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MushroomMommy
Myco N00b


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LC Master Solution Questions 1
#28543464 - 11/14/23 11:35 PM (2 months, 12 days ago) |
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I made (4) 300ml jars of LC solution with purified water (ran out of distilled) with a .18% LME, PCed at 15psi for 45m per Stipe-n-cap but forgot to add a marble (RIP wrists)
Added ~2cc of verified clean LC to each jar in SAB. One of the 4 jars is cloudy/contaminated, but I think that may have been my own carelessness. I was told to trash it, but I wanna give it a couple more days to see if the myc will win, I’m still n00b enough that it seems sad to throw things away.
The remaining 3 jars are just as clear as when I inoculated now 5 days in and swirling 1x per day.
Q1: how long should I be shaking the jar and is there any downside to shaking more or less often?
Q2: How much should I be letting this mycelium grow before stalling things by putting in fridge?
Q3: With a weaker nutrient solution than standard practice dictates for clarity, approximately how long until the mycelium run out of food?
Here’s a photo of one of the 3 viable jars
-------------------- 🅃 🄴 🄰 🄼 🄲 🄻 🄸 🄽 🄶 🅆 🅁 🄰 🄿 ABCD stands for Always Be Collecting Data “I don't know anything about anything, but I know enough about everything to know that you don't know what the fuck you're talking about”
Edited by MushroomMommy (11/14/23 11:36 PM)
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hellofresh
Strangler


Registered: 09/22/23
Posts: 215
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Hey!
So just want to leave this here since I think this is what you should try.
https://www.shroomery.org/forums/showflat.php/Number/23740857
You get to see how it grows out before you turn it into a lc and anything after it grows out falls into your sterile tek. This is how I went about making a lc in the past and in the future if I do it again.
When you inoculate use the smallest amount of lC possible so it's ideally clean.
-------------------- Don't panic
Edited by hellofresh (11/14/23 11:57 PM)
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cybork
Stdt mycology


Registered: 07/01/23
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Q1: how long should I be shaking the jar and is there any downside to shaking more or less often? >>The goal is to loosen up the mycelium, to create more contact area, so when that seems to be achieved, I stop. Could be about 15 seconds, or 30. Depending also on how easy you are swirling. PS: swirl, don't shake. When doing it by hand and you have a lot of jars, you will feel it in your wrist. That is downside 1 of shaking more often. I think you already know that because of the "(RIP wrists)". Tip: buy or build a magnetic stirrer. I use non-rusting screws in them as stirrer bars, as I find stirrer bars too expensive. Downside to too much stirring (especially by hand), is upping the accidental chance of making the gas exchange port wet and that then is dirty. You use micropore tape?
Q2: How much should I be letting this mycelium grow before stalling things by putting in fridge? >>The growing will stall by itself after some time. Depending on multiple factors like species, temp, nutrition. Latest batch I ran stalled after 2 weeks. Some of those jars are now in fridge (after QC on agar).
Q3: With a weaker nutrient solution than standard practice dictates for clarity, approximately how long until the mycelium run out of food? >>eh. Depending on multiple factors like species, temp, nutrition. I actually found darker (less clear) LC to be more informative. Anyways, let everybody has his own preferences . Latest completed batch I ran stalled after 2 weeks, and that had 4% honey (based on weight vs water). So also no peptides, LME or whatever.
PS: I found no benefit of adding a marble or whatever in a jar, when stirring by hand. But a magnetic stirrer saves your wrists and the gas exchange port. But with only a jar or 4, stirring 2 times a day, you will probably not get wrists injuries anyways.
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MushroomMommy
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Re: LC Master Solution Questions [Re: hellofresh]
#28543528 - 11/15/23 01:20 AM (2 months, 12 days ago) |
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Christ, I know PastyWhyte is God tier but, this sounds like a lot and I’m not so sure how I feel about water atop agar, I’ll re-read this write-up with a fresh mind tomorrow. Thanks Hellofresh!
-------------------- 🅃 🄴 🄰 🄼 🄲 🄻 🄸 🄽 🄶 🅆 🅁 🄰 🄿 ABCD stands for Always Be Collecting Data “I don't know anything about anything, but I know enough about everything to know that you don't know what the fuck you're talking about”
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hellofresh
Strangler


Registered: 09/22/23
Posts: 215
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It's just ghetto no pour agar in a jar and a ship port for water don't over complicate it.
-------------------- Don't panic
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MushroomMommy
Myco N00b


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Re: LC Master Solution Questions [Re: cybork]
#28543541 - 11/15/23 01:40 AM (2 months, 12 days ago) |
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Thank you for your direct responses! Sorry, I shouldn’t have said shake, I am indeed swirling. Yes there is a 6mm or ~1/4” hole drilled into the lid covered with .3u micron SF sticker. Additionally there are (2) 11mm or ~3/8” holes that are painstakingly fitted with 20mm SHIP with a 13mm stopper end. I suppose the SFS could be getting splashed, but I’m fairly careful in my swirling motions to avoid getting solution on the bottom/interior of lid. I was generally swirling to the point of breaking up the mass into ~1mm jellyfish? Since my budget tek is quickly turning into aesthetic-driven expensive tek, I’ll probably just go on and treat myself to a magnetic stirrer and set of bars. I don’t really plan to have more than 6 LC master pints in the fridge at any given time and once expanded I think I read to swirl them 1-2x per week? I don’t really have space to accommodate more than 4 66qt Monotubs at any given time so I won’t be drawing from these all too often.
Q: If I increase nutrients or get smaller jars, will they keep in refrigerator longer than 6 months?
Q: When you say stall, is it dying at that point if I don’t immediately get it in the fridge or is it just going dormant?
4%?! That’s massive! And with honey, how are you avoiding hideous sediment?
-------------------- 🅃 🄴 🄰 🄼 🄲 🄻 🄸 🄽 🄶 🅆 🅁 🄰 🄿 ABCD stands for Always Be Collecting Data “I don't know anything about anything, but I know enough about everything to know that you don't know what the fuck you're talking about”
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MushroomMommy
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Re: LC Master Solution Questions [Re: hellofresh] 1
#28543545 - 11/15/23 01:47 AM (2 months, 12 days ago) |
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Quote:
hellofresh said: don't over complicate it.
Hah! It’s me, MushroomMommy…I over complicate everything, my dude.
-------------------- 🅃 🄴 🄰 🄼 🄲 🄻 🄸 🄽 🄶 🅆 🅁 🄰 🄿 ABCD stands for Always Be Collecting Data “I don't know anything about anything, but I know enough about everything to know that you don't know what the fuck you're talking about”
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hellofresh
Strangler


Registered: 09/22/23
Posts: 215
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The reason I like pasty, milkboy, Yoshitrainer, beef, the_chosen_one, fahtster, Muad.Dweeb, etc.. is because I think they strip the whole thing down to the minimum in their own ways. It's easy to complicate things but the best content is simple and straightforward.
-------------------- Don't panic
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cybork
Stdt mycology


Registered: 07/01/23
Posts: 146
Last seen: 41 minutes, 41 seconds
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Q: If I increase nutrients or get smaller jars, will they keep in refrigerator longer than 6 months? >>I guess they will. I'm not aware whether smaller jars will stay less longer okay. Do be careful to store the jars in the fridge: make sure the fridge will not contaminate your jars. Use plastic or whatever.
Q: When you say stall, is it dying at that point if I don’t immediately get it in the fridge or is it just going dormant? >>It is stalling and just going dormant. Or at least I think it is, and my LC still gives life after being in the fridge with this method. Do not wait for weeks getting them in the fridge though.
4%?! That’s massive! And with honey, how are you avoiding hideous sediment? >>I don't get hideous content. I use clear looking honey. Actually malt extract gives me hideous sediment.
Edited by cybork (11/15/23 02:13 AM)
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stroopwafelman


Registered: 03/21/23
Posts: 375
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Re: LC Master Solution Questions [Re: cybork]
#28543558 - 11/15/23 02:20 AM (2 months, 12 days ago) |
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From my experience not having a marble (in my case it's a stirring bar) you end up with much bigger globs of mycelium that are a bit harder to aspirate with a syringe and take longer to break up.
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Shrimps
Traveler


Registered: 10/13/22
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just free pour that LC after a good shake
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Baba Yaga
♥ coir grower

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Re: LC Master Solution Questions [Re: Shrimps] 4
#28543568 - 11/15/23 02:58 AM (2 months, 12 days ago) |
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The easiest and most fool proof way to make LC is via the Josex Poke IMO
Make your LC broth with 0.1% LME and distilled or RO water and it will be clear as a bell.
Leave the lid unmodified and loosen a bit when PCing to avoid a vacuum, screw the lid shut when taking out of PC. Only fill the vessel half way and you will have enough oxygen for it to grow out. You can store the LC at room temp for at least a year IME. The mycelium will go dormant. Without a filter you can shake the hell out of the culture before inoculation.
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cybork
Stdt mycology


Registered: 07/01/23
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Yeah, but by hand the marble or whatever is in it, I can't get that object to 'touch' that glob anyways. The object basically makes rounds on the lower sides of the jar. Still, it will have some effect I guess. But to me it seems not that significant compared to using no object. And of course I 'm talking here in respect of swirling by hand. My magnetic stirrer + stir bar (actually screws) work best and break up the globs.
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normalperson
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Re: LC Master Solution Questions [Re: cybork]
#28544020 - 11/15/23 01:10 PM (2 months, 11 days ago) |
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i use stainless steel BBS, anyone see a downside to this?
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MushroomMommy
Myco N00b


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Re: LC Master Solution Questions [Re: Shrimps]
#28544678 - 11/15/23 10:56 PM (2 months, 11 days ago) |
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Quote:
Shrimps said: just free pour that LC after a good shake
Free pour?! Absolute madness! Hahaha
-------------------- 🅃 🄴 🄰 🄼 🄲 🄻 🄸 🄽 🄶 🅆 🅁 🄰 🄿 ABCD stands for Always Be Collecting Data “I don't know anything about anything, but I know enough about everything to know that you don't know what the fuck you're talking about”
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MushroomMommy
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Re: LC Master Solution Questions [Re: Baba Yaga]
#28544681 - 11/15/23 11:02 PM (2 months, 11 days ago) |
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@baba
I think you’re the answer I’ve been looking for. So you’re using .1? I was a bit afraid of .1 so I did .18, but you’re telling me it’ll eventually go dormant and I can leave it at room temp for a year and it’s still usable? How often are you breaking it up once it goes dormant?
Half full and no need for gas exchange?? I have to say, I’m intrigued. At .1% if things are still remaining stable a year later, why is it standard practice for folks to use 400x the amount of nutrients?!
Once I open it to free pour, would I need to use all of it at once? I would think free pour in a SAB vs Flowhood would be sketchy, no?
-------------------- 🅃 🄴 🄰 🄼 🄲 🄻 🄸 🄽 🄶 🅆 🅁 🄰 🄿 ABCD stands for Always Be Collecting Data “I don't know anything about anything, but I know enough about everything to know that you don't know what the fuck you're talking about”
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MushroomMommy
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Registered: 08/21/23
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Last seen: 8 hours, 57 minutes
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Quote:
stroopwafelman said: From my experience not having a marble (in my case it's a stirring bar) you end up with much bigger globs of mycelium that are a bit harder to aspirate with a syringe and take longer to break up.
Makes sense that they’d be harder to suck up. Thanks for the insight!
-------------------- 🅃 🄴 🄰 🄼 🄲 🄻 🄸 🄽 🄶 🅆 🅁 🄰 🄿 ABCD stands for Always Be Collecting Data “I don't know anything about anything, but I know enough about everything to know that you don't know what the fuck you're talking about”
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Baba Yaga
♥ coir grower

Registered: 09/13/20
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MushroomMommy said:@baba
I think you’re the answer I’ve been looking for. So you’re using .1? I was a bit afraid of .1 so I did .18, but you’re telling me it’ll eventually go dormant and I can leave it at room temp for a year and it’s still usable? How often are you breaking it up once it goes dormant?
The ones that go into long term storage will not get broken up. I only really break them up right before I use them.
Half full and no need for gas exchange?? I have to say, I’m intrigued. At .1% if things are still remaining stable a year later, why is it standard practice for folks to use 400x the amount of nutrients?!
That is correct, no gas exchange. The terms "standard practice" and "consensus" are only saying that a lot of people do the same thing for the same reason but it doesn't mean it is the be-all and end-all of things. Practices change all the time. All I can tell you is that I adopted this practice from mateja and that it works for me and you should try and see if you like it. So far it's working great and I have revived three out of three one year old LC's. Next year I will do tests with 2 and 3 year old LCs.
Once I open it to free pour, would I need to use all of it at once? I would think free pour in a SAB vs Flowhood would be sketchy, no?
If your technique is solid then it doesn't matter what you are working with, free pouring an LC in a SAB is not any different from dropping an agar wedge in a grain jar. I do use all at once or discard the leftovers. If I want to keep the culture around and I'm using the last bottle I will put some to new LC broth first before putting it to grain. I also only do small volumes like 60-120ml but I do several.
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MushroomMommy
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Re: LC Master Solution Questions [Re: Baba Yaga]
#28621465 - 01/14/24 08:11 PM (13 days, 11 hours ago) |
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I have since bought a FFU and am planning on switching my LC vessels from jars to unmodified media bottles. Can’t decide if I want 250ml or 100ml bottles. I’ll plan to fill them 50-60% to eliminate the need for mod thus allowing me to shake the hell out of it when needed.
As for the 4 master jars I started with the original post in this thread, about 20 days into the process the problematic jar cleared itself, I drew 10ml into a syringe via the port as well as one of the other. Once tested to agar they both had bacteria. I threw the syringes back in the fridge and then tested them again another 30 days later and the problematic one was still dirty with bact. BUT had a beautiful mycelium growth on it that was thicker and more robust than any I’ve ever had. So naturally I had to transfer it.

The other one that had tested dirty was now clean, but after a bit of humming and hawing I decided to toss both of these master jars as well as a 3rd one that I tested a few days ago and is thus far looking clean. I don’t know, since all these were made in SAB and 2 of 4 were dirty at one point I figure it’s better to just remake them all together in the new media bottles with the FFU as a safety.
-------------------- 🅃 🄴 🄰 🄼 🄲 🄻 🄸 🄽 🄶 🅆 🅁 🄰 🄿 ABCD stands for Always Be Collecting Data “I don't know anything about anything, but I know enough about everything to know that you don't know what the fuck you're talking about”
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MushroomMommy
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SSLC solution questions:
Trying my hand again at making master LCs. This time, instead of pint jars fitted with 2-port + gas exchange synthetic filter…I’m using 250ml media bottles that will be filled approximately 1/2 full (~125ml) and unmodified. So I can shake the hell out of them and not have to worry about contamination or ports/sfs popping off.
I have heard great things about SSLC for faster colonization time and am curious to see for myself. As most of my [limited] experience is with regular LC, I am debating whether I want to do everything as SSLC or just a bottle or two to see if I like it? Since I just bought (10) 250ml media bottles, I was considering making ~6, but maybe that’s too much given that I don’t know if I like SSLC or want to stick with regular LC. If I do not plan to use all of it right away is there any reason to prepare so many master LCs or does the long shelf life lean into a “fuck it, why not” attitude?
@baba_yaga said that once the myc in an LC has fully grown out it’ll stall. He additionally said LC can be stored indefinitely at room temp. Is this a higher risk of contamination rather than in the refrigerator?
I also have not decided if I will be free pouring or drawing the solution into syringes. Since the SSLC is thicker than normal LC from the addition of agar, would it require a 16g syringe or larger?—I currently have 18g. I honestly don’t love the idea of free pouring as my brain can’t really handle not having consistent measurements and data—plus additional moisture freaks me out. I hate seeing condensation of wet grains.
I had pretty good success with my first round of Master LCs at .18% LME, but have been told I can use as little as .1% — given that sslc is inherently cloudier than regular LC perhaps a lower nutrient content lending itself to a clearer solution where bacteria will be more visually obvious would be of benefit?
Q1: if bacteria is present in the inoculant for an LC, is a higher or lower nutrient percentage preferred?
Q2.1: For SSLC, I do not want to have to deal with the vessel having to sit on a stir plate for the duration of growth, partly because I only have 1 stir plate and partly because I just don’t want to have to have something electronic running for 2 weeks+ straight—Mr. Mommy would be none to pleased. I do not mind the idea of having to shake the bottles 1-3x daily.
Q2.2: Would a 1% agar (telephone brand) be enough to increase thickness/viscosity without the possibility of it solidifying with daily shaking? If I end up doing .18% LME, should I also do 1.8% Agar?
Q3: How far in advance can/should an LC nutrient solution be made prior to inoculation? Is there any reason I shouldn’t just make 6-8 media bottles of solution to have ready and sterilized whenever I decide to noc’ it—or is a sugary solution sitting around (at worst) asking for bacteria or at best evaporation that will lead to a more concentrated solution?
I’m thinking before I make a BUNCH of bottles, I should probably first, figure out if I like sslc and then second, dial in my ideal nute and agar percentages before prepping more than 1-2 bottles…
-------------------- 🅃 🄴 🄰 🄼 🄲 🄻 🄸 🄽 🄶 🅆 🅁 🄰 🄿 ABCD stands for Always Be Collecting Data “I don't know anything about anything, but I know enough about everything to know that you don't know what the fuck you're talking about”
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