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maryxmas
King of the Hippiecrates


Registered: 02/18/04
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Help. Need to fight bacteria on agar
#17794780 - 02/13/13 11:01 AM (11 years, 3 months ago) |
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I have some agar plates that I recently transplanted some pan cyan tissue onto.
the tissue is nice and fuzzy with fresh growth but the area around the tissue is contaminated with bacteria. I really need this clone so I don't want to pitch it into the trash.
is there a way to save the fungi or fight the bacteria so the fungi can at least out run it a little so I could get a sample to transfer to another plate.
could I maybe remove the tissue and dip it in hydrogen peroxide and transfer to another plate?
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mycofever
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Re: Help. Need to fight bacteria on agar [Re: maryxmas]
#17794835 - 02/13/13 11:12 AM (11 years, 3 months ago) |
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you have to transfer. you can try scraping from the fuzzy mycelium to do this. good luck.
-------------------- Patience will help you keep your sanity.It will insure your success if you are patient in all aspects of mushroom growing.When you rush you are prone to make mistakes and all of your efforts are wasted.
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Beefy1
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Re: Help. Need to fight bacteria on agar [Re: maryxmas]
#17798740 - 02/14/13 12:27 AM (11 years, 3 months ago) |
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I'm in the same situation.
I tried dipping in peroxide while transfering to a clean plate, Killed the myc but only some of the bacteria.
Next try will be gentamycin agar
If that doesn't work I'll try the hot pour method- basically place the tissue on a fresh agar plate and pour a layer of agar over it, hope the myc grows up through it and the bacteria stays trapped, at least long enough to get a clean sample.
Good luck.
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MonkeyJesusFresco
am i suspended in agar?



Registered: 10/09/12
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Re: Help. Need to fight bacteria on agar [Re: Beefy1]
#17798786 - 02/14/13 12:40 AM (11 years, 3 months ago) |
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out of curiosity, what advantage is there to "the hot pour" method versus laying a squar of medicated agar cut from a pre-poured dish on top of your myc. culture?
-------------------- LAGM v 2.024 - endo cabendo
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maryxmas
King of the Hippiecrates


Registered: 02/18/04
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Well I don't want to jinks it but one of my clones seems to be taking to the plate and there is no bacteria in sight on the plate..... Totally stoked if I can get one good plate to reproduce.
sadly of the three fruits I clone the plate that looks like it is going to make it was from the smallest of the three. But beggars can't be choosers and I am most def a beggar
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Beefy1
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Quote:
MonkeyJesusFresco said: out of curiosity, what advantage is there to "the hot pour" method versus laying a squar of medicated agar cut from a pre-poured dish on top of your myc. culture?
I think it's supposed to kinda seal it inside two layers.
If you just put a piece of premade agar on top I would guess the myc just grows between the two layers along with the bacteria instead of growing up through the agar
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mycofever
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Quote:
MonkeyJesusFresco said: out of curiosity, what advantage is there to "the hot pour" method versus laying a squar of medicated agar cut from a pre-poured dish on top of your myc. culture?
In theory A hot pour is to set the bacteria back and hope that a little mycelium will survive the event then grow out from the new layer as soon as it does you transfer the mycelium on top.I would not recommend this in a situation as vital as the one that maryxmas is in.It is also no good for molds this is only good for bacteria.
-------------------- Patience will help you keep your sanity.It will insure your success if you are patient in all aspects of mushroom growing.When you rush you are prone to make mistakes and all of your efforts are wasted.
Edited by mycofever (02/14/13 07:57 PM)
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maryxmas
King of the Hippiecrates


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Re: Help. Need to fight bacteria on agar [Re: mycofever] 1
#17808353 - 02/15/13 04:22 PM (11 years, 3 months ago) |
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My one good plate has a nickel sized white fluffy patch now.
Very gorgeous
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RogerRabbit
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Registered: 03/26/03
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Quote:
MonkeyJesusFresco said: out of curiosity, what advantage is there to "the hot pour" method versus laying a squar of medicated agar cut from a pre-poured dish on top of your myc. culture?
An enormous advantage.
The hot agar partly 'pasteurizes' the bacteria which slows it down, while leaving the mushroom mycelium unharmed so it can grow up through the new layer. The hot agar also completely surrounds the mycelium and bacteria rather than just laying on top.
When you simply make another slab of agar and plop it on top, you don't get this advantage. RR
-------------------- Download Let's Grow Mushrooms semper in excretia sumus solim profundum variat "I've never had a failed experiment. I've only discovered 10,000 methods which do not work." Thomas Edison
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Workman
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Re: Help. Need to fight bacteria on agar [Re: RogerRabbit]
#17812638 - 02/16/13 11:11 AM (11 years, 3 months ago) |
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In my experience, bacteria tends to slow down naturally in a sealed plate and the mycelium may eventually overgrow the perimeter of the bacteria. Once that happens, you should be able to snag some clean mycelium on the leading edge. Obvious this won't work if the bacteria covers the entire plate. But if the contamination isn't too bad, set the plates aside and see what happens.
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Edited by Workman (02/16/13 01:23 PM)
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MonkeyJesusFresco
am i suspended in agar?



Registered: 10/09/12
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Re: Help. Need to fight bacteria on agar [Re: RogerRabbit]
#17813107 - 02/16/13 01:06 PM (11 years, 3 months ago) |
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Cool, thank you so much. Guess I'm off to investigate more agar techniques! My agar work has been limited and dumb-luck has been a MAJOR contributing factor to any success I've had 
plus, just recently found reishi and (what appears to be lion's mane) on a walking trail near by, my plan is to snag tissue from them and transfer my ass off to get my techniques down and log some experience points ...also, reishi
-------------------- LAGM v 2.024 - endo cabendo
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RogerRabbit
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Good luck. Lion's mane is one of the harder mushrooms to clone due to the amount of bacteria and other fungi present.
Antibiotic agar will help, even though I hate to use it. When isolating wild cultures though, it can save the day. RR
-------------------- Download Let's Grow Mushrooms semper in excretia sumus solim profundum variat "I've never had a failed experiment. I've only discovered 10,000 methods which do not work." Thomas Edison
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Sillyputty67

Registered: 10/06/12
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Re: Help. Need to fight bacteria on agar [Re: RogerRabbit]
#17815292 - 02/16/13 08:07 PM (11 years, 3 months ago) |
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Recently ive found using inoculation loops work well for isolating from contamination. The loop allows you to grab the tiniest sample. Usually the myc grow faster than or away from the contamination. So a couple of rounds of this would be all it would require ime.
-------------------- 1) Everything I ever posted or say is a lie.
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Lennybernadino
Amazon grower


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Re: Help. Need to fight bacteria on agar [Re: Sillyputty67]
#17819617 - 02/17/13 02:24 PM (11 years, 3 months ago) |
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I usually get the job done with a series of hot pours.
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Chris893
The Lurker

Registered: 03/01/13
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Re: Help. Need to fight bacteria on agar [Re: Lennybernadino]
#18391399 - 06/09/13 07:11 AM (10 years, 11 months ago) |
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Sorry to bump an old thread but I thought it would be better than starting a new one plus I have a somewhat relevant question. I am new to mycology and microbiology in general and don't have any lab experience except for chemistry. I have recently assembled a 2x2 flow-hood and am using it to do agar work. I've had some bacterial contaminations on some of my plates along with healthy mycelial growth on the same plates. Is it safe to open these up in front of a flow hood to transfer away from? My concern is that bacterial spores will be blown all over my work space but I guess an alcohol spray and wipe following a transfer will be sufficient to kill any spores that may have contaminated my work space?
What do you all think? Recommend?
I've heard that transferring under a plastic bag is a safe route but I think that this would just give the bacterial spores the chance to contaminate my clean new agar plate which the mycelium is being transferred to. Any comments are appreciated
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Terry M
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Re: Help. Need to fight bacteria on agar [Re: Chris893]
#18391610 - 06/09/13 09:20 AM (10 years, 11 months ago) |
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Not all bacteria produce spores. And the ones that can only do so when food is exhausted and they are under stress to survive. I've gotten away with transferring bacteria contaminated plates using a flow hood. But if you want to be really safe, use a glove box.
-------------------- Liberté, égalité, humidité.
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micro
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Re: Help. Need to fight bacteria on agar [Re: Terry M]
#18391641 - 06/09/13 09:35 AM (10 years, 11 months ago) |
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That's not how the flow hood should work... The air is flowing in a way that shouldn't allow for contamination where you are working. It might get into the filters but assuming you change the filters when you need to, and you've wiped down the area with bleach and either keep it on or let the air flow for a while before using it you should really be fine.
This is assuming the flow hood works as it is supposed to.
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Chris893
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Re: Help. Need to fight bacteria on agar [Re: micro]
#18392300 - 06/09/13 12:45 PM (10 years, 11 months ago) |
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I found this:
" Always transfer healthy mycelium away from the contamination, not the other way around. In addition, never open a contaminated dish in front of a flow hood. Always shut it off, just before opening any contaminated product, or use a glovebox. RR "
Sounds safe enough provided I keep my dirty breath away from the plate and cause as little disturbance as possible to my surroundings while maintaining good sterile technique. I'm going to try this tonight to clean up a reishi and oyster culture.
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Terry M
Stranger in a Strange Land



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Re: Help. Need to fight bacteria on agar [Re: Chris893]
#18392529 - 06/09/13 01:48 PM (10 years, 11 months ago) |
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A flow hood will keep the nasty spores away from your work area. But do you really want them scattered all over your lab room to grow? I never, ever, so much as open a plate in the lab on which there is sporulating Trichoderma or Penicillium.
-------------------- Liberté, égalité, humidité.
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Sillyputty67

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Re: Help. Need to fight bacteria on agar [Re: Terry M]
#18392558 - 06/09/13 01:55 PM (10 years, 11 months ago) |
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One more reason a good Still Air Box is superior to a flow hood.
-------------------- 1) Everything I ever posted or say is a lie.
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