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OfflineBlue Helix
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Re: Some notes about LCs [Re: BrownBear]
    #27027410 - 11/07/20 06:47 PM (3 years, 3 months ago)

Quote:

Iambrownbear said:
Quote:

PitcherCrab said:
What brand of LME is that Demonic? Both my agar and LCs have been ending up with sentiment for as long as I can remember and it drives me nuts. The mushrooms don't care, but I'd love to make everything a little clearer if possible. Or if you have other tricks to get them clearer I'd love to heard them. Your plates are beautiful.




I use .5 grams of nutritional yeast in my LC's. I have setiment but after a few days at low speed on the stir plate the mycelium colonizes it all and it ends up crystal clear. Thats when you know its ready.




Yeah, I just bought that too.  I don't know if it works better or not yet (haven't tested it), but it does add a little more sediment.  I agree with you, though: when the sediment is gone, the LC is done!

One thing I should also mention is that it's kind of good to have the agar plates you are using have most of the same ingredients in them (at least have them share the malt).  That way the mycelium has the right enzyme profile right off the bat then you transfer it, so it'll have the jump on any bacteria in there.


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OfflineAsuraS
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Re: Some notes about LCs [Re: Blue Helix] * 1
    #27027461 - 11/07/20 07:31 PM (3 years, 3 months ago)

BH, that seems like an insane amount of nutrients to me but has me wondering
if I should do some tests with it. I do 1-2g of LME to 300ml water and I'm
still getting that fast growth for cyans. Might tie it in to the expansion
experiments I'm doing.


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InvisibleMateja
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Re: Some notes about LCs [Re: Blue Helix]
    #27027483 - 11/07/20 07:47 PM (3 years, 3 months ago)

Quote:

Blue Helix said:
It's probably bacteria, and it will probably resolve if you expand out the LC one more time.



Could you elaborate on this? I can't seem to make sense out of what is being "resolved". Cheers


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OfflineBlue Helix
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Re: Some notes about LCs [Re: Asura]
    #27027535 - 11/07/20 08:24 PM (3 years, 3 months ago)

Quote:

Asura said:
BH, that seems like an insane amount of nutrients to me but has me wondering
if I should do some tests with it. I do 1-2g of LME to 300ml water and I'm
still getting that fast growth for cyans. Might tie it in to the expansion
experiments I'm doing.




That is a 4% sugar solution, and it was the standard when I started across the entire Shroomery.  The honey recipes I've read use that same amount (of course with honey it's only 75% sugar per volume, so you have to adjust accordingly).  Having said that, I have read of people using only 1.5% sugar with good results.  I have not heard of anyone using 0.33 like what you said, and I am just going by what the mushroom farms use (4%).  I don't doubt it works using 0.3%, but I can tell you for certain that most recipes when I was searching around were 4% solutions.  It wasn't up for debate back then.  It was just the way it was.

I might knock mine down to 2% to give it a comparative whirl, and I'll let you know what I find.  Again, mine complete in 2 or 3 days, and they get so thick that when I allow them to settle, I see about 70% mycelium blobs in there.  They look 30 times or more as thick as what D3monic shows in that video, but I sort of assume that is bacterially contaminated or something because that is really wispy.  No constant-spun LC should be that wispy on use, and I don't even know if that would work for the inoculation of bulk substrates with low grain.  It'd probably work fine for spawn, though, since grains are easy to inoculate but I don't use spawn.


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OfflineBlue Helix
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Re: Some notes about LCs [Re: Mateja]
    #27027556 - 11/07/20 08:33 PM (3 years, 3 months ago)

Quote:

Mateah said:
Quote:

Blue Helix said:
It's probably bacteria, and it will probably resolve if you expand out the LC one more time.



Could you elaborate on this? I can't seem to make sense out of what is being "resolved". Cheers




Yes, of course,  When one starts an LC the first time is often slow and there is an extended period of time (as in a week) where the solution is cloudy.  My assumption is that during this time, there is a little bacteria in the solution (the key word being LITTLE).  I say this for two reasons: (1) LCs started from clear LCs clear up within 2 to 4 days, and (2) agar plates started from a cloudy LC will not colonize due to bacteria and stink to high heaven.

Having said all that, I've been informed by a trusted cultivator here that LCs always contain bacteria.  I do not believe that, and I don't have a very good scope to prove it either way (naturally during brewing everything is totally different since yeast are incredibly tenacious).  All I know is that if you open a cloudy LC, it smells off and you cannot start an agar plate with it usually.  If you use a clear LC, it will complete a new jar extremely fast (unless it's been chilled), the new jar will have ZERO bacterial smell it, and you can easily start an agar plate with it.

If something else is going on besides bacterial, I'd have to see actual proof.  Just someone asserting that on Shroomery doesn't matter to me since about a quarter of what I read on this site is not only false but easily proven incorrect.  Likewise, I don't have proof so what I'm saying is my own theory (i.e. that bacterially contaminated LCs stink and take awhile to clear up).

As for why expanding it to help it clear out, I have found that take a bit of a clear LC produces a more robust expanded LC.  I don't know why that is, but if it's cloudy or slow, doing an expansion often helps.


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OfflineBlue Helix
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Re: Some notes about LCs [Re: Asura]
    #27027578 - 11/07/20 08:43 PM (3 years, 3 months ago)

Quote:

Asura said:
BH, that seems like an insane amount of nutrients to me but has me wondering
if I should do some tests with it. I do 1-2g of LME to 300ml water and I'm
still getting that fast growth for cyans. Might tie it in to the expansion
experiments I'm doing.




Asura, if that is working for you, I'd stick with it.  The lower the sugar content, the longer they can store in the refrigerator.  In fact, I was going to lower my sugar content in the vacutainer to 0.5% to store it longer.  Also lower sugar might have other benefits too.  I'll give what you are doing a try.

With that extremely low sugar content, what is the shelf life of your LCs in a vacutainer in the refrigerator?  It must be several years, right?


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OfflineAsuraS
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Re: Some notes about LCs [Re: Blue Helix]
    #27027588 - 11/07/20 08:49 PM (3 years, 3 months ago)

I am only now getting into storing LC's for long term. Always been one and done
for me. But I have tons in storage now so I guess I will find out.


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OfflineBlue Helix
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Re: Some notes about LCs [Re: Asura]
    #27027605 - 11/07/20 09:04 PM (3 years, 3 months ago)

Quote:

Asura said:
I am only now getting into storing LC's for long term. Always been one and done
for me. But I have tons in storage now so I guess I will find out.




So I think I'll try to compared 0.4% to 4% sugar and get back with you on this thread.  This should be VERY interesting, but I am highly skeptical. 

Just to be clear, though, 4% IS the standard in mushroom farming.  It was not just some blurb form some kid on here that had a decent first flush so thinks they know it all.  If you do a search online you will find 4% repeatedly referenced in mushroom liquid culture formulas.

Here was something from some literature for professional mushroom farming which has, as expected, 4% sugar in the Excel sheet (see http://fongivore.bazaroccidental.org/en/interieure/vegetative/culture_liquide for the initial page and search for the recipe Excel sheet).  I copy and pasted it here (removed the fraction of a penny column since that doesn't matter so much for non-professionals I assume)

LC Media 1 h @ 15 psi
Water 600 ml
Premixed blend 28.20 g

Premixed blend:
Malt extract 24.0 g
Sawdust 2.4 g
Yeast 1.2 g
Calcium sulfate 0.6 g

PS - I sterilize only 30 minutes, but I also heat the mix to near boiling in the microwave before.


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InvisibleMateja
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Re: Some notes about LCs [Re: Blue Helix]
    #27027632 - 11/07/20 09:24 PM (3 years, 3 months ago)

Thanks for the reply! Idk if you've seen this post I made a few days ago where I inoculate two newly sterilized broths with 10ml of mold solution each and then I place one drop of bacterial solution in one of the broths and then the other broth I just gently touch on the surface with the bacterial needle tip. Both broths are crystal clear at inoculation and within 24h both turn completely turbid. What's interesting is that I was expecting exactly this to happen based on what the entire microbiology community says (as far as Ive been reading up on it) so it's hardy a surprise spectrophotometers are some of the more common tools used my microbiologists to estimate cell mass (bacterial prevalence) in a liquid media.


Ever so often Im being told that bacteria doesn't always cause turbidity or that some bacteria doesn't cause turbidity but I've not seen links posted that back this notion and I haven't seen any test documented either to back it back. Also I have yet to come across this information myself as I'm reading studies on bacterial activity almost daily and when I do tests out the things I read about how bacteria behaves in microbiology papers then I can easily confirm that what I'm reading online holds true (thus far). In case you're wondering so far I have a few different types of bacteria and mold colonies homogenized in liquid broths simply as a means to be able to perform tests in a more or less controlled setting (I can't identify and give you names on these homogenized microbes but I do know that I have them homogenized) besides the tests I'm performing I can of course link to numerous research papers confirming everything I'm saying all of the time regarding bacterial activity and more important bacterial activity in liquid media. (not saying that anything I say should be taken as gospel truth I'm just saying I can back every statement up with both links to peer reviews research papers as well as my own research that I'm documenting as I go about learning more about this intriguing topic.


You're definitely right about not believing anything said or argued unless it can somehow be substantiated with evidence that holds merit, this sounds like a totally reasonable and same approach I could only wish more growers would actually apply this standard when discussing these things. Btw I really dig the profile pic are those Pans? My very first Pan live culture is close to being ready to use as inoculate and the thrill is real :popcorn:


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OfflineGeinstein
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Re: Some notes about LCs [Re: verum subsequentis]
    #27027734 - 11/07/20 11:08 PM (3 years, 3 months ago)

Quote:

verum subsequentis said:
:whathesaid:

truth

Quote:

Verum aren’t you the one with the crazy modded half gallon jar and spray nozzle thing??


Such a great contraption.




yep. But i rarely use it anymore. Maybe I'll bust her out and give her another shot. Free pouring is just so much less hassle.






Does the tubing get PCde and if so what plastic are you using?


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Nothing breads nothing


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OfflineGeinstein
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Re: Some notes about LCs [Re: Geinstein]
    #27027861 - 11/08/20 01:53 AM (3 years, 3 months ago)

I'm prepping some LC jars 10 x 200ml of T2 and T3 plates(cleaned up from dirty ass print)
My LC ends up looking like this :



(Bad light making for bad photos, but basically looks like Cristal clear water)


Now the batch I made 2 days ago(I inoculate 3 days after PCing) have all got this piece of grain clump, I'd like to know if it'll still be fine to use



My recipe is:
1g BRF
0.5g Dextrose
10ml second brew black Tea
200ml water

My only difference with this one is I mixed everything together before PCing were normally I'd just add everything and then add water without mixing and PC it. Could the mixing be the reason for the clump and can it still be used?


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Nothing breads nothing


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OfflineBlue Helix
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Re: Some notes about LCs [Re: Mateja] * 1
    #27028243 - 11/08/20 09:37 AM (3 years, 3 months ago)

Quote:

Mateah said:
Thanks for the reply! Idk if you've seen this post I made a few days ago where I inoculate two newly sterilized broths with 10ml of mold solution each and then I place one drop of bacterial solution in one of the broths and then the other broth I just gently touch on the surface with the bacterial needle tip. Both broths are crystal clear at inoculation and within 24h both turn completely turbid. What's interesting is that I was expecting exactly this to happen based on what the entire microbiology community says (as far as Ive been reading up on it) so it's hardy a surprise spectrophotometers are some of the more common tools used my microbiologists to estimate cell mass (bacterial prevalence) in a liquid media.


Ever so often Im being told that bacteria doesn't always cause turbidity or that some bacteria doesn't cause turbidity but I've not seen links posted that back this notion and I haven't seen any test documented either to back it back. Also I have yet to come across this information myself as I'm reading studies on bacterial activity almost daily and when I do tests out the things I read about how bacteria behaves in microbiology papers then I can easily confirm that what I'm reading online holds true (thus far). In case you're wondering so far I have a few different types of bacteria and mold colonies homogenized in liquid broths simply as a means to be able to perform tests in a more or less controlled setting (I can't identify and give you names on these homogenized microbes but I do know that I have them homogenized) besides the tests I'm performing I can of course link to numerous research papers confirming everything I'm saying all of the time regarding bacterial activity and more important bacterial activity in liquid media. (not saying that anything I say should be taken as gospel truth I'm just saying I can back every statement up with both links to peer reviews research papers as well as my own research that I'm documenting as I go about learning more about this intriguing topic.


You're definitely right about not believing anything said or argued unless it can somehow be substantiated with evidence that holds merit, this sounds like a totally reasonable and same approach I could only wish more growers would actually apply this standard when discussing these things. Btw I really dig the profile pic are those Pans? My very first Pan live culture is close to being ready to use as inoculate and the thrill is real :popcorn:





That is a very interesting post.  Thank you.  One thing that is for absolute certain is that malt has malt solids that can make a solution appear turbid at first, but the big difference is that kind of turbidity, if not spun, will settle in several hours; bacterial turbidity will not.  Of course, the malt turbidity goes away as the liquid culture matures, but I could swear I've even seen the other type (bacteria) resolve too.  It doesn't always, but it has for me on some occasion.  I'm not sure what mechanisms are in play, but I assume most types of mycelium do have the ability to kill bacteria at some maximum concentration and below.  That's where the details are unknown to me.  How does mycelium do that and to what extent can it clear bacteria?  I don't know the answers for sure.

Yeah, my profile picture was pan cambos.  It was a picture contest on Mycotopia in the fall of 2008 I think.  That picture I called "Art of The Shroom" and won the picture contest.  Here are some related pictures:



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OfflineA.k.aM
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Re: Some notes about LCs [Re: Blue Helix]
    #27028553 - 11/08/20 12:51 PM (3 years, 3 months ago)

Do you guys notice your lc looks way less colonized in person than pictures? Mine always seem way less grown in then what I see people posting so I was gonna ask what would cause it to not colonize all the way.

But then I took pictures are they look much thicker in them.

Anyway the clear one is a year old cube lc, the yellow one is king oyster about two weeks in so I thought it would be like pudding by now the way oyster grows.



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LAGM2020


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OfflineBrownBear
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Re: Some notes about LCs [Re: A.k.a]
    #27028625 - 11/08/20 01:33 PM (3 years, 3 months ago)

Quote:

Blue Helix said:
Quote:

Iambrownbear said:
Quote:

PitcherCrab said:
What brand of LME is that Demonic? Both my agar and LCs have been ending up with sentiment for as long as I can remember and it drives me nuts. The mushrooms don't care, but I'd love to make everything a little clearer if possible. Or if you have other tricks to get them clearer I'd love to heard them. Your plates are beautiful.




I use .5 grams of nutritional yeast in my LC's. I have setiment but after a few days at low speed on the stir plate the mycelium colonizes it all and it ends up crystal clear. Thats when you know its ready.




Yeah, I just bought that too.  I don't know if it works better or not yet (haven't tested it), but it does add a little more sediment.  I agree with you, though: when the sediment is gone, the LC is done!

One thing I should also mention is that it's kind of good to have the agar plates you are using have most of the same ingredients in them (at least have them share the malt).  That way the mycelium has the right enzyme profile right off the bat then you transfer it, so it'll have the jump on any bacteria in there.




Thanks for the tip. I will give it a go with adding the NY to my agar and using that to transfer to my NY broth. Do you mind if I update my progress here?


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OfflineNurkurzda23
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Re: Some notes about LCs [Re: Mateja]
    #27028916 - 11/08/20 04:39 PM (3 years, 3 months ago)

hi
for how long do you autoclave?  or for how long would you autoclave 4 x 250ml?  20-30min or longer?


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Invisiblemushhead
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Re: Some notes about LCs [Re: Nurkurzda23]
    #27029220 - 11/08/20 08:47 PM (3 years, 3 months ago)

I PC my LC from 45min-1hr.
Usually its exactly 45min, but sometimes I forget my PC is running
I'm sure many think that's really overkill.


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InvisibleStipe-n CapMDiscord
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Re: Some notes about LCs [Re: Nurkurzda23] * 1
    #27029226 - 11/08/20 08:51 PM (3 years, 3 months ago)

Quote:

Nurkurzda23 said:
hi
for how long do you autoclave?  or for how long would you autoclave 4 x 250ml?  20-30min or longer?





15-25 is what youreaiming for. Liquids sterilize quickly at 15 psi, 15 mins is good enough. I do 20.


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OfflineNurkurzda23
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Re: Some notes about LCs [Re: Stipe-n Cap]
    #27029423 - 11/09/20 12:10 AM (3 years, 3 months ago)

I have read that a certain concentration of H2O2 in LC helps against contamination.  how much 3% solution do you use at approx 250 mL / 8.5 Oz?

are the jar lids are slightly open during they are autoclaved. (With tin foil over them) because I have a lot of contaminated jars... And don't know why


Edited by Nurkurzda23 (11/09/20 12:23 AM)


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OfflineBrownBear
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Re: Some notes about LCs [Re: Nurkurzda23]
    #27029474 - 11/09/20 02:19 AM (3 years, 3 months ago)

Quote:

Nurkurzda23 said:
I have read that a certain concentration of H2O2 in LC helps against contamination.  how much 3% solution do you use at approx 250 mL / 8.5 Oz?

are the jar lids are slightly open during they are autoclaved. (With tin foil over them) because I have a lot of contaminated jars... And don't know why




Are you using spore syringes to inoculate your lc's or are you germinating spores on agar and making a transfer before going to lc?


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InvisiblePastywhyteMDiscord
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Re: Some notes about LCs [Re: Stipe-n Cap]
    #27029860 - 11/09/20 09:28 AM (3 years, 3 months ago)

Quote:

p9hu7 said:
Quote:

Nurkurzda23 said:
hi
for how long do you autoclave?  or for how long would you autoclave 4 x 250ml?  20-30min or longer?





15-25 is what youreaiming for. Liquids sterilize quickly at 15 psi, 15 mins is good enough. I do 20.




I’ve never been able to sterilize any liquid media or agar for 25 min or less and not end up with bacteria later.


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