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OfflineGumby79
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Registered: 07/16/03
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WBS endospores
    #2392233 - 03/01/04 08:34 PM (12 years, 10 months ago)

SWIM has a question about WBS colonization. The last couple of times he has tried growing on WBS, the mycelium only colonized to ~80-90% while the rest of the substrate remained unchanged. Upon opening the jars he noticed a distinct fermentation smell. SWIM is pretty sure that the WBS contamination is due to bacterial endospores that survived PCing, even though he PCs for at least 1 hr at 15 psi.

The contaminated WBS was spawned to straw and it proceeded to colonize quickly. The fermentation smell disappeared and was replaced with the sweet, earthy smell of mushrooms. This has been the case the last two times he has grown.

Out of curiousity, SWIM placed a small sample of contaminated WBS spawn on an agar plate, and the mycelium quickly colonized the plate with no signs of contams.

So, I guess the question is how does the mycelium apparently conquer this contam on agar and on straw but not in the quart jars? Has anybody else had experience with this contam?


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OfflineSuntzu
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Re: WBS endospores [Re: Gumby79]
    #2392321 - 03/01/04 09:00 PM (12 years, 10 months ago)

If what you are saying is exactly the way things happened, the only thing I can come up with is *oxygen*.  Perhaps the mycelium was in a battle with the contam in a low oxygen environment [in the jars] but gained a significant advantage when the oxygen balance turned in its favor. 

Perhaps you can describe the jars/lids/filters you have employed?  How did you go about preparing the grain? 

Strange that mold did not take over the partially colonized/contaminated grain.  Keep an eye out for this, it seems like a distinct possibility while the bulk sub is colonizing. . .

It's tough to argue with mycelium taking off. . .I have never heard of this happening like you described, but quite likely most people pitch questionable jars before the spawning step.  Hesitantly, I have to say "go with it"  :smile:


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InvisibleJoshua
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Re: WBS endospores [Re: Gumby79]
    #2392347 - 03/01/04 09:10 PM (12 years, 10 months ago)

I have had similar problems. My guess regarding the differences may be due to aeration differences in the substrates. Perhaps you are dealing with an anaerobic bacteria, one that thrives in a sealed jar but is stalled in a more aerobic environment such as agar plates and straw.

I am colonizing some substrate right now with a similar story. I had jars that stalled a bit, but eventually colonized. I could not smell any strong odor from the millet jars so went ahead and spawned from it. I also took some remaining kernals from the spawn jar and placed them onto agar. The agar showed immediate signs of a bacterial contamination. The sample was taken from the bottom of the jar after shaking and pouring out the grain. The spawned compost did not colonize at all after 4 days. I then removed the container from the incubator and sprayed the surface with a 1.5% solution of peroxide. I left the lid cracked a bit to make the substrate environment more aerobic. I have checked on it every couple of days and have maintained it by spraying peroxide and fanning the substrate. The substrate has taken a remarkable recovery and appears to be about 85% colonized.

Bacteria can be elusive. If the bacteria was more anaerobic it would have a tendancy to form at the bottom of your jars. If this is where the stalling occurs, that would back-up the thought. If in turn you took your sample from the top of the jar for your agar plate, it would not be suprising if the sample were clean.

Also, lower temps usually favor mycelial growth while higher temps usually favor bacterial growth. A change in the environment, other than air exchange, may also play a role in the differences you see.

Joshua


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Invisiblekeyeghost
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Re: WBS endospores [Re: Gumby79]
    #2392358 - 03/01/04 09:16 PM (12 years, 10 months ago)

My friend also has had similar experiences. She has some WBS going right now that has that sour fermentation smell. She got it last time she did WBS, but the jars turned out ok, she thought the bacillus had gotten em good though. Did not.


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OfflineGumby79
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Re: WBS endospores [Re: keyeghost]
    #2392393 - 03/01/04 09:33 PM (12 years, 10 months ago)

I bet you guys are right about oxygen levels being the deciding factor on the growth of this particular bug. That thought hadn't occured to me.

Anyway, SWIM prepares his WBS by soaking in tap water for 12 hours, straining for about an hour, then sterilizing in a very nice autoclave for 1 hour. He has always used filters instead of polyfill. They are made out of nylon and have a pore size of 0.045 microns. He just recently discovered that the polypropylene backing on the filters begins to melt at about 248F, but I doubt that would have any effect on the integrity of the filters.

SWIM thinks he will try polyfill next time around to see if it makes any difference, but he suspects that endospores are the root of the problem and will increase PC time also.


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OfflineGumby79
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Re: WBS endospores [Re: Gumby79]
    #2392400 - 03/01/04 09:38 PM (12 years, 10 months ago)

Oh yeah, Joshua, SWIM can't recall from what part of the jar he took a sample to place on agar. However, the sample consisted of several grains with about 50/50 colonized to uncolonized substrate.


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OfflineSuntzu
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Re: WBS endospores [Re: Gumby79]
    #2392715 - 03/01/04 11:07 PM (12 years, 10 months ago)

The reason I was leaning towards non-anaerobic contamination is that 'fermenty' smell is more typical of aerobic activity.  True 'anaerobic' growth results in smells that are anything but nice [or at least wouldn't be described as fermenty].  Very distinctive, begs for some air freshener  :tongue:
There are gradients here. . .capnophilic or facultative anaerobic organisms, e.g.  Many/most Bacillus spp. are facultative anerobes, meaning that they can sort of 'make do' when the oxygen levels drop.  So perhaps there is a combination growth of Bacillus in this environment, where the predominating smells are from its aerobic activity, but it's eeking out an existence better ias the O2 continues to drop than the 'obligate aerobic' fungi. 

Just pontificating, I'd bet with a little extra rinsing before PC-ing you'd knock the endospore load down to where you can achieve sterilization at 1 hr.  I agree with you that the odds are very good that the contaminant is a remnant of the sterilization cycle.

One other question. . .do you think it's at all possible that the polyprop backing could have melted a little bit, compromising the porosity?  This could be a very good explanation.  You might have essentially created an air-tight jar if the surface of the filter melted at all.


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OfflineGumby79
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Re: WBS endospores [Re: Suntzu]
    #2392834 - 03/01/04 11:36 PM (12 years, 10 months ago)

Suntzu, I believe your hypothesis is probably right on the money. I assumed that the contam was a Bacillus sp. as they are one of the few bacteria that produce endospores. So, for this species at least, it appears that mycelium can beat the hell out of it in a head-to-head competition under aerobic conditions.

I don't think the porosity of the filters was compromised after PCing because SWIM just remembered that he could smell fermentation through them.

I really don't believe that this particular contam is a problem. SWIM has eaten some tasty shrooms grown from contamed WBS. Probably should have chunked them but I guess I like to live on the wild side.


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