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OfflineBlue Helix
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Re: vacutainers? [Re: MLPismyOPSEC] * 2
    #27006048 - 10/27/20 10:40 AM (3 years, 3 months ago)

Quote:

MLPismyOPSEC said:
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?




I almost always do that.  Most times it's from an LC stored in a vacutainer.  Here is the precise kind of vacutainer you need (do not just buy some random vacutainer since many kinds of chemicals in them or the wrong kind of septum).  If it's from a vacutainer, I use about 1 to 2 ml usually.  If I'm starting from another LC directly, I use a more - maybe 30 ml.  The whole beauty of an LC is--and I keep making this point over and over since I guess people don't know this--that once you have a viable LC, you don't have to worry about agar, still-air-boxes, flow hoods, clean techniques, etc.  It's all injection-based after that if you know what you are doing.  It's really like magic, and it makes the process of growing a lot more enjoyable and, in my opinion, higher-yielding.  I've done spawning many times, but in my opinion, LCs are a much better way to inoculate bulks substrates and store cultures.

Cultures can be stored in vacutainers containing the full 4% sugar solution in the refrigerator for about a year.  If you want much longer term storage, take about 1 ml of the LC (try to get it as dense as you can) and dilute it down by 9 parts sterile water in a vacutainer.  Store in cold storage as usual.  The lower the storage sugar content, the longer it'll stay good in storage (maybe 5 years?).  Research has shown that for labs that completely replaced the sugar solution with sterile water (this requires a centrifuge really which will set you back 30 to $100 on eBay), can store their LCs for TEN FULL YEARS, making LCs every bit as good of option as slants and, some would argue, spores too (and with LCs you can keep the phenotype which makes it even more powerful).

THIS IS NOT MY TECHNIQUE!  Nor is this some Shroomery thing.  LCs are often used in mainstream mushroom farming.  If you search for liquid mushroom cultures on YouTube, many mushroom farms will come up.  They use them direct to bulk substrates in the case of cordyceps almost exclusively.  In the case of other edibles, they often create spawn with them because spawn is the best technique for hundreds of pounds of mushrooms.  For the hobbyist grower, though, I think direct to sterile bulk substrate is the easiest way to produce as many mushroom as you want.  It's simple and fast, and for folks who don't like working under a flow hood or in a cramped still air box, it's ideal.


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Re: Some notes about LCs [Re: Nurkurzda23]
    #27007166 - 10/27/20 08:44 PM (3 years, 3 months ago)

Quote:

Nurkurzda23 said:
How do LC Quars look if they are contaminated?
Are here some pictures?
Ore do the contamination have a special look?





cloudy.  highly turbid.

Señor Helix: do you ever test your LC's on agar? Ever use blood agar to target bacterial contams?


Edited by nmd_myco (10/27/20 08:51 PM)


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OfflineBlue Helix
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Re: Some notes about LCs [Re: nmd_myco] * 1
    #27007386 - 10/27/20 10:47 PM (3 years, 3 months ago)

Quote:

nmd_myco said:
Quote:

Nurkurzda23 said:
How do LC Quars look if they are contaminated?
Are here some pictures?
Ore do the contamination have a special look?





cloudy.  highly turbid.

Señor Helix: do you ever test your LC's on agar? Ever use blood agar to target bacterial contams?




Yes, I have used agar to test them, but inevitably it's a waste of time.  If the LC is clear and you can see mycelium pieces, it's going to probably work.  I've had cases when it didn't but they are the vast minority.  I think if there is bacteria in an LC it's relatively low if the LC is clearish.  How low is something I'm trying to figure out with a scope.  I simply have never gotten a good answer, but I've been informed I'm a fool for thinking it isn't in there.  If anyone has some real data about this, let me know.

I have not used blood agar.  I generally use MEA or, recently, MYPA (malt/dextrose-nutritional-yeast-peptone agar).  Those two formulas seem to perform the same for me.  I've tried a couple antibiotics as well as 1% of 3% H2O2 in agar.  I can't say if those really slowed bacteria, though.  They certainly did not kill it.  The only technique I know to remove bacteria from a culture on agar is what this thread is about. That actually works.


Edited by Blue Helix (10/27/20 10:53 PM)


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InvisibleMateja
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Re: Some notes about LCs [Re: Nurkurzda23] * 1
    #27007435 - 10/27/20 11:40 PM (3 years, 3 months ago)

Thank you for the detailed explanation about LC growth rate!
I've never used stirrers before but I think I'll have to try before I speak about it :super:


Quote:

Nurkurzda23 said:
How do LC Quars look if they are contaminated?
Are here some pictures?
Ore do the contamination have a special look?



I'm experimenting with this right now https://www.shroomery.org/forums/showflat.php/Number/27007467/vc/1#27007467


Edited by Mateja (10/28/20 12:36 AM)


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OfflineMessiah of Savants
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Re: vacutainers? [Re: Blue Helix]
    #27007438 - 10/27/20 11:44 PM (3 years, 3 months ago)

Quote:

Blue Helix said:
I've also seldom seen them not work well in a properly-prepared bulk substrate.




Over the years here I've often seen people state that most of the nutrients that mushrooms use to grow were from the grains and that you needed the grains to get a good yield. Do you call bullshit on this? It never made a whole lot of sense to me because they naturally grow in the wild without grains.

I assume you sterilize your bulk substrates that you inoculate with LC? I tried knocking up a sterilized bulk sub with a LC once and it colonized fine, but I got very few fruits. Maybe the moisture content of my sub was low or just bad genetics.

I've always liked LC and the idea of skipping grains and using LC to bulk substrate.


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OfflineBlue Helix
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Re: vacutainers? [Re: Messiah of Savants]
    #27007503 - 10/28/20 01:27 AM (3 years, 3 months ago)

Quote:

Messiah of Savants said:
Quote:

Blue Helix said:
I've also seldom seen them not work well in a properly-prepared bulk substrate.




Over the years here I've often seen people state that most of the nutrients that mushrooms use to grow were from the grains and that you needed the grains to get a good yield. Do you call bullshit on this? It never made a whole lot of sense to me because they naturally grow in the wild without grains.

I assume you sterilize your bulk substrates that you inoculate with LC? I tried knocking up a sterilized bulk sub with a LC once and it colonized fine, but I got very few fruits. Maybe the moisture content of my sub was low or just bad genetics.

I've always liked LC and the idea of skipping grains and using LC to bulk substrate.




These species of mushrooms do not need grain.  The are secondary (compost/manure) and primary (grains for example) decomposers, so they can grow fine either way.  The idea idea that mushrooms derive most of their nutrition from grains supplemented in the substrate just doesn't match my experience at all.

In fact, if you look at the bulk substrate I recommend, the first thing you notice is how few grains it has.  That's not because I didn't try with many levels of grain.  I did and heavy grain mixes simply do not perform as well over all the flushes.  More grain sometimes increases the density of the first flush, but the subsequent flushes are much weaker than a manure-heavy mix.  As a result, the overall yield is considerably less with a grain-rich mix.  My theory of why is that manure holds more water than grains (65% versus 50%), the water is easier to access for the mycelium, and the substrate water can be replenished via the casing faster and more fully.  The grain-richer substrate does not equal a higher mushroom yield across all flushes, and I think I'm one of the few people on here qualified to say that since few seem to bother with biological efficiency measurements.  Instead I usually see a bunch of pictures of the first flush, which is just one of three to five flushes, right?  I'm more interested in the total yield as measured by biological efficiency.

You might wonder why I have grains at all in my substrate?  They might help by some minor extent with micronutrients or something, but the MAIN reason grain is in there is that liquid culture does not take that well to only manure, sawdust, or straw for that matter.  I don't know why that is, but it's true.  The grains serve as loci for the initial growth from a liquid culture.  And from each grain, the mycelium can easily spread to the surrounding manure.  In a sense that's exactly what spawn does too.

Yes, I do inoculate a bulk substrate with few grains with an LC.  If you had a low yield, I promise you it was not because you used an LC to inoculate a bulk substrate.  Mostly likely it was a water issue.  It's pretty easy to get the wrong water content in a spawn bag, and more often than too dry it's too wet.  You really have to follow a recipe like mine or do a microwave dehydration test.  You want that percentage of water to be about 65% without much variance.  Even 5% off that really reduces yield.


Edited by Blue Helix (10/28/20 01:38 AM)


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OfflineBlue Helix
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Re: vacutainers? [Re: Blue Helix]
    #27007523 - 10/28/20 01:48 AM (3 years, 3 months ago)

Since I brought it up, here is my simple bulk substrate I use for liquid culture inoculation (this is sterilized in a set of large spawn bags for 4 hours)

Quote:

Blue Helix's Manure-based Substrate
Horse Manure (totally dry, break up) - 44 ounces
WBS or rye (not cooked) - 10 ounces
Vermiculite - 1.5 liter
Water - 100 ounces
~10 pounds total in two spawn bags @ 65% moisture




PS - I recommend a simple fish scale to weigh things.  Here's a good one.  Make sure to turn the hold mode OFF to use it for weighing substrates, and you should probably invest $3 in a 5-gallon bucket with handle at Home Depot.  Whatever you do, do not try to use a human scale or luggage scale.  They won't work well.


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OfflineMessiah of Savants
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Re: vacutainers? [Re: Blue Helix]
    #27007563 - 10/28/20 03:02 AM (3 years, 3 months ago)

I'm going to be trying your dry weight recipe some time in the next few weeks.

I've used both field aged dried manure and store bought dehydrated manure pellets in the past. In my experience, they seemed to come out to different consistencies with the same amount of water. Maybe this doesn't really matter because it's all the same dry shit, literally. I will be using dehydrated manure pellets when I try it. Have you worked with these pellets before?

The dry field manure I have is a real pain in the ass to work with because it has a few rocks in it and also extremely hard chunks that are hard as rocks to break up even when it's been hydrated. I don't have a good grinder or way of breaking these chunks up.

I've always liked the idea of weighing out dry ingredients and measuring the water used. I like things to be precise for successful repeatability. With cubes I admit I mostly just mix everything up and judge it by feel/squeeze and had great success but I'm trying to perfect my techniques because I'm trying in a big way to get Pan Cyans down to a science.

Thanks for your experience!


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Re: vacutainers? [Re: Messiah of Savants]
    #27007591 - 10/28/20 03:59 AM (3 years, 3 months ago)

The thing about the nutrients coming from grains is definitely true for non manure grows.


I just made my first little Tupperware of manure wbs mix to try this out but forgot I only have oyster lc right now.

Guess it’ll be spore syringe for now til I get some pan lc running.


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OfflineNurkurzda23
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Re: vacutainers? [Re: A.k.a]
    #27008248 - 10/28/20 12:07 PM (3 years, 3 months ago)

I will take pictures tomorrow.
Maybe somebody is nice and can tell me if everything is going fine with my lc.

How long should they colonize before you put them in the refrigerator?


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OfflineBlue Helix
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Re: vacutainers? [Re: A.k.a]
    #27008352 - 10/28/20 01:07 PM (3 years, 3 months ago)

Quote:

A.k.a said:
The thing about the nutrients coming from grains is definitely true for non manure grows.


I just made my first little Tupperware of manure wbs mix to try this out but forgot I only have oyster lc right now.

Guess it’ll be spore syringe for now til I get some pan lc running.




Yes, if you are growing on a non-nutritive substrate like say vermiculite, grain is absolutely critical.  After all, mushrooms don't grow well on rocks, which is what vermiculite is.  I'd say for a low-nutritive substrate, you do need to think about how you intend to add nutrients to the substrate.  Grains or flour are as good of additions as any, but if you do that, you also need to be considering how your substrate will hold water.  Grains without some water reservoir like vermiculite will perform fairly poorly, so think about how your intend to deliver water to your mushrooms too.


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OfflineBlue Helix
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Re: vacutainers? [Re: Nurkurzda23]
    #27008380 - 10/28/20 01:18 PM (3 years, 3 months ago)

Quote:

Nurkurzda23 said:
I will take pictures tomorrow.
Maybe somebody is nice and can tell me if everything is going fine with my lc.

How long should they colonize before you put them in the refrigerator?




There is no hard number of days for how long you should let an LC colonize before storing.  If you let the mycelium blobs settle by not stirring it, I've stored them at 20% mycelium to maybe 75% or even more.  In any case, as long as you have 20% or so in there, you have plenty of mycelium to initiate a new LC.  I do not recommend using an LC that has been in cold storage for many months to inoculate a bulk substrate or even spawn.  Even though that would probably work, I'd recommend to instead expand it out again and use it then.  You want to keep the mycelium momentum up when you grow.  That helps avoid contamination and results in higher yields.


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OfflineBlue Helix
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Re: vacutainers? [Re: Messiah of Savants] * 1
    #27008449 - 10/28/20 01:48 PM (3 years, 3 months ago)

Quote:

Messiah of Savants said:
I'm going to be trying your dry weight recipe some time in the next few weeks.

I've used both field aged dried manure and store bought dehydrated manure pellets in the past. In my experience, they seemed to come out to different consistencies with the same amount of water. Maybe this doesn't really matter because it's all the same dry shit, literally. I will be using dehydrated manure pellets when I try it. Have you worked with these pellets before?

The dry field manure I have is a real pain in the ass to work with because it has a few rocks in it and also extremely hard chunks that are hard as rocks to break up even when it's been hydrated. I don't have a good grinder or way of breaking these chunks up.

I've always liked the idea of weighing out dry ingredients and measuring the water used. I like things to be precise for successful repeatability. With cubes I admit I mostly just mix everything up and judge it by feel/squeeze and had great success but I'm trying to perfect my techniques because I'm trying in a big way to get Pan Cyans down to a science.

Thanks for your experience!




I have not used the manure pellets, but if you do, you need to do at least one microwave dehydration test because you don't know how much water is in those highly-processed pellets.  To do this test, you'll need a scale like this one

To perform a microwave dehydration test, weigh out precisely 20.0g of final substrate.  Spread it on a microwave-safe ceramic saucer or plate (or whatever else you got that won't burn or melt and is microwave safe).  Microwave it for about 1 minute, mix it up a little, go another 30 seconds to a minute, mix it up, etc.  You probably will end up microwaving about 5 total minutes before the mix is truly totally dry.  You want it bone dry and basically right before it starts to carbonize (burn).  Reweigh the sample (let's call that 'x' grams).  Here's the formula

1 - (x/20) * 100 = % moisture

You want 65% moisture which means X should be 7.  Plus or minus a 2% isn't isn't necessarily horrible, but if your measurement varies more than about 2.5% (X is greater or less than 7 by more than 0.5), I recommend adding water or more manure to bring it in line.  Make a note of any adjustments you made so you don't have to do this test again.

You mentioned mixing up to a "feel/squeeze".  I used to do that too, but I don't recommend it anymore.  For one, it might have seemed like it worked great for you, but you well may have never had a tray look like the pictures below.  Also, it's not a very scientific way of measuring moisture content, and I've seen folks make mixes way out of whack using it (usually too high but sometimes too low too).  Just figure out what amount of water you need with your ingredients using the microwave test.  Write it down, and keep using it over and over.




Yes, breaking up manure is pretty annoying, but not everyone feels their time is equally valuable I guess.  I sure don't like breaking up manure, so that's why I bought a mulcher as the one here.  Whatever mulcher you buy, it must have a metal impeller and be able to handle rocks because they will be in there. If you get one, you need to buy canvas bags with it since the cheap bags that come with it will rip apart in less than one minute from the hard pieces in there.  Buy some zip-ties too in order to secure the bags.


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Re: vacutainers? [Re: Blue Helix] * 1
    #27009723 - 10/29/20 06:47 AM (3 years, 3 months ago)

I just found and used an lc that’s a year old, made from spores but tested clean a couple months after I made it, and has been sitting at room temp the whole time which for the last four months has been about 90.



Still looks good but we’ll see.


--------------------
LAGM2020


Edited by A.k.a (10/29/20 06:47 AM)


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Re: vacutainers? [Re: A.k.a]
    #27009941 - 10/29/20 09:24 AM (3 years, 3 months ago)

Does look good. Usually with my old LCs that haven’t been kept in the fridge I’ll let everything settle to the bottom and just give it a little swirl by hand to see if there’s bottom sediment that swirls up only with older LCs.

For my LC I do 1000ml water
15ml corn syrup/honey
10mldextrose
5ml LME
And small amount of peptone

There is sediment after the cook but I actually like sediment because it gives something for the mycelium to latch onto and chew on and I think it’s so cool to watch the mycelial cloud collect all this sediment and the once dark piss colored LC after just a couple weeks starts to turn clear. You can actually watch the mycelium clean up the LC of the nutrients. My LC will start dark yellow and end up completely clear with no sediment.


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Edited by Celestialexplorer1 (10/29/20 09:32 AM)


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InvisibleD3_Myc
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Re: vacutainers? [Re: Celestialexplorer1]
    #27010000 - 10/29/20 09:59 AM (3 years, 3 months ago)

If you’re doing LC I take it injection ports and syringe is a must? Or if you’re working in front of a hood is tip and pour acceptable similar to LI?


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Re: vacutainers? [Re: D3_Myc] * 1
    #27010005 - 10/29/20 10:05 AM (3 years, 3 months ago)

I’ve poured LC many times, it’s easy and I find often it recovers super fast when poured. I’ve seen recovery in 8 hours when pouring.


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Re: vacutainers? [Re: Pastywhyte]
    #27010041 - 10/29/20 10:21 AM (3 years, 3 months ago)

I want to store cultures 10+ years.. I go back to slants twice a year to regrow them on agar -almost lost cultures waiting a full year.  I'm going to try your vacutainer lc tek, maybe using a centrifuge to seperate the mycellium if i can figure that out.


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Re: vacutainers? [Re: trippleblack] * 1
    #27010045 - 10/29/20 10:24 AM (3 years, 3 months ago)

If you used wood in your slant it would last closer to two years before you need to transfer them. Just an FYI.


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Re: vacutainers? [Re: Pastywhyte]
    #27010056 - 10/29/20 10:31 AM (3 years, 3 months ago)

Quote:

Pastywhyte said:
I’ve poured LC many times, it’s easy and I find often it recovers super fast when poured. I’ve seen recovery in 8 hours when pouring.




Awesome thanks, just wanted to make sure I didn’t need to buy more equipment. What’s the PC time on that, same as agar 15-20 min? Safe to assume all the LC ingredients get PC?

I have kiro syrup and lme on hand.... off to do research!  🔎  🔬


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