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asdfasdf


Registered: 07/26/10
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Agar
#14033962 - 02/27/11 12:29 AM (12 years, 10 months ago) |
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I took a piece of mycelium from a colonized bulk substrate and dipped it in a 10% bleach solution for about a second, and stuck it on some agar. It is now blue and ugly and just unhealthy looking. Should I expect it to grow out?
-------------------- "FlexXx said: To answer some of your questions...If by "ms" you mean "magic shrooms" then yes that's what I'm trying to grow"
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dmonkey1
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bleach solution? The chloride ions prob killed a lot of the living mycelium. You should have dipped in a dilute hydrogen peroxide solution.
I'd consider that dish dead
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TheShroomJew23
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Registered: 11/29/07
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Bleach does not sterilize, it kills, well actually it does both but more of the latter.
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jokefox
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bleach , should NOT even ever be brought up around here, most noobs bring it up , but they love cancer i guess
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KingMob
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Registered: 10/24/10
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Don't get rid of it yet. Give it some time to see if it recuperates. The bluing is caused by the trauma but it doesn't mean the mycelium is dead. You don't necessarily need to dip the sample in a sterilizing solution. If there are contams present, the strongest mycelium could out run them and you can transfer a clean part of the growing mycelium. Thats one of the good things about agar; you can isolate away from contams.
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asdfasdf


Registered: 07/26/10
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"They have different purposes. Bleach is not effective against fungi. I regularly dip cultures from the wild into ten percent bleach before transferring to agar to kill mold spores and bacteria, but it doesn't harm the live culture. That's why spraying bleach on the black mold around your bathtub or window sills does nothing.
Iodine is very effective against microbes. When I clone, I wash the whole mushroom with iodine before cutting out a section to transfer, which is then dipped into ten percent bleach.
Peroxide will help prevent mold in your humidifiers. Many experienced mycologists also use peroxide to dip cultures before transferring to agar. I prefer not to use it for that purpose except for when transferring dry tissue because of the damage it causes at the cellular level, which the fungi must then recover from.
Alcohol is great for cleaning tools, tabletops and gloves before doing sterile work, but you should still flame sterilize scalpels and needles, etc. A well equipped lab will have a multitude of cleaning agents, as well as at least two fire extinguishers at opposite ends of the working area. I don't know of any particular one cleaning agent that will do it all. I hope this helps." RR
If I was transferring a wedge or the inner piece of a mushroom, something I know isn't too dirty, I wouldn't dip in anything, but because I was using myc that had been in fruiting conditions for over a week, I KNEW it was covered in contaminants, that's why I dipped. I know you can separate from contaminates, but that doesn't mean I'm going to willingly put some in there.
-------------------- "FlexXx said: To answer some of your questions...If by "ms" you mean "magic shrooms" then yes that's what I'm trying to grow"
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KingMob
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Some more specifics would be helpful to give you a clearer answer. For example, I didn't know you had introduced to fruiting conditions a week before you took the sample. I thought that you had taken some rizo myclium from the top before putting it out to fruit.
My earlier answer to wait before discarding is based on seeing the process of putting a primordia in a 10% bleach solution and then on agar.
I used your same source of info (RR)
The result was that the primordia looked unhealthy for a couple of days, then mycelium stared to grow really fast and covered the whole pin.
How long ago did you put it in the agar?
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RogerRabbit
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There's some horrible disinformation above. Bleach is one of the most effective and least toxic disinfectants used in cultivation.
Bleach is fine to use when cloning, if necessary. However, it's not going to nuke all the bacteria and mold in a second, so I suspect in a few days you're going to have a moldy mess on your hands.
You needed a sterile culture to transfer to agar. RR
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asdfasdf


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Thank you KingMob and RR. I will make sure I UPDATE MY THREAD. 2 days ago.
-------------------- "FlexXx said: To answer some of your questions...If by "ms" you mean "magic shrooms" then yes that's what I'm trying to grow"
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asdfasdf


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Ok so the piece I started with was about 1/4" in diameter. It's now covered in myc and the myc has spread to about 3/4" in diameter starting to get rhizomorphic on the edges. There are a few small spots of contamination near the myc, but none on the rest of the agar. So my sterile technique was good. I'm very satisfied.
-------------------- "FlexXx said: To answer some of your questions...If by "ms" you mean "magic shrooms" then yes that's what I'm trying to grow"
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KingMob
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Thanks for updating!
I'm glad it recuperated, I had a feeling that would happen.
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asdfasdf


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So I was just reading about anastamosis. Was what I did (take myc from colonized bulk and put on agar) a waste of time? As in, isn't the myc already an "isolate" because all the strains have all combined? Or am I not really understanding anastamosis? Thanks.
-------------------- "FlexXx said: To answer some of your questions...If by "ms" you mean "magic shrooms" then yes that's what I'm trying to grow"
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KingMob
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If you did not start with an isolate or a clone when you inoculated, then you do not have an isolate. When you inocculate with a multispore source, you are putting a bunch of spores in the grain or cake.
The mycelium each spore produces has to find a mate and go through anastomosis.
Rizomorphic mycelium is formed from the process of anastomosis.
There can be many different rizos from different mating spores in one substrate, sharing the substrate. And each strain of rizo will produce its own mushrooms.
Thats why people get clones from the mushrooms; they look for characteristics in the fruit (like size, shape, speed of growth, etc.) and use the flesh of the fruit (which they know comes from a single combination of two spores) to create that clone.
Thus, you have to isolate or clone to get a monoculture (rizo mycelium from one spore combination).
Since you used piece of substrate, you might have isolated one type of rizo, but then again, you might have to isolate some more to get a monoculture.
http://www.mushroomvideos.com/ (watch the video called "mushroom strain isolation")
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Doc_T
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Registered: 03/06/09
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Quote:
use the flesh of the fruit (which they know comes from a single combination of two spores)
Minor technical point in an otherwise great post- a tissue clone generally will have more than one substrain. Weird but true. It'll sector out on agar just like a spore inoculation. You start with fewer genetic lines, but it's still more than one.
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KingMob
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Re: Agar [Re: Doc_T]
#14074503 - 03/06/11 07:38 AM (12 years, 10 months ago) |
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Thanks Doc! Didn't know that important bit of info. You just saved me the trouble of making some culture slants without having made sure they were an isolate.
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Doc_T
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-------------------- You make it all possible. Doesn't it feel good?
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asdfasdf


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Quote:
KingMob said: If you did not start with an isolate or a clone when you inoculated, then you do not have an isolate. When you inocculate with a multispore source, you are putting a bunch of spores in the grain or cake.
The mycelium each spore produces has to find a mate and go through anastomosis.
Rizomorphic mycelium is formed from the process of anastomosis.
There can be many different rizos from different mating spores in one substrate, sharing the substrate. And each strain of rizo will produce its own mushrooms.
Thats why people get clones from the mushrooms; they look for characteristics in the fruit (like size, shape, speed of growth, etc.) and use the flesh of the fruit (which they know comes from a single combination of two spores) to create that clone.
Thus, you have to isolate or clone to get a monoculture (rizo mycelium from one spore combination).
Since you used piece of substrate, you might have isolated one type of rizo, but then again, you might have to isolate some more to get a monoculture.
http://www.mushroomvideos.com/ (watch the video called "mushroom strain isolation")
"One swipe of spores on agar will yield hundreds of strains. By selecting a dozen or so of the best rhizomorphic strains and fruiting each one separately, you can find the super performer that will cover every spot of your casing layer with healthy pins. It might take hundreds of multispore grows to find that strain, if ever, because multispore inoculated substrates usually end up with only one or two strains by the time they fruit because they've all combined. (anastomosis). This means the good fruiting and potent strains combine with the poor fruiting and bunk strains. You never know what you're going to get. Isolate on agar, then fruit separately and test each strain. When you find the best one, you can keep master slants in the refrigerator and grow that isolate forever."
RR
This makes me think that anastomosis is the combining of all the different strains, not the combining of 2 spores to make a strain.
-------------------- "FlexXx said: To answer some of your questions...If by "ms" you mean "magic shrooms" then yes that's what I'm trying to grow"
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KingMob
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Interesting.
I originally thought that anastomosis was only referring to the mating process, as I stated earlier, but upon reading some more I learned that anastomosis also refers to the fusion of cell walls between hyphae when creating the reticulate network.
Since this can happen between hyphae of the same or different origins(same or different spores), I see what RR is saying.
But what I don't understand then is how is it that only 1 or 2 strains produce fruit? I mean, what determines which strains will be the ones that eventually fruit? Is it an issue of strength? Like somehow one strain develops quicker or stronger than others and thus the other strains only end up providing more food and nurture for the stronger strains? or is there some kind of strain homogeneity that occurs from the combination of different strains into one mycelial network?
In any case, it is still mentioned in RR's comment that there IS more than one strain fruiting, and I would venture to think that the number of fruiting strains may not be limited to just one or two, so in the end, you might still have to isolate further to actually get a monoculture.
So your work, I would think, is not in vain my friend.
Doc T had also mentioned that there are different sub strains in the same mushroom, thus there is some isolation work that could be done even in a clone.
So don't give up yet. If anything you might have done some good work since you did start an isolate before fruiting.
If the tub gives good results, then you know that plate is worth saving
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asdfasdf


Registered: 07/26/10
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But see my goal was not to have an isolate immediately but have many strains that I could eventually isolate and test, so I could find a great one. If I have an isolate right now, then it was a waste of time. Especially because the tub I got the mycelium from, isn't fruiting.
-------------------- "FlexXx said: To answer some of your questions...If by "ms" you mean "magic shrooms" then yes that's what I'm trying to grow"
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biologys
Mycologist in Trainning




Registered: 12/21/09
Posts: 4,622
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you still will, once it starts growing out on agar it should start sectoring which you can remove different sectors and transfer to different dishes then grow each one out uto find your best product and go from there..
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