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Offlineragadinks
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problems cloning wild enoki ...
    #2244763 - 01/15/04 07:44 PM (13 years, 4 months ago)

hi,

I have tried to clone enoki ( flamulina velutipes ) found in the wild, but it was really very hard.
Every time I have put a piece of the mushroom tissue on agar I got some ( bacterial ? ) contamination. It was something that looked like a brown-yellow discoloration on the agar plate. Could only get rid of it by adding some antibiotics to the agar.
I also tried to germinate spores of them, but they either did not germinate or they had the same contamination when being soaked in distilled water before putting them on agar. I guess the bacteria/yeast? contamination must be a kind of parasite specific to enoki because it was present on spores and tissue of enoki from some different places.
Has anybody had similar experiences with enoki, or was it just an experience that occurred to me ?


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Invisibleeric_the_red
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Re: problems cloning wild enoki ... [Re: ragadinks]
    #2244855 - 01/15/04 10:06 PM (13 years, 4 months ago)

i've cloned many wild mushrooms. i do it just for fun, mostly.

the large, fat mushrooms are easy to clone. just break them open and you have a lot of tissue to choose from. i have found that smaller mushrooms, such as enoki, are much harder to clone. if you try to cut it open, the blade will most likely spread contams all over the inner tissue. it's also hard to break apart without spreading contams.

i have had the best results sinking the small mushrooms in a cup of peroxide for a while. at least wait for most of the bubbling to stop before continuing. after soaking it, pour a new cup of peroxide and hold the mushrooom under the surface. pull (not cut) it apart, keeping it under the whole time. then, still keeping the mushroom under the surface, grab a sterile scalpel and try to snag a piece of tissure around the junction of the stem and cap. try a few different areas if possible.

and yes, adding antibiotics may help out.

good luck.


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Offlinezeronio
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Re: problems cloning wild enoki ... [Re: ragadinks]
    #2245343 - 01/16/04 02:47 AM (13 years, 4 months ago)

Wild mushrooms are always contaminated. Enoki is even harder to clone because is so small and it's hard to get clean tissue from the inside. You have to spray the mushroom with peroxide and use several peroxidated agar plates to get clean growth . The success rate is quite low.


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Offlineragadinks
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Re: problems cloning wild enoki ... [Re: eric_the_red]
    #2246833 - 01/16/04 07:56 PM (13 years, 4 months ago)

eric, how strong is the dilution of your peroxid ?
I have got some mycelium on an agar plate with antibiotics now. But whenever I transfer it to another plate without antibiotics the contams comes back again. The contamination seems to stay on top of the mycelium itself and grow even on peroxidated plates. It's a pity that I have no good camera so that I can show you.


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Invisibleeric_the_red
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Re: problems cloning wild enoki ... [Re: ragadinks]
    #2246929 - 01/16/04 08:38 PM (13 years, 4 months ago)

i've never a problem similar to yours. if i did, here is what i would try first.
i've never tried this (on purpose) before, or tried it for the reason you may want to try it, so i don't know how well it will work. i'll just lay it out here and hopefully get some feedback from others on it.

i would totally encase the culture with antibiotic agar.
either (a) pour a dish and push the culture underneath the surface with a scalpel or (b) put the culture on a solidified dish, then pour another layer of antibiotic agar over the culture.
i get the feeling (b) might work better.

either way, the culture/mycelium should be totally surrounded by agar, forcing the mycelium to grow through the agar. this should cleanse every surface of the mycelium. any mycelium that grows up to the surface should shed its contaminants on the way up through the agar.

well, what do you all think? i've sunk cultures in agar before like this on accident and the plates turned out rather nice. seems like i also read about a similar technique recently.


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Anno cock? is that some kind of Greek liqueur? -Geo's All Knowing Sex Slave


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OfflineRohypnol
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Re: problems cloning wild enoki ... [Re: eric_the_red]
    #2247261 - 01/16/04 11:44 PM (13 years, 4 months ago)

Yes this is a good idea. Bacteria cannot grow in 3 dimensions. I may have an easier and more effective method. I have accidently seperated contamination from mycellium by banging on the petri dish while its upside down until the agar flips over. The mycellium has to grow through the agar, and sheds the contamination.


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Offlineragadinks
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Re: problems cloning wild enoki ... [Re: Rohypnol]
    #2247899 - 01/17/04 07:14 AM (13 years, 4 months ago)

I will try this and let you know how it worked out!


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Offlinecanid
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Re: problems cloning wild enoki ... [Re: Rohypnol]
    #2248744 - 01/17/04 05:55 PM (13 years, 4 months ago)

hrm, that's an interesting approach. two concerns i have are these:

transfers of wedges would be impossible; one would have to be careful about scraping tissue off for transfer [not too big of a problem at all].

not all bacteria are aerobc. anaerobes will grow through the agar more easily than the mycellia.


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



Attn PWN hunters: If you should come across a bluing Psilocybe matching P. pellicolusa please smell it.
If you detect a scent reminiscent of Anethole (anise) please preserve a specimen or two for study and please PM me.


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Invisibleeric_the_red
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Re: problems cloning wild enoki ... [Re: canid]
    #2249193 - 01/17/04 08:50 PM (13 years, 4 months ago)

>anaerobes will grow through the agar more easily than the mycellia.

definitely true with regular agar. ragadinks is using antibiots in his agar. wouldn't the antibiotics take care of the bacteria, killing it off and/or keeping it trapped within the agar?


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Anno cock? is that some kind of Greek liqueur? -Geo's All Knowing Sex Slave


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Offlinecanid
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Re: problems cloning wild enoki ... [Re: eric_the_red]
    #2249231 - 01/17/04 09:04 PM (13 years, 4 months ago)

in principle, yes, but if he is having contamination issues, his antibiotics are ont providing enough controll [given the high levels of bacterial exposure in tissue culture of species which colonise slowly].

anaerobes are not as likely to cause problems [given the prediminaltly aerobic conditions of the mushroom mycelia, but it is still a risk. in dead areas of the tissue, where oxygen is no longer being brought in, anaerobes may find footholds. they may then find thier way into the agar [ie. in the area between the tissue fragment used for inoculation and he agar surface; and area where i've noticed the tissue tends to die out, due i suspect to these anaerobic conditions].


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



Attn PWN hunters: If you should come across a bluing Psilocybe matching P. pellicolusa please smell it.
If you detect a scent reminiscent of Anethole (anise) please preserve a specimen or two for study and please PM me.


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Invisiblethescientist
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Re: problems cloning wild enoki ... [Re: Rohypnol]
    #2251459 - 01/18/04 11:41 PM (13 years, 4 months ago)

rohypnol, thats misinformation


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OfflineRohypnol
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Re: problems cloning wild enoki ... [Re: thescientist]
    #2253180 - 01/19/04 05:16 PM (13 years, 4 months ago)

Whats misinformation? I've read that bacteria cannot grow in 3d... if that is not true please correct me


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OfflineRohypnol
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Re: problems cloning wild enoki ... [Re: thescientist]
    #2253181 - 01/19/04 05:16 PM (13 years, 4 months ago)

Whats misinformation? I've read that bacteria cannot grow in 3d... if that is not true please correct me


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OfflineRohypnol
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Re: problems cloning wild enoki ... [Re: thescientist]
    #2253182 - 01/19/04 05:16 PM (13 years, 4 months ago)

Whats misinformation? I've read that bacteria cannot grow in 3d... if that is not true please correct me


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Offliner05c03
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Re: problems cloning wild enoki ... [Re: Rohypnol]
    #2253830 - 01/19/04 08:52 PM (13 years, 4 months ago)

Wow, three of the same post. I think we got it.

Where did you read that? It is important to be sure what your reference was talking about. If it was talking about a single bacterium, a single bacterial cell, then no bacteria cells are not able to grow in three dimensions (add girth, length, or whatever as they age like a tree, person, or mushroom). They go through fission and thats pretty much it. However, if you are talking about a bacterial colony where many thousands and millions of cells are dividing then the colony can increase in size in any direction as more and more cells are created. They will spread out in all direction as as long as they are not contstained by nutrient, physical or chemical barriers of some sort. You may be able to clean up a fungal culture by allowing the fungus to grow through the agar because the mycelium can penetrate the agar activiely, some thing that bacteria cannot do. Thus, fungi will grow through the agar first.


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