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OfflineBlue Helix
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What are people doing to select a substrain that shows rhizomorphic growth?
    #4311391 - 06/18/05 06:43 PM (16 years, 6 months ago)

I have had casing penetration problems time and again, and I am 100% certain my casing is perfect in every way. I have tested it for water content, pH, texture, etc. What I have noticed is that sometimes the casing colonizes like wildfire but MOST of the time I have trouble. I have tried different temperature and CO2 regimes, EVERYTHING. No one on here seems to have a clue about it either.

Having said all of that, I am really stuck. I am all the way back to the way that I start these substrains. Here is my procedure:

1) I take a tiny microdrop of spore solution or LC on agar.
2) I let the multiculture agar grow out over the agar plate
3) I scratch the surface under sterile water and suck up the pieces and solution.

This solution is made of a fluffy type of mycelium that shows absolutely no rhizomorphic growth at all. It is injected in spawn bags and used to make tons of seemingly healthy spawn.

I have no trouble making spawn in less than one week. I have no trouble colonizing horse manure that is well rinsed. I have no trouble doing anything other than getting the mycelium to TOUCH the casing. I am talking it doesn't even start to colonize it. It's like it is poison. I have this casing pH-balanced to 7.6 on the nose with a perfect water content as shown by drying analysis and a digital scale. The casing shows no rhizomorphic growth at all even after a week. The mycelium looks all white and fluffy but won't touch it, and the mycelium shows no rhizomorphic features either.

I am sick of this. Does ANYONE use the procedure I do above? When it works, it works perfectly, but usually, the mycelium lacks vigor to enter the casing layer.

What is happening?


Edited by Blue Helix (06/18/05 06:47 PM)


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Offlineshirley knott
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Re: What are people doing to select a substrain that shows rhizomorphic growth? [Re: Blue Helix]
    #4311625 - 06/18/05 08:16 PM (16 years, 6 months ago)

what is in the casing mixture?


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Invisiblebackupwards
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Re: What are people doing to select a substrain that shows rhizomorphic growth? [Re: shirley knott]
    #4311704 - 06/18/05 09:00 PM (16 years, 6 months ago)

check out the post: what am i doing wrong with my casings? by blue helix in this forum.
that explains alot.
peace


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Re: What are people doing to select a substrain that shows rhizomorphic growth? [Re: Blue Helix]
    #4311765 - 06/18/05 09:20 PM (16 years, 6 months ago)

Add 10% coir to your casing mix. That is somewhat like CAC'ing that some edible growers do.


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Re: What are people doing to select a substrain that shows rhizomorphic growth? [Re: backupwards]
    #4311794 - 06/18/05 09:29 PM (16 years, 6 months ago)

The current casing mix is:

20% coco coir (first time I have ever done this with peat)
35% peat moss
45% coarse vermiculite
2 cups oyster shell well rinsed
1/2 cup super fine pure calcium carbonate (not garden lime but pure calcium carbonate)

The mix ends up with a pH of about 7.6.

Today, I just took off nearly 75% of the casing (left about 1/4" on), took off the pin hole poked Saran wrap, and introduced the tray to fruiting conditions. Interestingly the core temperature suddenly increased drastically once put in these conditions! It was running about 83F before, but suddenly it shot up another 5F without any raise in ambient temperature.

Maybe the pin-hole-poked Saran wasn't a good idea? I only did that because I thought maybe that I was giving my trays too much fresh air by not putting anything over them before. Is it possible that pin holes every square inch was not enough gas exchange for a 3" deep substrate?

I don't know, but this temperature spiking in the tray is highly strange. It's up to 88F and the ambient temperature is only 76F. That is a delta of nearly 12F and I seldom see deltas that high once a tray is introduced to fresh air like this. I wonder if it was suffocating before. Maybe I went TOO far the other direction. I mean maybe I gave it not enough gas exchange this time and that stalled the casing run. I am hopelessly confused, but the sudden temperature rise is definitely unexpected.


Edited by Blue Helix (06/18/05 09:30 PM)


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OfflineKalix
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Re: What are people doing to select a substrain that shows rhizomorphic growth? [Re: Blue Helix]
    #4312608 - 06/19/05 01:51 AM (16 years, 6 months ago)

You could try cutting rhizomatic sections out of the agar plate, and innoculating with only the healthiest mycellium.. That's what Stamets recommends.


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OfflineBlue Helix
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Re: What are people doing to select a substrain that shows rhizomorphic growth? [Re: Kalix]
    #4313013 - 06/19/05 04:38 AM (16 years, 6 months ago)

Yeah, I could try that, Kalix, and I am well aware of what Stamets says. Stamets is hardly a home cultivator, though. That's a few months of work to get a pure isolate, but if that's what it takes, I might just do it. So, is that what you or your friend does?


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Re: What are people doing to select a substrain that shows rhizomorphic growth? [Re: Blue Helix]
    #4316025 - 06/20/05 04:18 AM (16 years, 6 months ago)

If you take tissue from a shroom will it always give rhizo growth? I have only done it once properly and the agar growth was amazing, pinned on the agar too.



I am asking this because I can buy commercial shrooms for ?20 which is about the same price as a commercial syringe (inc post).
I have read that you can store agar in distilled water in airtight containers for many years. Is the same true of mushroom tissue. I put some in water before and the tissue went blue and did not look very healthy. If you stab a syringe needle all the way through a shroom stem it cuts out a little core that can be injected into a jar of water. At a later date this could be injected with concentrated malt extract to start a LC. Does this sound viable?

I like the idea of cloning a commercial shroom since I presume that the professional grower would have gone to a bit of trouble to select a good fruiting strain, which I can now clone.


Edited by blackout (06/20/05 04:20 AM)


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Invisiblemycofile
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Re: What are people doing to select a substrain that shows rhizomorphic growth? [Re: blackout]
    #4326711 - 06/22/05 11:33 PM (16 years, 6 months ago)

Blue, I gotta say I just don't like your inoculation technique at all. If you are trying to get a pure culture it just has to have something going for it other than the fact that it's isolated. Now it doesn't sound like you are isolating true pure cultures, but you are definitely severely limiting the number of strains compared to just shooting with a spore syringe. And those limited strains just aren't being selected by anything other than chance (whatever ends up in the microdrop).

I say either clone, use lots more spores to give lots more strains, or isolate some aggressive rizo mycelium.

In fact, I can't even theorize a benefit of the way you are doing things as compared to say shooting a liquid media with a couple cc's of spore solution. At least in that case the strains can select themselves. What's the point of the agar step at all, just to make a liquid inoculate? Or are you trying to do a psuedo-isolation?

I think this thread sheds a lot of light on what the problem might be from your "casing" thread. If I were you I'd start a new batch using liquid inoculate (I like 4% dextrose, 96% water, pinch of powdered nutritional yeast, omit the yeast if you want it clear) with AT LEAST 1cc of spore solution per 500 cc of LM. While this batch grows out, you can do a real isolation on agar if you want, but I definitely would not waste grain on anything that didn't show aggressive rizo growth. I'd start the first plate with more than a microdrop of solution to get a decent number of spores going, then transfer the sectors to new plates. You can often get a good strain on the first or second transfer, using maybe only a dozen plates in all (counting the sectors you end up not using).
Personally though, I'd skip the agar stage alltogether. IME you'd have to go through several strains isolated like that to find a reallly decent strain. It's far easier to me to just clone from a multi-spore grow. You're nearly gauranteed a really decent strain and have a good shot at a great strain.

Bottom line, good genetics whether obtained naturally from multi-spore innoculations, cloning, or isolation on agar definitely should not have trouble racing through a casing but genetics obtained simply by limiting the amount of spores used to start the culture very well might.


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Re: What are people doing to select a substrain that shows rhizomorphic growth? [Re: Blue Helix]
    #4327608 - 06/23/05 06:59 AM (16 years, 6 months ago)

I am knew.. didn't think much about it... but CO/2 is the problem. You need better gas exchange and at the right times.

:confused:


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Re: What are people doing to select a substrain that shows rhizomorphic growth? [Re: Blue Helix]
    #4327611 - 06/23/05 07:01 AM (16 years, 6 months ago)

Moree... you are to worried about contams and don't give enough oxygen. You became to perfect in yur technique... mellow out and give em some air.


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Re: What are people doing to select a substrain that shows rhizomorphic growth? [Re: Blue Helix]
    #4328576 - 06/23/05 02:47 PM (16 years, 6 months ago)

Throw out everything but the peat and vermiculite. 50:50 add water until you can only squeeze out a drop or two. load into oven bag, and pressure cook at 15 psi for 45 minutes or more depending on the volume. Try and use this after it cools completely as a very thin casing layer less then 1/4 inch.

If that doesn't work, your peat is bad. or you are getting the moisture content wrong. You don't need PH meters, or moisture meters. Just add water and squeeze. If too much water is in it, add vermiculite slowly until you can get the content right.

If mycelium eats grain, or BRF, then it will go through the casing soil. Rhizo or cottony doesn't matter.

If you get the peat:vermiculite to work, then you can start adding lime in later mixes to deal with the green you might get. First try and get the mycelium to grow thorugh your mix, then adjust the mix to make it just right for your particulars.

1/4 inch of peat:vermiculite at the proper moisture, sterilized, cooled. NO lime of any kind.

Make sure your substrate is fully colonized, and actively growing when you case it.

If you fail with that. Just use sterile vermiculite laid shallow. If that fails, don't case. If that fails, get another hobby.


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Re: What are people doing to select a substrain that shows rhizomorphic growth? [Re: EonTan]
    #4330099 - 06/23/05 09:57 PM (16 years, 6 months ago)

mycofile, I agree with what you say entirely. I think what has happened is that I got a little too cleaver for my own good. By using the tiny microdrop on agar, there are few strains to begin with (perhaps a few dozen) and, thus, there is no real competition. By using an entire ml or two spores, I would guarantee more competition to get a stronger growth. I noticed my trouble with growing does seem to get worse every time I managed to reduce the number of strains more and more without any sort of selectivity.

So, I have gone back to the drawing board, and decided that instead of try to isolate on agar using rhizomorphic features, I would start using LC. I bought a mixer ($30 at the local lab surplus store) and am off to a new start:



I have started with a full CC of spores in 500 ml of dextrose/malt LC. It's being incubated by the mixer (ironically) at exactly 80-86F. The stirrer runs so hot that I had to insulate it from the plate even though it's a non-heating type!

I will use this to grow out, and if the results are good, I'll start another LC from a core sample of one of the largest fruits. If not I'll try another LC and another until I get a good fruit out. Once I have a good LC, I can separate it out and store it for years in sterile water in a vaccutainer in the refrigerator.



EonTan, that's for the reply but I think you are trying to take me back a little too far. Even the weakest mycelium in the world can make it through 1/4" casing because that's not really even a casing. I would call that a dusting. Such a thin dusting of casing material leaves gaps for the mycelium to go around. Still if the mycelium is not strong, green mold DOES set in. I have seen it happen time and again, and it has nothing to do with sterile procedure. Peat moss casings get green mold one way or the other, pasteurized or sterilized, unless the mycelium is strong.

When you say that any mycelium that eats through grain or brown rice flour can go through a properly prepared casing soil, my experience shows that to be dead wrong. I have never seen a mycelium not eat grains yet some of those will not penetrate a casing. However unusual it may be, that is, in fact, the exact phenomenon that I posted this thread over. It's also why I posted in the advanced forum because I realize that something I am doing is leading to a very unusual outcome with frequency. I believe it is the microdrop stuff that I had been doing, but I am not sure. I guess I'll know soon...


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Re: What are people doing to select a substrain that shows rhizomorphic growth? [Re: Blue Helix]
    #4330560 - 06/24/05 12:04 AM (16 years, 6 months ago)

My latest trays show typical super weak mycelium growth (sorry about the strange distortion but the picture was taken through vinyl):



Now you may say, "Hey, that looks fine!" It isn't fine. Do you see the baking soda patches? That's green mold patches each about 1/4" in diameter. And why mold on this tray? Answer: the mycelium never showed even the slightest sign of rhizomorphic growth and without any colonization mold will come in a two or three weeks. It happens EVERY TIME to me if the mycelium isn't strong.

What you see here is about 1/4" of casing material on THREE INCHES of colonized WBS/RYE/verimiculite. I should have cased with three times that amount without any trouble. Nearly all the casing material had to be taken off this tray after a week because there was no significant casing growth. After casing was stripped off and the tray put in the fruiter, very cotton-like mycelium with super weak growth eventually did take over the super thin casing and collapsed into what you see here (about ready to pin).

The mycelium substrate seemed vigorous from a temperature viewpoint as the core was seldom below 10F above ambient, but it's ability to colonize the casing was dismal.

I fully expect to see this tray fruit well in the next day--maybe not spectacular but it should be good--but it's not really what I'd call an ideal tray. It is NOT what an advanced grower should be growing. Since the mycelium didn't take the casing quickly and I had to take off the casing mostly, I don't expect to see a second flush without more mold. I certainly won't see a third before green mold takes it down.

I am no newbie with this stuff. I know what a champion mycelium run looks and acts like because luck has brought me such trays before, but this is not it. I want advanced users to tell me what I am missing. Right now I am going to give LCs a try, and I hope they can help.


Edited by Blue Helix (06/24/05 12:07 AM)


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Offlinescatmanrav
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Re: What are people doing to select a substrain that shows rhizomorphic growth? [Re: Blue Helix]
    #4330854 - 06/24/05 01:18 AM (16 years, 6 months ago)

You have the wierdest problems with casing layers lol...I havnt a clue...I dont do much different.

Once though..I read about isolates and such...and some isolates, rhizo or cottony, just dont pin...so taking a strain and doing a half assed isolate, say one transfer, where you have like 5 substrains in the wedge you take...then you cut off that plate and inoculate grain, it could actually be worse then doing a multispore injection..even though another few transfers to get a good fruiting isolate (seeing which pins on agar) can lead to a very aggressive strain and fruiter..the half isolate lacks genetic diversity and may or may not do as well...maybe your inoculation procedures is doing something to that effect..I know were not talking pinning here. That is the only thing you do differently then me or anyone else..the casing layer is obviously fine.

Even still, I couldnt image it just not colonizing the casing layer...I've never had that problem.


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Growing with bags, start to finish (including my new grain and substrate prep)
Anyone looking to start bulk tubs/mono tubs/shotgun hybrids? Good tubs to use..
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Turn your closet into a fruiting chamber
Casing layer colonization and overlay


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OfflineBlue Helix
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Re: What are people doing to select a substrain that shows rhizomorphic growth? [Re: scatmanrav]
    #4330883 - 06/24/05 01:30 AM (16 years, 6 months ago)

scatmanrav, I believe lack of diversity is the cause. If you start with few, it better be one or more that won out over others through a massive multi-spore race. If you restrict the diversity before any competition, you are likely to get mediocre strains. That is probably what's happened to me. I think the LC should solve this problem. It sure is easy! In 12 hours I see lots of growth already, although it was inoculated with 1 ml of someone else's active LC. If it continues at this pace, I think I've found the golden secret to end all problems.


Edited by Blue Helix (06/24/05 01:33 AM)


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Re: What are people doing to select a substrain that shows rhizomorphic growth? [Re: Blue Helix]
    #4336932 - 06/25/05 08:16 PM (16 years, 6 months ago)

Good. This has been bothering the hell out of me since the last thread you posted. I think that you'll have no trouble once you stop restricting the diversity on the front end. You just got a little too "advanced" with the micro-drop to agar step. I've "over-advanced" many things in the past, and it is often the culprit whenever experienced cultivators stumble. Keep us updated.


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I just looked at my profile and realized I had a website at one point in time on geocities, it's not there anymore and I have no idea what I had on it. Anybody remember my website from several years aga? PM if so please.


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Re: What are people doing to select a substrain that shows rhizomorphic growth? [Re: mycofile]
    #4342904 - 06/27/05 02:23 PM (16 years, 6 months ago)

Yeah... sometimes when we over-think things it spoils the fun, and usually ends up with us being pissed off, one way or the other.  Wait to do the fancy stuff when you have some shroomies to clone and play with. :smile:


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Re: What are people doing to select a substrain that shows rhizomorphic growth? [Re: Blue Helix]
    #4342958 - 06/27/05 02:32 PM (16 years, 6 months ago)

I use 1/22 of a 1/100 spore dilution and NEVER have the problem you are having.

I test individula dikaryons frequently, and never had one fail to colonize the casing if it colonized the substrate. I actually prefer to test single dikaryons over the multispore and clone method. You will get a far better picture of what the strain has to offer if you test them individually, it just takes more effort, and the rewards are negligible. Other then getting a VERY GOOD PICTURE of what diversity really means when talking about a Strain, and comparing substrains, or comparing substrains between strains.


Variety with substrains is based on variances in environmnetal tolerance. Some like more acid, some more base, some high temps, some low temps, etc... The majority fall within a average range, a few are out at the edges.

If you want somehting that completes its lifecycle in your particulars, multispore a jar, and clone the best looking fruit from your casing.

Then you have a single dikaryon that completes its' life cycle in your particulars, up to your standards.

I think too much emphasis is placed on substrain diversity when dealing with Lifecycel failures. EVERY SUBSTRAIN that was foermed by compatable monokaryons will fruit.


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Re: What are people doing to select a substrain that shows rhizomorphic growth? [Re: EonTan]
    #4344834 - 06/27/05 10:43 PM (16 years, 6 months ago)

Blue Helix, just to be clear, you started some LC with 1cc of SPORE solution in 500cc of LM, right?

I ask because your other thread was started with a cc of LM, which could theoretically be limiting the genes in the same way the microdrop was.

And Eontan may be right, that might not be the problem anyway. It just stands out as the one thing you are doing that isn't common, and that I would personally never do.

I guess a big difference will be if the new cultures show rhizo growth when they grow on the grain, which should be any day now.


--------------------
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PM me with any cultivation questions.

I just looked at my profile and realized I had a website at one point in time on geocities, it's not there anymore and I have no idea what I had on it. Anybody remember my website from several years aga? PM if so please.


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Re: What are people doing to select a substrain that shows rhizomorphic growth? [Re: Blue Helix]
    #4345127 - 06/28/05 12:17 AM (16 years, 6 months ago)

Yeah, EonTan, I don't think I've ever had a strain that wouldn't fruit either unless mold got to it first. But that is not what I am talking about. I am talking about strains that won't go through casings worth crap because they don't form rhizomophs. Mycelium like that is really a bummer. I am fruiting one of those right now:



I would consider this a poor tray for many reasons. For one the tray would not colonize the casing properly after incubation for seven days, so I had to shave most of it off except about 1/4" dusting. Secondly, the mycelium would not form rhizomorphs which left the casing open to trich, which I've had to cover in several places with baking soda. Third, the pin set is okay, as you can see, but not really spectacular, especially for Equators on a first flush. I've had EQ trays that were so dense that they barely left a two square inches open anywhere on the entire tray surface. A GOOD tray is like that.


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Re: What are people doing to select a substrain that shows rhizomorphic growth? [Re: Blue Helix]
    #4345161 - 06/28/05 12:28 AM (16 years, 6 months ago)

mycofile, I did start it from 1 CC of live culture. The LC was concentrated down and came from one of the best, and I know that the guy who started it often uses 2 to 3 ml of homemade spore solution to start up his LCs. And so this LC should already represent some degree of genetic selectivity. The difference will be that I am using about 20 times the amount I was before and the strains were in combat in a 3D space, so no losers could have hung around the sidelines out of the ring so to speak. So if this genetic diversity thing WAS the issue, I think that chances are I would see a definite difference.


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Re: What are people doing to select a substrain that shows rhizomorphic growth? [Re: Blue Helix]
    #4345202 - 06/28/05 12:41 AM (16 years, 6 months ago)

PS - It's been 12 hours since I injected the live culture and I already see my first sign of growth on one of the grains. That is the all time fasted I have ever seen a sign of growth in a non-incubated spawn bag from a mycelium injection. I really hope this is real cubensis and not something else. My gut tells me it is, though.


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Re: What are people doing to select a substrain that shows rhizomorphic growth? [Re: Blue Helix]
    #4354812 - 06/30/05 11:43 AM (16 years, 6 months ago)

UPDATE

I felt it was a little unfair to leave this thread the way it is. While the rhizomorphs of this these three trays were not good and I did have trouble with the casing soil penetration, the yield was fairly good in all areas except the baking soda-covered ones. In fact I destroyed most of the casing trying to remove the mushrooms. Here is HALF the harvest:



I think if I'd have gotten rhizomorphs, though, I could have managed the casing a lot better. I am not so sure anymore, though, that is was strain-related. I mean this harvest was nothing to scoff at.

In addition to going to liquid culture, I am going to give agar's suggestion of using a calcium hydroxide SOLUTION to balance my peat moss next time. I think a solution is a good idea since it lead to a more pH-uniform casing.


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Invisiblemycofile
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Re: What are people doing to select a substrain that shows rhizomorphic growth? [Re: Blue Helix]
    #4363673 - 07/02/05 06:15 PM (16 years, 6 months ago)

I still think it's strain related because 1) irregularity when you say nothing else changes, and 2) if it were uneven mixing in regards to casing pH, surely parts of the casing would colonize better. Seems to me the only thing that changes from colonized casings to non-colonized casings is the genes.

*edited to add*
I also like the idea of using basic solutions to adjust pH, I just don't think that's your problem.


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Edited by mycofile (07/02/05 06:17 PM)


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Re: What are people doing to select a substrain that shows rhizomorphic growth? [Re: Blue Helix]
    #4364305 - 07/02/05 09:49 PM (16 years, 6 months ago)

mycofile, I don't know. The LC mycelium is very fast. Although I don't see actual rhizomorphic growth, I do see very strong linear mycelium bridging gaps everywhere. I just did a final break up of the LC bags and they are on the last 48-hour recolonize before being laid down. I like to break it up in the bag once it's about 90% colonized and let it re-colonize for about 48 hours.

Today I mixed up the casing material. Usually I use only oyster shell and limestone powder or calcium carbonate flour for the pH balancing. Today I used a tiny, tiny bit of calcium hydroxide, like an 1/8th cup, and the pH shot through the roof. Mr. Hawke's 50/50 formula recommends a whole 1.2 CUPS of that stuff! If you use the amount he suggests, your casing pH will likely be above 10.5. I just don't have a clue what he is talking about. I think he's consumed one too many mushrooms himself.

ANYWAY back to the casing. I mixed it down to a pH of about 7.8 and added my regular oyster shell. I immediately noticed that the fuel-like smell that I thought was my peat moss is from the rinsed oyster shell. I don't know what that smell is, but I don't like it. Because it gives me bad vibes (the smell), I decided to ditch the oyster shell I have all together. I threw that casing out in the garden and mixed up another. This time I used aragonite crushed coral instead which I've used before and is a better buffer anyway since it dissolves easier.

So, the final mix was peat moss, vermiculite (about 60%), a few cups of aragonite crushed coral, and about 10% coco coir. I loaded that up in a cement mixing bag and rinsed the HELL out of the whole thing, using the cement bag as a big strainer of sorts and blowing gallons of water through it. I then squeezed the bag with my entire body weight to bring the moister to the right level, re-tested the pH, which stayed at about 7.8, and put it outside. I'm going to soak pasteurize the whole mix tomorrow.

You see I am not leaving anything to chance this time. If the mycelium doesn't like this casing soil, I don't know what to do. I will be finally out of ideas.


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