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InvisibleGhatti
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Continuing Contam issue
    #21337668 - 02/27/15 10:33 AM (9 years, 10 months ago)

So it seems I'm still having contamination issues to an extent. I am now usually able to get 1 flush out of my sub but soon after trich rears its head.

I am using spawn bags now (4qt a piece), pc'd 3 1/2 hrs, taken out in front of a hood  and innoc with LI from a clean agar plate.

I have a theory what's going on and I would like opinions. I am now leaning towards my spawn being infected when I break it up to spawn. When we damage mycelium it can give contams a chance to set in. This is the only other thing I can think of why I'm getting a flush but that's it.  It's obviously still the spawn that is bad because of the timing.

I'm not sure how to test or remedy this though. My hood isn't really big enough to spawn in front of and even if I did the tub has spores in it already. I never see anything off in my bags and they always smell/behave fine. It's only once in a sub that I see issues. I'm using straight sterilized coir and have the same issue no matter if it's sterilized, pasteurized, etc so I'm sure it's the spawn.

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OfflinehamloafM
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Re: Continuing Contam issue [Re: Ghatti]
    #21337717 - 02/27/15 10:40 AM (9 years, 10 months ago)

Yea, sounds like your spawn.  If your spawn is infected it won't matter if you spawn to bulk in front of a flowhood.  The contamination is already present, so it's going to show up during spawn run, at the end of spawn run, or before the first flush.

Your problem thus far is traced, and isolated into your inoculating, and expansion of culture procedures and techniques.  Care to expand a little bit on your sterile procedures, and techniques.  The source of inoculum, ect, please?


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InvisibleGhatti
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Re: Continuing Contam issue [Re: hamloaf]
    #21337888 - 02/27/15 11:11 AM (9 years, 10 months ago)

4qt bags pc'd 3 1/2 hrs, pc opened in front of my hood and bags inoculated via LI freshly made from a clean agar plate.

My LI is made same as anyone elses. PC water for 30mins, let cool, flame sterilize scalpel etc, cut out agar and drop in LI, blend, sanitize as best as possible, innoc bags, zip tie closed.

Maybe I should try to fruit a bag invitro and see what it does? Or mix coir/grain and pc it and never open the bag and innoc?

I don't like the idea of trying to fruit straight grains invitro though and if I add coir it compounds the results

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InvisibleGhatti
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Re: Continuing Contam issue [Re: Ghatti]
    #21337942 - 02/27/15 11:26 AM (9 years, 10 months ago)

So I actually just saw the tiniest speck of green in one of my bags so that kills my theory about damaged spawn. 

Is 3 1/2hrs not long enough for 6 bags @ 4qts a piece? I highly doubt my hood is causing the issue as all my plates are clean that I do in front of it.

So it's either improper sterilization or what I'm innocing with is dirty which I don't get since I only use agar.

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InvisibleGhatti
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Re: Continuing Contam issue [Re: Ghatti]
    #21339792 - 02/27/15 06:55 PM (9 years, 10 months ago)

To isolate the issue I'm going to pc 2 bags. One I will false inoculate and tie up and the other I will immediately seal when I pull out of the pc. If neither contaminate then it is how/what I am making my LI with. If the false innoc one contams then my hood/technique is at fault, if both contaminate then my pc time is not long enough.


Does this sound correct?

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OfflinetaGyo
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Re: Continuing Contam issue [Re: Ghatti]
    #21340560 - 02/27/15 09:47 PM (9 years, 10 months ago)

So you're saying it shows up after you massage the already developing mycelium?

That sounds like an inoculation problem. Try it again with a different plate you think is clean. Take a picture of the plate before you do and post it here, maybe we'll spot something you don't.

Does it show up near the mycelium or away from it, off to the corner on a grain? If it shows up near the mycelium then it's probably a ride-along contam, if a grain then I would PC for longer.

I don't know bag times, a user named BW86 (Or something like that) does bags all the time though.


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InvisibleGhatti
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Re: Continuing Contam issue [Re: taGyo]
    #21340991 - 02/27/15 11:40 PM (9 years, 10 months ago)

Today was the first sign I saw a hint of it in my actual bags. It usually show up right before, during, or right after first flush.

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OfflinehamloafM
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Re: Continuing Contam issue [Re: Ghatti]
    #21341608 - 02/28/15 05:45 AM (9 years, 10 months ago)

I do 50-100 4-8 quart spawn bags per month and your spawn is contaminated.  My bet is that you are cross-contaminating somewhere in between the making of your LI, and the inoculation of your spawn bags, or both.  If your inoculm was dirty, and, or your sterilizer wasn't sterilizing properly the contamination would have showed up a lot sooner.


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Offlinebw86
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Re: Continuing Contam issue [Re: hamloaf]
    #21341660 - 02/28/15 06:15 AM (9 years, 10 months ago)

Quote:

THE_LOAF said:
I do 50-100 4-8 quart spawn bags per month and your spawn is contaminated.  My bet is that you are cross-contaminating somewhere in between the making of your LI, and the inoculation of your spawn bags, or both.  If your inoculm was dirty, and, or your sterilizer wasn't sterilizing properly the contamination would have showed up a lot sooner.





move on to g2g and you will not have this problem.Funky things live in LCs'LI's

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OfflinehamloafM
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Re: Continuing Contam issue [Re: bw86]
    #21341789 - 02/28/15 07:33 AM (9 years, 10 months ago)

Going to have to strongly disagree with you here, bw86.  "Funky things" can live in grains too undetected.  Cross-contamination can also occur during the performance of grain to grain transferring techniques.  Great success can be had with LC and LI at the cost of extra attention paid to the details of your sterile procedure.  Furthermore, grain to grain transfers have been a staple in my normal cultivation regimen for years.


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Offlinebw86
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Re: Continuing Contam issue [Re: hamloaf]
    #21341800 - 02/28/15 07:46 AM (9 years, 10 months ago)

thats true funky things can live anywhere:yesnod:
i would still personally suggest g2g

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OfflinetaGyo
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Re: Continuing Contam issue [Re: hamloaf]
    #21341867 - 02/28/15 08:14 AM (9 years, 10 months ago)

Yup,

It's your LI process or your plate.

Post a pic of the next plate you use and verify it's clean and we'll rule out that vector.


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InvisiblePastywhyteMDiscord
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Re: Continuing Contam issue [Re: taGyo]
    #21341940 - 02/28/15 08:39 AM (9 years, 10 months ago)

Quote:

taGyo said:
Yup,

It's your LI process or your plate.

Post a pic of the next plate you use and verify it's clean and we'll rule out that vector.




I agree. Often molds meshed in with the myc can be hard to spot. Also a vector often not discusssed is the culture itself. Weak cultures that are less agressive are more prone to contam during spawning. The state of a culture before inoculation can have a big impact on the success of the project.

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Re: Continuing Contam issue [Re: Pastywhyte]
    #21341967 - 02/28/15 08:47 AM (9 years, 10 months ago)

50 to 100 grain bags a month can we see a pic that would be epic thats alot of work


--------------------
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Offlinebw86
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Re: Continuing Contam issue [Re: tripdawg420]
    #21341985 - 02/28/15 08:53 AM (9 years, 10 months ago)

you do 50 -100 bags with LI/LC??

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Offlinetwistedty
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Re: Continuing Contam issue [Re: bw86]
    #21342002 - 02/28/15 08:59 AM (9 years, 10 months ago)

try gettin away from LCs, if you have a clean culture.

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InvisibleGhatti
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Re: Continuing Contam issue [Re: twistedty]
    #21342042 - 02/28/15 09:10 AM (9 years, 10 months ago)

A note, same issue when using just agar wedges and not LI. I can push a flush out but only 1 and sometimes I get hit right before pinning.

So are we agreed that the sterilization times are adequate and can be ruled out?

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OfflinetaGyo
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Re: Continuing Contam issue [Re: Ghatti]
    #21342052 - 02/28/15 09:13 AM (9 years, 10 months ago)

That's my question as well.

Like I said though, post a pic of your next plate and we'll verify it clean.


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OfflineKizzle
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Re: Continuing Contam issue [Re: tripdawg420]
    #21342066 - 02/28/15 09:15 AM (9 years, 10 months ago)

I would try to isolate a single strain. Hidden Trichoderma will cause uneven growth so if you can get a nice monoculture that could help you rule out the cultures as a source.


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Re: Continuing Contam issue [Re: Ghatti]
    #21342132 - 02/28/15 09:25 AM (9 years, 10 months ago)

Quote:

Ghatti said:
A note, same issue when using just agar wedges and not LI. I can push a flush out but only 1 and sometimes I get hit right before pinning.

So are we agreed that the sterilization times are adequate and can be ruled out?




Have you left some grain uninoculated for the same time your grains take to colonize? That will be the only way to rule that out.

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InvisibleGhatti
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Re: Continuing Contam issue [Re: Pastywhyte]
    #21342278 - 02/28/15 09:49 AM (9 years, 10 months ago)

Since switching to bags, no.

I proposed a plan to isolate and rule things out above but didn't receive any feedback.

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InvisibleGhatti
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Re: Continuing Contam issue [Re: Ghatti]
    #21342288 - 02/28/15 09:51 AM (9 years, 10 months ago)

Quote:

Ghatti said:
To isolate the issue I'm going to pc 2 bags. One I will false inoculate and tie up and the other I will immediately seal when I pull out of the pc. If neither contaminate then it is how/what I am making my LI with. If the false innoc one contams then my hood/technique is at fault, if both contaminate then my pc time is not long enough.


Does this sound correct?



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OfflinetaGyo
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Re: Continuing Contam issue [Re: Pastywhyte]
    #21342295 - 02/28/15 09:52 AM (9 years, 10 months ago)

Quote:

Pastywhyte said:

Have you left some grain uninoculated for the same time your grains take to colonize? That will be the only way to rule that out.




Sterilize some bags and leave them out for a spawn run. Don't inoculate them. If they contaminate before 2-3 weeks then you're not PCing long enough. Pasty answered that.


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Re: Continuing Contam issue [Re: taGyo]
    #21342336 - 02/28/15 10:00 AM (9 years, 10 months ago)

bag are differant than jars you really cant leave them out they will start sucking in air thur the top of the bag


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InvisibleGhatti
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Re: Continuing Contam issue [Re: tripdawg420]
    #21342472 - 02/28/15 10:32 AM (9 years, 10 months ago)

Lol I'm sure he meant to seal it.

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Re: Continuing Contam issue [Re: Ghatti]
    #21342493 - 02/28/15 10:36 AM (9 years, 10 months ago)

Of course I did :lol:

I think he's saying even if it's sealed it will leak in.

BW does that look like good sterilization times to you? I know you do a lot of bags too.


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InvisibleGhatti
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Re: Continuing Contam issue [Re: taGyo]
    #21342506 - 02/28/15 10:38 AM (9 years, 10 months ago)

Since when will sealed bags leak?

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Re: Continuing Contam issue [Re: Ghatti]
    #21342512 - 02/28/15 10:39 AM (9 years, 10 months ago)

Ask trip,

I don't use bags. I think it's in your spawn honestly.

:shrug:


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InvisibleGhatti
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Re: Continuing Contam issue [Re: taGyo]
    #21342519 - 02/28/15 10:41 AM (9 years, 10 months ago)

I'm 100% positive its my spawn. The question is if its incomplete sterilization, dirty culture, or faulty hood work.

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Re: Continuing Contam issue [Re: Ghatti]
    #21342522 - 02/28/15 10:42 AM (9 years, 10 months ago)

lol 3.5 hours is well enought pc time :thumbup: i would say its your li :shrug: but i use grain spawn to innoc grain bags might wana try that are you washing the jar down


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InvisibleGhatti
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Re: Continuing Contam issue [Re: tripdawg420]
    #21342550 - 02/28/15 10:46 AM (9 years, 10 months ago)

As in after I make the LI, yes.

Also seem to have the one flush issue when just using wedges as well. I thought maybe PC time since the contam seems to take its time manifesting. Maybe I juuussstttt about killed it and it takes that long to recover and present itself.

I guess the only way to be certain is run the control test I stated.

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Re: Continuing Contam issue [Re: Ghatti]
    #21342557 - 02/28/15 10:48 AM (9 years, 10 months ago)

:thumbup:


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coir tek
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results :thumbup:

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Re: Continuing Contam issue [Re: tripdawg420]
    #21342566 - 02/28/15 10:50 AM (9 years, 10 months ago)

Post a pic of what you would consider a clean plate and clean spawn. Do a jar run and leave some uninoculated. Don't even open them. See what they look like in a few weeks.

I would also suggest you run some muda bottles. They can teach a lot about your grain prep. Even if you don't fruit them you can learn things.

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OfflinetaGyo
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Re: Continuing Contam issue [Re: tripdawg420]
    #21342568 - 02/28/15 10:51 AM (9 years, 10 months ago)

And to show us pics of your next plate before you LI it.

EDIT:

:whathesaid:

Some molds are tricky.


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Edited by taGyo (02/28/15 10:51 AM)

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InvisibleGhatti
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Re: Continuing Contam issue [Re: taGyo]
    #21342582 - 02/28/15 10:54 AM (9 years, 10 months ago)

I will leave a bag to sit for a few weeks and post a pic of the next plate I plan to use. Prolly be a week or more.

I'm seriously tired of throwing shit out. Its nice to at least get a flush but I wanna strangle every noob on their 5th flush lol.

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Re: Continuing Contam issue [Re: Ghatti]
    #21342589 - 02/28/15 10:55 AM (9 years, 10 months ago)

:laugh2:


--------------------
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Re: Continuing Contam issue [Re: tripdawg420]
    #21342646 - 02/28/15 11:08 AM (9 years, 10 months ago)

Keep in mind that some cultures may only be good for one flush. I have a culture that is only good for one or two at most but I get 150% BE with it so its worth it. If I get two dry oz or more per quart of spawn used first flush, I really don't care if I see a second.

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Re: Continuing Contam issue [Re: Ghatti]
    #21342654 - 02/28/15 11:10 AM (9 years, 10 months ago)


Op, your issue is cross-contamination in your procedures of the step of inoculating of your sterile grain mediums.  If your issue was dirty inoculm, and, or your sterilizer wasn't sterilizing properly, contamination would show up within 2-3 days. 

IF you feel like your sterilizer isn't sterilizing properly, you can order indicator test strips that you run through the sterilizer with your grainmasters.  The test strips won't tell you how long the 15psi was sustained for, but they will indicate if 15 psi is even being achieved by your PC.


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Edited by liquidmyce (02/28/15 11:23 AM)

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Re: Continuing Contam issue [Re: Pastywhyte]
    #21342659 - 02/28/15 11:12 AM (9 years, 10 months ago)

go from agar to grain master to spawn bag i wouldnt mess with any thing in between :thumbup:


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

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

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Re: Continuing Contam issue [Re: hamloaf]
    #21342667 - 02/28/15 11:14 AM (9 years, 10 months ago)

:hatsoff:


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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:

Edited by tripdawg420 (02/28/15 11:20 AM)

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OfflinetaGyo
Strainiac/AMU
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Registered: 10/16/14
Posts: 18,802
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Re: Continuing Contam issue [Re: tripdawg420]
    #21342675 - 02/28/15 11:16 AM (9 years, 10 months ago)

I think he's talking to me? No idea.


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InvisibleGhatti
Totally not a Federal Agent
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Registered: 06/23/14
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Re: Continuing Contam issue [Re: taGyo]
    #21356505 - 03/03/15 01:40 PM (9 years, 10 months ago)

So today I ran a batch of bags with no sleeves. When the cycle was over I sat my PC in front of my hood to cool and depressurize.

Once cool I pulled a bag out and immediately ziptied it without opening in any way. (This bag will indicate if sterilization is incomplete)

The next bag I opened and mock innoced it then ziptied it up. (This bag should tell if it is my hood being faulty or my technique)

If neither one of these bags contaminate I will feel confident saying that it is my culture that is weak/dirty

Agree?

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InvisiblebodhisattaMDiscordReddit
Smurf real estate agent
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Registered: 04/30/13
Posts: 61,916
Loc: Milky way
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Re: Continuing Contam issue [Re: Ghatti]
    #21356513 - 03/03/15 01:42 PM (9 years, 10 months ago)

make a few plates and then plate your culture. the only way to be positive is to eliminate the variables. like the fact that maybe 1 in 6 of your grain bags would have gone bad anyway because you're right on the edge of sufficient sterilization.

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InvisibleGhatti
Totally not a Federal Agent
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Registered: 06/23/14
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Re: Continuing Contam issue [Re: bodhisatta]
    #21356553 - 03/03/15 01:50 PM (9 years, 10 months ago)

I have several plates growing out at the moment that won't be ready for a week or so. I'll post pics when the time comes. I would think 3 1/2hrs at 15psi would be sufficient for 6 4qt bags though but I'm probably Wong.

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