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OfflineBlue Helix
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Some notes about LCs * 8
    #26977498 - 10/09/20 07:08 PM (3 years, 5 months ago)

As anyone who's done searches on my grow log knows, I'm a big proponent of LCs, and I've taught many people to not fear them so much.  But, on the other hand, I understand why they have the reputation they do.  In some cases, it's just easier to go from spore to agar plate and stop there rather than go the additional LC step.  But once you have a good LC that contains exactly what you want, there is no need for agar, flow hoods, still air boxes, clean techniques, etc.  I mean it doesn't hurt to be clean, but I routinely grow LCs in places that have mold around.  I store them in inexpensive vacutainers as well in the refrigerator, where they last about a year in a full-sugar solution or many years in a highly diluted sugar solution (say 1 ml of dense LC in a normal sugar solution to 9 mls sterile water)  Everything with LCs is based on injections for me, and injections just don't allow mold and bacteria in new LCs or sterile spawn bags unless you fail to sterilize the needle.  I like to get to a viable LC as soon as I can so I can stop messing around with agar plates and clean stuff.

One thing I've noted lately, though, is that agar to LC can kill a tiny bit of mycelium if you immediately spin the LC even if it is spun at a fairly low RPM (200 RPM or so).  I use a still air box and commercial sterile swabs (rather than a scalpel) to transfer a very tiny bit of mycelium from a promising section of agar plate to an LC.  The piece is often no bigger than a few periods on a piece of paper, so this may not apply to those who use agar wedges which are dozens of times larger.  With such a tiny bit, I've had times when the piece is killed in the LC by the spinning (at least I think that is the issue).  So I recommend sometimes allowing the LC to sit a little bit after introducing a very tiny mycelium fragment to give it a little time--say a couple days--to grow before the spinning starts (if you use spinning at all).  I like spinning them because they propagate faster and become denser, but spinning a tiny fragment of mycelium from the start can kill it in my experience.  In contrast, when inculcating a new LC from a vacutainer of stored LC, I typically use only a couple ml of LC in the inoculation and start the spinning immediately.

Another tip I'd like to give is to not give up on a cloudy LC.  Cloudy LCs are normal if one uses malt even from day one because the malt has solids form from the heat (no, I don't know the chemistry behind this).  But if the cloudiness doesn't resolve right away (say a few days), it still might be fine if you see mycelium blobs forming.  The reason is that mycelium can kill very minute amounts of bacteria.  In the end, a good LC is not cloudy, though, since the mycelium will sequester all the malt sugar fragments and kill all bacteria that may be in there.  I have had cloudy LCs work that I am positive had either bacteria or yeast growing in them (from a smell test), but the chances of stalling the bulk substrate (or spawn if you go that route) is a lot higher with a cloudy LC. 

Anyway, I'd love to open this post up for other little tips that people have of successful LCs.

And, of course, I have to include a little LC grows porn. Each of these grows started either as a multi-spore on agar or an LC direct from CLEAN spores (few people even know what a clean print means so you probably can forget about trying that unless you have a very good spore vendor).  I do not fuss too much about sectoring away "bad" growth on agar nor do I believe it is possible to do so without actually growing out the phenotype (you only get a very crude idea by the agar projection's appearance).  I don't even bother with clones because I personally don't see the point given how dense my grows often are without one. but if you are a clone believer, go for it I guess.  All grows in the pictures used an LC to inoculate direct to sterile bulk in a spawn bag:


Edited by Blue Helix (10/09/20 07:33 PM)

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InvisibleMateja
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Re: Some notes about LCs [Re: Blue Helix]
    #26977532 - 10/09/20 07:39 PM (3 years, 5 months ago)

:takingnotes:


I snapped these pics just now, two 10 days old broths consisting of 0.1% LME.
One broth (the two top pics) is becoming progressively more turbid as one or more different microbes are happily expanding their colonies at a fast rate. Consequently becaude of the competing environment the cubensis colony has slowed down its growth to a practical halt. The other LC has a broth that's as clear on incubation day 10 as it was on day one, only difference is the broth is being rapidly colonized by this healthy looking Cubensis culture. I'll use this LC in a moment to start a few new ones and I'll dump what's left onto some recently sterilized rye jars and a few BRF cakes I have laying around as well. Thanks for posting :hatsoff:

Steadily rising on the turbidity gradient



Now this is my preference on the good looks of a broth :popcorn: will definitely be expanding handsome fella


--------------------
Cakes inside Water Tub

Edited by Mateja (10/09/20 07:40 PM)

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OfflineBlue Helix
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Re: Some notes about LCs [Re: Mateja]
    #26977557 - 10/09/20 07:58 PM (3 years, 5 months ago)

Quote:

Mateah said:

Now this is my preference on the good looks of a broth :popcorn: will definitely be expanding handsome fella




Yeah, I love the picture of the good LC!  That's how they will look without spinning.  With spinning, they'll appear more like this:



These pictures show on a very young LC can have some cloudiness and still be fine (they will clear up when mature):



Once you get a good LC, stirred or not, you should store a little of it before use so you don't have to mess with the annoying agar plates again.  I recommend vacutainers, but not all vacutainers are the same!  Many have additives and clotting factors that you don't want.  Many do not have the kind of septum that you want (red silicone ones that can also be used to create my LC Kerr jar lids).  The best place to buy the ones you DO want that I know of is from Walmart's Online PetRx Pharmacy.  You won't find the additive-free ones that you want cheaper than that anywhere, including eBay.

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OfflineHikeadellic
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Re: Some notes about LCs [Re: Blue Helix]
    #26977567 - 10/09/20 08:06 PM (3 years, 5 months ago)

Nice write up! Perfect timing too because I was planning on researching this in depth tonight anyway!

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Offlineverum subsequentis
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Re: Some notes about LCs [Re: Mateja]
    #26977575 - 10/09/20 08:10 PM (3 years, 5 months ago)

:splooge:

The pics are amazing.

When you say "sterile bulk in a spawn bag" what exactly do you mean.

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OfflineBlue Helix
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Re: Some notes about LCs [Re: verum subsequentis]
    #26977601 - 10/09/20 08:29 PM (3 years, 5 months ago)

Quote:

verum subsequentis said:
:splooge:

The pics are amazing.

When you say "sterile bulk in a spawn bag" what exactly do you mean.




If you do a search for my alias Blue Helix, you'll see grow logs that talk about it, but in a nutshell, I put a piece of gaffers tape opposite the filter patch (tape cannot be the type that melts), load up large spawn bags (the kind you seal with an impulse sealer and can get cheap on eBay) with a manure/WBS/vermiculite/water mix (about 4 to 7 pounds per bag), impulse seal the bags, pressure cook the bags for 4 or 5 hours, let them fully cool, inject the LC through the tape (I use 140 ml syringe but you can use less), put a blob of hot glue on the tape to sterilize and seal the hole left by the injection, and knead the bag a little.  Once the substrate has fully colonized (about 8 days usually), I open the bag, lay my trays with the crumbled substrate, carefully even the top, let the trays recover overnight covered with hole-poked syran wrap, and remove the wrap and case them the next day.  This basically is all there is to my grow technique, but I do have a pretty nice growing chamber admittedly (I can control humidity and temperature precisely).  You can read about the details of these steps and where to get the materials by searching for Blue Helix.  I go back over 17 years on this site and Mycotopia, so there is plenty of grow log examples describing the grows more fully.

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InvisibleMateja
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Re: Some notes about LCs [Re: Blue Helix]
    #26977614 - 10/09/20 08:42 PM (3 years, 5 months ago)

2yesr old experiment. Never been swirled even once, none of them. Completely untouched virgins.
65day incubation resulting in all broths colonizing fully. All of em eventually pinned from the colonized broth surface. By the end of this all the jars ended being homogenized by the same clone culture suspended in broths as clear as the day they were sterilized.

Quote:

Mateah said:
:hi:

I made 5 liquid cultures in varying strengths of ELME at 0.1%, 0.2%, 0.4%, 0.8%, and 1.5% strength.
I inoculated all LCΒ΄s with the same clone using The Biopsy Method by Josex.

Updates

12 days after inoculation Jan 3
                0.1%                                0.2%                                  0.4%                                0.8%                                1.5%                                                                   

18 days after inoculation Jan 9

25 days after inoculation, Jan 16

31 days after inoculation, Jan 22

38 days after inoculation, Jan 29

43 days after inoculation Feb 12

64 days after inoculation Mar 5




Other experiments
WBS hydration methods -[EXPERIMENT]-
WBS Hydration/Colonization Times -[EXPERIMENT]-
Water absoprtion in BRF cakes -[EXPERIMENT]-




A couple of stone producers suspended in crystal clear view



--------------------
Cakes inside Water Tub

Edited by Mateja (10/09/20 08:45 PM)

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Invisiblemushhead
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Re: Some notes about LCs [Re: Mateja]
    #26977621 - 10/09/20 08:50 PM (3 years, 5 months ago)

I've only ever done one LC grow of Gulf Coast recently.
It was a very amateur attempt, but was quite successful.
I plan to do more
:takingnotes:

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Offlineverum subsequentis
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Re: Some notes about LCs [Re: mushhead]
    #26977630 - 10/09/20 09:01 PM (3 years, 5 months ago)

Quote:

manure/WBS/vermiculite/water mix




That's all i really wondered about but thanks for the info.

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InvisibleNobler Hino
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Re: Some notes about LCs [Re: verum subsequentis]
    #26977639 - 10/09/20 09:07 PM (3 years, 5 months ago)

:studmuffin: gunna check this out after work ladies


--------------------

"The sacred mushroom takes me by the hand and brings me to the world where everything is known. It is they, the sacred mushrooms, that speak in a way I can understand. I ask them and they answer me.”
– Maria Sabina

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Re: Some notes about LCs [Re: Blue Helix]
    #26977657 - 10/09/20 09:20 PM (3 years, 5 months ago)

@Blue Helix,

Have you ever tried using grain flour to reduce your pc time? I did 90 minutes with wild bird seed flour in a presto with 3 bags. Nocced with 60 ccs of LI 5 days ago and shook it up 2 days ago. 5 hours seems really really long.

These bags are wbs flour, verm, and water via spongiforms tek.



--------------------
πŸ…ƒ πŸ„΄ πŸ„° πŸ„Ό    πŸ„² πŸ„» πŸ„Έ πŸ„½ πŸ„Ά πŸ…† πŸ… πŸ„° πŸ„Ώ

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OfflineBlue Helix
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Re: Some notes about LCs [Re: JHOVA] * 1
    #26977733 - 10/09/20 10:36 PM (3 years, 5 months ago)

Quote:

JHOVA said:
@Blue Helix,

Have you ever tried using grain flour to reduce your pc time? I did 90 minutes with wild bird seed flour in a presto with 3 bags. Nocced with 60 ccs of LI 5 days ago and shook it up 2 days ago. 5 hours seems really really long.

These bags are wbs flour, verm, and water via spongiforms tek.






The reason my bags take that long is not because I use grains.  In fact, that has nothing to do with it, but it is true that flour might make a difference (but I'll get to the pros and cons later).  The grain bags take that long because they are about twice the size of the bags you show in those pictures and the pressure cooker is full.  It's not uncommon for me to sterilize two 7-pound bags (about 14 pounds) in an AA 921 cooker.  To get heat to penetrate to the center of the bags, even if they are properly separated, just takes a lot of time.  Most bags I've seen on Shroomery are about 3 or 4 pounds, and those can sterilize just fine in 2.5 or 3 hours. 

I have tried 10-pound bags before (two of them) but the packing is so tight and they are so close together that even 5 hours was not enough CONSISTENTLY (it worked sometimes).  It's not like they develop mold or anything weak like that; mold is very easy to kill.  The bacterial endospores, though, in the center of the bags can germinate once the bags cool, and the bag will stall.  It might not smell weird or anything--it's not like that kind of bacteria--but they will not fully colonize.  That's why I don't go over 7 pounds usually.

I think if I had cooked the grain prior and allowed it to sit overnight, the bags could probably be sterilized in far les time since the bacterial endospores would germinate, and live bacteria is far weaker than the endospores of course.  And shortening the sterilization time might be an advantage to flour too since I suspect it's typically bleached and pasteurized whereas wild bird seed or rye for feed usually is not.  Still, I don't like the idea of flour because it doesn't release its nutrients as well over time.  Grains are more of a timed-release affair, and that's good for big and more flushes.

Edited by Blue Helix (10/10/20 01:08 AM)

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Re: Some notes about LCs [Re: Blue Helix]
    #26977795 - 10/10/20 12:15 AM (3 years, 5 months ago)

Can you explain why it's better to take the same volume in 2 big bags instead of 3 or 4 smaller ones over 5 hours? You could easily do 2 or 3 cycles at 90 minutes each with flour.

Respectfully I know this method works for you and has for 2 decades almost. All im saying is maybe improvements can be made in your workflow to save you time.

Ive had many and good flushes with flour alone. I cant say it was better or worse than grain itself. If i remember correctly the highest biological efficiency was with cakes not monos. I dont think flour makes one suffer yield.

Specifically to your style of growing take a look at this Russian pan cyan grower that uses NO GRAIN at all though he uses straw and manure which i suspect is grain/grass humus. He gets 3 nice flushes. My point being grain is over rated for pans and possibly not needed at all. If grain is the reason you have to pc 5 hours perhaps theres a way to reduce that time without sacrificing anything.



https://www.shroomery.org/forums/showflat.php/Number/26293989

Quote:

angam said:
Greetings brothers Grover! I apologize for my English.Please understand.



Hay-manure was prepared, placed in jars as airy as possible.

Sterilized in a pressure cooker.

In gloveboxes shifted by a small piece overgrown with mycelium in each jar.

Then in incubator on 30 degrees in during 18 days.



Mycelium-snow-white cotton wool!


The container is larger, for a wider area of fouling.

Volume of 10 liters.



I taped the outside of it with metallic light tape.

Sterilized 4 cans 0.7 l. with a cover in a pressure cooker.

In an isolated box laid a layer of 2 cm. Moistened.



Well overgrown eight cans laid an air layer over the cover and sprayed liberally.



Covered the remaining cover as thin as possible, closer to 0.5 cm and also moistened.



Wrapped with foil and put on fouling in an incubator 30 degrees.



After a day and a half, the mycelium sprouted through the cover.



Put it in the greenhouse.Temperatures on fruiting fielded 28gr.





On the third day the little ones appeared…




On the fourth day have grown up..



On the fifth-go to the forest to harvest Panaeolus!






Harvest 214 grams of raw weight for the first flash.




In the drying-10.6 gr.

Waiting for the second flash.



Three days later, we collect the second flash.



On yields-slightly less the first.



Passed still three days and the third wave of assembled.



The next waves can not be expected.Mushrooms will gradually grow and gather.






https://www.shroomery.org/forums/showflat.php/Number/12826882
These pics are all in one bags with 3 quarts verm, 1 quart water, 1.5 flour. 90 minute pc cycle from spongiform.








--------------------
πŸ…ƒ πŸ„΄ πŸ„° πŸ„Ό    πŸ„² πŸ„» πŸ„Έ πŸ„½ πŸ„Ά πŸ…† πŸ… πŸ„° πŸ„Ώ

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Offlinetripdawg420
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Re: Some notes about LCs [Re: JHOVA]
    #26977813 - 10/10/20 12:36 AM (3 years, 5 months ago)

:takingnotes:


--------------------
HUSTLER
How U Survive This Life Everyday Resourcefully
epic GT mono tub
http://www.shroomery.org/forums/showflat.php/Number/17277772

wbs tek
http://www.shroomery.org/forums/showflat.php/Number/11525679
coir tek
http://www.shroomery.org/forums/showflat.php/Number/11917410
results :thumbup:

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InvisibleFunky Monkey
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Re: Some notes about LCs [Re: Blue Helix]
    #26977819 - 10/10/20 12:46 AM (3 years, 5 months ago)

:threadmonitor:

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OfflineBlue Helix
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Re: Some notes about LCs [Re: JHOVA] * 1
    #26977850 - 10/10/20 01:38 AM (3 years, 5 months ago)

Quote:

JHOVA said:
Can you explain why it's better to take the same volume in 2 big bags instead of 3 or 4 smaller ones over 5 hours? You could easily do 2 or 3 cycles at 90 minutes each with flour.

Respectfully I know this method works for you and has for 2 decades almost. All im saying is maybe improvements can be made in your workflow to save you time.

Ive had many and good flushes with flour alone. I cant say it was better or worse than grain itself. If i remember correctly the highest biological efficiency was with cakes not monos. I dont think flour makes one suffer yield.

Specifically to your style of growing take a look at this Russian pan cyan grower that uses NO GRAIN at all though he uses straw and manure which i suspect is grain/grass humus. He gets 3 nice flushes. My point being grain is over rated for pans and possibly not needed at all. If grain is the reason you have to pc 5 hours perhaps theres a way to reduce that time without sacrificing anything.






I find it interesting that you assume I haven't thought about these things.  Believe me, I've thought of almost everything over the years and tried most all of them.  I use the bigger bags for few reasons.  One is that they can actually fit in the pressure cooker more easily with less issues.  For example two bags can touch the bottom where the heat is originating, which I've found help with sterilization (when I'd stack them, the ones on top of other bags would often not be sterilized without extra time).  Another reason is that I hate doing the injections.  I use 140 ml, and it's the one thing that is difficult to do (as in you kind of have to be sober at least) because it's extremely easy to poke through the other side of the bag doing it.  It's the one time you introduce the potential for contamination more than any other (and again, I don't do clean techniques), so I want to limit injections and kneading multiple bags.  Lastly, the bags are like 70 cents each.  Why would I want to waste that much time and money on bags when electricity is dirt cheap?  Have you actually done the numbers on it?  It's ridiculously cheap to keep a pressure cooker hot (much more expensive to heat one up).  You can run a large electric burner at half heat for 5 hours at 20 cents a kilowatt hour for less than a dollar or the price of one and a half bags.  And as for a gas burner, forget it; gas probably costs a third the price or less compared to electricity!  It's not like it's hard to keep the cooker going longer or something.  In fact, to me it's ten times as annoying putting together a whole bunch of bags, doing sets of them, heating and cooling the cooker repeatedly, putting new bags in, pressurizing the cooker, and then injecting them all separately.  The entire point of using bags was to get away from that.  I want things easy, cheap, fast, and, maybe most importantly, very high-yielding.  And if I could do it all in ONE bag, I would do it that way even if it took 6 hours.  I just can't though because I've tried it and it doesn't work.

As for the point about the grain, I think you must have misunderstood why I use grain at all.  Again, I've tried the bags without grain, without vermiculite, without horse manure, with more grain, with less grain, etc.  The problem with no grain is simply that the LC won't take to the substrate a lot of times.  Grain provides a nutrient source that even a weak LC can take off on - straw and manure, not so much.  The reason I use so little grain is because high grain mixes don't yield well.  The grain doesn't hold as much water (50% water versus manure's 65%) and it doesn't soak up water from the casing as well, so the substrate isn't refreshed as much between flushes.  Now I do like straw but not because it's very nutritious or holds water well--it doesn't do either of those things--but because it opens the substrate up to better airflow and provides pathways for the mycelium to colonize.  But there is no way in hell I would take the time to collect it and cut it up.  I know you can use a weed whacker and all that in a trash can to chop it up, but I don't want to buy a weed whacker or collect straw on top of the manure too.  Again, less is more for me.  The fewer ingredients, steps, bags, cooking cycles--you name it--the better.  It's like the difference between American and Japanese cars in the 80s.  The Japanese destroyed the American car industry for one main reason - they used fewer parts with lower complexity designs which made their cars more reliable, easier to fix, and last longer than the American offerings at the time.

Here is the final formula I came up for my substrate.  This makes 10 pounds:

Quote:

Manure-based Substrate (grain-to-manure wet volume ratio 1:6.5, moisture content 65%)
Manure 44 oz
WBS 10 oz
Vermiculite 1.5 L
Water       ~100 oz
~10 pounds total (4 x 13"x9" trays)



Edited by Blue Helix (10/10/20 01:44 PM)

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Re: Some notes about LCs [Re: Blue Helix]
    #26977992 - 10/10/20 05:44 AM (3 years, 5 months ago)

Thanks for the tip about the walmart vacutainers Blue!
When i used to work with pans and cubes i did them full cycle without agar, spores to lc, clones to lc. There are a couple of Mexican species however that don’t clone well to lc for me and I've had to go back to agar for them.

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OfflineAtomHeart
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Re: Some notes about LCs [Re: Rumblestrip]
    #26978012 - 10/10/20 06:18 AM (3 years, 5 months ago)

Cheers!!



--------------------
You once ripped a man's jaw off...I seent it!


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Offlineel gordo
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Re: Some notes about LCs [Re: AtomHeart]
    #26978016 - 10/10/20 06:22 AM (3 years, 5 months ago)

very nice thread and pictures, thanks!

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Offlinepolyflakes
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Re: Some notes about LCs [Re: AtomHeart] * 1
    #26978017 - 10/10/20 06:23 AM (3 years, 5 months ago)

I have always thought using a LC was so much less trouble then g2g and its the same idea mycelium not spores.


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