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zrow
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Registered: 11/26/12
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PF de-contam
#18053684 - 04/03/13 05:42 PM (11 years, 1 month ago) |
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I would like to share an experience that might be helpful to somebody else.
I ordered a Psilocybe Cyanescens syringe that turned out to be contaminated. About two months and 35 jars later I had tried most combinations of alder and oak chips, sawdust, rye, cardboard, agar, and agar with antibiotic – all refused to grow anything but bacteria. I had two PF pint jars made with brf/verm 1:4. One with just a filter disk top exposed to RH 40% at 72 F. In two weeks that jar dried up so that there was a 1/8 inch gap between the jar and the cake. Four small mycelium spots were growing in that gap. I opened the jar and transferred the mycelium to 10 agar jars. Seven were contaminated and three worked. I guess mycelium can handle drought better than bacteria.
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MonkeyJesusFresco
am i suspended in agar?



Registered: 10/09/12
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Re: PF de-contam [Re: zrow]
#18054066 - 04/03/13 06:53 PM (11 years, 1 month ago) |
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if I'm not mistaken Psilocybe Cyanescens are grown outdoors; the prints collected from them are pretty much contaminated, you have to clean them up on agar first
-------------------- LAGM v 2.024 - endo cabendo
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zrow
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I tried the agar first and couldn't do it. The one thing that may work with agar is to incubate at 4C. I am trying that as well, but its pretty slow going. Agar jars did show a little white after they were sealed long enough for the oxygen to be used up, but then they recontaminate when the jar is opened. If I was hard core I would try a CO2 filled glove box.
The antibiotic agar seems to only support one contaminant while the plain agar had several.
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MonkeyJesusFresco
am i suspended in agar?



Registered: 10/09/12
Posts: 3,308
Loc: South East USA
Last seen: 5 days, 23 hours
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Re: PF de-contam [Re: zrow]
#18057589 - 04/04/13 01:13 PM (11 years, 1 month ago) |
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yeah, agar work sounds like a hassle (especially if all ya got's an upside down tub and bulky jars )
again, if I'm not mistaken, I think I read somewhere that "medicated agar" isn't the best for starting spores on;
you might fare better inoculating SEVERAL un-medicated petris, hoping one or two sprout germinate, then transferring that culture to medicated agar??? I dunno, I was kind'a hoping more pros would chime in;
I really love a good thread about agar work and outdoor/contaminated spore/culture samples
-------------------- LAGM v 2.024 - endo cabendo
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zrow
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Update [Re: zrow]
#18112683 - 04/15/13 12:39 PM (11 years, 1 month ago) |
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I transferred the mycelium in the three good jars to 7 more agar jars. All the agar jars developed contamination but contained pale whitish contaminated mycelium as well. I cut circles of soaked cardboard the size of the bottom of the jars and pressed it into the agar. One jar now has several white spots on top of the cardboard. The rest show no progress. The next step is to inoculate more agar from the cardboard growth.
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fastfred
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Registered: 05/17/04
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Loc: Dark side of the moon
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Re: Update [Re: zrow]
#18116053 - 04/15/13 10:53 PM (11 years, 1 month ago) |
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Sounds like a lot of work.
If you dilute your spore solution right and streak some plates properly you should have a clean culture in one or two steps.
Imagine salt and pepper mixed, and all you have is a licked finger to pick up the grains. You have to spread the components out until your fat finger can snap up either the salt or pepper without getting the other.
-------------------- It drinks the alcohol and abstains from the weed or else it gets the hose again. -Chemy The difference between the substances doesn't matter. This is a war on consciousness, on our right to the very essence of what we are. With no control over that, we have no need to speak of freedom or a free society. -fireseed "If we are going to have a war on marijuana, the least we can do is pull the sick and the dying off the battlefield." -Neal Levine (MPP) I find the whole "my drug should be legal but yours should be illegal" mindset disgusting and hypocritical. It's what George Bush and company do when they drink a cocktail and debate the best way to imprison marijuana users. -Diploid
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zrow
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Registered: 11/26/12
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Thanks for the response. It is a lot a work, but I'm in the first learning stages so its all interesting to me and I don't mind.
This was a commercial spore syringe and I still have a half cc of solution left. Following the clue you gave me, I will dilute it and try again.
Now if there are more grains of pepper than salt, and you need two or more grains of salt to combine before the pepper can multiply enough to overtake it, things may get a little dicey. Still, it should be interesting.
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mycoelf
Agent Of Chaos



Registered: 06/26/09
Posts: 555
Loc: hyperspace
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Re: Update [Re: zrow]
#18135074 - 04/19/13 11:51 AM (11 years, 1 month ago) |
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spores wont work on agar containing gentomyacin sulfate, you have to use a no medicated agar to germinate, then transfer away from the contam's and after dicaryotic myc has formed you can use the gentomyacin to work against the bacteria. Remember that myc can migrate through a layer of agar, while bacteria can not. Try the "agar sandwich" method, wheby you transfer an inoculum to a clean plate then pour a medicated agar over the wedge and covering the plate. The myc will emerge, and yo ucan carefully scrape a few cells form the top and transfer to a new dish.
Bacteria are easy to beat, you just need a fast moving myc and a few tricks. Without the advantage of agar skills, you are going to have a tough time getting an isolate
-------------------- Mycoelf Sterility is a process that can be likened unto infinity, which is a long walk, the closer to the end you start before beginning, the more achievable the goal of infinity becomes. Remember, cleanliness in next to goddessness
      
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fastfred
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Quote:
spores wont work on agar containing gentomyacin sulfate, you have to use a no medicated agar to germinate,
Why would you think that? Spores germinate just fine on every antibiotic agar I've heard of, including gentamicin.
-FF
-------------------- It drinks the alcohol and abstains from the weed or else it gets the hose again. -Chemy The difference between the substances doesn't matter. This is a war on consciousness, on our right to the very essence of what we are. With no control over that, we have no need to speak of freedom or a free society. -fireseed "If we are going to have a war on marijuana, the least we can do is pull the sick and the dying off the battlefield." -Neal Levine (MPP) I find the whole "my drug should be legal but yours should be illegal" mindset disgusting and hypocritical. It's what George Bush and company do when they drink a cocktail and debate the best way to imprison marijuana users. -Diploid
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mycoelf
Agent Of Chaos



Registered: 06/26/09
Posts: 555
Loc: hyperspace
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this is my own experience, I have tried to germinate on the Fungi perfecti (MEPA) with Gentomyacin, standard agar and got nothing, it was like to monocaroyte myc died immediately after it come out. I poured a no medicated agar and form the same spore print, no trouble. Maybe I am wrong but I have only dong MSG's on no med agar since.
-------------------- Mycoelf Sterility is a process that can be likened unto infinity, which is a long walk, the closer to the end you start before beginning, the more achievable the goal of infinity becomes. Remember, cleanliness in next to goddessness
      
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fastfred
Old Hand



Registered: 05/17/04
Posts: 6,899
Loc: Dark side of the moon
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There's no reason to think the gentamicin prevented germination.
You used an undefined media and got random results. No real surprise there. You never know what you're going to get when you order mystery media from a pseudo-scientific company.
-FF
-------------------- It drinks the alcohol and abstains from the weed or else it gets the hose again. -Chemy The difference between the substances doesn't matter. This is a war on consciousness, on our right to the very essence of what we are. With no control over that, we have no need to speak of freedom or a free society. -fireseed "If we are going to have a war on marijuana, the least we can do is pull the sick and the dying off the battlefield." -Neal Levine (MPP) I find the whole "my drug should be legal but yours should be illegal" mindset disgusting and hypocritical. It's what George Bush and company do when they drink a cocktail and debate the best way to imprison marijuana users. -Diploid
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zrow
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Registered: 11/26/12
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Last seen: 10 years, 11 months
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I didn't have a problem germinating the spores on the medicated agar.
I've never had a problem isolating with oysters, cubes, or reishi. It seems like those myc are a lot faster than cyan. This is the first I heard about the "agar sandwich", although I notice that there is myc on the bottom of two cardboard topped jars that migrated downward.
Thanks a lot for the tip.
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