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Offlineagentjrr
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Registered: 01/14/04
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Loc: Mountain West
Last seen: 12 years, 20 days
How to prevent bacteria?
    #2585043 - 04/21/04 12:51 AM (13 years, 7 months ago)

Ok, having successfully fruited a few casings I would like to improve my success rate. Thus far I have had only two problems, jars drying out and a bacteria characterised by wet looking areas, a sour old sock type smell, and little to no mycelial growth. One was easily fixed, the other I have not been able to get rid of. It claims about 60%-70% of my jars. It has occurred in both BRF and Popcorn. With steaming and pressure cooking (Popcorn at 12 PSI for 80 minutes). And with mycelial and multi-spore inncoculation. It occurs in multiple jars in each batch in spite of flame sterilizing followed by an alcohol wipe between each jar. The popcorn has almost no burst kernels and is prepared according to the tek on this site.

Has anyone else had this kind of problem with bacteria. If so how did you beat it? Its getting kind of frustrating seeing as how this is the only type of contam I have gotten and the fruiting stage goes very smoothly once I get there. If it helps at all my germination times seem longer than average at 5+ days for visible growth with mycelial inccoculation in a tub in tub incubator at 82 degrees.

Any advice would appreciated.


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Invisible@cro
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Re: How to prevent bacteria? [Re: agentjrr]
    #2586659 - 04/21/04 01:33 PM (13 years, 7 months ago)

PCing at 12 PSI for 80 mins should be sufficient unless your using gallon size jars. So the bacteria is most likely being introduced afterwards, I would guess from your syringes. Homemade syringes?
Bacteria will flourish in too wet jars and hot environments. You could try altering your water concentration or lowering your temps but the ultimate factor is to stop introducing bacteria into the jars.
I've found almost 90% of prints I get in trades will have some sort of contam and usually it's bacteria, if you don't have access to agar your best bet would be to stick with vendor syringes and work with your own prints made from these in sterile environments.

peace - @cro


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OfflineSuntzu
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Re: How to prevent bacteria? [Re: agentjrr]
    #2586676 - 04/21/04 01:40 PM (13 years, 7 months ago)

Was 12 PSI used on the BRF jars too?

It sounds like incomplete sterilization, perhaps more PSI would make a difference. Are the outsides of the jars moist at all in the incubator? Bacteria can swim through paths of moisture to the insides of jars. What kind of filter system do you have on the lids?
Negative controls might help you figure out where the problems are coming from. Keep one jar aside and incubate with the rest. Take a second and do everything for an innoculation except actually innoculating. Since it's only 70% of the jars that go bad, these are no guarantee to pinpoint the problem, but if your 'untouched' negative control goes bad, that can tell you something.

Good general ways to combat bacteria:
1. Filter disc system/Polyfill.
2. Cooling the PC in as clean of a place as possible.
3. Try not to get grain goo on the outsides of the jars while filling them---after PC'ing, you can get a nice sterile nutrient film on the outsides of the jars, perfect starting grounds for contams. Dry the outsides of the jars and incubate dry.
4. Moisture content of the grain.
5. Increasing sterilization PSI.


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Offlineagentjrr
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Re: How to prevent bacteria? [Re: Suntzu]
    #2586881 - 04/21/04 02:46 PM (13 years, 7 months ago)

The filter is double layer tyevk. This problem has occurred with both vendor spore syringes and home made liquid culture. The spore syringes I can rule out because they came from a reputable vendor and I have had several batches with no contams that failed mainly due to to insufficient water content.

I have had this problem in my control jars also. With BRF I usually let the jars sit for 2-3 days before innoculating to see if anything will develop. And sometimes the contam will show up before inncoulation, despite a variety of lid/tyvek combos and PC times from 60-80 minutes. In between attempts I clean the jars with bleach solution and wash in the dishwasher followed by an alcohol wipe shortly before use. I have also tried pre-sterilizing the vermiculite/rice flour/water.

Innocultion is done in room air in a small room completely washed down (floor, walls, countertops) with bleach and lysol. The heating vent is sealed over with plastic and the room is left at least an hour without major air movement.

When I increase sterilization times or decrease water content, I get some bacterial contam but more commonly the jars just stall from drying out.

The incubator is completely dry inside, and I've had this same contam at temps from 74-86. Its just noticeable sooner at higher temps.

The only thing I can think of is that there is usually a lot of condensation built up under the foil after PCing, perhaps the tyvek is getting wet between the layers or something.

I going to try PCing longer (90+ minutes) because my cooker only does 12 PSI. But in the past increasing sterilization times has just led to overly dry jars that stall.

Also, maybe the longer germination times are hurting me. The syringes I'm using are the remains of a batch I bought 4 months ago and in popcorn they seem to take 2 weeks from innoculation with spores to first signs of growth. With liquid culture (dextrose) it still takes 10 days to see any sign of growth. Its just frustrating because I seem to have good success culturing in dextrose (excellent growth 100% of the time, milky looking in 7-10 days) and with fruiting (6 flushes on the average 1/2 pint of BRF) and I have no contams but the one type of bacteria (no molds, nothing, even when I have badly botched the sterility of casings).

Is there any additional step I couild be employing either in preparation or innoculation? Could something be hiding in the lids/bands that arrives after bleaching/washing?


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Invisible@cro
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Re: How to prevent bacteria? [Re: agentjrr]
    #2587199 - 04/21/04 04:09 PM (13 years, 7 months ago)

Quote:

agentjrr said:
The only thing I can think of is that there is usually a lot of condensation built up under the foil after PCing, perhaps the tyvek is getting wet between the layers or something.




There ya go. I actually had the same problem for a while. The condensation on the tyvek will lead to a good breeding ground for bacteria. Take the foil off it won't lose water all that fast, you should be fine.
With mine I found a control sitting out would be fine, even the jars colonizing would be fine, until I shook them. This moved the bacteria on and within the tyvek to incorporate into the substrate, causing bacteria contamination then.
I'm not sure what your setup is but you could try using the tyvek with the metal ring and no foil. It should work quite a bit better not allowing the condensation to build up. Or, I like plastic lids, I have swithced the tyvek from under the lid to the outside so it doesn't build up condensation also I've tried using breathable tapes over the holes for filters both seem to work.

Peace


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Invisiblesakura
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Registered: 02/08/04
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Loc: Japan
Re: How to prevent bacteria? [Re: agentjrr]
    #2587563 - 04/21/04 06:14 PM (13 years, 7 months ago)

When using honey water, I have found h2o2 to be useful (haven't had a baccilus since I started using it :wink: )

I have a few units already in my sterile syringe before I suck up my myc...  I shake and deliver immediately to the jars (I use popcorn a lot) and shake the jars immediately to distribute.

Also, I add some verm to my popcorn before sterilising (I like to use a lot of liquid myc for fast colonisation, but don't want a lot of fluid laying around in my jars).

Seems to be working just great for me anyway...


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Shrooms aren't everyone's cup of tea... (Some folks just eat 'em)


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Offlinecformaldehyde
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newbie [Re: agentjrr]
    #2588803 - 04/21/04 11:33 PM (13 years, 7 months ago)

k well im new here and dont really know how to work things here, so i clicked on random things to post this and hopefully get some replies. Im buying some shrooms this weekend for the first time. Anything i should watch out for (types that are poisonous, death caps). And how would i be able to tell if these shrooms are really good? thanks


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Offlineagentjrr
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Loc: Mountain West
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Re: newbie [Re: cformaldehyde]
    #2589035 - 04/22/04 12:14 AM (13 years, 7 months ago)

You really picked the wrong forum to post this in. Move your thread to something besides the contamination forum. Try posting on the forum for experiencing mushrooms. You will probably get replies there.

The answers to your questions can be found in the FAQs and by reading the forums.


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OfflineMAIA
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Re: newbie [Re: agentjrr]
    #2591048 - 04/22/04 02:49 PM (13 years, 6 months ago)

I agree with Suntzu about the timings when sterilizing. Try PCing longer or use the fractional sterilization procedure.
Anyway, you seem to have covered almost all the possibilities but there are other more subtile details like the water and the substrate. Try changing either one or both factors, change the water you're using and also give it at try to other substrate brands.

MAIA


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Spiritual being, living a human experience ... The Shroomery Mandala



Use, do not abuse; neither abstinence nor excess ever renders man happy.
Voltaire


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OfflineMAIA
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Re: newbie [Re: cformaldehyde]
    #2591093 - 04/22/04 02:58 PM (13 years, 6 months ago)

Welcome to the shroomery ...
Now and then people ask in this forum if their bought mushrooms are poisonous or contaminated, that's ok if you are a newbie and you don't know about the rules. Try posting in the General Discussion forum.
As for now, there you have something to read about a similar question
http://www.shroomery.org/forums/showflat...ev=#Post2392350

MAIA


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Spiritual being, living a human experience ... The Shroomery Mandala



Use, do not abuse; neither abstinence nor excess ever renders man happy.
Voltaire


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