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OfflineSherlock Shrooms
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What is this contam? Can't trace the source of it, tried everything...
    #26730852 - 06/09/20 09:13 AM (3 years, 8 months ago)

Hey friends. It's been a while since I've posted. Looking for some assistance, maybe someone has seen this specifically or can guide me through troubleshooting it. All my cultures look fine on agar when I'm transferring them, yet when I pull them out of the fridge after 4 or 6 months they look like the pictures below. These are all different varieties started at different times and even grown on different agar recipes yet the same weird contam grows around the edges consistently on all of them.








For my go to agar prep I mix 500ml distilled water, 10g malt extract, and 10g agar in pyrex media bottles then put in PC vent for 20 minutes and PC for about 35 minutes. I make a lot of plates at a time using sleeves of Nest Scientific pre sterilized plastic plates and wrap cling wrap around the edges of the unused ones and store them in the sleeve they originally came out of. I do all this work in front of a flow hood. The unused plates will sit around for as long as 10 or 12 weeks before I use them and I NEVER see even one of them contaminate. Not bragging but really I don't. I am crackheadedly sterile with all my procedures.

I've started many different cultures from spores. Some spores I've collected myself using the double printing method, and some spores I've gotten from friends or vendors. I use a propane torch for my scalpel, work right in front of the hepa filter, and everything one can do to keep things clean as far as I know. And again, I can't remember the last time I saw an obvious contamination on any of my plates. It's happened but its been several months, and even then it was 1 plate out of hundreds that I made. This is what my cultures look like before they go into jars for expanding or the fridge for later use.






I think there is a small amount of hidden bacteria in some of those plates indicated by the lifting rhizos near the edge of some of the cultures and near the middle of one culture. I have used antibiotic agar, sandwiched it, taken as many as 8 or 10 transfers when it's dime sized growth to try and out run it yet there always seems to be a little bit of bacteria there, I can't escape it. If Mad Seasons hidden contam thread is correct and what I'm seeing is truly hidden bacteria.. I've started many new cultures from spores and end up at the same exact place. My grain masters don't have any wet spot bacteria at all, they look perfect from what I can tell.

The problem is that I'm not getting good results. I lose many tubs before the first flush has come in and the others all crap out somewhere in the second flush. I only do 1 grain expansion to bags. I used to do all jars but recently upgraded to bags and it didn't change my results at all. I grow in a 100% coir substrate using the bucket/cooler tek. I also clean the shizz out of my tubs between cycles, first hosing them out with hot water and after they dry I vigorously wipe every inner surface with iso. I use fresh micropore tape on the innermost layer of the holes and I reuse old micropore tape from previous rounds for the outer layers on every hole. And I load the tubs in front of my flow hood after its run for a while (in case of a high spore load in my home) wearing gloves and a surgical mask. I know most say that's unnecessary but I just keep trying to eliminate variables until I see better results. This has been going on for a loooong time. I've started new cultures and always end up at the same place. I really can't figure this out. There's more I could share but I'll just let folks ask questions about what I'm doing. Hope that wasn't too long winded. I would very much appreciate any thoughts people are willing to share, please help I'm losing hope! Thanks guys!


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"When you pass through the waters, I will be with you; And through the rivers, they shall not overflow you. When you walk through the fire, you shall not be burned, Nor shall the flame scorch you."


Edited by Sherlock Shrooms (06/09/20 09:19 AM)


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OfflineSherlock Shrooms
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Re: What is this contam? Can't trace the source of it, tried everything... [Re: Sherlock Shrooms]
    #26730903 - 06/09/20 09:33 AM (3 years, 8 months ago)

The only thing I can imagine is there are hidden contams in all my cultures but I can't figure out from where. I've done test plates and test jars doing blank transfers in each to check all my procedures and they never contaminate. If I had a bad batch of presterilized plates then I'd surely see something in the unused plates I pour that sit around for months. I also have replaced all my synthetic filter disks to rule that out. I wrap the ring of every jar with cling wrap and put colonizing jars and plates in cleaned out empty totes to keep the spore load on everything low. Everything colonizes at around 70 degrees consistently in a climate controlled room. All my grains get PC'd for 2 hours and 30 minutes for jars and 3 hours and 15 minutes for bags. Then they get loaded right from the PC directly into the flow hood. I use rye berries because they seem to end up with less bacteria than oats, but again I never see any visible bacteria in my masters. After the first grain expansion I might see a spot of bacteria here and there but very little.

When I bucket tek I pre warm the cooler with a pot of boiling water then dump it out and put the bricks in and add the measured amount of boiling water that brings it to just a touch under field capacity. I wear heat resistant gloves and break up the coir chunks while it's piping hot to make sure that any existing mold or spores would get killed throughout the coir.


--------------------
"When you pass through the waters, I will be with you; And through the rivers, they shall not overflow you. When you walk through the fire, you shall not be burned, Nor shall the flame scorch you."


Edited by Sherlock Shrooms (06/09/20 09:36 AM)


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InvisiblePastywhyteMDiscord
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Re: What is this contam? Can't trace the source of it, tried everything... [Re: Sherlock Shrooms]
    #26730905 - 06/09/20 09:33 AM (3 years, 8 months ago)

I’m gonna be honest, many of those plates look fine to me. But I would be more interested in hearing more about the bags. I know when I was doing G2G with masters to bags that the culture needs to be very vigorous. Bags go anaerobic very easily IME, especially when the volume of grain is 4 quarts or more. I would try expanding the grain master to quart jars instead of a bag and see what happens.


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OfflineSherlock Shrooms
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Re: What is this contam? Can't trace the source of it, tried everything... [Re: Pastywhyte]
    #26730918 - 06/09/20 09:38 AM (3 years, 8 months ago)

Thanks Pasty. I can go back to jars, this last run was my first full go with bags. But I have always had this same experience even when I was using all jars. Unfortunately this has been going on for a few years and I've just dealt with it. I'd like to get 2 full flushes from most of my tubs instead of throwing half of them out every time. Good to know the cultures look clean though. I'll have to trouble shoot elsewhere.


--------------------
"When you pass through the waters, I will be with you; And through the rivers, they shall not overflow you. When you walk through the fire, you shall not be burned, Nor shall the flame scorch you."


Edited by Sherlock Shrooms (06/09/20 09:43 AM)


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OfflineSherlock Shrooms
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Re: What is this contam? Can't trace the source of it, tried everything... [Re: Sherlock Shrooms]
    #26730926 - 06/09/20 09:39 AM (3 years, 8 months ago)

What about that weird white spotting around the edges of the plates that were in the fridge for several months in the first string of pictures? Is that normal behavior of healthy myc or some cold tolerant contam creeping out over time?


--------------------
"When you pass through the waters, I will be with you; And through the rivers, they shall not overflow you. When you walk through the fire, you shall not be burned, Nor shall the flame scorch you."


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OfflineSherlock Shrooms
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Re: What is this contam? Can't trace the source of it, tried everything... [Re: Sherlock Shrooms]
    #26730931 - 06/09/20 09:41 AM (3 years, 8 months ago)

In the bags I use 4 quarts of grain and use half of a grain master to inoculate each bag. They are unicorn bags with the .2 mil filters. I also dry the surface of the grains thoroughly before filling the bags. I spread them out on screens in front of fans for at least 3 or 4 hours. I was thinking about using a full grain master next go at it to speed up colonization..


--------------------
"When you pass through the waters, I will be with you; And through the rivers, they shall not overflow you. When you walk through the fire, you shall not be burned, Nor shall the flame scorch you."


Edited by Sherlock Shrooms (06/09/20 09:46 AM)


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Re: What is this contam? Can't trace the source of it, tried everything... [Re: Sherlock Shrooms]
    #26731385 - 06/09/20 12:54 PM (3 years, 8 months ago)

I pretty much had a similar experience. Bags were really hit and miss for me until i started to use huge amounts of inoculate to speed up colonization. If it took a week or more to colonize it was a coin flip. If it colonized in 4 days it did great.

I started using huge amounts of LC that was so thick it was practically slurry. It would start to recover in 12 hours and be finished in 4 days or less.


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OfflineSherlock Shrooms
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Re: What is this contam? Can't trace the source of it, tried everything... [Re: Pastywhyte]
    #26731890 - 06/09/20 04:20 PM (3 years, 8 months ago)

Cool I'll definitely up the inoculant. Ya know the last time I had a good run I previously fogged the room out with a botanical disinfectant called decon 30. I wonder how much spore load effects things. I know the thing about clean spawn but I'm ridiculously careful about that and haven't seen a contam in any plate or jar in ages. Even done test plates and jars and nothing. I wonder what else my problem is. I grew some microgreens with coir from this last run and the trays molded over in a week. I've been  growing microgreens for years and really never see that. It happened in several different trays I grew with different seed varieties. It was from seeds that I've grown fine a hundred times...


--------------------
"When you pass through the waters, I will be with you; And through the rivers, they shall not overflow you. When you walk through the fire, you shall not be burned, Nor shall the flame scorch you."


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