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InvisiblemushboyMDiscord
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Re: Some notes about LCs [Re: AtomHeart]
    #26996489 - 10/21/20 02:12 PM (3 years, 3 months ago)

That recovery bruh:awesomenod:


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OfflineBlue Helix
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Re: Some notes about LCs [Re: AtomHeart]
    #26996699 - 10/21/20 04:37 PM (3 years, 3 months ago)

Quote:

AtomHeart said:
Here we are, not quite 72 hours after inoculating this liquid culture into half gallon jars.  So much faster colonization than agar wedges.  This is why I use LC or at least LI.  With half gallon jars you need the speed.






Yes, it is flexible like that.  You make spawn and I make the final fruiting substrate with it.  Lots of options with a LC!


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Re: Some notes about LCs [Re: Blue Helix]
    #26996988 - 10/21/20 08:35 PM (3 years, 3 months ago)

I'm very interested in the potential of storing cultures as LC for the long term.

In this scenario, what vessel and lid setup would you use?


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Re: Some notes about LCs [Re: bongoman] * 2
    #26997136 - 10/21/20 10:55 PM (3 years, 3 months ago)

Quote:

bongoman said:
I'm very interested in the potential of storing cultures as LC for the long term.

In this scenario, what vessel and lid setup would you use?




I would use and do use Becton Dickinson's 366430 (BD 366430) 10ml, red-stopper, additive-free vacutainers (and don't just buy some random vacutainers since they differ a lot).  Here is the best deal I have ever found online for them if you consider the delivered price.

Liquid cultures at full sugar (I use 4%) can store for about a year in the refrigerator.  If you'd like to store them many years, you need to bring the sugar content way down.  One way is to add 1ml of the liquid culture to 9mls of sterile water or use a centrifuge if you happen to have one (I don't) to aspirate off the sugar solution entirely and replace it with sterile water.  Just remember the sugar concentration is what matters for storage, not the density of the LC.  You can always expand out almost any density LC later, but the sugar concentration determines if the metabolic processes stop before it drowns in its own toxic waste.  Mycelium in sterile water can be stored for about 10 years in a refrigerator according to the scientific research I found (although I cannot locate the paper right now).  As such it's probably comparable to slant oil immersion in a freezer.


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Re: Some notes about LCs [Re: Blue Helix]
    #26997154 - 10/21/20 11:15 PM (3 years, 3 months ago)

Good to know, I've been nervous about not using my spore syringes and they are nearly a year old (in a fridge).
Started some agar to get some fresh genetics to store.

I think I'll try some LC's, it's been a while but they're just so useful :grin:


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OfflineBlue Helix
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Re: Some notes about LCs [Re: Psilosion]
    #26997163 - 10/21/20 11:25 PM (3 years, 3 months ago)

Quote:

Psilosion said:
Good to know, I've been nervous about not using my spore syringes and they are nearly a year old (in a fridge).
Started some agar to get some fresh genetics to store.

I think I'll try some LC's, it's been a while but they're just so useful :grin:




It's hard to say how long a spore syringe will last.  There are several reasons for that too.  For one it's a spore syringe and syringes have a pathway to the unsterile air through the needle.  With time I suspect simple diffusion would bring in contamination.  Of course, the real question is how much contamination because spore syringes are often not sterile to begin with.  I know that because if you put a bit of that solution on agar, they'll often bacterially contaminate (not always but often).  If they were just spores in sterile water, that wouldn't happen.  Honestly, they should be selling spore vacutainers with an empty sterile syringe.  A new vacutainer is all of 20 cents, so what's the problem?  That would last a lot longer and would not allow bacteria to diffuse in it during storage.  But spore syringe vendors probably don't much mind that your old spore syringes go bad since they can sell you a new one then, right?


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Re: Some notes about LCs [Re: Blue Helix]
    #26997176 - 10/21/20 11:43 PM (3 years, 3 months ago)

Yeah it does feel like a waste not even using 1cc for agar and have 9+ cc's
left in a syringe, then having it open to more contams until you use it next.
I'd be happy with little 1cc spore syringes for single use as well as vacutainers.


Edited by Psilosion (10/21/20 11:44 PM)


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Re: Some notes about LCs [Re: Psilosion]
    #26998551 - 10/22/20 07:23 PM (3 years, 3 months ago)



And here is day 5 after LC inoculation.  All six half gallon jars are at 99% colonization.

By the one week mark they will be thickened up and white...and ready for monotubs.

You simply can't do that with A2G.


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Edited by AtomHeart (10/22/20 07:29 PM)


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Re: Some notes about LCs [Re: AtomHeart] * 1
    #26998566 - 10/22/20 07:32 PM (3 years, 3 months ago)


First time trying RGS(mixed with a little wheat) Day 3
Mexicana, pans and a semilanceata jar


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Invisible210cg
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vacutainers? [Re: Blue Helix]
    #27002519 - 10/25/20 10:31 AM (3 years, 3 months ago)

The vacutainers, do you use them in a similar way you would use agar plates? TIA:confused:


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OfflineBlue Helix
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Re: vacutainers? [Re: 210cg]
    #27002558 - 10/25/20 11:12 AM (3 years, 3 months ago)

Quote:

210cg said:
The vacutainers, do you use them in a similar way you would use agar plates? TIA:confused:




It's more like the way one might use an agar slant really in storage of a culture.  An agar plate is often used to create a 2D projection of growth in hopes to delimit the contamination or to steer desirable growth in some direction through sectoring.  You really cannot do that with a LC in a vacutainer, although you can do some of it in a slow-spun LC (I think I covered this).  Vacutainers can also be used to create agar slants too--I've done it--but I think they are bit small for that purpose really.

Here are the Becton Dickson 366430 vacutainers you want to store an LC in the refrigerator (at the best price I know - even cheaper than eBay expired ones).  There are many types of vacutainers, and I do not recommend random types.  Many have a clotting agent, separator, or preservation chemicals in them for other types of biological samples or do not have the desired kind of septum (i.e. silicone "cork" you can do sterile injections through).  These have a red silicone septum that can be beautifully repurposed to make LC wide-mouth canning jar lids, which is a second benefit that is every bit as important as for storage.


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Re: vacutainers? [Re: Blue Helix]
    #27002570 - 10/25/20 11:24 AM (3 years, 3 months ago)

Quote:

Blue Helix said:
Quote:

210cg said:
The vacutainers, do you use them in a similar way you would use agar plates? TIA:confused:




It's more like the way one might use an agar slant really in storage of a culture.  An agar plate is often used to create a 2D projection of growth in hopes to delimit the contamination or to steer desirable growth in some direction through sectoring.  You really cannot do that with a LC in a vacutainer, although you can do some of it in a slow-spun LC (I think I covered this).  Vacutainers can also be used to create agar slants too--I've done it--but I think they are bit small for that purpose really.

Here are the Becton Dickson 366430 vacutainers you want to store an LC in the refrigerator (at the best price I know - even cheaper than eBay expired ones).  There are many types of vacutainers, and I do not recommend random types.  Many have a clotting agent, separator, or preservation chemicals in them for other types of biological samples or do not have the desired kind of septum (i.e. silicone "cork" you can do sterile injections through).  These have a red silicone septum that can be beautifully repurposed to make LC wide-mouth canning jar lids, which is a second benefit that is every bit as important as for storage.




Can you reuse it, sterilizing again in PC? Do they support it?


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Re: vacutainers? [Re: diegosf]
    #27002842 - 10/25/20 02:17 PM (3 years, 3 months ago)

Quote:

diegosf said:

Can you reuse it, sterilizing again in PC? Do they support it?




They are very strong and designed to be used many times.  The septum probably will break apart after several hundred needle pokes, but that's about it.  For the price (20 cents each) they are a steal.  Of course, anything actually made for this hobby like that would cost $5 each or some ridiculous thing.  Most everything I use in the hobby is designed for something else, though.


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OfflineMLPismyOPSEC
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Re: vacutainers? [Re: Blue Helix]
    #27003253 - 10/25/20 05:58 PM (3 years, 3 months ago)

Fuckin love LC's, i think mine don't do as well as they should because of hidden bacteria. Was thinking of trying a hot pour or black tea agar to see if they will get any better.


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Re: vacutainers? [Re: MLPismyOPSEC]
    #27003320 - 10/25/20 06:27 PM (3 years, 3 months ago)

Quote:

MLPismyOPSEC said:
Fuckin love LC's, i think mine don't do as well as they should because of hidden bacteria. Was thinking of trying a hot pour or black tea agar to see if they will get any better.




I believe that bacteria does not hide very well in LCs.  I am buying a microscope and going to use syringe filters to prove that as well, but that's going to take some weeks to get set up.  The reasons I think that is because of bacteria's high motility within a liquid.  It is perfectly understandable how bacteria might hide out on an agar surface with mycelium, but I don't see how that works in a liquid culture - that is being stirred no less!  Anyone who can explain to me better how that might happen with a mycelium-based LC (please do not refer to yeast brewing stuff because it does not apply), please do.

At any rate, I've done hundreds of LCs over the years (some 20+ years if memory serves me) of a wide variety of species - many edibles, some bioluminescent, and some psychoactive.  During that time, I seldom have seen LCs fall to bacteria.  I've also seldom seen them not work well in a properly-prepared bulk substrate.

But not everyone is using properly-prepared substrates.  Many are using absolutely ridiculous tests for water content like the so-called "squeeze test" for example.  As a result, people don't have the kind of consistent success I usually do.  If you have too much water in the substrate--and almost always it's too much because the right amount doesn't seem like enough at first--then you can't expect it to work that well. 

This is the current substrate I use for nearly all psychoactives.  It was carefully water-balanced through computations to accept two 140 ml injections in two spawn bags.  Even if you use only half that LC, it should be about right.

Quote:

Manure-based Substrate (grain-to-manure wet volume ratio 1:6.5)
Manure 44 oz
WBS 10 oz
Vermiculite 1.5 L  (1.5L * 4.41oz/L = ~6.6oz)
Water       ~100 oz
~10 pounds total split into two large spawn bags




So, if you ask me, I highly doubt your LC is at fault for your yield troubles.  It's almost always the water content of the substrate the poor temperature or humidity control or something like that.  Without knowing how you grow, though, it's hard to know.  For example, I hate spawning, so I don't do it.  I can't help you with it either because I don't think it yields as well usually.  I go LC to bulk substrate directly and have for decades, and if you do that, then I can help.


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Re: vacutainers? [Re: Blue Helix]
    #27003777 - 10/25/20 10:33 PM (3 years, 3 months ago)

Just to emphasize what a LC that is already going is like, this LC was started about 36 hours ago from the 25ml of the clean one above.  As you can see, it's within a day of being plenty thick to use (actually it could be used now really even while it still needs a good 6 to 12 hours to flocculate the malt solids which will clear it up). 



Also when I open this one, there will be no bacterial smell, and that's because once you expand a good clear LC, bacteria is completely gone.  There is zero bacteria as can easily be established via microscope or agar plate.  It'll never smell bacterial because it's not there.

So I expanded 25 to 500ml within a day and a half, but you could just as easily expand and 500ml LC into several gallons in two days if you wanted.  It's really endless, which is one reason I like LCs; they are fast.


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Re: vacutainers? [Re: Blue Helix]
    #27005129 - 10/26/20 06:02 PM (3 years, 3 months ago)

I thought about asking this when i first read through this thread but now i need to; you do an LC to LC expansion? I don't see why it wouldn't work. Any rate of expansion to follow?


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Re: vacutainers? [Re: Blue Helix]
    #27005184 - 10/26/20 06:46 PM (3 years, 3 months ago)

Quote:

Blue Helix said:
this LC was started about 36 hours ago from the 25ml of the clean one above.  As you can see, it's within a day of being plenty thick to use

So I expanded 25 to 500ml within a day and a half.



LC's can grow that fast? That's an increase in mass by the rate of 20X in 36h :eek:
So theoretically if you started with 1ml LC you'd get 20ml in 36h and after 72h you'd have 400ml of LC? My LC's colonize probably 10times slower, so what's your secret? :takingnotes:


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Re: vacutainers? [Re: Mateja]
    #27006006 - 10/27/20 10:19 AM (3 years, 3 months ago)

Quote:

Mateah said:
Quote:

Blue Helix said:
this LC was started about 36 hours ago from the 25ml of the clean one above.  As you can see, it's within a day of being plenty thick to use

So I expanded 25 to 500ml within a day and a half.



LC's can grow that fast? That's an increase in mass by the rate of 20X in 36h :eek:
So theoretically if you started with 1ml LC you'd get 20ml in 36h and after 72h you'd have 400ml of LC? My LC's colonize probably 10times slower, so what's your secret? :takingnotes:




Well, one secret to an LC developing that fast would be, unfortunately, mold.  And normally I would suggest this is mold, but I'm positive this time it's not.  I went back and looked at the syringe and I probably added closer to 35 ml (It was a 40ml syringe I believe), so it wasn't as quickly as I said. 

Here are some things that speed up an LC:

1) LC is from a fresh LC that is already in the process of growing
2) LC has the same sugar content (type and concentration) as the LC you are tryting to expand
3) LC for the inoculation is the same temperature of the one you are adding it to
4) LC is constantly spun.  LCs that are not spun are much slower - 2, 3 or even 4x slower in fact.

Here are some factors that slow down LC development:

1) An LC with inoculum being taken out of cold storage, will not develop that fast. 
2) LCs started from spores can take a two weeks if the spores are old and dehydrated (I generally do not recommend LCs direct from spores unless you are very certain those spores are clean; no vendor consistently sends out clean spores that I know, so making sure that spore print is pristine that would be on you). 
3) LCs started from a piece of mycelium from agar are much slower than a fresh LC, but they still should be done in about six or seven days
4) Agar that has roughly the same sugar mix as the LC helps speed them up a little.
5) Obviously the more inoculum you use, the faster.  LCs develop exponentially at first, though, so it's not quite the effect you might assume.

So the answer is that in this case, everything was just right.  Normally, you should not expect an LC to finish in 2 or 3 days, and if they do, its feasible they contain mold if you don't know more about it.  Mold usually does not form balls of mycelium, though, and they look like a fast-spun LC even when they are slow-spun (that's one reason I do not recommend very rapidly spinning of an LC: you cannot tell mold from desirable growth)


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Re: Some notes about LCs [Re: Blue Helix]
    #27006046 - 10/27/20 10:40 AM (3 years, 3 months ago)

How do LC Quars look if they are contaminated?
Are here some pictures?
Ore do the contamination have a special look?


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