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OfflineBigBrassBed
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Microbiologist here!
    #5794113 - 06/26/06 11:50 PM (14 years, 4 months ago)

Hi there. You can call me brass. I'm a microbiologist here in the USA. My personal field of experience is in bacteria however, as that's what our company mainly deals with. I recently became interested in growing some mushrooms, and came across this site. I have access to both an autoclave as well as a laminar flow hood. I'll be doing all work after hours, obviously.

I'm figuring that my best course of action is to use 16 ounce nalgene plastic fungal culture pots. Have any of you had bad results with plasticware? I figure that i can drill a few larger holes into the lid of the pot, inoculate, and then use .2 micron filters to cover the holes, allowing for better gas exchange. Anyone forsee a problem with this setup?

I'll be using mixture of commercially available Malt Extract Broth base and PDA agar as my food source. These are very nutrient dense, and one of my concerns is with osmotic balance. Have any of you had issues of this sort?

Also, seeing as how this is my first post, I'd like to just say hello to my fellow shroomers. May your pots be plentiful.


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InvisibleTomandjerry58
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Re: Microbiologist here! [Re: BigBrassBed]
    #5794146 - 06/26/06 11:59 PM (14 years, 4 months ago)

hello...welcome to the shroomery ....good luck


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Offlinesplifner180
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Re: Microbiologist here! [Re: BigBrassBed]
    #5794149 - 06/27/06 12:00 AM (14 years, 4 months ago)

Hi Brass.

I can't answer any of your questions 'cept maybe mention RTV lids. Self-sealing, durable, cheap.

Welcome to the Shroomery. There are a lot of good people here.

splif


--------------------
First Grow: Ecuador -> LC -> HPoo/Straw -> Monotub
Build a Do-It-Yourself Magnetic Stirrer in thirty minutes with no money.


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InvisibleOmnicracker
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Re: Microbiologist here! [Re: splifner180]
    #5794205 - 06/27/06 12:17 AM (14 years, 4 months ago)

so youre making a liquid culture, right? you can use almost any complex carb as a food medium for an LC. a ratio of 1.5-2tablespoons per liter of water is typical. but thats just step one, what do you have planned for substrate?


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




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Invisibleliamtheloser
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Re: Microbiologist here! [Re: Omnicracker]
    #5794217 - 06/27/06 12:21 AM (14 years, 4 months ago)

welcome to the shroomery... there's a lot of nice people here, as splif said... but then there's the jerks like me...

just playin, have fun with your new hobby, i hope you share some of your knowledge of bacteria with us!


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


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OfflineBigBrassBed
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Re: Microbiologist here! [Re: liamtheloser]
    #5794235 - 06/27/06 12:28 AM (14 years, 4 months ago)

I'll start with a little hint on managing contamination.

Keeping your media's Ph lower, around 5.5 or so, will prevent several typical bacterial contaminants from taking root. This can be done by adding dilute acid to the water for your substrate.

Thanks for the welcome everyone!


Edited by BigBrassBed (06/27/06 12:30 AM)


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OfflineSnaggletooth
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Re: Microbiologist here! [Re: BigBrassBed]
    #5794236 - 06/27/06 12:28 AM (14 years, 4 months ago)

Hey and welcome to this little community :grin:

You sound like you have a good start, and a good head on them shoulders

I doubt that most here can not answer that for ya man, but there are a few that certainly can, just might take some times until they chime in.

Starting with agar, is a great way to start, I have not yet played with it, mainly cause of no flowhood, yet :grin:

The only suggestion that I can give at this point, is to document what you can, Pics will certainly help and share it here.


Anyway man, hope you have a good grow  :thumbup:


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


Atheist Chat


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InvisibleRoadkillM
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Re: Microbiologist here! [Re: BigBrassBed]
    #5794265 - 06/27/06 12:39 AM (14 years, 4 months ago)

Welcome to the Shroomery!~

I hope that you enjoy your stay!~

:smile:


I have never used any nalgene plastic...is it autoclaveable?

But I have used other types of autoclaveable plastics.
They work fine.

I'd suggest making an injection port into the lids to inoculate with.
If you have access to any vacutainers...use the cap for your injection port.
like this..

then you could use the micron filter instead of the bandaid that you see in my picture.

I use PDYA and MEA agar for culture work.
I like to switch back and forth between them when working with cultures.


If you need any help...

just ask...or do some reading.


tc


--------------------
Laterz, Road

Who the hell you callin crazy?
You wouldn't know what crazy was if Charles Manson was eating froot loops on your front porch!


Brainiac said:
PM the names with on there names, that means they have mushrooms for sale.



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Invisiblecreamcorn
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Re: Microbiologist here! [Re: BigBrassBed]
    #5794271 - 06/27/06 12:40 AM (14 years, 4 months ago)

Quote:

BigBrassBed said:
I'll start with a little hint on managing contamination.

Keeping your media's Ph lower, around 5.5 or so, will prevent several typical bacterial contaminants from taking root. This can be done by adding dilute acid to the water for your substrate.

Thanks for the welcome everyone!




yeah but low pH encourages mold growth, which is another major enemy in this hobby, and is far from ideal for fruiting. there are many types of bacteria that grow over a wide range, and a lower pH might prevent some but encourages others. i did some pH "torture tests" of my own a ways back to see what exactly mycelium would tolerate, and found that it can tolerate a rather large range, and as a side effect found bacterial infections to be consistently much more prevelant with lower pH substrates... neutral pH is a better bet all-around, and buffering to be slightly above neutral is common practice in mycology because mycelium excretes acidic metabolites.


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InvisibleRoadkillM
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Re: Microbiologist here! [Re: BigBrassBed]
    #5794274 - 06/27/06 12:41 AM (14 years, 4 months ago)

Quote:

BigBrassBed said:

I'll start with a little hint on managing contamination.

Keeping your media's Ph lower, around 5.5 or so, will prevent several typical bacterial contaminants from taking root. This can be done by adding dilute acid to the water for your substrate.

Thanks for the welcome everyone!





And check out our Contamination forum...

I'm sure you will enjoy that one!~

:smile:


tc


--------------------
Laterz, Road

Who the hell you callin crazy?
You wouldn't know what crazy was if Charles Manson was eating froot loops on your front porch!


Brainiac said:
PM the names with on there names, that means they have mushrooms for sale.



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InvisibleOmnicracker
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Re: Microbiologist here! [Re: Roadkill]
    #5794324 - 06/27/06 12:57 AM (14 years, 4 months ago)

im still not clear what youre trying to do. if youre working with agar, why not use an agar dish? im sure you access to those. let us know more about what your are doing if you really want help.


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




xxx..Learn Something..xxx


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OfflineSnaggletooth
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Re: Microbiologist here! [Re: BigBrassBed]
    #5794334 - 06/27/06 01:00 AM (14 years, 4 months ago)

oh yeah you can check out this link, not home made but,

https://www1.fishersci.com/Coupon?cid=1328&gid=88815&details=Y


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


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Offlinemakaveli8x8
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Re: Microbiologist here! [Re: Snaggletooth]
    #5794840 - 06/27/06 05:01 AM (14 years, 4 months ago)

hmm, wounder if he is more interested in the edible feild ?


--------------------
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OfflineBigBrassBed
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Re: Microbiologist here! [Re: makaveli8x8]
    #5794923 - 06/27/06 06:44 AM (14 years, 4 months ago)

I'm not more interested in the edible field. I'm using agar in a very small amount to stiffen the media. If you use agar and vermiculite together, you get a very aerated, fluffy consistency. It will improve gas exchange.

I'm also making a myc. culture to inoculate my substrate. Seeing as how I have access to the aseptic techniques neccessary for such a thing, it makes more sense to me. I can easily store my culture at work without any fear.


Edited by BigBrassBed (06/27/06 06:47 AM)


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Offlinemakaveli8x8
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Re: Microbiologist here! [Re: BigBrassBed]
    #5794927 - 06/27/06 06:53 AM (14 years, 4 months ago)

well i personally think you should start out with edibles then. because doin that stuff at work, well there could be hidden cameras anywhere
anything unexpected could happen,


--------------------
We were sent to hell for eternity :hellfire: Ø:omgawesome:h®
We play on earth to pass the time :foreheadslap:

Over-population the root of all Evil-brings the Elites Closer to the gates.


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OfflineBigBrassBed
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Re: Microbiologist here! [Re: makaveli8x8]
    #5794934 - 06/27/06 07:13 AM (14 years, 4 months ago)

You've been doing this too long if you think there's hidden cameras in every reputable place of employment.


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Offlinemakaveli8x8
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Re: Microbiologist here! [Re: BigBrassBed]
    #5795033 - 06/27/06 09:16 AM (14 years, 4 months ago)

they sell them the size of a quarter, and its legal for bisness to tap your computers.

hidden cameras in bis. is a good idea to catch theifs that seem to be everywhere....highly highly possible.

with all these viruses being used by terror....another reason for them.


--------------------
We were sent to hell for eternity :hellfire: Ø:omgawesome:h®
We play on earth to pass the time :foreheadslap:

Over-population the root of all Evil-brings the Elites Closer to the gates.


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OfflineTheFish
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Re: Microbiologist here! [Re: makaveli8x8]
    #5795362 - 06/27/06 12:13 PM (14 years, 4 months ago)

Welcome to the boards. I envy your access to such a wonderful environment to work in. I might remodel my bathroom just to keep innoculating and spawning more fun.  :smirk:


--------------------
"Comes a time when the blind man takes your hand,
Says, 'Don't you see?'
Gotta make it somehow on the dreams you still believe.
Don't give it up.
You got an empty cup only love can fill."

Jerry Garcia


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Offlinecoda
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Re: Microbiologist here! [Re: TheFish]
    #5795913 - 06/27/06 03:41 PM (14 years, 4 months ago)

welcome to the shroomery!

A lot of people would love to gain access to the type of lab equipment you have.  Since you're already versed in microbiology cultivating mushrooms should be a snap for you. :smile:


--------------------
To get really high is to forget yourself. And to forget yourself is to see everything else. And to see everything else is to become an understanding molecule in evolution, a conscious tool of the universe. And I think every human being should be a conscious tool of the universe. . . .

-JG

i really am glad you came back to us instead of taking the other path. *hug*

-A_S (RIP your final words to me will never be forgotten)



Don't fuck with the laughing jesus.


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InvisibleJim
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Re: Microbiologist here! [Re: Roadkill]
    #5795922 - 06/27/06 03:46 PM (14 years, 4 months ago)

those lexan nalgene bottles can take anything you throw at them....


--------------------
Use the Fucking Reply To Feature You Lazy Pieces of Shit!

afoaf said:
Jim, if you were in my city, I would let you fuck my wife.


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OfflineBigBrassBed
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Re: Microbiologist here! [Re: Jim]
    #5796495 - 06/27/06 07:19 PM (14 years, 4 months ago)

Just so you all can see what i'm talking about, here's a link

http://www.nalgenelabware.com/common/images/products/211697aL.JPG


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Invisiblefastfred
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Re: Microbiologist here! [Re: BigBrassBed]
    #5796821 - 06/27/06 08:51 PM (14 years, 4 months ago)

Welcome Brass. The shroomery is a pretty interesting place. Lots of stuff to learn. Growing mushrooms is one of the most rewarding things you can do.

Don't let the paranoids here freak you out. They've probably been smoking a little too much grass. Most people don't really realize how the lab environment works. Nobody questions what you're doing. They've all learned that questioning a scientist about what they're doing results in a long and animated but extremely boring discussion of their research interests, what they're doing, why and how they're doing it, etc.. "None of your business" and "you wouldn't understand" are also common responses. You won't be doing anything that would look suspicious anyway.

The plastic culture pots should work fine, but are kind of a waste of money/supplies. Mason jars are cheaper and work just as well, but might look a little strange. Any standard methods would work well. An erlenmeyer with foil on top or a foam plug would also work well and look pretty standard. .2 filters should work fine, otherwise use polyfill plugs, tyvek, or any of the other methods.

MEA and PDA work the best. MEY and PDY also work well. You would think Saboraud's would work well, and it works OK, but not as well as MEA and PDY. MEA is a little better than PDA IME. No need to worry about osmotic balance as long as you use normal concentrations (2-4%). 4% will work best.

A couple of other tips that you'll probably find useful... Filter sterilization will be your best friend unless you like waiting for the autoclave. Get a cannula or two, they're useful for reaching the myc at the bottom of your liquid media and for breaking it up. You'll probably find it handy to add antibiotics to your liquid media. AMP works well at standard concentrations, haven't really tried any others. LB and LB/AMP will grow myc, but only very poorly. You probably have a bunch of that around. It might be useful for storage.

Hmm... What else. Your edelbach(sp?) might be handy for blending up cultures. You might try using a bioreactor for liquid cultures, but I haven't tried this. You might try using some buffered media for testing growth rates at different pHs.

Hopefully you'll have good results with your first run and it will really get you into mycology. It did for me. You can find everything you need to know about practical aspects here, and hopefully you'll want to give something back by doing some much needed research.

You might try doing some RFLPs on different strains. There is a post in advanced about that. You also might spec some spore solutions to help with the spore color discussion. Check the Redboy post in advanced to see some that I did. "Essential Fungal Genetics" is a good book that I'm currently reading, you might want to check it out. You might try some fungal transformation experiments since you probably have some cool plasmids laying around. Maybe even have some fun with a YAC.

Anyhow, I think you'll love working with fungi. Much more fun than bacteria and a good step up in complexity. Being the simplest of the eukaryotes they're a good first step into eukaryotic genetics. Feel free to PM me if you have questions on any specifics.


-FF


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Offlinehyphae
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Re: Microbiologist here! [Re: BigBrassBed]
    #5797046 - 06/27/06 09:48 PM (14 years, 4 months ago)

Quote:

BigBrassBed said:
I'll start with a little hint on managing contamination.

Keeping your media's Ph lower, around 5.5 or so, will prevent several typical bacterial contaminants from taking root. This can be done by adding dilute acid to the water for your substrate.

Thanks for the welcome everyone!



Welcome Brass! Heres a little hint for you higher pH's are what you want neutral to slightly alkaline is by far your best bet. You have a good background but there is much to learn here so absorb all you can and success can only follow! Experience is our best teacher. GL my friend


--------------------
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OfflineBigBrassBed
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Re: Microbiologist here! [Re: fastfred]
    #5800554 - 06/28/06 09:51 PM (14 years, 4 months ago)

I'll be using the lab's supplies. That's the best part.. all i have to pay for is spores.

Thanks for the idea to add antibiotics to my media, as obvious as that seems, I hadn't thought of it. I've got access to all of the standard penicilins and several broad spectrums.

I wonder if i could add an anti fungal as well, maybe i should test some of ours to see if they don't damage the myc. I've got access to ketoconazole, fluconazole, itraconazole, Amphotericin B, Flucytosine, and a few others. Maybe i could actually do some active contribution to the community in this way. Granted, antibiotics in the media would mean antibiotics in the shrooms, so i'd have to be careful. Some people have allergies.

I was thinking about running some extractions and calculating the potency of my cultures. I've decided to start with Golden Teacher and the koh samui super strain. I'm thinking about doing panaeolus cyan. next. Thanks for all the encouragement and help.



Quote:

fastfred said:
Welcome Brass. The shroomery is a pretty interesting place. Lots of stuff to learn. Growing mushrooms is one of the most rewarding things you can do.

Don't let the paranoids here freak you out. They've probably been smoking a little too much grass. Most people don't really realize how the lab environment works. Nobody questions what you're doing. They've all learned that questioning a scientist about what they're doing results in a long and animated but extremely boring discussion of their research interests, what they're doing, why and how they're doing it, etc.. "None of your business" and "you wouldn't understand" are also common responses. You won't be doing anything that would look suspicious anyway.

The plastic culture pots should work fine, but are kind of a waste of money/supplies. Mason jars are cheaper and work just as well, but might look a little strange. Any standard methods would work well. An erlenmeyer with foil on top or a foam plug would also work well and look pretty standard. .2 filters should work fine, otherwise use polyfill plugs, tyvek, or any of the other methods.

MEA and PDA work the best. MEY and PDY also work well. You would think Saboraud's would work well, and it works OK, but not as well as MEA and PDY. MEA is a little better than PDA IME. No need to worry about osmotic balance as long as you use normal concentrations (2-4%). 4% will work best.

A couple of other tips that you'll probably find useful... Filter sterilization will be your best friend unless you like waiting for the autoclave. Get a cannula or two, they're useful for reaching the myc at the bottom of your liquid media and for breaking it up. You'll probably find it handy to add antibiotics to your liquid media. AMP works well at standard concentrations, haven't really tried any others. LB and LB/AMP will grow myc, but only very poorly. You probably have a bunch of that around. It might be useful for storage.

Hmm... What else. Your edelbach(sp?) might be handy for blending up cultures. You might try using a bioreactor for liquid cultures, but I haven't tried this. You might try using some buffered media for testing growth rates at different pHs.

Hopefully you'll have good results with your first run and it will really get you into mycology. It did for me. You can find everything you need to know about practical aspects here, and hopefully you'll want to give something back by doing some much needed research.

You might try doing some RFLPs on different strains. There is a post in advanced about that. You also might spec some spore solutions to help with the spore color discussion. Check the Redboy post in advanced to see some that I did. "Essential Fungal Genetics" is a good book that I'm currently reading, you might want to check it out. You might try some fungal transformation experiments since you probably have some cool plasmids laying around. Maybe even have some fun with a YAC.

Anyhow, I think you'll love working with fungi. Much more fun than bacteria and a good step up in complexity. Being the simplest of the eukaryotes they're a good first step into eukaryotic genetics. Feel free to PM me if you have questions on any specifics.


-FF




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InvisiblePrisoner#1
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Re: Microbiologist here! [Re: BigBrassBed]
    #5800824 - 06/28/06 11:03 PM (14 years, 4 months ago)

Quote:

BigBrassBed said:
I'm figuring that my best course of action is to use 16 ounce nalgene plastic fungal culture pots. Have any of you had bad results with plasticware?





I've had great luck with these



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Invisiblefastfred
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Re: Microbiologist here! [Re: Prisoner#1]
    #5801864 - 06/29/06 04:10 AM (14 years, 4 months ago)

> Thanks for the idea to add antibiotics to my media, as obvious as that seems, I hadn't thought of it.

No prob. That's what we're here for.

> I wonder if i could add an anti fungal as well, maybe i should test some of ours to see if they don't damage the myc.

My guess is that it would not work very well. However, this is the kind of basic research that is lacking in this field. If you could find an anti-fungal that it tolerates that would be a great. It would be pretty handy to develop a fool-proof LC that people couldn't contaminate. It would probably sell pretty well too.

Thiophanate methyl is used with Agaricus bisporus, but lowers yields. Mertect, Topsin, Benlate, Benzimidazole, and Bravo (Chlorothalonil) are also used. You might want to try those first.


> Granted, antibiotics in the media would mean antibiotics in the shrooms.

The standard concentration of ampicillin is 50-100 ug/ml. A standard human dose of ampicillin is 250-500 mg. So assuming that you started a 500 mL LC with 100 ug/ml amp that would total only 50 mg in the entire culture. So if you drank the entire LC you would only be getting 1/5 - 1/10 of a standard dose. I don't know that much about amp allergies, but I'd guess that 1/5 of the minimum oral dose wouldn't present much risk. Further, if you took that LC and injected say 5cc into a 1/2 pint (236 mL) PF style jar you would have 100 ug/mL * 5cc = 500 ug = .5 mg per jar. 0.5 mg/236 mL = 0.002 mg/ml substrate concentration. If your 1/2 pint cake produces 1/8 of dried fruit, and the fruit had the same concentration as the substrate, then your eighth (3.5g) of edible mushrooms would only have around 0.0074 mg of ampicillin in them.

You might want to check my math, there are some shaky weight to volume conversions in there. But I don't think 0.0074 mg of amp is going to set off even the most sensitive person in the world. I don't think you could even detect that concentration with a HPLC or GC/MS.

> I was thinking about running some extractions and calculating the potency of my cultures.

Good idea. That would be a major contribution. Check out this paper:
The Measure of the Mushroom (from PM&E Volume Five)

It would be nice to calibrate the p-DMAB test with an HPLC.


-FF


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Mushrooms, Mycology and Psychedelics >> Mushroom Cultivation

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