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Invisibleriflecityjesus
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Registered: 02/21/16
Posts: 21
Ethanol to induce rhizomorphic growth?
    #22980079 - 03/06/16 05:05 PM (8 years, 24 days ago)

Hello all,

I'm a beginner in mycology--- I know, this forum is for advanced mycologists---but I do have a degree in biology with a concentration in microbiology, so I'm familiar with sterile technique, agar work, and, well, you know, biology. I've been learning more about fungi and in my reading discovered that ethanol and other small alcohols were known to have induced rhizomorphic growth.

Has anyone heard of this? Or maybe put it to practice?

I read this in Fungal Biology by J. W. Deacon, although I'd love to find the actual published research that reported this finding.

Anyhow, if no one has tried this, I was thinking how helpful it would be to have a method of using some type of small alcohol to induce rhizomorphic growth, perhaps on agar. This would save lots of time when starting with a spore print or syringe, if dialed in correctly. Instead of isolating from several plates, perhaps agar supplemented with ethanol could cut your isolation time in half or better.

Any ideas? Or first hand knowledge of this? Is this an idea worth pursuing or a waste of time? Opinions?

Cheers.
------------------------------------------------------
UPDATED 3/16/16

Here is my process so far:

Scope: The effect on growth and rhizomorph formation of 12 alcoยญhols at different concentrations has been examined by researchers (ALLERMAN et al.) for isolates of Armillaria melea and Clitocybe geotropa. The fungi were cultivated for 28 days on a synthetic, liquid glucose medium with the alcohols as supplement. In their study, they determined the ideal concentration for EtOH supplemented agar to be between 30-40mM for overall growth; rhizomorph formation was induced and unhindered in concentrations ranging from 10 to 150mM. The goal of this study will be to determine whether or not ethanol (EtOH) supplemented potato dextrose agar (PDA) will help induce desirable rhizomorph development in another species of fungi. For the purposes of this study, [EtOH] of ~35mM was aimed for.

Materials:
PDA agar
500mL Erlenmeyer flask
(12)Sterile Petri Dishes
Sterile Syringe
Golden Grain (GG) 190 proof (95% ABV)
Aluminum Foil
Pressure Cooker
Microwave
Distilled Water
Spore Syringe or Live Tissue Sample
Flame Source

Methods:
1. Prepare PDA Solution
-dissolve 10 gram PDA powder in 250mL distilled water in an Erlenmeyer flask and stir.
-microwave until boiling (take care not to boil over)

2. Sterilize PDA Solution
-cover flask with aluminum foil
-place in pressure cooker for 15 minutes @15psi (121 C)

3. Supplement Agar with EtOH
-pre fill sterile syringe with ~0.51 mL GG and recap.
-once agar solution is cooled enough to handle comfortably (50 C), remove tip from sterile syringe and inject GG through aluminum foil into the agar solution and stir.
-immediately pour agar solution. 250mL will fill 5 or 6 plates.

4. Prepare Agar Solution (no EtOH)
-repeat steps 1, 2, and 3 without addition of GG. Agar can be poured at ~50 C.

6. Ensure Agar Sterility
-Let plates sit overnight at ~21 C (or room temperature).
-If no contaminants are visible on agar, put plates in refrigerator (2-5 C) until ready to inoculate.

7. Inoculate Agar Plates
-Using sample source of choice (multi-spore syringe or live tissue sample), inoculate all plates using sterile technique. (4 EtOH and 4 unsupplemented plates were inoculated with ~0.2cc multi-spore solution.)

8. Incubate Agar Dishes
-incubate dishes at 25 C, checking daily for growth characteristics.

9. Document Growth Characteristics
-Compare EtOH plates to non-supplemented plates.


Results:

-5th day of incubation showed first real signs of germination and very slight growth. No real difference other than the non-supplemented agar plates have slightly more growth than EtOH supplemented plates. Slight condensation and small amounts of growth made taking clear pictures problematic.

Edited by riflecityjesus (03/23/16 11:55 AM)

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Invisibleriflecityjesus
Road Captain


Registered: 02/21/16
Posts: 21
Re: Ethanol to induce rhizomorphic growth? [Re: riflecityjesus]
    #22980098 - 03/06/16 05:11 PM (8 years, 24 days ago)


Edited by riflecityjesus (03/06/16 08:40 PM)

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Invisibleriflecityjesus
Road Captain


Registered: 02/21/16
Posts: 21
Re: Ethanol to induce rhizomorphic growth? [Re: riflecityjesus]
    #22981415 - 03/07/16 12:36 AM (8 years, 24 days ago)

Well, it doesn't seem like there is a lot of interest in this.

So, I'm going to make up some agar with an EtOH concentration of 35 mM and do multispore inoculations and compare them to MS inoculations on agar with no ethanol.

I think I might also make some MS BRF cakes and use a 35 mM EtOH solution instead of straight distilled water for the cake recipe and compare the rhizomorph development to their non-EtOH counterparts.

35mM solution is roughly 1.36 mL or 1.07 g of Everclear in 500mL water, so this could end up being a really easy way to get aggressive rhizomorphic growth or it could be nothing.

Any advice, ideas, comments, criticism etc welcome!


--------------------
Whoever coined the phrase 'a man's got to play the hand that was dealt him' was most certainly one piss-poor bluffer.

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OfflineLogicaL ChaosM
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Re: Ethanol to induce rhizomorphic growth? [Re: riflecityjesus]
    #22982264 - 03/07/16 11:09 AM (8 years, 23 days ago)

Sounds interesting.

Take pics as progress happens (or doesnt happen).

Good luck :popcorn:


--------------------
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OfflineNewZooReview
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Re: Ethanol to induce rhizomorphic growth? [Re: riflecityjesus]
    #22982411 - 03/07/16 12:24 PM (8 years, 23 days ago)

Quote:

riflecityjesus said:
Well, it doesn't seem like there is a lot of interest in this.





Interesting to me for sure. Ethanol would cut down on bacterial contams as well, I expect. It is funny if alcohol is as helpful to fungal love making as it is to human.


--------------------

Happiness is a rack of clean jars.

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Invisible36fuckin5
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Registered: 08/11/03
Posts: 12,083
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Re: Ethanol to induce rhizomorphic growth? [Re: NewZooReview]
    #22982855 - 03/07/16 02:46 PM (8 years, 23 days ago)

When you heat it, the alcohol will come out of solution and create a fire hazard. I wouldn't do it.


--------------------
Redd Foxx said:
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Pat The Bunny said:
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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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Invisibleriflecityjesus
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Registered: 02/21/16
Posts: 21
Re: Ethanol to induce rhizomorphic growth? [Re: 36fuckin5]
    #22983310 - 03/07/16 05:05 PM (8 years, 23 days ago)

I'm not so sure about that. I thought about this, but 150 proof Everclear has an EtOH concentration of 12.84 mol/L. The solution I'm talking about making is 0.035 mol/L. We're talking about 0.27% alcohol by volume (ABV). I think an EtOH solution has to be like 50% ABV to burn. Perhaps I'll mix it in agar after it cools slightly. Just to minimize alcohol evaporation.


--------------------
Whoever coined the phrase 'a man's got to play the hand that was dealt him' was most certainly one piss-poor bluffer.

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Invisibleriflecityjesus
Road Captain


Registered: 02/21/16
Posts: 21
Re: Ethanol to induce rhizomorphic growth? [Re: 36fuckin5]
    #22983322 - 03/07/16 05:09 PM (8 years, 23 days ago)

Plus, these researchers did it. I can't think of another way to supplement agar with alcohol other than exactly how you think it would be done - ~1.36mL Everclear in 500mL agar solution.

Edited by riflecityjesus (03/07/16 06:08 PM)

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Invisibleriflecityjesus
Road Captain


Registered: 02/21/16
Posts: 21
Re: Ethanol to induce rhizomorphic growth? [Re: riflecityjesus]
    #22983359 - 03/07/16 05:22 PM (8 years, 23 days ago)

Although, the flash point *is* pretty low for ethanol (61F). I'll just take extra precautions. I mean spraying a glove box with isopropyl alcohol is a pretty big fire hazard as well. I'll just be extra careful. Science is dangerous business. I've worked with way more dangerous chemicals with success. But good safety grabber!


--------------------
Whoever coined the phrase 'a man's got to play the hand that was dealt him' was most certainly one piss-poor bluffer.

Edited by riflecityjesus (03/07/16 09:56 PM)

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Invisiblemicro
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Re: Ethanol to induce rhizomorphic growth? [Re: riflecityjesus]
    #22983501 - 03/07/16 05:57 PM (8 years, 23 days ago)

Hmm, this is neat!

I wonder if it is along the same premise as all those journal articles linking an oxidative state to fruitbody production.


--------------------
Any research paper or book for free
(Avatar is Maxxy, a character by Mizzyam, RIP)

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Invisibleriflecityjesus
Road Captain


Registered: 02/21/16
Posts: 21
Re: Ethanol to induce rhizomorphic growth? [Re: micro]
    #22983524 - 03/07/16 06:03 PM (8 years, 23 days ago)

I'm not sure, I haven't studied mycology long. Like I said, I'm a newb to mycology, so my knowledge in the field is limited, but I am familiar with general lab work, research, and microbiology. I'll have to read those papers when I get a chance!


--------------------
Whoever coined the phrase 'a man's got to play the hand that was dealt him' was most certainly one piss-poor bluffer.

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Offlinemyceliups
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Re: Ethanol to induce rhizomorphic growth? [Re: riflecityjesus]
    #22983936 - 03/07/16 08:01 PM (8 years, 23 days ago)

Maybe add the ethanol with a sterile swab AFTER the petri dishes are cooked and cooled. The only reason I can see for that is ethanol is probly sterile in that high of a concentration.. Dry the ethanol over calcium chloride. Just a thought.

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Invisibleriflecityjesus
Road Captain


Registered: 02/21/16
Posts: 21
Re: Ethanol to induce rhizomorphic growth? [Re: myceliups]
    #22984022 - 03/07/16 08:34 PM (8 years, 23 days ago)

The only problem with that is it would be impossible to control concentration. The paper I linked finds that the rhizomorphic growth was strongest when the agar EtOH concentration was between 30 and 40 mM (millimolar).


--------------------
Whoever coined the phrase 'a man's got to play the hand that was dealt him' was most certainly one piss-poor bluffer.

Edited by riflecityjesus (03/07/16 08:38 PM)

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Offlinemyceliups
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Re: Ethanol to induce rhizomorphic growth? [Re: riflecityjesus] * 1
    #22984118 - 03/07/16 09:16 PM (8 years, 23 days ago)

Mess around with it. Put it in a mister or use a q tip. try multiple different concentrations. Do some experiments lol

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Invisibleriflecityjesus
Road Captain


Registered: 02/21/16
Posts: 21
Re: Ethanol to induce rhizomorphic growth? [Re: myceliups] * 1
    #22984241 - 03/07/16 10:05 PM (8 years, 23 days ago)

I'll definitely experiment a bit!! After my current project is over (~3 weeks), I'll start looking into this and update this thread with methods, pictures, and progress (or lack thereof)


--------------------
Whoever coined the phrase 'a man's got to play the hand that was dealt him' was most certainly one piss-poor bluffer.

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Invisiblewoodrow
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Posts: 142
Re: Ethanol to induce rhizomorphic growth? [Re: riflecityjesus]
    #22984588 - 03/08/16 01:41 AM (8 years, 23 days ago)

I recall reading somewhere that shrooms produce volatile compounds (alcohols?) that are trapped when a casing layer is applied and this stimulates the growth of certain microbes that are instrumental in stimulating the formation of fruiting bodies. This is the purpose of a casing layer.

I am curious to know if ethanol can stimulate the growth of mycelium in a substrate or if it can promote fruiting. Perhaps one could toss in a bit of yeast to a pasteurized substrate to see if it has any benefit. Yeasts and shrooms are naturally found together and the red Rhodotorula yeasts have been known to stimulate spore germination.

I would save the Everclear in favor of a more natural environment and better uses.

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Invisibleriflecityjesus
Road Captain


Registered: 02/21/16
Posts: 21
Re: Ethanol to induce rhizomorphic growth? [Re: woodrow]
    #22984640 - 03/08/16 02:46 AM (8 years, 23 days ago)

Well, I'm going to try to prepare 5 plates with EtOH supplemented agar at the ideal concentration discussed in the research article and 5 without EtOH and inoculate all 10 from a MS syringe. I'm mainly interested in seeing if this helps speed up the process of isolating rhizomorphic mycelium. If the first series of plates show strong rhizomorph development, this would be a great help in saving time and resources.

Making a very dilute EtOH solution (35 millimolar or 0.27% ABV) and using in place of water for BRF recipes or bringing bulk substrates to field capacity was sort of an afterthought. I'll see how my first experiment goes.


--------------------
Whoever coined the phrase 'a man's got to play the hand that was dealt him' was most certainly one piss-poor bluffer.

Edited by riflecityjesus (03/08/16 02:53 AM)

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Invisible36fuckin5
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Re: Ethanol to induce rhizomorphic growth? [Re: riflecityjesus]
    #22985087 - 03/08/16 09:07 AM (8 years, 22 days ago)

Quote:

riflecityjesus said:
I'm not so sure about that. I thought about this, but 150 proof Everclear has an EtOH concentration of 12.84 mol/L. The solution I'm talking about making is 0.035 mol/L. We're talking about 0.27% alcohol by volume (ABV). I think an EtOH solution has to be like 50% ABV to burn. Perhaps I'll mix it in agar after it cools slightly. Just to minimize alcohol evaporation.




It'll distill off, just like any liquid would.

Quote:

riflecityjesus said:
I mean spraying a glove box with isopropyl alcohol is a pretty big fire hazard as well.




That's why we don't reccomend that you spray a SAB with alcohol.


--------------------
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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Invisiblemicro
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Re: Ethanol to induce rhizomorphic growth? [Re: myceliups]
    #22985150 - 03/08/16 09:30 AM (8 years, 22 days ago)

Ethyl alcohol burns somewhere around 70%
Quote:

myceliups said:
Maybe add the ethanol with a sterile swab AFTER the petri dishes are cooked and cooled. The only reason I can see for that is ethanol is probly sterile in that high of a concentration.. Dry the ethanol over calcium chloride. Just a thought.




You want to use magnesium sulfate for alcohol, or another compatible desiccant.

Calcium chloride can have hydroxides and stuff that react with the alcohol.

Definitely add it *after* you pressure cook or you'll vaporize the alcohol :o


--------------------
Any research paper or book for free
(Avatar is Maxxy, a character by Mizzyam, RIP)

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Invisibleriflecityjesus
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Posts: 21
Re: Ethanol to induce rhizomorphic growth? [Re: 36fuckin5]
    #22985842 - 03/08/16 02:10 PM (8 years, 22 days ago)

http://wiki.bugwood.org/Alcohol_agar

Preparation of alcohol agar.

Mix components of agar.

Sterilize.

Add alcohol to solution before pouring.


--------------------
Whoever coined the phrase 'a man's got to play the hand that was dealt him' was most certainly one piss-poor bluffer.

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