too. Though if you colonize quickly, they won't be a problem.


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No trees were killed in the sending of this message. However, a large number of electrons were terribly inconvenienced.

Edited by Mad Season (07/24/16 11:11 AM)

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Re: Strain Isolations on Agar, Pics and Questions [Re: Mad Season]
    #23483958 - 07/27/16 09:40 PM (7 years, 7 months ago)

makes perfect sense, thanks mad. exactly, thats what makes this place such an awesome resource for both amateur and professional mycologists alike :smile:

btw

UPDATE:

I have finished a few more series of transfers and the cultures are starting to look MUCH more organized than the earlier plates. Here are a few shots of a few of the ones that look interesting:

20 X (Orissa):

20 Y (Orissa):

20 Z (Orissa):

100 X (Ecuador):

100 Y (Ecuador):

100 Z (Ecuador):


the pics dont look as good as they do irl, but its amazing how much different they are starting to look from the earlier plates!! I have a lot of others going as well, and am one transfer past this on some AA+ and Orissa.

I cut back the yeast and peptone to McKenna's recipe instead of stamets for these pictured (red agar), and in the current series i am only using half strength MEA (purple agar;, will post pics after further colonization

Any feedback on these? Much obliged my friends :grin:

warm regards

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Re: Strain Isolations on Agar, Pics and Questions [Re: c10h12n2o]
    #23538160 - 08/13/16 02:33 PM (7 years, 7 months ago)



Took 2 transfers from the first plate today. :dancer:


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Re: Strain Isolations on Agar, Pics and Questions [Re: weetsie]
    #23563299 - 08/21/16 03:44 PM (7 years, 7 months ago)

hello friends, just wanted to post an update to this project

I have several hundred plates going now, going through about 3 sleeves of transfers per day. I am working with lots more than what is posted here, including cordyceps m., p. micro. (plastic digesting), and a bunch of different edibles and medicinals. I absolutely love playing with agar :smile: so much faster than gardening

Anyways, here are a few shots of the underside of plates that seem to show the exact same growth on their last 3 transfers (same rate, same shape, same basic characteristics, in the last three series of transfers), so i figure they must be getting close. any feedback is much appreciated, im definitely still getting my feet wet with isolations




a couple of these have been showing the same growth since the 4th and 5th transfer, so they became isolate candidates and i kept transferring them to see if they showed any new sectoring, so maybe i got lucky! though i suppose it would be more lucky if it was more aggressive. I have some really aggressive rhizo cultures in the works right now, i cant wait to see how they look when i get them isolated :smile: i will post the better looking cultures as soon as i have some not showing clear sectoring after 2-3 transfers, should have something sexy soon :smile:

though i gotta say, i have been amazed at how often i will have a beautiful rhizo sector that i am confident will be close to an isolate, or at least rhizo on the next plate, only to find that there was indeed a jungle of strains on top of each other, even when transferring a single rhizo tip smaller than a mm. it is also amazing how myc can go from rhizo to linear to tomentose and back again

i really appreciate all the good info guys!! thanks a bunch


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

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


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"Convictions are more dangerous enemies of truth than lies"
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Re: Strain Isolations on Agar, Pics and Questions [Re: c10h12n2o]
    #23563321 - 08/21/16 03:54 PM (7 years, 7 months ago)

nice plates my dude! where did you get the microspora??

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Re: Strain Isolations on Agar, Pics and Questions [Re: MysticMoteToter]
    #23563365 - 08/21/16 04:20 PM (7 years, 7 months ago)

thanks buddy! i cant wait to post the sexy ones rhizo ones, they just keep sectoring!! will be beautiful though eventually :smile:

I was trading a bunch of cultures getting ready to get back into the hobby, and a few weeks after our trade, a guy sent me p. micro. and cord. m. as a thankyou! i thought it was a pretty fair trade to begin with, but gotta love folks like that :smile: i sent him the rest of what i had as a dbl thankyou :smile: i think he really dug the AA+

lol now to try to make this sumbeech salt water tolerant and turn some of those plastic islands in the pacific int islands of mushrooms :laugh: (obviously kidding, mushies hate salt, but a nice thought :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"
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Re: Strain Isolations on Agar, Pics and Questions [Re: c10h12n2o]
    #23563394 - 08/21/16 04:28 PM (7 years, 7 months ago)

Quote:

c10h12n2o said:
lol now to try to make this sumbeech salt water tolerant and turn some of those plastic islands in the pacific int islands of mushrooms :laugh: (obviously kidding, mushies hate salt, but a nice thought :laugh: )



It may be possible through genetic modification, like if you took goldenrod or some other salt tolerating species and put that into p. microsp.

That'd be amazing


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Re: Strain Isolations on Agar, Pics and Questions [Re: c10h12n2o]
    #23563587 - 08/21/16 05:33 PM (7 years, 7 months ago)

Where'd you get the zip bags for your petri's?  That's a good idea.  Tedious with all the plates I'm getting into but it would make them easily examinable.  I have 100 mm dishes now but I ordered 100 60 mm dishes, 10 per sleeve and I can easily fit them in my glovebox.


--------------------
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Re: Strain Isolations on Agar, Pics and Questions [Re: Kenetic]
    #23563616 - 08/21/16 05:44 PM (7 years, 7 months ago)

certainly makes for a fun species to experiment with, my mote toting friend :smile: and an inspiring thought :smile:

Quote:

kenetic said:
Where'd you get the zip bags for your petri's?  That's a good idea.  Tedious with all the plates I'm getting into but it would make them easily examinable.  I have 100 mm dishes now but I ordered 100 60 mm dishes, 10 per sleeve and I can easily fit them in my glovebox.




I had them left over from another project, i got them from Uline (best prices on any kind of bag or containers) and since some perfectly fit the petris i put them to use :smile:

definitely got tedious though. i eventually got a big roll of parafilm from my lab supply place and have been loving it! i got the 4" kind and cut a 1" strip that easily fits around the edge, works beautifully, like they were made for each other :smile: i still follow up by putting them in sandwich bags just for an extra layer of protection while i ogle them

nice! i love those little petris, but just dont forget one in the incubator, itll be solid white in no time lol

i actually just finished building a big stealth SAB so that i could do g2g and 3 sleeves (60) plates at a time :smile: working great so far!! here is a link


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

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: bodhisatta]
    #23571003 - 08/23/16 10:22 PM (7 years, 7 months ago)

Quote:

Trusted Cultivator said:
Staments is a great researcher, great grower, but a miserable scientist. He touts mushrooms as a legitimate cure for cancer, and has never cured a single person




I have heard him say in one of his seminars that his mother's cancer was cured with the help of mushroom capsules. Now, that's just what he said, but I don't think I would think he would lie about something like that. I have a lot of respect for Stamets and the great ways he uses mycology to help the world.
I wouldn't really doubt that mushrooms helped cure his mother and I wouldn't doubt that they could help others. I've heard from RR about the powerful anti-cancer properties of certain mushrooms and believe that, well idk, it could be a thing.


--------------------
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Re: Strain Isolations on Agar, Pics and Questions [Re: TedTheHighlighter]
    #23572436 - 08/24/16 10:31 AM (7 years, 7 months ago)

Quote:

TedTheHighlighter said:
Quote:

Trusted Cultivator said:
Staments is a great researcher, great grower, but a miserable scientist. He touts mushrooms as a legitimate cure for cancer, and has never cured a single person




I have heard him say in one of his seminars that his mother's cancer was cured with the help of mushroom capsules. Now, that's just what he said, but I don't think I would think he would lie about something like that. I have a lot of respect for Stamets and the great ways he uses mycology to help the world.
I wouldn't really doubt that mushrooms helped cure his mother and I wouldn't doubt that they could help others. I've heard from RR about the powerful anti-cancer properties of certain mushrooms and believe that, well idk, it could be a thing.




yeah, i COMPLETELY disagree with bodhi on that account. I think nothing short of arrogance and naivety could lead someone who probably doesnt have a single academic paper published to look down on one of the hardest working, most innovative researchers in the field of mycology, with dozens of studies to his name

he absolutely does not "tout mushrooms as a legitimate cure for cancer" , he researches the potential mycological compounds might have in treating various types of cancer. And he has good reason for doing so, there has been some really amazing research lately. the fact that someone is smart enough to recognize the potential for these compounds is flat out incompatible with an oversimplification like that. stamets is brilliant, humble, and dedicated and we are lucky to have him

a few of his studies to put this nonsense in context:
We're sitting on a mould mine.
Stamets, Paul
Source:
New Scientist. 2/13/2016, Vol. 229 Issue 3060, p28-29. 2p. 3 Color Photographs.

Truly Magical MUSHROOMS.
Authors:
Baker, Linda
Source:
Utne. Mar/Apr2003, Issue 116, p78. 5p. 3 Color Photographs.

MUSHROOM MANIFESTO. (cover story)
Authors:
MILLER, KENNETH
Source:
Discover. Jul/Aug2013, Vol. 34 Issue 6, p38-47. 10p.

Mushrooms Proposed for Gulf Spill Cleanup.
Source:
Oil Spill Intelligence Report. 11/11/2010, Vol. 33 Issue 47, p4-4. 1/3p.

Mushrooms: Ancient Allies for Modern Medicine.
Authors:
Stamets, Paul
Source:
Share Guide. Nov/Dec2010, Issue 112, p20-29. 3p.

Earth's Natural Internet.
Authors:
Stamets, Paul
Source:
Whole Earth. Fall99, Issue 98, p74. 4p. 5 Black and White Photographs.

Novel Antimicrobials from mushrooms.
Authors:
Stamets, Paul
Source:
HerbalGram. Spring2002, Issue 54, p28-33. 6p.

Crude Extracts of Marine-derived and Soil Fungi of the Genus Neosartorya Exhibit Selective Anticancer Activity by Inducing Cell Death in Colon, Breast and Skin Cancer Cell Lines.
Authors:
Ramos, Alice Abreu1
Castro-Carvalho, Bruno1,2
Prata-Sena, Maria1,2
Dethoup, Tida3
Buttachon, Suradet1,4
Kijjoa, Anake1,4
Rocha, Eduardo1,2 erocha@icbas.up.pt
Source:
Pharmacognosy Research. Jan-Mar2016, Vol. 8 Issue 1, p8-15. 8p.

Potential of four marine-derived fungi extracts as anti-proliferative and cell death-inducing agents in seven human cancer cell lines.
Authors:
Ramos AA; Interdisciplinary Center for Marine and Environmental Research (CIIMAR), CIMAR Associate Laboratory (CIMAR LA), University of Porto (U. Porto), Rua dos Bragas, nº 289, 4050-123 Porto, Portugal.
Prata-Sena M; Interdisciplinary Center for Marine and Environmental Research (CIIMAR), CIMAR Associate Laboratory (CIMAR LA), University of Porto (U. Porto), Rua dos Bragas, nº 289, 4050-123 Porto, Portugal; ICBAS - Institute of Biomedical Sciences Abel Salazar, University of Porto (U. Porto), Rua de Jorge Viterbo Ferreira, nº 228, 4050-313 Porto, Portugal.
Castro-Carvalho B; Interdisciplinary Center for Marine and Environmental Research (CIIMAR), CIMAR Associate Laboratory (CIMAR LA), University of Porto (U. Porto), Rua dos Bragas, nº 289, 4050-123 Porto, Portugal; ICBAS - Institute of Biomedical Sciences Abel Salazar, University of Porto (U. Porto), Rua de Jorge Viterbo Ferreira, nº 228, 4050-313 Porto, Portugal.
Dethoup T; Department of Plant Pathology, Faculty of Agriculture, Kasetsart University, Bangkok, Thailand.
Buttachon S; Interdisciplinary Center for Marine and Environmental Research (CIIMAR), CIMAR Associate Laboratory (CIMAR LA), University of Porto (U. Porto), Rua dos Bragas, nº 289, 4050-123 Porto, Portugal; ICBAS - Institute of Biomedical Sciences Abel Salazar, University of Porto (U. Porto), Rua de Jorge Viterbo Ferreira, nº 228, 4050-313 Porto, Portugal.
Kijjoa A; Interdisciplinary Center for Marine and Environmental Research (CIIMAR), CIMAR Associate Laboratory (CIMAR LA), University of Porto (U. Porto), Rua dos Bragas, nº 289, 4050-123 Porto, Portugal; ICBAS - Institute of Biomedical Sciences Abel Salazar, University of Porto (U. Porto), Rua de Jorge Viterbo Ferreira, nº 228, 4050-313 Porto, Portugal.
Rocha E; Interdisciplinary Center for Marine and Environmental Research (CIIMAR), CIMAR Associate Laboratory (CIMAR LA), University of Porto (U. Porto), Rua dos Bragas, nº 289, 4050-123 Porto, Portugal; ICBAS - Institute of Biomedical Sciences Abel Salazar, University of Porto (U. Porto), Rua de Jorge Viterbo Ferreira, nº 228, 4050-313 Porto, Portugal. Electronic address: erocha@icbas.up.pt.
Source:
Asian Pacific Journal Of Tropical Medicine [Asian Pac J Trop Med] 2015 Oct; Vol. 8 (10), pp. 798-806. Date of Electronic Publication: 2015 Sep 25.

Sloth hair as a novel source of fungi with potent anti-parasitic, anti-cancer and anti-bacterial bioactivity.
Authors:
Higginbotham S; Smithsonian Tropical Research Institute, Panama, Republic of Panama.
Wong WR; Department of Chemistry and Biochemistry, University of California Santa Cruz, Santa Cruz, California, United States of America.
Linington RG; Department of Chemistry and Biochemistry, University of California Santa Cruz, Santa Cruz, California, United States of America.
Spadafora C; Instituto de Investigaciones Científicas y Servicios de Alta Tecnología, Panama, Republic of Panama.
Iturrado L; Smithsonian Tropical Research Institute, Panama, Republic of Panama.
Arnold AE; School of Plant Sciences, University of Arizona, Tucson, Arizona, United States of America.
Source:
Plos One [PLoS One] 2014 Jan 15; Vol. 9 (1), pp. e84549. Date of Electronic Publication: 20140115 (Print Publication: 2014).

Aurovertin-Type Polyketides from Calcarisporium arbuscula with Potent Cytotoxic Activities against Triple-Negative Breast Cancer.
Authors:
Zhao, Hong1
Wu, Rui2
Ma, Lie-Feng2
Wo, Li-Ke1
Hu, Yuan-Yuan1
Chen, Chao1
Zhan, Zha-Jun2,3
Source:
Helvetica Chimica Acta. Jul2016, Vol. 99 Issue 7, p543-546. 4p.

Taxol produced from endophytic fungi induces apoptosis in human breast, cervical and ovarian cancer cells.
Authors:
Wang X; Key Laboratory of Microbiology, School of Life Science, Heilongjiang University, Harbin, China E-mail : zybin395@126.com.
Wang C
Sun YT
Sun CZ
Zhang Y
Wang XH
Zhao K
Source:
Asian Pacific Journal Of Cancer Prevention: APJCP [Asian Pac J Cancer Prev] 2015; Vol. 16 (1), pp. 125-31.

Cytotoxicity Induced by Extracts of Pisolithus tinctorius Spores on Human Cancer and Normal Cell Lines—Evaluation of the Anticancer Potential.
Authors:
Alves, Ricardo1
Preto, Marco2
Vasconcelos, Vitor2,3
Oliveira, Rui S.1,4,5
Martins, Rosário1,2
Source:
Journal of Toxicology & Environmental Health: Part A. 2015, Vol. 78 Issue 13/14, p840-847. 8p.

Toward a Cancer Drug of Fungal Origin.
Authors:
Kornienko A; Department of Chemistry and Biochemistry, Texas State University, San Marcos, TX, 78666.
Evidente A; Dipartimento di Scienze Chimiche, Università di Napoli Federico II, Complesso Universitario Monte S. Angelo, Via Cintia 4, 80126, Napoli, Italy.
Vurro M; Institute of Sciences of Food Production, National Research Council, Via Amendola 122/0, 70126, Bari, Italy.
Mathieu V; Laboratorie de Cancérologie et de Toxicologie Expérimentale, Faculté de Pharmacie, Université Libre de Bruxelles (ULB), Brussels, Belgium.
Cimmino A; Dipartimento di Scienze Chimiche, Università di Napoli Federico II, Complesso Universitario Monte S. Angelo, Via Cintia 4, 80126, Napoli, Italy.
Evidente M; Dipartimento di Scienze Chimiche, Università di Napoli Federico II, Complesso Universitario Monte S. Angelo, Via Cintia 4, 80126, Napoli, Italy.
van Otterlo WA; Department of Chemistry and Polymer Science, University of Stellenbosch, Private Bag X1, Matieland, 7602, South Africa.
Dasari R; Department of Chemistry and Biochemistry, Texas State University, San Marcos, TX, 78666.
Lefranc F; Service de Neurochirurgie, Hôpital Erasme, Université Libre de Bruxelles (ULB), Brussels, Belgium.
Kiss R; Laboratorie de Cancérologie et de Toxicologie Expérimentale, Faculté de Pharmacie, Université Libre de Bruxelles (ULB), Brussels, Belgium.
Source:
Medicinal Research Reviews [Med Res Rev] 2015 Sep; Vol. 35 (5), pp. 937-67. Date of Electronic Publication: 2015 Apr 08.

Destruxin B Isolated from Entomopathogenic Fungus Metarhizium anisopliae Induces Apoptosis via a Bcl-2 Family-Dependent Mitochondrial Pathway in Human Nonsmall Cell Lung Cancer Cells.
Authors:
Chun-Chi Wu
Tzu-Hsiu Chen
Bing-Lan Liu
Li-Chen Wu
Yung-Ching Chen
Yew-Min Tzeng
Shih-Lan Hsu
Source:
Evidence-based Complementary & Alternative Medicine (eCAM). 2013, Vol. 2013, p1-11. 11p. 2 Diagrams, 4 Graphs.

The Marine-Derived Fungus Clonostachys rosea, Source of a Rare Conjugated 4-Me-6E,8E-hexadecadienoic Acid Reducing Viability of MCF-7 Breast Cancer Cells and Gene Expression of Lipogenic Enzymes.
Authors:
Dias AC; Faculty of Pharmacy, University of Nantes, MMS; 9, Rue Bias, 44000 Nantes, France. acamiladias@gmail.com.
Ruiz N; Faculty of Pharmacy, University of Nantes, MMS; 9, Rue Bias, 44000 Nantes, France. nicolas.ruiz@univ-nantes.fr.
Couzinet-Mossion A; Faculty of Pharmacy, University of Nantes, MMS; 9, Rue Bias, 44000 Nantes, France.
Bertrand S; Faculty of Pharmacy, University of Nantes, MMS; 9, Rue Bias, 44000 Nantes, France. samuel.bertrand@univ-nantes.fr.
Duflos M; Faculty of Pharmacy, University of Nantes, IICiMed, 9 Rue Bias, 44000 Nantes, France. muriel.duflos@univ-nantes.fr.
Pouchus YF; Faculty of Pharmacy, University of Nantes, MMS; 9, Rue Bias, 44000 Nantes, France. yves-francois.pouchus@univ-nantes.fr.
Barnathan G; Faculty of Pharmacy, University of Nantes, MMS; 9, Rue Bias, 44000 Nantes, France. gilles.barnathan@univ-nantes.fr.
Nazih H; Faculty of Pharmacy, University of Nantes, MMS; 9, Rue Bias, 44000 Nantes, France. el-hassane.nazih@univ-nantes.fr.
Wielgosz-Collin G; Faculty of Pharmacy, University of Nantes, MMS; 9, Rue Bias, 44000 Nantes, France. gaetane.wielgosz-collin@univ-nantes.fr.
Source:
Marine Drugs [Mar Drugs] 2015 Aug 06; Vol. 13 (8), pp. 4934-48. Date of Electronic Publication: 2015 Aug 06.

I could go on for quite a while, i have literally hundreds of these bookmarked, and would suggest anyone who seeks to form an opinion on the validity of anti-cancer fungal research (or stamets brilliance for getting so many started researching in that direction) should be required to actually read some of the literature.

CONTEXT CONTEXT CONTEXT, anyone who actually keeps up with the research on this stuff knows that stamets contribution is massive, and would be grateful that he is so committed to learning more about these things


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Re: Strain Isolations on Agar, Pics and Questions [Re: c10h12n2o]
    #23572537 - 08/24/16 11:07 AM (7 years, 7 months ago)

those that know stamets well understands his points but have a problem with the way he uses his position to earn $$ instead of focusing on the works.
stamets is also well known to exaggerate his truths and statements to an extent where he can't back his claims up. that doesnt take away all his done for the community tho, but that's not what's being said either.

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Re: Strain Isolations on Agar, Pics and Questions [Re: spacechildo]
    #23572651 - 08/24/16 12:06 PM (7 years, 7 months ago)

i think someone would need to contribute a WHOLE LOT to the subject of mycology and medicine before they could look down on stamets, in any respect

i get what you are saying, he certainly has the propensity to get excited about his work, and he is definitely guilty of tryng to dumb things down enough in public speaking events that the DoD guy asking "so does each spore turn into a mushroom?" and the people who would lose interest if he said the word "cytotoxic" can get the gist of what he is saying.

his big thing lately has been saying that the old growth forest should be saved as a matter of national defense, which is certainly a radical idea that many a defense contractor would find laughable. but if you look at the work he has been doing, and more importantly, the studies that have been citing him and advancing this line of research, it is easy to see how he could get excited

it bugs me when someone has to take something out of context to criticize it, its just not valid


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Re: Strain Isolations on Agar, Pics and Questions [Re: c10h12n2o]
    #23572854 - 08/24/16 01:35 PM (7 years, 7 months ago)

saving old forest and peat moss ground isn't a "new thing" its been known for "ever" but the industry just doesnt give a flying fuck about the environment.

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Re: Strain Isolations on Agar, Pics and Questions [Re: spacechildo]
    #23572948 - 08/24/16 02:06 PM (7 years, 7 months ago)

The environment doesn't actually exist.


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Re: Strain Isolations on Agar, Pics and Questions [Re: Kenetic]
    #23572968 - 08/24/16 02:14 PM (7 years, 7 months ago)

muda earth then..

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Re: Strain Isolations on Agar, Pics and Questions [Re: spacechildo]
    #23573007 - 08/24/16 02:27 PM (7 years, 7 months ago)

Uhhhh, gaia?

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Re: Strain Isolations on Agar, Pics and Questions [Re: Rooster Cogburn]
    #23573073 - 08/24/16 02:46 PM (7 years, 7 months ago)

I have a lot of respect for Stamets due to the fact I learned nearly everything about cultivating oysters from GGMM and nothing else, but I feel the main reason people LOVE hating him is because his company is ridiculous!Fungi Perfecti is a company that takes advantage of new growers, not only that but their cultures really suck, they are on the bottom of the chain for commercial cultures which is the ONE thing they should have right.
There was that thread recently where that old timer Parecelusgold said Stamets was an asshole. I got a kick out of that, says nothing about his skill set and research, but he does have that feel to him.
One last thing, people always say, "Stamets doesn't grow cubes", this right here, considering he had research grants and legally grew them and documented them, I can guarantee he grew more cubes than we will ever see between his researching and his college days. Asshole, horrible business owner, turkey tail cancer curer or not, he is a good cultivator and writer.

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Re: Strain Isolations on Agar, Pics and Questions [Re: Rooster Cogburn]
    #23573164 - 08/24/16 03:08 PM (7 years, 7 months ago)

lol i bet he can come off that way :smile: most professor-types can

ive never ordered spawn or anything like that from them, they definitely have decent prices on hepa filters though (and crazy prices on assembled hoods lol), but if their commercial cultures are anything short of amazing that would certainly be a let-down, like you said, thats one thing they should have right. Very lame if they are using his name to sell shitty cultures

FP is a big company, i was amazed to see how many employees are pictured in their current catalog


I doubt he has a whole lot to do with running it these days, probably spends most of his time on research projects, many of which seem to be classified these days

right there with you codburn, mad respect

UPDATE: here are a couple of the more rhizo plates i mentioned. one has grown out, and the other 2 are more current. one seems to be an isolate :smile:


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Re: Strain Isolations on Agar, Pics and Questions [Re: Rooster Cogburn]
    #23573171 - 08/24/16 03:09 PM (7 years, 7 months ago)

Quote:

Rooster Cogburn said:
I have a lot of respect for Stamets due to the fact I learned nearly everything about cultivating oysters from GGMM and nothing else, but I feel the main reason people LOVE hating him is because his company is ridiculous!Fungi Perfecti is a company that takes advantage of new growers, not only that but their cultures really suck, they are on the bottom of the chain for commercial cultures which is the ONE thing they should have right.
There was that thread recently where that old timer Parecelusgold said Stamets was an asshole. I got a kick out of that, says nothing about his skill set and research, but he does have that feel to him.
One last thing, people always say, "Stamets doesn't grow cubes", this right here, considering he had research grants and legally grew them and documented them, I can guarantee he grew more cubes than we will ever see between his researching and his college days. Asshole, horrible business owner, turkey tail cancer curer or not, he is a good cultivator and writer.



Thanks for your insight. I am surprised whenever I see people hating on Stamets on The Shroomery. After seeing a few of his talks, I instantly idolized him and figured other mushroom lovers and hobbyists would do the same.
I can see how you can lose some respect for him due to fungi perfecti. Personally, I don't know a whole lot about it (other than I wanted to buy Mycelium Running from it) so I can't form an opinion on it, but if what you are saying about it is true, then that is disappointing.
Regardless however, I still think people should respect Stamets and listen to the things he has to say. If his claims are exaggerated then I want to see people prove it. Unfortunately, it seems like a lot of scientists ignore mushrooms research too often. I think fungi could make enourmous positive difference.


Quote:

Rooster Cogburn said:
their cultures really suck, they are on the bottom of the chain for commercial cultures.



Which commercial cultures are at the top of the chain?


Sorry that my post is way off the oriinal topic. This is a really good thread though, lot of stuff going on


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Re: Strain Isolations on Agar, Pics and Questions [Re: TedTheHighlighter]
    #23573213 - 08/24/16 03:20 PM (7 years, 7 months ago)

Quote:

TedTheHighlighter said:
After seeing a few of his talks, I instantly idolized him and figured other mushroom lovers and hobbyists would do the same.





this is exactly why:


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Re: Strain Isolations on Agar, Pics and Questions [Re: c10h12n2o]
    #23573264 - 08/24/16 03:33 PM (7 years, 7 months ago)

Quote:

c10h12n2o said:
UPDATE: here are a couple of the more rhizo plates i mentioned. one has grown out, and the other 2 are more current. one seems to be an isolate :smile:





the outgrown one? because none of the rest looks isolated if you mean a strain/monoculture and notjust isolated somewhat or whatever they say nowaday :lol:

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Re: Strain Isolations on Agar, Pics and Questions [Re: TedTheHighlighter]
    #23573280 - 08/24/16 03:36 PM (7 years, 7 months ago)

Ted, the best commercial cultures come from Aloha culture bank, Amycel, Sylvaan, and I'm sure I'm missing a few but IMO Aloha is #1. I have bought both spawn KO1 Eryngii(which I saved by taking grains to plates>slants) and have 2 traded cultures from them, shiitake 3782, and their white oyster.
I grow grey oysters mainly for extra cash so I use Amycel 3015, from Amycel, it is the best oyster culture I have found, you can only order it by phone though and send a money order or COD, and takes quite a bit of time to get it, it comes in 20# spawn bags something like a buck a pound, and is WAY worth it if you get into pumping out oysters, but also I have that saved and stored from taking grains to plates>slants, but I would never discourage anyone from buying more spawn when needed.

c10, I have a purchased FP culture, Enoki, and also have a traded Enoki that blew FP's out of the water, and also have a traded FP Shiitake that sucks ass compared to the Aloha one. I don't know anyone who grows FP cultures by choice:shrug: if you do I would love to hear it.
If you guys havn't checked it out the Aloha Culture Bank is just a an awesome website to go check out in general, lot's of information and medicinal studies. http://www.alohaculturebank.com/grain-spawn.html#.V74QjPkrLIU

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Re: Strain Isolations on Agar, Pics and Questions [Re: Rooster Cogburn]
    #23573338 - 08/24/16 03:52 PM (7 years, 7 months ago)

no i was thinking the first one might be getting close, if not there. note these are shots of underside of the plates. showed symetrical, even growth on the plate it came from, and is exactly the same on this plate. what specifically makes you so sure its not?

bear in mind these are my first isolations, so im definitely still learning

much obliged rooster, thats some killer info. I havent ordered any cultures in a while, but i love aloha for cheap filter disks! they will definitely be my next vendor for cultures :thumbup:

everything i am currently working with has either come from hawk's eye or trade


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Re: Strain Isolations on Agar, Pics and Questions [Re: c10h12n2o]
    #23573349 - 08/24/16 03:55 PM (7 years, 7 months ago)

its far from a perfect circle. how many transfers in are you?

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Re: Strain Isolations on Agar, Pics and Questions [Re: spacechildo] * 1
    #23573386 - 08/24/16 04:07 PM (7 years, 7 months ago)

Tonight I'm making slants for the first time. I don't have parafilm as I've never used it....would micropore tape be a fine substitute for taping around the caps?



Once I PC these I'll put them on the bench at a slight angle and drop clean wedges in once they solidify. I wish they had a wider mouthed, its going to be a pain getting wedges out when I need them again,  but I'm working with what I've got.

I plan on keeping them stored in a ziplock inside of jar with SFD filter inside the fridge.

Tips?


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Re: Strain Isolations on Agar, Pics and Questions [Re: spacechildo]
    #23573417 - 08/24/16 04:16 PM (7 years, 7 months ago)

its perfectly symmetrical to the agar i transferred though, and using calipers to measure from the edge of the transferred agar to the leading edge of the myc it is almost dead even all the way around. this plate is either 11 or 12 transfers in

btw i had been meaning to ask you something spacechildo: when you have a plate that is heavily sectored showing 2 distinct kinds of myc, and there are 3-4 sections of aggressive rhizo myc and 3-4 linear sectors, and some or all of the rhizo sectors look similar, or are even connected, do you transfer all the good looking sectors, even though there is a good chance that some of them are the same? especially late into a series of transfers, like 10-15th plate.

sometimes lately it has seemed like i am transferring the same thing twice on the same plate, what appears to be 4 sectors might only be 1 "sector" that is on top/under another sector that is diving it (not to say that this single "interrupted sector" is an individual strain, of course there could be numerous compatible strains forming the same "sector")

i suppose there is no harm in labeling and testing them all separately, but im working on so much right now that i would like to minimize wasted time/space on testing strains X, Y, and Z against each other only to discover/suspect that they are indeed only one strain.

let me know if i need to clarify anything. i would really like to hear your thoughts on this and potential ways to negate this possibility without missing out on potentially valuable strains

warm regards


ps:re: ben
probably better than nothing, but you should get some parafilm brother! that stuff is the bomb, and a little bit goes a LONG way if you use it right. hell, PM me and i will send you some. I lucked out and found a lab supply store that stocks the old kind that was even better :laugh:


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Edited by c10h12n2o (08/24/16 04:19 PM)

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Re: Strain Isolations on Agar, Pics and Questions [Re: c10h12n2o]
    #23573439 - 08/24/16 04:21 PM (7 years, 7 months ago)

Quote:

weetsie said:
Quote:

Supalemonhaze said:
Quote:

weetsie said:
Even when you don't dilute spores and start with a big black dot  your first plate you can still get isolates in less than 5 transfers.





I'd like to see you try and pull this off and document it.




I'll take you up on that.

Watch this space.





:standingby:


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Re: Strain Isolations on Agar, Pics and Questions [Re: c10h12n2o]
    #23573461 - 08/24/16 04:27 PM (7 years, 7 months ago)

Quote:

c10h12n2o said:
1) its perfectly symmetrical to the agar i transferred though, and using calipers to measure from the edge of the transferred agar to the leading edge of the myc it is almost dead even all the way around. this plate is either 11 or 12 transfers in

2) btw i had been meaning to ask you something spacechildo: when you have a plate that is heavily sectored showing 2 distinct kinds of myc, and there are 3-4 sections of aggressive rhizo myc and 3-4 linear sectors, and some or all of the rhizo sectors look similar, or are even connected, do you transfer all the good looking sectors, even though there is a good chance that some of them are the same? especially late into a series of transfers, like 10-15th plate.

3) sometimes lately it has seemed like i am transferring the same thing twice on the same plate, what appears to be 4 sectors might only be 1 "sector" that is on top/under another sector that is diving it (not to say that this single "interrupted sector" is an individual strain, of course there could be numerous compatible strains forming the same "sector")

4) i suppose there is no harm in labeling and testing them all separately, but im working on so much right now that i would like to minimize wasted time/space on testing strains X, Y, and Z against each other only to discover/suspect that they are indeed only one strain.

let me know if i need to clarify anything. i would really like to hear your thoughts on this and potential ways to negate this possibility without missing out on potentially valuable strains

warm regards




1, well try just 1 more transfer and make it real small, I still doubt it but I've been wrong before :shrug:

2, just look for fast growing strong looking myc. all your plates seem nice and healthy so just pick the fastest growing sectors as that's the only trait you can see on agar.

3, that's confusing :lol: its perfectly normal that sectors grow together and what looks like 1 is really 10 which you'll find out later on as you keep transferring.

4, yeah that's a bitch, in a perfect world you'd test them all but :shrug: do the amount of searching you can/have time for.

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Re: Strain Isolations on Agar, Pics and Questions [Re: PsilocyBen17]
    #23573528 - 08/24/16 04:45 PM (7 years, 7 months ago)

Quote:

PsilocyBen17 said:
Tonight I'm making slants for the first time. I don't have parafilm as I've never used it....would micropore tape be a fine substitute for taping around the caps?



Once I PC these I'll put them on the bench at a slight angle and drop clean wedges in once they solidify. I wish they had a wider mouthed, its going to be a pain getting wedges out when I need them again,  but I'm working with what I've got.

I plan on keeping them stored in a ziplock inside of jar with SFD filter inside the fridge.

Tips?




Those are plenty wide, you want to put a tiny piece of mycelium in there.
My biggest tip that makes it simple is getting your sample with your right hand and unscrewing the top between your pinky and ring finger of the transfer hand and doing the transfer while holding the cap like that then it's easy to screw back on.
The most valuable tool I think for making slants is a test tube holder, simply fill the tubes in the holder, screw caps on tight and throw the whole thing in the PC, pull it out and lay the rack at an angle and wait to cool, then it's really nice for doing the actual work.
The only thing I think about the micropore tape is it would dry the culture out faster over time, but better than nothing I guess, you do need GE in those things. Im not sure a jar is necessary for storing, is there a specific reason for doing this?

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Re: Strain Isolations on Agar, Pics and Questions [Re: spacechildo]
    #23573768 - 08/24/16 06:19 PM (7 years, 7 months ago)

Quote:

spacechildo said:
If I had a dollar for every time a noob said he'd prove something wrong and even went as far as updating his thread I'd still be a poor son of a bitch!



If I had a dime for every time a noob said he'd prove something wrong and even went as far as updating his thread I'd have ZERO DIMES


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Re: Strain Isolations on Agar, Pics and Questions [Re: Munchauzen]
    #23573787 - 08/24/16 06:26 PM (7 years, 7 months ago)

Quote:

Munchauzen said:
Quote:

spacechildo said:
If I had a dollar for every time a noob said he'd prove something wrong and even went as far as updating his thread I'd still be a poor son of a bitch!



If I had a dime for every time a noob said he'd prove something wrong and even went as far as updating his thread I'd have ZERO DIMES






Well, weetsie has begun his experiment and already updated the thread soooo i guess someone owes you ten cents
Props to weetsie for actually carrying out the experiment


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Re: Strain Isolations on Agar, Pics and Questions [Re: TedTheHighlighter]
    #23573803 - 08/24/16 06:31 PM (7 years, 7 months ago)

Quote:

TedTheHighlighter said:
Quote:

Munchauzen said:
Quote:

spacechildo said:
If I had a dollar for every time a noob said he'd prove something wrong and even went as far as updating his thread I'd still be a poor son of a bitch!



If I had a dime for every time a noob said he'd prove something wrong and even went as far as updating his thread I'd have ZERO DIMES






Well, weetsie has begun his experiment and already updated the thread soooo i guess someone owes you ten cents
Props to weetsie for actually carrying out the experiment



he hasn't updated shit. a random picture of an empty plate from over a month ago is not an update, its a waste of a post and our time. so, I'm sticking with NO DIMES.

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Re: Strain Isolations on Agar, Pics and Questions [Re: Munchauzen]
    #23573809 - 08/24/16 06:33 PM (7 years, 7 months ago)

Quote:

Munchauzen said:
Quote:

spacechildo said:
If I had a dollar for every time a noob said he'd prove something wrong and even went as far as updating his thread I'd still be a poor son of a bitch!



If I had a dime for every time a noob said he'd prove something wrong and even went as far as updating his thread I'd have ZERO DIMES






:philososloth:

havent seen any updates myself :shrug: should get its own thread for sure if its happening!

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Re: Strain Isolations on Agar, Pics and Questions [Re: spacechildo]
    #23573813 - 08/24/16 06:35 PM (7 years, 7 months ago)

this is what dude considered an 'update'

Quote:

weetsie said:
Scraped up at least half a square inch of spores and dumped them in a pile on a plate.



Feeling confident :dancing:





https://www.shroomery.org/forums/showflat.php/Number/23468297#23468297

I could have made 4 transfers and finished this shit within a month. No need to let the plates grow out. You could probably get to the 4th transfer in 2 weeks, if you actually tried.

Edited by Munchauzen (08/24/16 06:35 PM)

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Re: Strain Isolations on Agar, Pics and Questions [Re: weetsie]
    #23573826 - 08/24/16 06:39 PM (7 years, 7 months ago)

Quote:

weetsie said:


Took 2 transfers from the first plate today. :dancer:




Is this not an update?


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Re: Strain Isolations on Agar, Pics and Questions [Re: weetsie]
    #23573890 - 08/24/16 07:02 PM (7 years, 7 months ago)

Quote:

weetsie said:


Took 2 transfers from the first plate today. :dancer:



:lol: I blame space for priming me with bad info

still, dont know why he is waiting for it to grow out to the edge of the plate to make a transfer. Weetsie, stop pussy-footin' around and get crackin'!

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Re: Strain Isolations on Agar, Pics and Questions [Re: Munchauzen]
    #23573914 - 08/24/16 07:09 PM (7 years, 7 months ago)

IMO its a terrible excuse for an update. horrible pics and no telling whats going on. but yeah seems I got ma dollah as well!

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Re: Strain Isolations on Agar, Pics and Questions [Re: Rooster Cogburn]
    #23574225 - 08/24/16 09:06 PM (7 years, 7 months ago)

Quote:

Rooster Cogburn said:
Quote:

PsilocyBen17 said:
Tonight I'm making slants for the first time. I don't have parafilm as I've never used it....would micropore tape be a fine substitute for taping around the caps?



Once I PC these I'll put them on the bench at a slight angle and drop clean wedges in once they solidify. I wish they had a wider mouthed, its going to be a pain getting wedges out when I need them again,  but I'm working with what I've got.

I plan on keeping them stored in a ziplock inside of jar with SFD filter inside the fridge.

Tips?




Those are plenty wide, you want to put a tiny piece of mycelium in there.
My biggest tip that makes it simple is getting your sample with your right hand and unscrewing the top between your pinky and ring finger of the transfer hand and doing the transfer while holding the cap like that then it's easy to screw back on.
The most valuable tool I think for making slants is a test tube holder, simply fill the tubes in the holder, screw caps on tight and throw the whole thing in the PC, pull it out and lay the rack at an angle and wait to cool, then it's really nice for doing the actual work.
The only thing I think about the micropore tape is it would dry the culture out faster over time, but better than nothing I guess, you do need GE in those things. Im not sure a jar is necessary for storing, is there a specific reason for doing this?




Cool, thanks kindly for your response...

No reason for the jar really, just a convient way to store them. They all fit in there nicely, why not throw an sfd on there just cuz...

I DID forget to prehydrate the popsicle sticks :facepalm3:. Hopefully they don't dry out super fast cuz of this....worst case senario I can transfer to new slants 6 months down the road.


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Re: Strain Isolations on Agar, Pics and Questions [Re: spacechildo]
    #23574881 - 08/25/16 02:06 AM (7 years, 7 months ago)

Quote:

spacechildo said:
IMO its a terrible excuse for an update. horrible pics and no telling whats going on. but yeah seems I got ma dollah as well!




No telling what's going on?

I transferred two small pieces of mycelium from the leading edge of the plate on the left to the two on the right. But that much was obvious, what else is there to say. :lol:

I'll make the 2nd transfer in a couple of hours and post some photos with the lids off.


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Re: Strain Isolations on Agar, Pics and Questions [Re: weetsie]
    #23575096 - 08/25/16 06:07 AM (7 years, 7 months ago)

your initial plate looks like shit but I really cant zoom in on it.
and just for the sake of argument, lets say you did get an isolate in 5 transfers. are you gonna use this thread to show it can be done?
"hey dudes, just look for my posts somewhere in between c12's posts" :confused2:

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Re: Strain Isolations on Agar, Pics and Questions [Re: spacechildo] * 1
    #23575109 - 08/25/16 06:17 AM (7 years, 7 months ago)

i got an MS isolate in 8 transfers (so the ninth plate if u include the initial germination plate) only one time.



pure luck


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Re: Strain Isolations on Agar, Pics and Questions [Re: blindingleaf]
    #23575245 - 08/25/16 07:57 AM (7 years, 7 months ago)

Quote:

blindingleaf said:
i got an MS isolate in 8 transfers (so the ninth plate if u include the initial germination plate) only one time.



pure luck




Is that isolate a Pan cambo? Looks like thats what you wrote on the plate. If so, its an interesting growth form for a Pan.
The reason I ask is I'm working on a cambo Sandos isolate right now.
Have you had success taking Pan isolates to fruiting?

Edited by mary fairchild (08/26/16 06:18 AM)

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Re: Strain Isolations on Agar, Pics and Questions [Re: mary fairchild]
    #23575380 - 08/25/16 09:03 AM (7 years, 6 months ago)

No, that's not panaeolus mycelium.

This is



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Re: Strain Isolations on Agar, Pics and Questions [Re: Mad Season]
    #23575527 - 08/25/16 09:55 AM (7 years, 6 months ago)

It looks kinda more "fibrous" than most Pans- but my cambo Sandos looks that way too.


Edited by mary fairchild (08/25/16 09:56 AM)

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Re: Strain Isolations on Agar, Pics and Questions [Re: mary fairchild]
    #23575561 - 08/25/16 10:03 AM (7 years, 6 months ago)

:creepylurker:


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Re: Strain Isolations on Agar, Pics and Questions [Re: weetsie]
    #23575651 - 08/25/16 10:34 AM (7 years, 6 months ago)

Quote:

weetsie said:
Quote:

spacechildo said:
IMO its a terrible excuse for an update. horrible pics and no telling whats going on. but yeah seems I got ma dollah as well!




No telling what's going on?

I transferred two small pieces of mycelium from the leading edge of the plate on the left to the two on the right. But that much was obvious, what else is there to say. :lol:

I'll make the 2nd transfer in a couple of hours and post some photos with the lids off.




Wheetsie, your going to set off a shit storm if you get this! It's definitely gonna need it's own thread man.

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Re: Strain Isolations on Agar, Pics and Questions [Re: Rooster Cogburn]
    #23576120 - 08/25/16 01:34 PM (7 years, 6 months ago)

That's a pretty big 'if' thou. Good on him for trying but I give it a pretty slim chance. prove me wrong, plz.

Edited by Munchauzen (08/25/16 07:43 PM)

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Re: Strain Isolations on Agar, Pics and Questions [Re: Munchauzen]
    #23576354 - 08/25/16 02:56 PM (7 years, 6 months ago)

All the plates used so far:





One of the transfers resulted in a very circular cottony growth which I don't plan to continue with. The other has a large rhizomorphic sector which I made two more transfers from.

A photo of said plate with the lid off:






Quote:

Munchauzen said:

still, dont know why he is waiting for it to grow out to the edge of the plate to make a transfer. Weetsie, stop pussy-footin' around and get crackin'!




Because the goal is to do it in as few transfers as possible.

The rhizomorphic mycelium I'm after takes time to develop, if I transferred too soon I might not see a rhizomorphic sector or any sectoring after the first transfer.

The wedge I transfer will also be relatively smaller the more I let the plate grow out which I think is key to what I'm trying to do.

I imagine letting the plate grow out more will also thin the heard so to speak.


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Re: Strain Isolations on Agar, Pics and Questions [Re: weetsie] * 1
    #23576475 - 08/25/16 03:31 PM (7 years, 6 months ago)

This thread is bangin


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Re: Strain Isolations on Agar, Pics and Questions [Re: TedTheHighlighter]
    #23576488 - 08/25/16 03:34 PM (7 years, 6 months ago)

I think even if you took a small chunk you'd still get tons of genetic diversity.


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Re: Strain Isolations on Agar, Pics and Questions [Re: Kenetic]
    #23577463 - 08/25/16 07:44 PM (7 years, 6 months ago)

I guess we'll see

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Re: Strain Isolations on Agar, Pics and Questions [Re: Munchauzen]
    #23578444 - 08/26/16 02:37 AM (7 years, 6 months ago)

Quote:

spacechildo said:
Quote:

c10h12n2o said:
1) its perfectly symmetrical to the agar i transferred though, and using calipers to measure from the edge of the transferred agar to the leading edge of the myc it is almost dead even all the way around. this plate is either 11 or 12 transfers in

2) btw i had been meaning to ask you something spacechildo: when you have a plate that is heavily sectored showing 2 distinct kinds of myc, and there are 3-4 sections of aggressive rhizo myc and 3-4 linear sectors, and some or all of the rhizo sectors look similar, or are even connected, do you transfer all the good looking sectors, even though there is a good chance that some of them are the same? especially late into a series of transfers, like 10-15th plate.

3) sometimes lately it has seemed like i am transferring the same thing twice on the same plate, what appears to be 4 sectors might only be 1 "sector" that is on top/under another sector that is diving it (not to say that this single "interrupted sector" is an individual strain, of course there could be numerous compatible strains forming the same "sector")

4) i suppose there is no harm in labeling and testing them all separately, but im working on so much right now that i would like to minimize wasted time/space on testing strains X, Y, and Z against each other only to discover/suspect that they are indeed only one strain.

let me know if i need to clarify anything. i would really like to hear your thoughts on this and potential ways to negate this possibility without missing out on potentially valuable strains

warm regards




1, well try just 1 more transfer and make it real small, I still doubt it but I've been wrong before :shrug:

2, just look for fast growing strong looking myc. all your plates seem nice and healthy so just pick the fastest growing sectors as that's the only trait you can see on agar.

3, that's confusing :lol: its perfectly normal that sectors grow together and what looks like 1 is really 10 which you'll find out later on as you keep transferring.

4, yeah that's a bitch, in a perfect world you'd test them all but :shrug: do the amount of searching you can/have time for.




Makes sense, thanks man. I will post a pic tomorrow of some plates that show the kind of growth that makes me wonder about that, just for talking points

Quote:

Munchauzen said:
still, dont know why he is waiting for it to grow out to the edge of the plate to make a transfer. Weetsie, stop pussy-footin' around and get crackin'!




Yo munch, can you elaborate on why there is no need to let it grow out? I'm curious if I'm misunderstanding something, but shouldn't letting it grow out allow more chance for sectors to show, and give more opportunity for strains to outpace each other? I usually end up making transfers about every 3-5 days or as soon as I see something sexy, but if the goal is to get an isolate in the fewest possible transfers, why wouldn't he want to give strains time to outpace each other?

I for one appreciate weetsie's experiment and think it is right on topic for the thread, will certainly be useful to anyone with similar questions who reads in the future

Wow blindingleaf, killer plate, beautiful. Did you start with diluted spores?

Also, I have some pan tropicalis on agar right now about 8 transfers in, looks far whisper though, I will post a pic tomorrow

Edit: looking nice weetsie!! Fun experiment, very helpful for readers regardless of "proving" anything

Edited by c10h12n2o (08/26/16 02:39 AM)

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Re: Strain Isolations on Agar, Pics and Questions [Re: c10h12n2o]
    #23578582 - 08/26/16 04:51 AM (7 years, 6 months ago)

no, just normal spore swipe.

it was just random that it happened in 8 transfers..its not something I expect.

I let mine grow out too once I'm on later plates, like 3-5+ xfers.  not all the way to the edge, but about the size of that picture.  I don't know if it technically helps, but i personally find it easier to spot where I wanna pull from later rather than sooner.


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Re: Strain Isolations on Agar, Pics and Questions [Re: blindingleaf]
    #23579152 - 08/26/16 09:45 AM (7 years, 6 months ago)

Whats the advantage of using a spore swipe rather than a syringe?


--------------------
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Re: Strain Isolations on Agar, Pics and Questions [Re: TedTheHighlighter]
    #23579164 - 08/26/16 09:50 AM (7 years, 6 months ago)

not having excess water on your plate. squirt some spore water on the inoc-loop if you only have syringes. or just "spill" some on the tip of the needle and swipe that.

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Re: Strain Isolations on Agar, Pics and Questions [Re: spacechildo]
    #23580207 - 08/26/16 02:01 PM (7 years, 6 months ago)

Okay, here's another noob question
Whats the advantage in swiping the spores in a "Z" or "L" shape?


--------------------
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Re: Strain Isolations on Agar, Pics and Questions [Re: TedTheHighlighter] * 1
    #23580238 - 08/26/16 02:10 PM (7 years, 6 months ago)

Z is for potency and L is for colonization speed.


Not really, it's just to spread them evenly.  Increases the chances of clean germination by giving a wider area for the (hopefully few) contaminants to be separated from the myc.


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Re: Strain Isolations on Agar, Pics and Questions [Re: Inocuole]
    #23580474 - 08/26/16 03:24 PM (7 years, 6 months ago)

Ahhhhh thanks for the responses


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Re: Strain Isolations on Agar, Pics and Questions [Re: TedTheHighlighter]
    #23580572 - 08/26/16 03:45 PM (7 years, 6 months ago)

here is a killer video explaining how to streak plates for isolating bacterial colonies:


obviously we are working with fungus instead, but same principles apply.

i have started doing it this way, it is VERY helpful for isolating away from contaminants in a syringe or print, and also useful for having literally thousands of potential colonies to choose from on your initial plate


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Re: Strain Isolations on Agar, Pics and Questions [Re: c10h12n2o]
    #23582212 - 08/27/16 12:12 AM (7 years, 6 months ago)

something that hasn't been mentioned in this thread but I have heard others mentioned when isolating agar is the size of the agar plate.
those 110mm plates are specially good (IMO) for isolating because given that you give the mycelium enough space to colonize the easier it will be to pick out different sectors so IMO it should be possible to achieve an isolate in less than 10 plates or so (specially the big ones)... just my 2c
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Re: Strain Isolations on Agar, Pics and Questions [Re: MMG]
    #23582515 - 08/27/16 04:57 AM (7 years, 6 months ago)

I just got a bunch of 60mm ones.  Should be easier to pour in a GB.


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Re: Strain Isolations on Agar, Pics and Questions [Re: Kenetic]
    #23583335 - 08/27/16 12:06 PM (7 years, 6 months ago)

Good point MMG, makes sense to me, i need to get some variety rather than just the standard 90mm

Even more to the point i was asking munch, why he said there is no need to let them grow out for what weetsie is trying to do. could you clarify on that my friend? i think i must be missing something

UPDATE:

here are a few plates i made transfers from yesterday, some of the more rhizomorphic examples of GT and AA+ cultures:




and here is a pan tropicalis (pan cyan?) that is on its 9th or 10th transfer. like i mentioned before, it  has been looking a lot whispier than other peoples' pan myc. also, it has been perfectly round on almost all of those whispy plates, showing no sectors at all, very frustrating. but i just checked my most recent transfer in that series and it looks a lot more like its supposed to. in this pic are some of the older ones and  the new one. never worked with this species before




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Re: Strain Isolations on Agar, Pics and Questions [Re: c10h12n2o]
    #23585370 - 08/27/16 10:22 PM (7 years, 6 months ago)

Hello guys, pardon my ignorance, but I have been wondering about this for a while and have failed to solve the problem with the search function. I see a lot of people talking about isolating cultures and making monocultures, but I have not read of any benefits of doing so. What is the benefit of creating a monoculture instead of getting a clone and leaving it at that?

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Re: Strain Isolations on Agar, Pics and Questions [Re: herrenvolk]
    #23585394 - 08/27/16 10:34 PM (7 years, 6 months ago)

It's even more consistent.  You can run actual meaningful tests on it since it is a control at that point.


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Re: Strain Isolations on Agar, Pics and Questions [Re: Inocuole]
    #23585406 - 08/27/16 10:40 PM (7 years, 6 months ago)

Are there more benfits to it? Will isolating a choice strain make for better pinsets, growth, and yields than a clone?


--------------------
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Re: Strain Isolations on Agar, Pics and Questions [Re: TedTheHighlighter]
    #23585420 - 08/27/16 10:44 PM (7 years, 6 months ago)

If that isolate does in fact have those features.  It could just as easily not.  Actually that's a misrepresentation...  It's highly unlikely that a randomly chosen isolate is going to be a better than a well chosen clone.  Isolating from a clone could produce better or worse results than the clone as it was originally, as well.


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Re: Strain Isolations on Agar, Pics and Questions [Re: TedTheHighlighter]
    #23585423 - 08/27/16 10:45 PM (7 years, 6 months ago)

Maybe change the alkaloid production by isolating on water soluable cbd and thc to help with anticarcinogenic effects and keep cloning.. i was actually going to do this and see how strong i could get it and try it on a sick animal

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Re: Strain Isolations on Agar, Pics and Questions [Re: Cameron1996]
    #23585428 - 08/27/16 10:47 PM (7 years, 6 months ago)

Quote:

Cameron1996 said:
Maybe change the alkaloid production by isolating on water soluable cbd and thc to help with anticarcinogenic effects and keep cloning.. i was actually going to do this and see how strong i could get it and try it on a sick animal




.... What?  Are we still talking about mushrooms here?  Mushrooms getting cancer isn't something we really worry about..  You may come to find that not much in the world can actually be fixed by weed, as nice as that would be from an idealists perspective.

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Re: Strain Isolations on Agar, Pics and Questions [Re: Inocuole]
    #23585447 - 08/27/16 10:57 PM (7 years, 6 months ago)

Quote:

Inocuole said:
It's even more consistent.  You can run actual meaningful tests on it since it is a control at that point.




Ah, so clones can inconsistent results too? I have observed atleast two distinctly different phenotypes in my clone, but chalked it up to growing conditions.

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Re: Strain Isolations on Agar, Pics and Questions [Re: herrenvolk]
    #23585514 - 08/27/16 11:21 PM (7 years, 6 months ago)

Clones can have quite a few sets of genetics present still, yes.


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Re: Strain Isolations on Agar, Pics and Questions [Re: Inocuole]
    #23585520 - 08/27/16 11:22 PM (7 years, 6 months ago)

No not the mushrooms getting cancer silly, i ment feed the mushrooms things that fight cancer like water soluable thc and cbd with medical mushrooms like how they can biosynthesize things from different fungi such as ergot stroma tissue with tryptophan and amino acids or how they biosynthesize thc with yeast.. mainly see how growing on a heavy anticarcinogenic chemical content will effect the mycelium and fruit bodys, and clone both so the mushrooms and mycelium dont lose there traits and keep doing that to see if i can get them strong or to produce a whole new chemical in general...try it on pets or animals that are already dieing from cancer and if its successful without the cancer coming back maybe add it to chemo treatments..i have another project i am working on that i dont want to disclose to much because i dont want people trying it and fucking thereselves up but it involves a single spore isolation, do u think i should filter, dilute, then use a glass needle or is there a easyer method? Im going to be working with molds not mycelium at that point

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Re: Strain Isolations on Agar, Pics and Questions [Re: Cameron1996]
    #23585527 - 08/27/16 11:23 PM (7 years, 6 months ago)

So this is on topic... how?  :confused2:


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Re: Strain Isolations on Agar, Pics and Questions [Re: Cameron1996]
    #23585538 - 08/27/16 11:27 PM (7 years, 6 months ago)

Okay, I understand how most monocultures will be weaker than a choice clone. You can visibly see the best mushroom to clone but it takes luck and testing to find the best monoculture after months of isolating.
So what are the chances you get that monoculture that yields consistant, excellent grows that are even better than grows from a nice clone?
Just wondering how beneficial isolating actually is


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Re: Strain Isolations on Agar, Pics and Questions [Re: TedTheHighlighter]
    #23585547 - 08/27/16 11:30 PM (7 years, 6 months ago)

About x%.  :shrug:  The chance is there, but there's no cut and dry way to say for sure how long you could spend isolating cultures before you find something that makes it worthwhile.


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Re: Strain Isolations on Agar, Pics and Questions [Re: TedTheHighlighter]
    #23585554 - 08/27/16 11:32 PM (7 years, 6 months ago)

The chances of getting a good isolate with good yield and potency? Uh I highly doubt anyone will have an answer. Also depends on what you call good yield and potency. For my standards, I'd say I'd be looking at 1 in 50 isolates would be what I want.


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Re: Strain Isolations on Agar, Pics and Questions [Re: Mad Season]
    #23585588 - 08/27/16 11:47 PM (7 years, 6 months ago)

My definition of good enough would be better than any clone you could take.
Simoly because, while isolating a strain is an awesome thing, I don't see how it is worth the months of work if you can just clone and get better results


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Re: Strain Isolations on Agar, Pics and Questions [Re: TedTheHighlighter]
    #23585619 - 08/28/16 12:08 AM (7 years, 6 months ago)

And that's why I've always done clones. Isolating is something I'd do if you have a lot of spare time to go genetics hunting


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Re: Strain Isolations on Agar, Pics and Questions [Re: TedTheHighlighter]
    #23586052 - 08/28/16 07:53 AM (7 years, 6 months ago)

Quote:

TedTheHighlighter said:
My definition of good enough would be better than any clone you could take.
Simoly because, while isolating a strain is an awesome thing, I don't see how it is worth the months of work if you can just clone and get better results




Isolates like that exist, but that's not to say another clone couldn't crop up in the future that's better still.


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Re: Strain Isolations on Agar, Pics and Questions [Re: Inocuole]
    #23586451 - 08/28/16 10:37 AM (7 years, 6 months ago)

Soooooo

Say you isolate 100 strains and find the ideal one. Is that ideal strain likely to be a better producer than an ideal clone from a MS grow?

Basically, is an awesome monoculture better than an awesome clone?


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Re: Strain Isolations on Agar, Pics and Questions [Re: TedTheHighlighter]
    #23586608 - 08/28/16 11:36 AM (7 years, 6 months ago)

It could be, until you find a better clone, and then that could be the best until you find a better isolate.  There is no ceiling of how "awesome" something can be, so, without knowing what measurements you're using to gauge awesomeness, it's hard to say.  All that anyone could say for sure is that they'll be different in some way.

If you want something more consistent than a clone, which will sometimes do different things, then you isolate it further.  If you don't mind a little bit of variability, you stick with clones.

It's all far too random for anyone to deifinitively answer any questions like that about it though, because things change, life keeps mutating and crossing and doing its own thing.


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Re: Strain Isolations on Agar, Pics and Questions [Re: Inocuole]
    #23586684 - 08/28/16 12:03 PM (7 years, 6 months ago)

Yeah I understand.
I was just wondering because cloning is much simpler than isolating so I figured that isolating will eventually give you your best possible grows. Otherwise, it seems not worth it when you can just clone and get the same results


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Re: Strain Isolations on Agar, Pics and Questions [Re: TedTheHighlighter]
    #23586807 - 08/28/16 12:40 PM (7 years, 6 months ago)

Overall I don't recommend isolating unless you have a goal in mind that requires it. :shrug:  But there are merits and everybody should probably do it at least once.


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Re: Strain Isolations on Agar, Pics and Questions [Re: Inocuole]
    #23587224 - 08/28/16 03:04 PM (7 years, 6 months ago)

I'm currently doing transfers from MS plates, hoping to clean the mycelium of bacterial contaminations from the dirty spore print. But I'm wondering if this process is also isolating "strain groups", strains that go well together and run intertwined on the agar plate. The cultures were transferred 3 times. (I did the agar way too soft for the 2 first transfers).

I've made some bottle/v-tek to test my subcultures (3 replicates for each of the 8 partial isolates) and maybe get some clones. The results should be a bit more consistent than straight MS jars, right ?

Maybe I should have tried some MS BRF cakes, fruit the possibly contaminated cakes, then clone ? What are the advantages of going the agar way, beside learning the technique ?

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Re: Strain Isolations on Agar, Pics and Questions [Re: Cameron1996]
    #23587310 - 08/28/16 03:32 PM (7 years, 6 months ago)

Quote:

Cameron1996 said:
No not the mushrooms getting cancer silly, i ment feed the mushrooms things that fight cancer like water soluable thc and cbd with medical mushrooms like how they can biosynthesize things from different fungi such as ergot stroma tissue with tryptophan and amino acids or how they biosynthesize thc with yeast.. mainly see how growing on a heavy anticarcinogenic chemical content will effect the mycelium and fruit bodys, and clone both so the mushrooms and mycelium dont lose there traits and keep doing that to see if i can get them strong or to produce a whole new chemical in general...try it on pets or animals that are already dieing from cancer and if its successful without the cancer coming back maybe add it to chemo treatments..i have another project i am working on that i dont want to disclose to much because i dont want people trying it and fucking thereselves up but it involves a single spore isolation, do u think i should filter, dilute, then use a glass needle or is there a easyer method? Im going to be working with molds not mycelium at that point




Very confusing/confused post my friend.

1. Neither THC nor CBD are water soluble, at all. That's the whole principle behind most hash making procedures

2. THC and even CBD don't really have anti carcinogenic effects, that is a misunderstanding. THC and CBD are often used to treat the nausea and anorexia associated with cancer, certainly not anti carcinogenic though

3. The compounds that have shown some potential for actually treating certain types of cancer are not the psychoactive compounds, but rather the 270ish other nonpsychoactive compounds found in cannabis, with more being discovered all the time

4. Lol genetic engineering, a la splicing the ability to produce spider silk into goats, etc, is a LOT more complicated than that. Every time it has been done it involved a whole team of PhDs with gov grants (nearly infinite resources and brain power), and geneticists competing for the Nobel prize. There is some amazing potential in engineering molds to produce hard-to-manufacture compounds, but it would require a few genetic engineering degrees to even scratch the surface on

5. "Single spore isolation" is not a thing (well, not beyond monokaryotic isolates ). Spore reproduction processes basically are analogous to sexual reproduction, with genetic material from 2 spores. I highly doubt you want a monokaryotic isolate, I think you are confused

6. Mold is still mycelium, just a different species. Like how you and a naked mole rat are different species, but both have blood

Read Paul Stamets books TMC and Mycelium Running, should clear up a lot and will fill your brain with wonder :smile:

Quote:

TedTheHighlighter said:
Yeah I understand.
I was just wondering because cloning is much simpler than isolating so I figured that isolating will eventually give you your best possible grows. Otherwise, it seems not worth it when you can just clone and get the same results




The "same", definitely not. It's similar, but the distinguishing feature lies in that an isolate is a single strain, and clones usually have multiple strains present. It depends what you are trying to do and how you define "good". If you are just trying to grow some good mushrooms, isolates are altogether unnecessary and clones are much preferable. But if your intention is to run meaningful tests or have cultures you could bet on, isolates are essential

Mad and inocuole, great info, very helpful for all involved :highfive1:

Quote:

Piezo said:
I'm currently doing transfers from MS plates, hoping to clean the mycelium of bacterial contaminations from the dirty spore print. But I'm wondering if this process is also isolating "strain groups", strains that go well together and run intertwined on the agar plate. The cultures were transferred 3 times. (I did the agar way too soft for the 2 first transfers).

I've made some bottle/v-tek to test my subcultures (3 replicates for each of the 8 partial isolates) and maybe get some clones. The results should be a bit more consistent than straight MS jars, right ?

Maybe I should have tried some MS BRF cakes, fruit the possibly contaminated cakes, then clone ? What are the advantages of going the agar way, beside learning the technique ?




1. These strain groups you are referring to are fascinating to me as well, I have had a couple of cultures which were perfectly round for the first 10 transfers, only started showing sectors on the 11th. These groups are good in that they mean compatible strains that can network together, but a pain in the ass trying to isolate

2. Partial isolate isn't a thing, it's either an isolate or not. What you're referring to is what many call "limited MS" because the herd of strains has been thinned from the initial MS. More consistent? Not necessarily, because you never know which strain(s) will become most established or how well they will play with the other strains present.

3. Don't fool with contaminated cakes, waste of time. Figure out where the failure was and fix it. You say the spore print was dirty, so probably here.  And most importantly, "the agar way" is not an alternative to "learning the technique "; agar is no substitute for technique. If you have bad technique on agar it will get old fast. Agar requires even more technique than no agar

4. There are many uses for agar: doing clones, isolations, cleaning cultures, etc. just like any other tool or technique, the advantage lies in how you employ it, not the tool itself. In your case, agar is perfect for cleaning up a dirty spore print. You will want to take transfers from the leading edge of the myc until you are sure you have a clean culture, only then would you inoculate


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Edited by c10h12n2o (08/28/16 03:59 PM)

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Re: Strain Isolations on Agar, Pics and Questions [Re: c10h12n2o]
    #23587358 - 08/28/16 03:46 PM (7 years, 6 months ago)

:firstladyofapproval:

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Re: Strain Isolations on Agar, Pics and Questions [Re: c10h12n2o]
    #23587619 - 08/28/16 05:04 PM (7 years, 6 months ago)

Thanks for the reply.
Quote:

c10h12n2o said:
1. These strain groups you are referring to are fascinating to me as well, I have had a couple of cultures which were perfectly round for the first 10 transfers, only started showing sectors on the 11th. These groups are good in that they mean compatible strains that can network together, but a pain in the ass trying to isolate

2. Partial isolate isn't a thing, it's either an isolate or not. What you're referring to is what many call "limited MS" because the herd of strains has been thinned from the initial MS. More consistent? Not necessarily, because you never know which strain(s) will become most established or how well they will play with the other strains present.

3. Don't fool with contaminated cakes, waste of time. Figure out where the failure was and fix it. You say the spore print was dirty, so probably here.  And most importantly, "the agar way" is not an alternative to "learning the technique "; agar is no substitute for technique. If you have bad technique on agar it will get old fast. Agar requires even more technique than no agar

4. There are many uses for agar: doing clones, isolations, cleaning cultures, etc. just like any other tool or technique, the advantage lies in how you employ it, not the tool itself. In your case, agar is perfect for cleaning up a dirty spore print. You will want to take transfers from the leading edge of the myc until you are sure you have a clean culture, only then would you inoculate





2. Yeah, by "partial isolate" I was referring to those "strain groups" obtained from limited MS. If I understand correctly, the replicated jars from the same limited MS plate could give different results because the strain demographics might diverge in each jar ?

3. The print was made in a hurry in a septic and humid environment... I haven't tried to inoculate a jar with it yet.
That's why i thought it would be a good time to learn agar work. By "learning the technique" I was referring to the agar work. Like you said in 4, I thought this situation would be the perfect time to learn it.

I think my agar plates are clean now (I will take pictures during the next transfer). However, I'm worried about my genetic choices. They must be supported by test runs and that's a lot of work and time to be spent...

The alternative I was mentioning is to let the nature to do its natural selection thing in the jar inoculated with MS, and then clone the best looking fruit body. It seems to be much less work...

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Re: Strain Isolations on Agar, Pics and Questions [Re: Piezo] * 1
    #23587735 - 08/28/16 05:42 PM (7 years, 6 months ago)

if you are worried about "too much work" then definitely avoid isolates lol in the past couple of months i have been through about 800 plates and still dont have any true isolates lol... which is why many of us end up making LI or the like out of good looking clean plates at some point (limited MS) and inoculating with it, fruiting it, and make some clones to play with while we are working on isolates

2. right, thats another fascinating dynamic of MS, all the different aspects which can be influenced by environment, genetics, survivability, etc. I often wonder how much of the same genetics are present in different sectors of a MS plate, and also between fruits of a MS grow. i have started to suspect that some strains are really good at intermingling with other strains, making it very hard to isolate either of them until they split up for some reason, or grow different directions on agar. i know there is a TON of genetic diversity within a MS grow, but i wonder how much genetic similarity there is, ie. how many sectors share strains, and how many fruits are from the same strain and combination of strains. truly fascinating stuff

3. what do you mean by inoculating a jar with a print? one or both of us is confused haha... btw most prints are dirty, rare for them not to be, thats why we use agar to clean up the culture rather than making a dirty spore syringe, etc, and wondering what went wrong when our jars smell sour or turn green.  ahhhh rereading it i think i am starting to understand your post a little better, just a little strangely worded. This may be exactly what you are doing, i am just stating it clearly for the benefit of you and anyone working on something similar who might come across the thread

It can actually be tricky sometimes to tell if plates are clean. there is the obvious stuff to look for, but then there is also bacteria that can get down inside the culture in a way that it does not grow separately and does not distinguish itself from the myc on agar. this can be damn tough to catch before jars contam. definitely post pics and we can tell you if we see anything obvious though

but you shouldnt be worried about your genetic choices at this point, thats for sure. really, you havent made any genetic choices yet, since there could be hundreds to thousands of strains present in each initial ms transfer, and you have no way to distinguish one from the other, much less select one. At this point you should just be worried about transferring clean growth to new plates, then let it grow out and do it again, until you are relatively sure its clean. then let a clean plate colonize fully and make a LI, or go agar to grain if you are more patient than me

with MS there really isnt any genetic choice involved, it is 100% luck of the draw

you could of course keep on doing transfers until sectors start to show, and then transfer the aggressive rhizo sectors, and then use that to make LI, and that would likely be more aggressive/rhizo than a less limited MS. but less than 8-10 transfers, it is not really limited at all, just MS.

also bear in mind, you could make an LI from a limited MS rhizo plate and then for some reason the rhizo myc could be totally outcompeted by some tomentose or linear myc that happens to be tangled up in the rhizo myc that was invisible to you during the transfers. there are just so many variables with MS

you could also make a limited MS culture, make a bunch of spawn from it, make 10 monotubs, and have all 10 monotubs turn out ENTIRELY different. some could be albino, some could look like dinosaurs, some could look like pug dogs, but they will most definitely all be different. even the fruits within the same tub will be different, with a potentially high degree of genetic diversity no matter how limited the culture seemed to be, since it was still MS. this is why i say you should not worry about your genetic choices because you really havent made any yet

cloning desirable fruits is definitely the fastest and easiest way to a culture that is more predictable than MS, and more likely to obtain full canopy flushes. take multiple clones and test them separately

the amount of work involved in creating and testing isolates makes it more of a specialized thing, definitely not the easiest. but for those of us who want to collect truly meaningful data points, it is essential


EDIT:

btw i had a question for yall vets:

is there any reason to isolate strains found within a clone? for example, i took several clones from a recent AA+ grow, and they are grown out enough that i could transfer them today. ive noticed several sectors on each clone culture, some rhizo some not so much. is there any reason or benefit in trying to isolate strains from each of these sectors? and if so should they be tested as isolated strains or as subsets of a clone? how do yall usually handle clone plates showing sectors?

im still pretty new to both agar and clones, so dont assume i know anything, assume i am a dumbass and school me  :dunce:


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Edited by c10h12n2o (08/28/16 06:06 PM)

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Re: Strain Isolations on Agar, Pics and Questions [Re: c10h12n2o]
    #23588589 - 08/28/16 10:00 PM (7 years, 6 months ago)

In response to Pleze:

2. Unless you are using an isolated strain, yes, you will get various results throughout a grow, especially if it is multi-spore
3. If you are trying to transfer healthy mycelium away from contams then you are using agar for a great purpose
4. Yeah, if you want guaranteed genetics then you will have to isolate a bunch of strains, test them all, and choose
5. Yeah, that's why I was wondering is isolating is generally worth it when you can clone easier.
My guess to this problem is clone once you grow and if you want to then start isolating as a side projectI. I just wonder how likely it is to be worth it


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Edited by TedTheHighlighter (08/28/16 10:01 PM)

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Re: Strain Isolations on Agar, Pics and Questions [Re: TedTheHighlighter]
    #23588801 - 08/28/16 11:32 PM (7 years, 6 months ago)

I'm not too into isolating much so I can't really answer your specific questions on the clone isolation but
I would like to tell you that you don't have to fruit out a whole mono to trial different isolates...check out Violets small container grows or pasty's invitro pods too. Doing small grows in containers like the ones shown in these teks makes testing out different isolates for potential winners much easier than doing a bulk grow.:thumbup:


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Re: Strain Isolations on Agar, Pics and Questions [Re: MMG]
    #23589072 - 08/29/16 04:32 AM (7 years, 6 months ago)

you shouldn't test your cultures on cased grains in cups if you plan to do monotubs with them, they'll behave very differently on cakes,subs,bottles etc.

so you might be throwing away a kickass culture just because you didnt grow it the way it wanted to.

get to know your cultures, what's their likes and dislikes, thats how you can really take advantage.

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Re: Strain Isolations on Agar, Pics and Questions [Re: spacechildo]
    #23589751 - 08/29/16 11:33 AM (7 years, 6 months ago)

thanks for the info MMG!

that was exactly my thought space, very succinctly put

when i first started this project my plan was to test each in its own little bucket a la otto, which has actually been working alright, but i figured since my main method will probably be monotubs for a bit (until i get my automated room built) so i didnt want to miss out on a killer culture that just happens to need a 3" substrate depth or quits the job if we dont give it straw and manure on a trashbag-lined platter

EDIT: here is a pic of one of those buckets btw:


makes sense to test cultures the same way you would ideally fruit them

that said, we are talking a little bit theoretically. mini grows to test isolates could be very useful if someone is limited on space, and i think the odds of finding an isolate that does great in a monotub but sucks on smaller methods is probably VERY small. but since that is one more set of variables i can control for, im gonna be testing in monotubs & bulk trays

Also, for the guy who was talking about cleaning up cultures on agar, here are a few cultures i am in the process of cleaning up, a p. microspora and a cordyceps militaris. these plates are on their second transfer, and are still pretty dirty.



you can see in the cord. m. especially, the line of bacteria all the way down the plate and all the way around in a ring. probably from liquid rolling around. In these, i am taking transfers from the leading edge of the cleanest looking, healthiest myc and transferring to new plates until there is no more visible bacteria, then will make LI. this is the same thing i would suggest doing to clean up a dirty spore print


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Edited by c10h12n2o (08/29/16 03:22 PM)

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Re: Strain Isolations on Agar, Pics and Questions [Re: c10h12n2o]
    #23589860 - 08/29/16 12:26 PM (7 years, 6 months ago)

Quote:

c10h12n2o said:
if you are worried about "too much work" then definitely avoid isolates lol in the past couple of months i have been through about 800 plates and still dont have any true isolates lol... which is why many of us end up making LI or the like out of good looking clean plates at some point (limited MS) and inoculating with it, fruiting it, and make some clones to play with while we are working on isolates




Now that I'm pouring agar with stackable PP5 containers, i can do two dozens of plates in one batch. They fit better than plasty plates in my tiny pressure cooker. Short term project is a bulk grow, so isolates will wait.


Quote:

c10h12n2o said:
2. right, thats another fascinating dynamic of MS, all the different aspects which can be influenced by environment, genetics, survivability, etc. I often wonder how much of the same genetics are present in different sectors of a MS plate, and also between fruits of a MS grow. i have started to suspect that some strains are really good at intermingling with other strains, making it very hard to isolate either of them until they split up for some reason, or grow different directions on agar. i know there is a TON of genetic diversity within a MS grow, but i wonder how much genetic similarity there is, ie. how many sectors share strains, and how many fruits are from the same strain and combination of strains. truly fascinating stuff




Yes, with this observations, the genome evolution dynamics are also interesting. At the level of a single line of descent, we have haplogroups, groups of genes inherited together. At the "social" level we may have co-evolved strains that grow and sporulate together in carpophores.
A bit more than half of the pack needs to sporulate to pass most of the genetic diversity.  This only works with the hypothesis of isolated groups, but strains are not species. So if this is real, there must be some mechanisms acting against the genetic drift, like mycelium segregation or the fact that some strains overpower others for a given environment.

I'm also curious about the redundancy of strains in sectored growths. For example, two strains intertwined (A+B) could grow more than two sectors if they separate in a stripes (CW: A, B, A, ..., B). Even with no redundancy, the combined strains could grow in a third sector : (A, A+B, B). With more strains the number of combinations explodes.

I also stand to be corrected here, I'm on my third agar project, and the first two were not really successful...


Quote:

c10h12n2o said:
3. what do you mean by inoculating a jar with a print? one or both of us is confused haha... btw most prints are dirty, rare for them not to be, thats why we use agar to clean up the culture rather than making a dirty spore syringe, etc, and wondering what went wrong when our jars smell sour or turn green.  ahhhh rereading it i think i am starting to understand your post a little better, just a little strangely worded. This may be exactly what you are doing, i am just stating it clearly for the benefit of you and anyone working on something similar who might come across the thread




Sorry about that, not native, still learning. :crazy:
Yes, I'm currently using agar to clean MS culture, but considering to try MS in small cakes for making clones directly. As said by MMG, it should be possible to contain contaminations with isolated growth (bottle teks). Then the clone is passed through some plates to clean it or to isolate strains.


Quote:

c10h12n2o said:
with MS there really isnt any genetic choice involved, it is 100% luck of the draw

you could of course keep on doing transfers until sectors start to show, and then transfer the aggressive rhizo sectors, and then use that to make LI, and that would likely be more aggressive/rhizo than a less limited MS. but less than 8-10 transfers, it is not really limited at all, just MS.




Maybe MS mycelium do less sectoring than clones because its strains weren't subjected to bulk grow and fruiting ?  I have not seen rhizomorphic growth yet in my limited MS plates. It's seems easier to get rhizomorphic from clones. Plasticity and/or selection ?

As you said, changes in characters also happen with liquid inoculants. The environmental pressures from the spore germinations until fruiting in bulk could be decisive on the results, maybe more than the original set of genetics.
That's the exciting part in getting contaminations and see them eaten by the cubensis mycelium.


Quote:

c10h12n2o said:
Also, for the guy who was talking about cleaning up cultures on agar, here are a few cultures i am in the process of cleaning up, a p. microspora and a cordyceps militaris. these plates are on their second transfer, and are still pretty dirty.



you can see in the cord. m. especially, the line of bacteria all the way down the plate and all the way around in a ring. probably from liquid rolling around. In these, i am taking transfers from the leading edge of the cleanest looking, healthiest myc and transferring to new plates until there is no more visible bacteria, then will make LI. this is the same thing i would suggest doing to clean up a dirty spore print



Ok thanks for the illustrated example.

Practical question : sometimes i transfer aerial mycelium with a needle without touching the agar surface. It's a bit tricky to peel of the mycelium from the needle, but I think it transfer less bacteria. Am i the only one doing this ?

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Re: Strain Isolations on Agar, Pics and Questions [Re: Piezo]
    #23595825 - 08/30/16 10:40 PM (7 years, 6 months ago)

Quote:

Piezo said:

Now that I'm pouring agar with stackable PP5 containers, i can do two dozens of plates in one batch. They fit better than plasty plates in my tiny pressure cooker. Short term project is a bulk grow, so isolates will wait.




if i am not mistaken, the definition of a pasty plate" would be using commonly available PP5 containers and no-pour agar instead of petris (which arent available to some people) and pouring (which some people either struggle with or would rather avoid), i think thats the main idea he was trying to introduce people to with that tek. (your stackable pp5 containers are probably still pasty plates)

But for what you are trying to do, i would suggest doing what i did: streak some plates with spores, transfer healthy myc to new plates, let it grow out a bit, and either do a few more transfers or go ahead and make a LI (liquid inoculant) using the fresh plate once it is colonized a bit. then use the LI however you like, and clone some fruits. after that, isolation work is a matter of preference, sounds like clones would be best suited for your purpose

Quote:


Maybe MS mycelium do less sectoring than clones because its strains weren't subjected to bulk grow and fruiting ?  I have not seen rhizomorphic growth yet in my limited MS plates. It's seems easier to get rhizomorphic from clones. Plasticity and/or selection ?




something like that lol... the first few plates in a series of isolation transfers (starting with MS) are almost never rhizomorphic in my experience. it is mainly a matter of the jungle of strains present growing on, through, between, and beside each other. I am not sure why such large numbers of strains group together at this point and form circular growth (rather than, say, 400,000 little sectors), where a smaller number of strains would likely show clear sectoring

I am working on isolating strains from several races. i started out with 4 races of cubes and 1 pan, and have since started 4 more races. out of these, only 2 races have shown rhizomorphic growth at all, even 12 transfers in most of them are either linear growth or tomentose, and often perfectly round and maybe a few cells thick, just wimpy.

on the other hand, by about the 4th or 5th transfers, those some of the plates from those 2 races started showing rhizo sectors, and i started focusing on transferring those. after a few more transfers the plates in those series were very aggressive with several rhizo sectors each, totally different than the type of growth i was seeing on the earlier plates and on other races i am working on

when i started using these very rhizomorphic plates to make LIs, my colonization times went from weeks to days (one of these monotubs literally colonized in 4 days, where my initial monotub of the same race took about 2 weeks). if i had only been working with a few plates/races, i might have never gotten rhizo growth, or i might just not have gotten it yet. you might have to transfer a hundred times just to see clear sectors :/ thats why it makes sense to dilute spores

the reason why it is easier to get rhizo growth from clones is probably because of the MUCH smaller number of total strains present in the culture/biopsy

i think you are severely underestimating the number of strains present (as i did, and might still be)

Quote:

That's the exciting part in getting contaminations and see them eaten by the cubensis mycelium.




i agree, they are fun to watch, i used to keep every contammed sample just to watch it.

BUT make sure you separate it from your serious projects, and dont get attached to the jars/bags/dishes.

Focus on learning how to obtain clean cultures and figuring out the point of failure and resolving it, that would benefit you the most in the long run. you want to be sure a culture/spawn is 100% clean before using it for g2g, or betting on it for anything serious. personally, i want to do everything i can to avoid having a tub full of rotten feet smell, and clean spawn is the least we can do to ensure success

Quote:


Practical question : sometimes i transfer aerial mycelium with a needle without touching the agar surface. It's a bit tricky to peel of the mycelium from the needle, but I think it transfer less bacteria. Am i the only one doing this ?




why would you do that? i did it a few times, in my early plates where i wanted to see rhizo myc soooooo bad but there just wasnt. any. there.

i was able to fool myself into thinking "hey, there in the middle, that looks rhizomorphic! that big fuggin thing coming out of the top of the agar i transferred!"... but i think i was bullshitting myself. there was no rhizo growth on those plates

what there was was a piece of retarded myc that grows straight in the only direction without agar. aerial myc is not a good quality, certainly not a feature you should try to transfer.

also, if you are doing what i was doing, then you are probably grabbing aerial myc that is very near the origin of the dish's growth, maybe even growing off of the agar you transferred, not the agar you transferred to. if this is the case (as it was for me), you are misunderstanding what you are trying to accomplish by using agar. the whole point is to have controlled growth across the surface of the agar so that you can distinguish various characteristics and transfer desirable sectors. if you are pinning growth from your initial transfer, that did not even grow across the agar at all, it throws away all the advantages of using agar for this purpose

at least that is what made sense to me, so i stopped doing that


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Edited by c10h12n2o (08/30/16 10:42 PM)

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Re: Strain Isolations on Agar, Pics and Questions [Re: spacechildo]
    #23595880 - 08/30/16 11:03 PM (7 years, 6 months ago)

I see your point there. bottle growing aren't just cased grains, when I do bottles I spawn at around 1:5 or so ratio (spawn to sub) mixing the spawn together with the bulk sub (cvg) and then I case them.
You can eliminate some of the unknowns or type 1 errors (discarding a culture given that it was a good one) by doing multiple bottles, say 4 or 5 per isolate.
Still less hazzle than doing a mono and it will give you a good idea of how the culture responds. if 3 or 4 out of the 5 show good results then you can say with a good probability that it's a keeper or one to keep testing on.
TBH I hadn't heard of mushrooms being picky over how much sub they're fruiting on. :hehehe: types of sub, and growth parameters yes (but space - no)
if you're doing the same cvg sub for bottles or a mono (keeping the sub the same) then I don't see how it would affect performance given that fruiting parameters are kept on check, illuminate me if I'm wrong here.
edit: sub depth probably does play some role in the development (for example pans liking more shallow subs) but in general for cubes I don't think it would matter much (a good culture will perform well regardless), again this is my 2c

edit 2: at OP - that's so cool that you're cultivating p. microspora, do you plan on using it to digest plastics or just doing it for the novelty?


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Edited by MMG (08/30/16 11:41 PM)

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Re: Strain Isolations on Agar, Pics and Questions [Re: MMG]
    #23598450 - 08/31/16 06:17 PM (7 years, 6 months ago)

great info MMG :smile:

i actually just harvested a otto bucket today, one HUGE cluster! :laugh:



im about to clone one of those monsters that poked its head out  :clone:


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Re: Strain Isolations on Agar, Pics and Questions [Re: c10h12n2o]
    #23598485 - 08/31/16 06:28 PM (7 years, 6 months ago)

damn! that's a nice cluster right there. is this one of the isolates you've been working on or MS? looks like the hard work is paying off! :rockon:


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Re: Strain Isolations on Agar, Pics and Questions [Re: MMG]
    #23598554 - 08/31/16 06:44 PM (7 years, 6 months ago)

sorry, i forgot to answer your question about p. microspora: yes, i have various projects in mind! :laugh: so excited about that one. i know it is stupid, but i have been fantasizing about making it saltwater tolerant, or building some kind of platform that would allow it to digest some of those plastic islands in the ocean... i know its dumb for at least a dozen reasons, but its a thought haha... also plan to experiment with various trash-to-food projects :smile:

thanks buddy!! that bucket is actually one of the first things i did when i started this project, was planning to work out a good system for testing isolates and figured i would practice with MS and clones. the plan was to use these little pails with 1qt of spawn, and fruit them like otto or monos. they ended up taking a lot longer to colonize than i had hoped, but this AA one finished faster than the others

so no, that bucket is just MS, LI wasnt even really made from a good looking plate if i recall... i cant wait to start fruiting some of the AA tubs i spawned 5 and 6 days ago, they are looking GREAT! much more rhizo, much more limited genetics, and the monotubs were solid white in 5 days!!! crazy stuff.. i almost fruited them today, but there is one tiny spot on each that is still a little uncolonized, about the size of a pinhead, and i figure letting it colonize another day or two certainly wont hurt

I plan to make 4 big spawn bags today from an AA clone culture from my last monotub, and i am about to clone one of those monsters in that giant cluster :laugh:


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Re: Strain Isolations on Agar, Pics and Questions [Re: c10h12n2o]
    #23599114 - 08/31/16 09:18 PM (7 years, 6 months ago)

Quote:

c10h12n2o said:
sorry, i forgot to answer your question about p. microspora: yes, i have various projects in mind! :laugh: so excited about that one. i know it is stupid, but i have been fantasizing about making it saltwater tolerant, or building some kind of platform that would allow it to digest some of those plastic islands in the ocean... i know its dumb for at least a dozen reasons, but its a thought haha... also plan to experiment with various trash-to-food projects :smile:





Doesn't sound dumb to me! I think it would be awesome for you to dig into what you can do in mycology, especially if you look into beneficial stuff like your ideas


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Re: Strain Isolations on Agar, Pics and Questions [Re: TedTheHighlighter]
    #23599226 - 08/31/16 09:53 PM (7 years, 6 months ago)

I second that, no idea that is pro-world/life is dumb in my book!
speaking about remediation I wanna restart my efforts on decomposing cigarette butts (cellulose acetate fiber) with oysters. I plan on "training" the myc by slowly exposing it to the filter material (along with grain) until I secure a good candidate for the application...one of the things I was debating was the feasibility of using the mushroom mycelium directly on dirty filters which is probably null (proper sterilization is most likely necessary) so I don't know how cost-efficient or sustainable it would be. It'd have to be like kilos of cigarette butts to justify the sterilization but it might be worthwhile...guess I'll have to get to work on it and report back!
so wait, the decomposed material from the P. Microspora is edible? that's pretty crazy haha
in the case of the cig butts, given that they're carcinogenic (a tleast when utilized) the mushrooms fruits (if any are produced at all) are non edible or deemed unsafe for human consumption. I'd be happy to just decompose the waste at a much faster rate than it does in landfill or even worse in the ocean for the case of cig waste. then composting it of course
back on topic...@op, that's the thing with isolates man, sometimes (at least in my case) isolating yielded worse or basically the same results that an MS culture did so I haven't done too much isolating because for my purposes I don't see it as a necessity. if it's clean I'm happy :biggrin: and if I get a big ass cluster like the one you did then awesome, just clone some tissue from it and isolate a bit from there and re-test again to see how it performs.


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Re: Strain Isolations on Agar, Pics and Questions [Re: MMG]
    #23600110 - 09/01/16 06:45 AM (7 years, 6 months ago)

@MMG
Sounds like a great idea. Report back on that project if you start it up


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Re: Strain Isolations on Agar, Pics and Questions [Re: TedTheHighlighter]
    #23619533 - 09/06/16 07:58 PM (7 years, 6 months ago)

So does weetie have any updates for us?


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Re: Strain Isolations on Agar, Pics and Questions [Re: TedTheHighlighter]
    #23620705 - 09/07/16 04:25 AM (7 years, 6 months ago)

Quote:

TedTheHighlighter said:
So does weetie have any updates for us?



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Re: Strain Isolations on Agar, Pics and Questions [Re: Supalemonhaze]
    #23637272 - 09/11/16 09:53 PM (7 years, 6 months ago)

Does the OP have updates?


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Re: Strain Isolations on Agar, Pics and Questions [Re: TedTheHighlighter]
    #23657331 - 09/18/16 11:13 PM (7 years, 6 months ago)

hey guys sorry for the late reply, had a small series of minor emergencies come up with work and family and health, been trying to get caught up. meant to update this a while back, i will post a bunch of relevant pics shortly as soon as i have time to organize em

Quote:

MMG said:
so wait, the decomposed material from the P. Microspora is edible? that's pretty crazy haha
in the case of the cig butts, given that they're carcinogenic (a tleast when utilized) the mushrooms fruits (if any are produced at all) are non edible or deemed unsafe for human consumption.



yep, apparently they are! here is a page where someone has made a really cool little self contained system. it actually looks pretty sweet, a very different approach than most projects ive seen


also, i am curious about the oysters picking up toxins, i know they are really good about concentrating heavy metals (or bad, depending on whether you are trying to remove heavy metals from a substrate or eat mushrooms), but in Mycellium Running Stamets mentions a project where the government was testing several ways to clean up oil spill type stuff, testing some bacteria, algae, and a few other means of breaking down the hydrocarbons. Stamets said that at the end of the trial, the other piles were rotten, but the oyster pile had produced a mountain of beautiful fruits that were safe to eat. between concentrating heavy metals, and what you are saying about cigarette toxins, it seems amazing (or questionable) that Stamets petro oysters were really safe to eat

Quote:

MMG said:back on topic...@op, that's the thing with isolates man, sometimes (at least in my case) isolating yielded worse or basically the same results that an MS culture did so I haven't done too much isolating because for my purposes I don't see it as a necessity. if it's clean I'm happy :biggrin: and if I get a big ass cluster like the one you did then awesome, just clone some tissue from it and isolate a bit from there and re-test again to see how it performs.




oh goodness! im worried about exactly that! i often wonder if a well organized culture of compatible strains might perform than certain isolates. i guess it just underscores the need for extensive testing and good notes huh

I did take several clones that are lookin good :smile: I have about 10 i am working with right now, isolating some out further, and have 2 particular clone cultures currently colonizing tubs, and a TON of grainspawn colonizing from 2 different clone cultures. one of the clone cultures ive inoculated with comes from one of those huge clusters, the other from a pin, and about 8 others im isolating further. Im really excited about testing some of the clones and isolates!! i look forward to testing clone cultures that have most of the strains present in the initial biopsy vs those that have been further isolated (the rhizo sectors)

Quote:

TedTheHighlighter said:
Does the OP have updates?



sorry for the delay my friend... life got a little crazy! but i have stayed on top of my projects though

I have been through a little over 1000 plates since i began this project, and boy have i learned a lot + destroyed a few of my delusions hahaha...

when weetsie started his project, i did the same thing, 20 different initial plates, with varying amounts of spores on each one (some had a ton like weetsies, some had far less, some were streaked, some were dilluted), using 3 races of cubes and one p. sub..

Out of all 20 different series, i dont think any of them are anywhere near an isolate, even as we speak. The different series are on between their 9th and 13th transfers, and there are multiples of quite a few from each generation after 3rd. RR might be able to get isolates in just a couple of transfers, but apparently i have a LOT of learning left to do before i can pull off something like that haha....

it has been an informative experience though, thats for sure. like someone mentioned at various points before (i think it was bodhi and supalemonhaze and space), sometimes you will have a VERY rhizomorphic culture that looks like there could be "at most 1-3 strains present", but you would be totally wrong. made me want to scream the first dozen or so times i transfered a tiny little sliver from what i believed would contain only 1 highly rhizo strain, only to have it turn completely tomentose and have a 8+ sectors on the next plate  :facepalm:

I have also been working with lots of clone cultures in addition to the isolations im working on, and they have been fascinating. I expected to see 1-3 sectors in clones, but to my surprise, there are often many more. and when i transfer a rhizo sector from a clone culture, expecting it to be an isolate, it often turns cottony, or shows many previously unseen sectors

so yeah, every time i have thought i had an isolate, further transfers have revealed that there were many, many strains present

I will update with a bunch of pictures, from different parts of the process, and will post updates as i get closer to isolates

lol hopefully weetsie is having better luck than me, and if so i hope he enlightens me!! i appreciate everyone who has contributed info and advice, you guys rock

btw, i think it was supalemonhaze who told me that trying to work with isolating multiple races at the same time is a hellacious amount of work, and BOY WAS HE RIGHT. this project quickly became like a job, and now that i am working with 7 races of cubes and 6 other species, it is downright ridiculous. ive been keeping up to the best of my ability, but i certainly would not recommend working with this many different races at a time, even if you do have a ridiculous work ethic, it ends up being an insane amount of work (and if you just want predictable crops the same end can be achieved a lot easier; me personally though, i cant wait to start collecting some meaningful datapoints testing isolates)

EDIT:
BTW, i meant to ask, do yall have any good tips for disposing of old plates? i am cleaning up, and have 2 black trashbags full of plates that are from earlier in these transfers. that is a TON of plates, most are clean but i just have no use for them, since i am much later in these isolation processes now. it seems like a VERY sketchy 2 bags of trash, that many labeled petri dishes might get the wrong kind of attention if it was ever noticed.

obviously, would want to make sure no mail or anything with my name, address, etc is in there with it, and dispose of it at a random dumpster, id burn it if i wasnt in a city. i usually throw away small amounts of sketchy stuff through regular trash pickup, but this many dishes looks seriously sketchy. any advice?

much obliged my friends


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

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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Edited by c10h12n2o (09/19/16 01:46 AM)

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OfflineTedTheHighlighter
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Re: Strain Isolations on Agar, Pics and Questions [Re: c10h12n2o]
    #23658046 - 09/19/16 07:52 AM (7 years, 6 months ago)

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


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Re: Strain Isolations on Agar, Pics and Questions [Re: TedTheHighlighter]
    #23658062 - 09/19/16 08:01 AM (7 years, 6 months ago)

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?


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Re: Strain Isolations on Agar, Pics and Questions [Re: Mad Season]
    #23658744 - 09/19/16 01:12 PM (7 years, 6 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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Re: Strain Isolations on Agar, Pics and Questions [Re: Supalemonhaze]
    #23658750 - 09/19/16 01:13 PM (7 years, 6 months ago)

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

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Re: Strain Isolations on Agar, Pics and Questions [Re: bodhisatta]
    #23658910 - 09/19/16 02:02 PM (7 years, 6 months ago)

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

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Re: Strain Isolations on Agar, Pics and Questions [Re: Mad Season]
    #23659375 - 09/19/16 04:54 PM (7 years, 6 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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Re: Strain Isolations on Agar, Pics and Questions [Re: c10h12n2o]
    #23659391 - 09/19/16 04:59 PM (7 years, 6 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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Re: Strain Isolations on Agar, Pics and Questions [Re: bodhisatta]
    #23659432 - 09/19/16 05:16 PM (7 years, 6 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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Re: Strain Isolations on Agar, Pics and Questions [Re: spacechildo]
    #23659448 - 09/19/16 05:20 PM (7 years, 6 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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Re: Strain Isolations on Agar, Pics and Questions [Re: spacechildo]
    #23660024 - 09/19/16 08:55 PM (7 years, 6 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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Re: Strain Isolations on Agar, Pics and Questions [Re: c10h12n2o]
    #23660061 - 09/19/16 09:10 PM (7 years, 6 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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Re: Strain Isolations on Agar, Pics and Questions [Re: Boogieman47]
    #23660118 - 09/19/16 09:39 PM (7 years, 6 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


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


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

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