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ballsalsa
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A question about selective media
#23669528 - 09/22/16 09:38 PM (7 years, 4 months ago) |
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Is it possible to select for alcohol tolerance in mushrooms(not cubensis necessarily, but any mushrooms)by using a high abv agar medium? or would something like that require a transfer of genes from yeast or some such? Seems like a high tolerance to ethanol would erase contam problems overnight. just soak your substrate in some cheap vodka and you're good to go. yes? no? maybe so?
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36fuckin5
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Re: A question about selective media [Re: ballsalsa]
#23669680 - 09/22/16 10:28 PM (7 years, 4 months ago) |
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Probably not, simply because the alcohol would at minimum suck all the water out of the mycelium. But I've never seen it tried.
-------------------- Redd Foxx said: If you're offended I don't give a shit and don't come see me no more. Pat The Bunny said: A punk rock song won't ever change the world, but I can tell you about a couple that changed me. bodhisatta said: i recommend common sense and figuring it out. These are the TEKs I use. They're all as cheap and easy as possible, just like your mom.
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drake89
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Re: A question about selective media [Re: 36fuckin5]
#23670265 - 09/23/16 07:27 AM (7 years, 4 months ago) |
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Wut? I'm sure there is some resistance but not like it's gonna grow on some moonshine mash.
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JanuaryWolf
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Re: A question about selective media [Re: ballsalsa]
#23671440 - 09/23/16 03:35 PM (7 years, 4 months ago) |
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Interesting ideas! Could you test for this on agar plates, maybe dropping vodka in with some mycelium and observing and selecting for the plates/mushrooms least effected?
What is abv agar medium?
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ballsalsa
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Re: A question about selective media [Re: JanuaryWolf]
#23671568 - 09/23/16 04:15 PM (7 years, 4 months ago) |
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abv stands for alcohol by volume. I was thinking something along the lines of mixing up the agar with some percentage alcohol replacing water, like a jello shot. Then inoculate the plates with spores from multiple sources and start isolating any that manage to grow. then make a new batch of agar with a slightly higher percentage abv. rinse and repeat until you have something that can tolerate something like 20%+ the way high gravity yeast can
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drake89
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Re: A question about selective media [Re: ballsalsa]
#23671863 - 09/23/16 06:30 PM (7 years, 4 months ago) |
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Pressure cooker will evap some of your alcohol.
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ballsalsa
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Re: A question about selective media [Re: drake89]
#23672308 - 09/23/16 09:20 PM (7 years, 4 months ago) |
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Quote:
drake89 said: Pressure cooker will evap some of your alcohol.
hmmmm...good point. a PC filled with hot ethanol vapor sounds pretty dangerous.
suggestions?
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dankington
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Re: A question about selective media [Re: ballsalsa] 1
#23672447 - 09/23/16 10:12 PM (7 years, 4 months ago) |
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Well, if you were so inclined, you could pour alcohol from a sealed bottle (say an airplane bottle) into your agar vessel in your SAB or whatever. You know, before you were to begin pouring. Then you could take it out to swirl the solution, wipe it down, and carefully begin pouring.
That said, I don't believe the alcohol would be beneficial in any way.
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ballsalsa
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Re: A question about selective media [Re: dankington]
#23672525 - 09/23/16 10:40 PM (7 years, 4 months ago) |
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its not supposed to be beneficial. its supposed to prevent the growth of the vast majority of spores you try to inoculate it with in the hopes of finding a few that grow under those adverse conditions.
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drake89
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Re: A question about selective media [Re: ballsalsa]
#23673777 - 09/24/16 11:59 AM (7 years, 4 months ago) |
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It's kind of doubtful you're going to get anywhere and for what? To grow on infected substrate that you could orherwise avoid with good techniques?
-osmotic pressure- is the force you're working against. Pretty basic physics.
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ballsalsa
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Re: A question about selective media [Re: drake89]
#23675033 - 09/24/16 07:42 PM (7 years, 4 months ago) |
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I guess it would mostly just be for fun. It would be like a search for yeast analogue genes in mushrooms. There are plenty in humans. There are even guys who have successfully shoved human genes into yeast in place of their analogues and the yeast was functional! you might end up with something that you can just spritz with booze to knock out any contam problems you encounter rather than toss the shit out.
http://femsyr.oxfordjournals.org/content/femsyr/6/5/744.full.pdf
Quote:
This study revealed that ethanol, 1-pentanol and 1-octanol penetrated the intracellular space and damaged various organelles in a similar way. According to a cDNA microarray analysis, exposure to these alcohols led to a number of up-regulated genes being classified into certain functional categories, and to the grouping of some gene expression profiles into a congeneric hierarchical cluster. These results suggest that yeast cells may deploy a similar defense mechanism against various alcohols with different log Pow values. In other words, alcohols may exert a cytotoxic effect on analogous targets, regardless of their lipophilicity. In order to elucidate the cellular events of this mechanism, further studies on the genetic pathways and/or mechanisms are needed. Several genes and pathways required for ethanol tolerance have been carefully studied. Some researchers have suggested that ethanol resistance originates from the lipid composition and fluidity of the plasma membrane (Alexandre et al., 1998; You et al., 2003). Five of the genes, BEM2, PAT1, ROM2, VPS34 and ADA2, identified as required for ethanol tolerance, are related to the integrity of the cell wall (Takahashi et al., 2001). Mutants lacking mitochondrial manganese-superoxide dismutase (MnSOD) are sensitive to ethanol, indicating that SOD2 is essential for ethanol tolerance (Costa et al., 1997).
http://www.sciencedirect.com/science/article/pii/S016816561000266X
Quote:
The economic production of biofuels from renewable biomass using Saccharomyces cerevisiae requires tolerance to high concentrations of sugar and alcohol. Here we applied an inverse metabolic engineering approach to identify endogenous gene targets conferring improved alcohol tolerance in S. cerevisiae. After transformation with a S. cerevisiae genomic library, enrichment of the transformants exhibiting improved tolerance was performed by serial subculture in the presence of iso-butanol (1%). Through sequence analysis of the isolated plasmids from the selected transformants, four endogenous S. cerevisiae genes were identified as overexpression targets eliciting improved tolerance to both iso-butanol and ethanol. Overexpression of INO1, DOG1, HAL1 or a truncated form of MSN2 resulted in remarkably increased tolerance to high concentrations of iso-butanol and ethanol. Overexpression of INO1 elicited the highest ethanol tolerance, resulting in higher titers and volumetric productivities in the fermentation experiments performed with high glucose concentrations. In addition, the INO1-overexpressing strain showed a threefold increase in the specific growth rate as compared to that of the control strain under conditions of high levels of glucose (10%) and ethanol (5%). Although alcohol tolerance in yeast is a complex trait affected by simultaneous interactions of many genes, our results using a genomic library reveal potential target genes for better understanding and possible engineering of metabolic pathways underlying alcohol tolerance phenotypes.
http://science.sciencemag.org/content/346/6205/71
Quote:
Ethanol toxicity in the yeast Saccharomyces cerevisiae limits titer and productivity in the industrial production of transportation bioethanol. We show that strengthening the opposing potassium and proton electrochemical membrane gradients is a mechanism that enhances general resistance to multiple alcohols. The elevation of extracellular potassium and pH physically bolsters these gradients, increasing tolerance to higher alcohols and ethanol fermentation in commercial and laboratory strains (including a xylose-fermenting strain) under industrial-like conditions. Production per cell remains largely unchanged, with improvements deriving from heightened population viability. Likewise, up-regulation of the potassium and proton pumps in the laboratory strain enhances performance to levels exceeding those of industrial strains. Although genetically complex, alcohol tolerance can thus be dominated by a single cellular process, one controlled by a major physicochemical component but amenable to biological augmentation.
i dunno, just seemed like a cool thing to do, if it could be done at all.
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Pastywhyte
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Re: A question about selective media (moved) [Re: ballsalsa]
#23684762 - 09/27/16 10:14 PM (7 years, 4 months ago) |
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This thread was moved from Advanced Mycology.
Reason: Moved at OP request. . .
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ballsalsa
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Re: A question about selective media (moved) [Re: Pastywhyte]
#23687292 - 09/28/16 03:54 PM (7 years, 4 months ago) |
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being able to use an ethanol solution for LCs and spore syringes might be nice.
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dankington
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Re: A question about selective media (moved) [Re: ballsalsa]
#23689957 - 09/29/16 10:09 AM (7 years, 3 months ago) |
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I don't think you're understanding what we're saying.
Take a nice clean plate of mycelium and wash it with say, a 10% ethanol solution. See what happens?
I think I understand what you're trying to do, but why not make a ridiculously alkaline agar solution? You may have better luck (like what they do with straw log pasteurization).
But if you really wanna try, you're going to have to add the alcohol after PC'ing it like I said.
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Psilicon
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Re: A question about selective media (moved) [Re: dankington]
#23690004 - 09/29/16 10:22 AM (7 years, 3 months ago) |
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I like the idea. If the genes are there, it may be possible to do.
It's true that this is rendered useless by good aseptic technique, but if you can lower the bar a little bit why not do so?
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bodhisatta 
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Re: A question about selective media (moved) [Re: Psilicon]
#23690040 - 09/29/16 10:31 AM (7 years, 3 months ago) |
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Alcohol tolerance has to do with cell membrane and those genes, yeast can be bred to have higher alcohol tolerance to a point.
Either way it's stupid. Doing something more complicated to lower the bar to do something already incredibly easy.
If you can't do agar without a handicap then you're not about to be the one working on this project. If you can do agar well this won't be interesting for you at all.
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Psilicon
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Re: A question about selective media (moved) [Re: bodhisatta] 1
#23690353 - 09/29/16 12:09 PM (7 years, 3 months ago) |
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bodhi, you come off as such a dick.
Higher fungi have cell membranes, too. The genes might have been conserved, and they could be expressed and selected for. You only need to do this once and stabilize for everyone to get the benefit of it. It's probably futile, but I've done way dumber projects.
OP, if I were going to do this I'd start with a print as close to wild as possible, so the genetics aren't limited by generation upon generation of selection.
Edited by Psilicon (09/29/16 12:38 PM)
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morty422
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Re: A question about selective media (moved) [Re: Psilicon]
#23690399 - 09/29/16 12:28 PM (7 years, 3 months ago) |
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Quote:
van der griegen said:
bodhi, you come off as such a dick.

We just talked about this bodhi...
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bodhisatta 
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Re: A question about selective media (moved) [Re: morty422]
#23690408 - 09/29/16 12:30 PM (7 years, 3 months ago) |
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oh well. Most any fungi should be able to be bred to a higher tolerance to ethanol but it only really makes sense for things like yeast that make and bathe in alcohol
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ballsalsa
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Re: A question about selective media (moved) [Re: bodhisatta]
#23691629 - 09/29/16 06:38 PM (7 years, 3 months ago) |
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Quote:
bodhisatta said: Alcohol tolerance has to do with cell membrane and those genes, yeast can be bred to have higher alcohol tolerance to a point.
i know, thats why linked some papers with some of the most important genes for ethanol tolerance in yeast listed. in addition, i linked a paper about a process to bolster existing ethanol tolerance by increasing the KCl and KOH in the medium, though that might be irrelevant if it will convey the same ability to bacteria and other contams as well.
Quote:
van der griegen said:
bodhi, you come off as such a dick.
Higher fungi have cell membranes, too. The genes might have been conserved, and they could be expressed and selected for. You only need to do this once and stabilize for everyone to get the benefit of it. It's probably futile, but I've done way dumber projects.
OP, if I were going to do this I'd start with a print as close to wild as possible, so the genetics aren't limited by generation upon generation of selection.
maybe even multiple wild prints from different locations. thanks for the advice.
Quote:
bodhisatta said:
oh well. Most any fungi should be able to be bred to a higher tolerance to ethanol but it only really makes sense for things like yeast that make and bathe in alcohol
Its alright, i often come off as a dick as well. I didn't take offense.
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bodhisatta 
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Re: A question about selective media (moved) [Re: ballsalsa]
#23692016 - 09/29/16 08:15 PM (7 years, 3 months ago) |
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I wouldn't be surprised if cubensis tolerated higher alcohol than yeast anyway. I've seen mold growing in 40% 80proof liquor bottles. Not badly but its grown
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ballsalsa
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Re: A question about selective media (moved) [Re: bodhisatta]
#23692110 - 09/29/16 08:35 PM (7 years, 3 months ago) |
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really? eating residual sugars maybe. or using the alcohol as a carbon source even? thats disheartening though in a sense because that means one could accidentally select for resistant contams. i didn't think about resistant molds.
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Pastywhyte
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Re: A question about selective media (moved) [Re: ballsalsa]
#23692132 - 09/29/16 08:39 PM (7 years, 3 months ago) |
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Shit. . . Who here hasn't misted their sub with alcohol at least once. . .
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bodhisatta 
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Re: A question about selective media (moved) [Re: ballsalsa]
#23692240 - 09/29/16 09:08 PM (7 years, 3 months ago) |
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Quote:
ballsalsa said: really? eating residual sugars maybe. or using the alcohol as a carbon source even? thats disheartening though in a sense because that means one could accidentally select for resistant contams. i didn't think about resistant molds.
Only in brandy have I seen it. Also with fruit flies in the bottle
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ballsalsa
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Re: A question about selective media (moved) [Re: bodhisatta]
#23692314 - 09/29/16 09:31 PM (7 years, 3 months ago) |
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oh, ok, then the mold might have been eating dead flies or castoff pupae casings or some such. Drosophila melanogaster (a common lab species of fruit fly) select strongly for alcohol tolerance and are commonly found in wine cellars, much more so than other Drosophila spp.
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morty422
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Re: A question about selective media (moved) [Re: Pastywhyte]
#23692674 - 09/29/16 11:38 PM (7 years, 3 months ago) |
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Quote:
Pastywhyte said: Shit. . . Who here hasn't misted their sub with alcohol at least once. . .

You have gained 1,000 Morty points for your comment.
Your Morty points can be redeemed at a later time... Enjoy!!!
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ballsalsa
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Re: A question about selective media (moved) [Re: bodhisatta]
#23692819 - 09/30/16 01:13 AM (7 years, 3 months ago) |
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was the mold growing on the surface of the brandy, or up above in or around the neck of the bottle?
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bodhisatta 
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Re: A question about selective media (moved) [Re: ballsalsa]
#23694633 - 09/30/16 05:04 PM (7 years, 3 months ago) |
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Quote:
ballsalsa said: oh, ok, then the mold might have been eating dead flies or castoff pupae casings or some such. Drosophila melanogaster (a common lab species of fruit fly) select strongly for alcohol tolerance and are commonly found in wine cellars, much more so than other Drosophila spp.
it doesn't matter what it was eating.. what matters that it was growing in 40% ethanol
it was growing IN the brandy, not on the cap. pour spouts (hence how flies got in)
small sphere colonies probably a good 10-20 of them all about 0.5-1cm in diameter. floating in the liquid not on the surface
Edited by bodhisatta (09/30/16 05:10 PM)
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ballsalsa
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Re: A question about selective media (moved) [Re: bodhisatta]
#23695232 - 09/30/16 08:00 PM (7 years, 3 months ago) |
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damn
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