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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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Offlinetripdawg420
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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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InvisiblePastywhyteMDiscord
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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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