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OfflineCliftonGK1
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From poor spores to great grains; a short story
    #19185552 - 11/25/13 06:08 AM (10 years, 2 months ago)

Recently moved across the country and in doing so I stopped all my growing.  Before packing all my equipment, I checked to see that I had prints remaining so I could start back up after the move.

Move went well, and I finally got set up here and ready to start things going again, so I made up a batch of BRF cakes and a syringe.  I let my syringe sit to rehydrate for 5 days while watching the condition on the cakes; I like to have them sit for 5 - 7 days before using them, just to assure sterility.  Everything was good to go, and I inoculated 4x 1/2pt jars and put them to colonize... and they only came up at 50% positive.  2 of them just didn't do squat for some reason.  :confused:

One of the two, for whatever reason, decided to start fruiting in the jar after only 50% colonization, so I chalked that one up as a loss as well.  These things happen, right?  Still have the 1 good one left, and I decided to make that one worth its weight in gold.
I've never done WBS to bulk before, but what better time to try it, eh?  I used Doc34's WBS Tek prep and made up a dozen pint jars (I've got a small PC and can't fit quart jars in it).

In my glove box, I did a sterile transfer of a dissected piece from the top of the colonized BRF cake into a single WBS jar.  I gave it a mighty shake and set it to colonize.  In a closet I have a variable temperature aquarium heater mat under a layer of coroplast, to keep from having direct contact with the jars while still maintaining a reasonable temperature since I keep my apartment pretty cold.  Within 3 days I had a reasonable looking amount of grain colonization going on, and at day 5 I gave the jar another shake.  By day 10 it was fully colonized, and I did a straight G2G transfer.

That was 3 days ago, and the remainder 11 jars I inoculated are coming along nicely.  I plan on using a pasteurized 50/50 coir/verm bulk in minitubs to fruit, and eventually creating isolate slants from there. 
Pix to follow, as I get things uploaded.


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Offlinekrypto2000
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Re: From poor spores to great grains; a short story [Re: CliftonGK1]
    #19185742 - 11/25/13 07:51 AM (10 years, 2 months ago)

So you G2G'd the one jar into 11 or you inoculated 11 and made a transfer of the one? How do you plan to fruit em?


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OfflineCliftonGK1
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Re: From poor spores to great grains; a short story [Re: krypto2000]
    #19185998 - 11/25/13 09:09 AM (10 years, 2 months ago)

Overall steps were:

Print to syringe
Syringe to 4x 1/2pt BRF
1 section of colonized BRF to 1 WBS jar
G2G from 1 colonized WBS jar to 11 jars

Fruiting plan in original post.


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OfflineCliftonGK1
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Re: From poor spores to great grains; a short story [Re: CliftonGK1]
    #19243790 - 12/08/13 06:36 AM (10 years, 2 months ago)

Thanks go out to all the cultivators who have put the time and effort into designing and posting all the different teks.  Even with all my laboratory background, without your help I'd still be floundering.  Instead, I've got what's looking to be a successful minitub.


So I started out with a print, made a syringe, and ended up with 1 really nice looking cake.


I took sections of that colonized cake and made some grain jars.


Which I crumbled.


And made into more jars.


To spawn into minitubs which now look like this.


I realized all too late that I didn't use a liner, and that may lead to some issues.  Live and learn, remember it for the next time.
More pictures of the progress to follow.


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Invisiblegrainbrain
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Re: From poor spores to great grains; a short story [Re: CliftonGK1]
    #19244155 - 12/08/13 09:39 AM (10 years, 2 months ago)

Not to rain on your parade, but photo #2 clearly shows bacterial infection which you G2G out to more jars & spawned to a tub.  You'll likely get some fruits, but your myc is competing against a contam.


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Stop cold shocking your mycelium!
Hot Spawn - Get It On



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OfflineCliftonGK1
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Re: From poor spores to great grains; a short story [Re: grainbrain]
    #19244335 - 12/08/13 10:31 AM (10 years, 2 months ago)

:frown:
What is it that looks like contaminant to you?

Picture is poorly lit and the glass shows a lot of reflection from the yellow t-shirt I was wearing, but I didn't see any issues while these were growing up, and didn't notice any foul odor when spawning them out.

The biggest issue that I noticed during growth was that I know I had my stove on too high when I was simmering the first round of seeds, and so there were quite a few which cracked and starched out.

*edit*
I'm making sure to leave a jar from that batch I made from the one in pic #2 to just sit for a while so I can monitor and see if something comes up skunky.


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Edited by CliftonGK1 (12/08/13 10:34 AM)


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Offlinekrypto2000
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Re: From poor spores to great grains; a short story [Re: CliftonGK1]
    #19244522 - 12/08/13 11:20 AM (10 years, 2 months ago)

Yeah, I don't see any bacteria in that jar, what are you referring to exactly? Also ime a little bit of bacteria in a grain jar isn't so bad, if you're using a ms syringe it's almost impossible not to get *some* bacteria as it's almost always going to be present in vendors syringes ime, just not enough to cause trouble. I'm talking a few grains in the bottom of the jar which the myc normally will overtake. Additionally after g2ging such a jar the bacteria tends to not show back up, and ime actually helps by reducing the total amount of bacteria and allowing the mycilium to either metabolize it or simply covering it up before the spawn run, thus once spawned all it has to focus on it overtaking the substrate. Should be on par with a jar that never had bacteria. Again, this is for a trivial amount though, often not even visible, if you have more than a few grains with visible bacteria I would not use it.

edit: I can only guess he's referring to the grains pressed against the wall btw, but that's not bacteria from what I can tell, just grains pressed against the wall :shrug:.


Edited by krypto2000 (12/08/13 11:23 AM)


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InvisiblePastywhyteMDiscord
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Re: From poor spores to great grains; a short story [Re: CliftonGK1]
    #19244536 - 12/08/13 11:24 AM (10 years, 2 months ago)

Quote:

CliftonGK1 said:





The melted ice cream look to the mycelium in this pic is often a clear indicator of bacterial contam. I would not have used this jar for anything IMO.


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Offlinekrypto2000
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Re: From poor spores to great grains; a short story [Re: Pastywhyte]
    #19244564 - 12/08/13 11:29 AM (10 years, 2 months ago)

That does happen with bacterial contaminated jars, but it might also just simply be well colonized, it's hard to tell the difference unless you've watched it grow.


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OfflineCliftonGK1
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Re: From poor spores to great grains; a short story [Re: krypto2000]
    #19244683 - 12/08/13 12:09 PM (10 years, 2 months ago)

Quote:

Pastywhyte said:

The melted ice cream look to the mycelium in this pic is often a clear indicator of bacterial contam. I would not have used this jar for anything IMO.




I agree that the melted look isn't the most visually appealing of mycelium.  Moving forward I'm going to try to alleviate that by cloning out to agar and selecting something more rhizomorphic (if this turns out not to be a contaminated batch; crossing fingers).  Since I was starting from MS and had a very limited batch of starter culture to work with, I was in the "meh, take what I can get" stage of things.  From the get-go in the original BRF jar, it wasn't a ropey culture; but, looking back at that BRF jar which I held onto as a contamination control indicator, it still doesn't show signs of any problems, so I've got at least a moderate assurance that my instruments were sterile and my work materials were properly cleaned before introduction to the glove box.
Time shall tell, I suppose.
 
Quote:

krypto2000 said:
That does happen with bacterial contaminated jars, but it might also just simply be well colonized, it's hard to tell the difference unless you've watched it grow.




It could be a product of the colonization state.  That jar was just about to the point of consolidation and starting to present a significant amount of accumulated moisture as the mycelium pulled away from the wall of the jar.
During the initial growth, I didn't see anything that appeared to be bacterial contamination with that awful tell-tale non-colonizing section of a jar.  The mycelium spread evenly from all initialization points and didn't exhibit any metabolite discolouration during the growth.

So, still keeping my fingers crossed on this one and hope that I'm correct.


I worked for a couple decades in cell culture labs, but never did much mycological work aside from QC sample identification if we had a contamination in the lab.  I worked with A. niger and A. fumigatus for a brief stint with one lab, but most of my work was with tissue culture in flasks and roller bottles, or bacterial propagation in small and mid-scale bioreactors. 
So I'm really glad to have a place like this where there are so many people with knowledge of both laboratory and home-laboratory science, and the experience that everyone brings to the table. 
After spending a few too many years on the engineering and medical devices side of things towards the recent end of my career, I really began to miss the hands-on laboratory time.  This hobby helps to fill that void.  :smile:


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Invisiblegrainbrain
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Re: From poor spores to great grains; a short story [Re: krypto2000]
    #19245735 - 12/08/13 03:59 PM (10 years, 2 months ago)

Quote:

krypto2000 said:
That does happen with bacterial contaminated jars, but it might also just simply be well colonized, it's hard to tell the difference unless you've watched it grow.




Those wet grains pushed against the glass are a dead giveaway.


--------------------
Stop cold shocking your mycelium!
Hot Spawn - Get It On



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OfflineCliftonGK1
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Re: From poor spores to great grains; a short story [Re: grainbrain]
    #19245928 - 12/08/13 04:27 PM (10 years, 2 months ago)

Quote:

grainbrain said:
Those wet grains pushed against the glass are a dead giveaway.




What I'm hoping for right now is that those are more a product of improper hydration technique.  I let the stove get too hot when simmering the grains and ended up with a bunch of blowouts.
I know that Bacillus can cause a damp uncolonized area as well, but doesn't that usually manifest itself as a sour smelling gloop?

I'm really only familiar with what it looks like in an EMEM liquid culture, which ends up cloudy all throughout and if you look at it under a scope you can identify the budding chains of the little bastards.


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OfflineCliftonGK1
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Re: From poor spores to (not so) great grains; a short story [Re: CliftonGK1]
    #19333162 - 12/27/13 02:16 PM (10 years, 1 month ago)

Changed the thread title...

Pastywhyte and grainbrain == WINNER!

Yep, turned out to be a latent contamination hiding in with the mycelial growth.  Not totally evident to a new cultivator, since the mycelium grew OK and there wasn't some sort of immediate yellow/brown/green contaminant growth. 
But, over the course of spawning out to bulk, the mycelium eventually lost to the contaminant and nothing fruited.
I took a sample from a colonized, non-fruiting tub and plated it to MEAY, and there was a heavy bacterial growth around the sample within 24 hours.  By 36 - 48 hours, the bacterial growth had spread, and there was only the faintest trace of mycelial growth near the edge of the bacterial colony.  After 72 hours, there is a definite battle for nutrients between the mycelium and the contaminant; while the mycelium growth progresses, it is obviously hindered by the contaminant (although, not fully overtaken by it.)

Anyhow, live and learn.  Thanks to everyone who pointed out the things I missed as a new cultivator.  I've learned this lesson, and moved on from it as one should:  Take the information to heart, and try again, differently, incorporating the advice you've gotten.

I've restarted from an old syringe to MEAY plates, ended up with a clean growth from there, then passaged out to more MEAY and PDYA plates (variety to see if either acts as a faster medium).  Within 24 hours of passaging to new plates, there is already good recovery and mycelial growth from the transferred section to the new plate (on both varieties of plate.)

I also found another B+ print (I'm pretty sure it's a B+ since I didn't have any other really large capped varieties in my last batch) in my supplies collection, so I inoculated some additional MEAY plates to see if I get anything from an MS startup with a faster colonization or stronger rhizomorphic quality than the current culture I am passaging.

Thanks again to everyone for the help, advice, criticism, etc.  Every bit helps me learn to do better.  Eventually, I'll get this right.  :smile:
Until then (and even after then), it's all about the enjoyment of hands-on science for me.


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Invisiblegrainbrain
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Re: From poor spores to (not so) great grains; a short story [Re: CliftonGK1]
    #19333607 - 12/27/13 04:46 PM (10 years, 1 month ago)

Thanks for coming back to update the thread.  You'll look at that photo in a year from now & it'll make you cringe.  It takes some time to really get familiar with the myc, you've got a great attitude & will be there sooner rather than later.


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OfflineCliftonGK1
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Re: From poor spores to (not so) great grains; a short story [Re: grainbrain]
    #19334350 - 12/27/13 08:18 PM (10 years, 1 month ago)

Quote:

grainbrain said:
Thanks for coming back to update the thread.  You'll look at that photo in a year from now & it'll make you cringe.  It takes some time to really get familiar with the myc, you've got a great attitude & will be there sooner rather than later.




Heh, I imagine I will look back at this and wonder WTF was I thinking.  For now, I can only look at it as part of the learning curve.  Shit happens, learn from it, move on.
It's good having supportive forum members who are willing to post all their hard work for the rest of us newbs to learn from.  And if I've learned nothing else from decades of lab work, it's not to be an argumentative dick when someone offers criticism.  It's OK to offer your opinion as a rebuttal, but the true test is the test of time; and these cultures failed. 
I'd take pictures, but they're so ugly I might break the server trying to upload them. 
I will, instead, start a new thread with pix starting from the plate pours and onward as I get more photos.

Thanks again, everyone!


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