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Offline3SIXTY5
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Bacteria Problem, Old Culture, Or Something Else?
    #26907735 - 08/30/20 12:08 PM (3 years, 4 months ago)

Hey, so I had a big spawn run and all of my bulk substrates got a bacterial contamination, or so it seems, sour smelling, almost chemically, no signs of visual contamination. I noticed the smell around day 3 of colonization. I'll explain my processes, and then I have a few questions. All of my agar work is done in front of a flow hood, as far as dishes getting contaminated I have a less that 2% contamination rate, so I highly doubt that has anything to do with it.

GRAIN PREP

So I fill up my cooker with wheat kernels, fill with hot water, scoop floaters, drain and fill 3 timesish until the water is mostly clear. Then I soak in cold water for 12-18 hours, then I cook until hydrated and spin them dry before bagging, like dry, dry, and completely cooled. Then I bag the grains 5lbs per 14a filter bag, I pre seal them before sterilizing. Then I load them in my sterilizer, get them up to 180f-190f and cook them for about 18 hours. Unload, let them fully cool, inoculate each bag with a ( non iso, BRF cloned culture on the 8th transfer currently) full agar dish cut into wedges. They are fully colonized by day 14.

SUBSTRATE PREP

I sterilizer my bulk substrate in a sort of modified insulated bucket Tek style bin, I boil water in the bin, wait until it's over 200f, dump in my bulk ingredients (780 grams coco + 225 grams verm + a handful of gypsum + a little more than 3 litres of water) let it sit for about 2 hours, mix, let it sit for another 2 hours or so, then mix again. The bin stays above 180f for like 12 hours, I usually have to keep mixing and waiting for 2-3 days before the substrate is back under 100F.

SPAWNING PROCEDURE

I mix my substrate and spawn using a mixer just because I have it and it's easier. Before I spawn to bulk I clean the mixer with vinegar, scrub, dry, and then H2O2, scrub and dry. I open air mix my substrate with the spawn, I keep the windows closed, but it's just regular household air, no filtration. I use 9-10lbs substrate per 5lb bag of spawn, which works out to about a 1:3-1:2 volumetric spawn to sub ratio. 
I put the spawned substrate into lined trays (24"L * 20"W * 3"D)which I then put inside a contractors garbage bags And no tie them closed, normally I cut slits in the bags and put 2 layers micropore over them. I let them colonize at 70f-75f they're almost always done by day 10-12, but I let them sit for about 4-5 days before I troducing to fruiting conditions.

I've had 4 successful runs with my current setup, no major contamination issues, lost a few substrates to trich, but less than 10%. This last run I did two things differently that I can think of.

1. I didn't slit the bags and micropore them, I only loosely tied the bags.

2. I let the grain bags sits fully colonized for two weeks before I spawned to bulk because of issues with my fruiting chamber.

So my questions are

1. Could bacteria be growing because my culture is just getting old, or is a 7th transfer from a clone not old enough for that to be happening?

2. Could the bacteria be growing because I didn't put GE holes even tho I loosely tied the bags and there was still probably a 1/2" Gap at the end.

3. Could the fully colonized bags sitting for two weeks be the culprit for bacterial growth?

I'm sure I can figure this out with a few more bulk runs, but if my culture is getting old, if rather just use a new culture than to waste my time and money on more grain bags and substrate. Thanks in advance for any answers!


Edited by 3SIXTY5 (08/30/20 12:10 PM)


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Offlineredhandmat
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Registered: 05/09/19
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Re: Bacteria Problem, Old Culture, Or Something Else? [Re: 3SIXTY5]
    #26907911 - 08/30/20 01:29 PM (3 years, 4 months ago)

Quote:

3SIXTY5 said:
Then I load them in my sterilizer, get them up to 180f-190f and cook them for about 18 hours.




Say what? Where did you read that that is enough to sterilize grains? IMO this is one of the biggest sources for your contams. We want to be as close to 15 PSI as possible for 90 minutes (steam temp is around 250F at this pressure). I have like 12-13PSI and I PC for 150 minutes to be on the safe side (havent had contams).

Quote:

3SIXTY5 said:

1. I didn't slit the bags and micropore them, I only loosely tied the bags.

2. I let the grain bags sits fully colonized for two weeks before I spawned to bulk because of issues with my fruiting chamber.

So my questions are

1. Could bacteria be growing because my culture is just getting old, or is a 7th transfer from a clone not old enough for that to be happening?

2. Could the bacteria be growing because I didn't put GE holes even tho I loosely tied the bags and there was still probably a 1/2" Gap at the end.

3. Could the fully colonized bags sitting for two weeks be the culprit for bacterial growth?





I know of growers (trusted cultivators too) that let jar lids be a little loose and dont use holes or MP or anything and its fine. But they have jar lids, which makes it a little hard for contams to get inside. I think this may be another vector for contams to enter your bags.

Letting a clean and fully colonized jar sit, especially for a couple weeks, wont make it contam.

1-no. contam is contam.
2- very possible
3-no.

But my money is on both the loose tie and the (bad?) sterilization.


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Offline3SIXTY5
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Re: Bacteria Problem, Old Culture, Or Something Else? [Re: redhandmat]
    #26908097 - 08/30/20 02:59 PM (3 years, 4 months ago)

I don't use grain jars, I use 14a unicorn bags, I think you're confused.

it's atmospheric pressure sterilization, you don't need to use a PC to sterilizer grains, I have an AA41 PC, but when you're doing bulk grains the PCs aren't realistic. look up a drum sterilizer if you haven't heard of this before. The bags are pre sealed prior to going in the sterilizer I cook them for 18 hours and start the timer once the grains have reached 180F.

I just read Kizzles guide on identifying contamination and read that a lack of GE creates an anaerobic environment which is a breeding ground for bacteria, so I'm thinking that's probably my issue.

I'm more worried about an aging culture though, because that happened to me a year or two ago.

Thanks for your response, I've gotten enough views on the thread but only one response hopefully some other mfers chime in.


Edited by 3SIXTY5 (08/30/20 03:03 PM)


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