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OfflineBigBrassBed
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Registered: 06/26/06
Posts: 33
Last seen: 12 years, 4 months
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


--------------------
Getting the most out of your casings!, A pinning strategy.
Oyster Shell "Flour" $2 for 1lb. a hell of a deal :wink:
Not what is overlay but rather what overlay is
Gas Exchange vs. FAE

"We all have priorities. I used a closet once setup a nice little lab trouble was all the shit that was in there ended up in the bedroom that pissed off the GF then I ended up dumping her as she was getting in the way of my sterile culture technique! Ya I got priorities too!!!"


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OfflineBigBrassBed
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Registered: 06/26/06
Posts: 33
Last seen: 12 years, 4 months
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
Even Dumber ThanAdvertized!
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Registered: 01/22/03
Posts: 193,665
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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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