agar and printing questions

[polyarcturus]

well my ghetto agar tek, worked and well at that! got some uncontaminated B+ and some contaminated GT which leads me to believe they where contaminated in the syringe which makes sense because GT has had the most problems with contamination, fortunately i have 2 clone jars that are uncontaminated.

so whats the best tool to pull some of the good mycelium out of the contaminated GT jars and into fresh agar jars?

how do i turn one of the B+ into some LC out of the agar?(a single yellow spot has formed amongst white, does not look alive assuming its a metabolite?)

how to take some spore prints? would it matter if the fruit(thinking about GT) i used came from the contaminated jar i got to fruit?(pulled about a 1g of dry mushrooms so far, right after veils broke)

 

[canndo]

well my ghetto agar tek, worked and well at that! got some uncontaminated B+ and some contaminated GT which leads me to believe they where contaminated in the syringe which makes sense because GT has had the most problems with contamination, fortunately i have 2 clone jars that are uncontaminated.

so whats the best tool to pull some of the good mycelium out of the contaminated GT jars and into fresh agar jars?

how do i turn one of the B+ into some LC out of the agar?(a single yellow spot has formed amongst white, does not look alive assuming its a metabolite?)

how to take some spore prints? would it matter if the fruit(thinking about GT) i used came from the contaminated jar i got to fruit?(pulled about a 1g of dry mushrooms so far, right after veils broke)

Somewhere in this area is a complete series of teks on the use of agar that I wrote. I don't know where it is and I seem to recall that there was a period where posts were lost so it might not be as complete as I imagine.

If you want to isolate your best instruments are wax sculpting tools. You must be very timely and you cannot use a hood. Still air in this situation is your only friend. The entire point of agar is it's two dimensionality and there is where you get to play. Take the smallest bit of mycelium you can from the side most opposite and furthest away from the contamination. Try your DAMNEST to get that bit of mycelium before your contamination begins it's first round of sporulation. If you cannot do so you can lay another "disk" of agar over your established one forcing all growth to be linear. there will be no arial growth and many contaminations will not spore without that ability. Then you have only to "race" your good and bad mycelium to a point where you can easily cut a bit of the good stuff from this mycelial "sandwich".

You (as I just wrote in another thread) have only to get a single hyphae into your liquid culture and agitate that culture with a mag stirer in order to get a superior liquid culture innoculant solution. You can also grow out a decent petri dish, and dump the entire dish into a sterile blender with sterile waterin it. Pulse that sucker for no more than about 20 seconds - then if you wish you can let it sit. Draw your solution up into a clean syringe - the larger agar particles will have fallen to the bottom and the smallest of the hyphae will still be in suspension. Doing your work this way is ultra efficient as even a single ml of your solution will heavily innoculate a jar or bag of grain. There is no time wasted waiting for spores to germinate and your result is most likely a monoculture. In general monocultures will yield far more than multi culures, they will fruit in a concerted way and your flushes will all be simultaneous from flush to flush. You sir, are on to the real secrets of getting the very best from your growing endeavors and I assure you that if you bring this sort of thing to fruition if you don't mind the pun - you will be astounded at your results. Some of the old pictures I posted of ultra dense, concerted flushes were done this way.



One more thing - alcohol sterilization or electric heating sterilization only - open flame will agitate your environment and tend to put those contam spores into the air over your work.

 

[polyarcturus]

thank you so much for the info i will immediatly get to work on my agar culures, unfortunaly i do not have a blender or a mag stirrer, but i can make things work in place i beleive, i will get started timmorow as to prevent further contam on my GT and i will convert a B+ agar to a LC in syringe to inoc some jars.( im gonna do a little comparision for myself too, piece of agar vs the LC)

 

[testtime]

thank you so much for the info i will immediatly get to work on my agar culures, unfortunaly i do not have a blender or a mag stirrer, but i can make things work in place i beleive, i will get started timmorow as to prevent further contam on my GT and i will convert a B+ agar to a LC in syringe to inoc some jars.( im gonna do a little comparision for myself too, piece of agar vs the LC)

While those tools (sterile blender and mag stirrer) are nice, they are not required. A few BIG and a few SMALL pieces of glass in a quart jar with a shakable lid (RTVed latex stuffed with poly in my case(bought at surgical supply, they are the same tubes used for slingshots and exercise strength pulling), binder paper clamp at the base when shaking).

When you prepare your LC jars create 2 sets with 2 different formulas, along with some grain jars to be used as testers.

In the primary set use a karo recipe, going light on the nutrients. Do your initial myc drop into that, the SMALLEST bit possible. That will take a couple of days to start growing out. Give it a few shakes a day. The karo LC will be clear and you will at least get a chance of seeing any contams. In a couple of days (really, you only need the tiniest of growth) use a sterile needle with sterile prep,shake the LC jar hard, suck a bit of liquid from the karo LC.

Don't worry if you don't see any myc in your needle, it is in there. Also, don't worry about slow karo LC growth, you WANT it slow.

In the secondary set of jars use LME/DEX/a bit of nutritional yeast powder. Noc up those jars with the tiny bit of karo LC. Also noc up a few tester grain jars. The issue here it is next to impossible to identify certain LC contams, and you really don't know if it is any good until you do a couple of jars and only see white myc growth.

The reason for the LME/DEX jars is because they grow a LOT faster than karo jars. And it seems (someone else can prove/disprove) that the resulting myc goes through grain faster than the karo based one. But they are tough to see into, so you want to knock out the possibility of the initial contams in the karo LC step. So you can have a couple of LME/DEX jars ready in a week (a shake a day), capable of noccing up hundreds of jars, and you will be a bit more confident you are not about to blow the whole run on contams.

You might want a couple of tester jars before committing to a large run from the LME/DEX jars. Good idea, but not required, at least for me so far.

After the 1st week, no shaking is required in the LC jars. You'd rather have the growth slow down at that point. You probably want them in the fridge anyway.

A month later, when you are ready for your next pass, there might be some serious hard myc masses in the jars. A single piece of glass or a few small ones might not enough to break it up. The multiple pieces of glass that I described will be able to shred them.

If the LME/DEX goes bad (and it will sooner or later), you can go back to the karo jars (which will go bad as well, but easier to see and less nutrients to support the contams) and run the process again without taking too much of the original karo LC.

If you have an ozone heavy environment like me (not where I breath, but glove box/wall and some storage shelves), you want to minimize how much time the LC jar is in it if you used latex tubes for your GE. Ozone will eat right through that shit and leave an open hole at the top of your jar where the tube fell out.

 

[canndo]

It really isn't fair for me to talk about mag stirers and hoods - but you CAN get a conventional blender for only a 20 where the base fits into a mason jar! If you do this, then you can put your blender blades in the mason jar and tighten it down with the standard mason ring - if you sterilize the entire rig you can dump your dish in there quite easily and it doesn't cost as much as a metal blender rig.

 

[testtime]

It really isn't fair for me to talk about mag stirers and hoods - but you CAN get a conventional blender for only a 20 where the base fits into a mason jar! If you do this, then you can put your blender blades in the mason jar and tighten it down with the standard mason ring - if you sterilize the entire rig you can dump your dish in there quite easily and it doesn't cost as much as a metal blender rig.

Yeah, but you will have to buy the base for each LC jar you have, and then how do you pull from it? You have to open it up.

I will do almost anything that allows sterile needle transfers over any other type of transfer (petri slices, g2g), which in this case means I need the jar lid with the injection port.

And those spinning bases add up. When I do LC, I do 3 primary and 3 secondary for each myc I'm working with (yes, I'm paranoid, I always want to cover my ass), so I might have 4 mycs (at the moment) x 6 = 24 jars. That's a lot of blender bases (not the mechanics, I know you didn't mean that) for me to buy. Maybe fine for someone with a few less jars.

But I AM quite envious of your resources and experience!

 

[canndo]

Yeah, but you will have to buy the base for each LC jar you have, and then how do you pull from it? You have to open it up.

I will do almost anything that allows sterile needle transfers over any other type of transfer (petri slices, g2g), which in this case means I need the jar lid with the injection port.

And those spinning bases add up. When I do LC, I do 3 primary and 3 secondary for each myc I'm working with (yes, I'm paranoid, I always want to cover my ass), so I might have 4 mycs (at the moment) x 6 = 24 jars. That's a lot of blender bases (not the mechanics, I know you didn't mean that) for me to buy. Maybe fine for someone with a few less jars.

But I AM quite envious of your resources and experience!

No no, in this scenario, you do not grow out your liquid culture. You use a single quart jar and dump your petri dish into it. Now you have a full quart of innoculant, plenty to do what ever you wish with. It is pretty easy to fill your 60 ml syringes from your quart in a glove box or even in a still air space. Doing it this way insures that your contamination is at most a few spores per syringe and you are doing 5 mls per innoculation, the numbers are to your advantage.

My resources come from many years of collecting and improving and.... of course... justifying. It is hard to show my wife a large handful of shaggy manes and say "see? wasn't that 150 dollar ultrasonic cleaner worth it?


btw - if you want to experiment with single spores from a dychariotic organism, you want to dilute your spore solution so that squirting a ml or so will only introduce one or two spores to your dish. the spores of this variety tend to clump together but! placing your syringe in an ultrasonic cleaner for a few minutes tends to separate the spores.

 

[canndo]

Yeah, but you will have to buy the base for each LC jar you have, and then how do you pull from it? You have to open it up.

I will do almost anything that allows sterile needle transfers over any other type of transfer (petri slices, g2g), which in this case means I need the jar lid with the injection port.

And those spinning bases add up. When I do LC, I do 3 primary and 3 secondary for each myc I'm working with (yes, I'm paranoid, I always want to cover my ass), so I might have 4 mycs (at the moment) x 6 = 24 jars. That's a lot of blender bases (not the mechanics, I know you didn't mean that) for me to buy. Maybe fine for someone with a few less jars.

But I AM quite envious of your resources and experience!

Now there is nothing better than a mag stirer for growing out your lc - the problem is that if you want to use your same quart or pint mason jars, you will have a tough time getting the mag bar to balance on the natural convex bottom of the jar. In this sort of case, your using glass and shaking every now and again is probably as good because you will need one base per jar. I always used wood screws - but they quickly get rusty. The stirers are pretty cheap though if you get used and you find that it is superior for making multi-branched hypae in a short period of time.

The mycologist's real friend is the old glass baby bottles - best for all lc, best for slants, best for storage - the tops have inserts that can hold down a fiber disk and the opening is small to begin with.


You mention pulling from your jar - there are several approaches, the one I find works best is to use as large a syringe as you can - replacing tips as you go. That way you don't have your jar open for very long. I don't like flaming the needles as it ruins the temper and tends to make the needle surface rough which binds in your ports (if you use ports).

 

[testtime]

No no, in this scenario, you do not grow out your liquid culture. You use a single quart jar and dump your petri dish into it.

Ok, understood. I don't do any open air anything (even in my incredibly poisonous ozone chamber) if I can avoid it.

I have done lots of successful center myc pull to grain jar, and lots of g2g, and they were good for the most part. But it seemed there was the inevitable random contam.

Not a lot, 3 out of 100 or so, but it was there.

Some may accept this, and SHOULD. I'm a crazy fucker. I know it. but those contams got to me (mentally).

So when I design any process, I will avoid open transfers of any type if possible.

I have yet to hit a situation where petri dishes are required (I have a sterile stack, along with all the agar and nutrients imaginable), along with the evil tape (I hate using it with gloves on, but it is in the chamber). All my initial spore needles grew cleanly and when I "cloned" straight into grain the next was fine, so I never needed to cleanup anything, and I have yet to use any of my spore prints. I'm sure I'll need to go back to prints sooner or later, so of course I WILL do what is required in the open, but not until then or some other condition forces me to.

 

[polyarcturus]

quick question, wht are my mushrooms matureing only a handfull at a time some faster than others, it feels like im harvesting a mushroom daily, this okay? what could be considered a flush 2 weeks fruiting and harvesting? so maybe let my tubs go 4 weeks?

and why did my mushroom get slime on them? i think its bacteria(no biggie so long as i dry fast) but is this from me harvesting with an unclean hand probably?

 

[polyarcturus]

alright then wish me luck made a single LC(only had one syringe shoddy try at best IMO will retry again later, threw one of the agar jars into the fridge for later to try again.

transferred a section of Gt mycelium to clean agar jars. and made a clone of a fruit in grain using inner tissue of a good fruit from GT for shits and giggle.

 

[testtime]

quick question, wht are my mushrooms matureing only a handfull at a time some faster than others, it feels like im harvesting a mushroom daily, this okay? what could be considered a flush 2 weeks fruiting and harvesting? so maybe let my tubs go 4 weeks?

and why did my mushroom get slime on them? i think its bacteria(no biggie so long as i dry fast) but is this from me harvesting with an unclean hand probably?

It used to be harvest was dictated by grabbing as the veil broke, and for sure before spores dropped. Some people think the spores cause problems on next flush, others feel the the additional growth on the mushroom after open isn't very active. Not me, at least not anymore.

The spores will never have a chance to germinate and grow, so it is essentially like dropping a dust storm on top of the baby pins. I have heard (but this could be myth repeating, I certainly never did side by side grows to be sure) that this causes some additional aborts.


It should make no difference if you are doing a hydration step since you are washing the sub clean at that point.


On the other hand, I've neglected tubs, gotten some serious spore dumps (so many spores they crusted on your finger when you wiped a lower cap with it), and then gotten great next flushes.


The less times you harvest, the less times you have disturbing the pins "next-door", so they have a better chance of survival from that aspect. So I prefer a full open flush.


But that also means leaving 1/2 size aborts to rot for a couple of days, possibly impacting others.


On the other other hand, depends on which strain/sub-strain.


PE doesn't drop, or doesn't drop much, or is barely visible when it does. AND it keeps growing, filling out the sides, adding weight and height LONG after the caps fully open, so I delay picking those as long as possible. Usually one of the mature shrooms will start to rot and deflate, and that is the signal to grab EVERYTHING at that point (even the underdeveloped ones) so I can hydrate and prime fur a full next flush, rather than pull 1 at a time over 2 weeks and delay the next flush, widening the contam window.

The slime is probably a bit of bacteria on an overly moist cap. You should attempt to air out/dry caps with an hour or so of a heavy spray to avoid it. But if it slimes a bit one day and you harvest the next, you should be fine. You do NOT want to leave that more than a day or so.

I try to use gloves and mask if I'm forced to pick anything on an ealry tub (if it seems to have a weeks left for the rest of them), but when I do a full harvest, no gloves, maybe I sneeze on the sub, don't care, then it goes in the tub for a hydration pass with a bit of bleach.