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 LastBreath
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 Mycelium won't colonize my agar plate 
#16661415 - 08/07/12 03:56 PM (11 years, 8 months ago) |
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I have potato dextrose based Yeast & Mold agar that I flaked some dry spores onto, which hydrated and grew, but it's been several weeks and they haven't grown more than a few white specs on the plate. Anyone know why it won't colonize? The strain is Mexicana btw. I'm thinking it might need water and that I should drop some sterile water on and around the mycelium to get it to grow, would this be advisable?
Edited by LastBreath (08/07/12 05:54 PM)
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Terry M
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Re: Mycelium won't colonize my agar plate [Re: LastBreath]
#16661946 - 08/07/12 05:31 PM (11 years, 8 months ago) |
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Looks to me like you've got a plateful of contaminants.
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Liberté, égalité, humidité.
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LastBreath
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Re: Mycelium won't colonize my agar plate [Re: Terry M]
#16662094 - 08/07/12 05:54 PM (11 years, 8 months ago) |
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Whoops i put up the wrong pic, fixed now.
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 36fuckin5
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Re: Mycelium won't colonize my agar plate [Re: LastBreath]
#16662381 - 08/07/12 06:43 PM (11 years, 8 months ago) |
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Still looks like you have a plate of contams. WTF are those black spots?
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i recommend common sense and figuring it out.
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LastBreath
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Re: Mycelium won't colonize my agar plate [Re: 36fuckin5]
#16662637 - 08/07/12 07:24 PM (11 years, 8 months ago) |
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Those are spores that didn't germinate (yet).
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cloudpersona
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Re: Mycelium won't colonize my agar plate [Re: LastBreath]
#16662664 - 08/07/12 07:28 PM (11 years, 8 months ago) |
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Quote:
LastBreath said:
Those are spores that didn't germinate (yet).
wat.
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“The shaman is not merely a sick man, or a madman; he is a sick man who has healed himself.”
– Terence McKenna
“If the words ‘life, liberty, and the pursuit of happiness’ don’t include the right to experiment with your own consciousness, then the Declaration of Independence isn’t worth the hemp it was written on.”
-Terence McKenna
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LastBreath
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Re: Mycelium won't colonize my agar plate [Re: cloudpersona]
#16662719 - 08/07/12 07:35 PM (11 years, 8 months ago) |
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Does anyone have any advice on how to get the mycelium to colonize the agar? You may not be able to tell, but these dishes only have a very thin film of agar on the bottom as that is how they make them in the lab I got them from. I know other people usually use quite thick wedges of the stuff when they put them in their jars, I'm thinking i might have to get some plates with more agar in them, but I still don't see why it won't colonize it this dish even with a thin layer. 
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cloudpersona
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Re: Mycelium won't colonize my agar plate [Re: LastBreath]
#16662750 - 08/07/12 07:38 PM (11 years, 8 months ago) |
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Quote:
LastBreath said:
Does anyone have any advice on how to get the mycelium to colonize the agar? You may not be able to tell, but these dishes only have a very thin film of agar on the bottom as that is how they make them in the lab I got them from. I know other people usually use quite thick wedges of the stuff when they put them in their jars, I'm thinking i might have to get some plates with more agar in them, but I still don't see why it won't colonize it this dish even with a thin layer.
Is it wet? Can you turn it on it's side and see liquid?
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“The shaman is not merely a sick man, or a madman; he is a sick man who has healed himself.”
– Terence McKenna
“If the words ‘life, liberty, and the pursuit of happiness’ don’t include the right to experiment with your own consciousness, then the Declaration of Independence isn’t worth the hemp it was written on.”
-Terence McKenna
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LastBreath
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Re: Mycelium won't colonize my agar plate [Re: cloudpersona]
#16662773 - 08/07/12 07:40 PM (11 years, 8 months ago) |
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Well now it is since I dripped some sterile water onto it and shook it up a bit.
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Cyber
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Re: Mycelium won't colonize my agar plate [Re: LastBreath]
#16662902 - 08/07/12 07:58 PM (11 years, 8 months ago) |
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Quote:
LastBreath said:
I have potato dextrose based Yeast & Mold agar that I flaked some dry spores onto, which hydrated and grew, but it's been several weeks and they haven't grown more than a few white specs on the plate. Anyone know why it won't colonize? The strain is Mexicana btw. I'm thinking it might need water and that I should drop some sterile water on and around the mycelium to get it to grow, would this be advisable?
So this is PDA (Potato Dextrose Agar)
First, I would only work with dry spores if you know the print is less and 2 years old. Otherwise, I tend to rehydrate them in a syringe for a week before putting them on agar. But that is just me.
At this point I would isolate out the mycelia you have onto new agar plates. One plate per clump of mycelia or select the best looking clumps to transfer, and see how they do. They may take off. I would also make sure you are incubating them at around 84f I have found that it improves growth on agar a lot more than it does in BRF jars. A am assuming that it is because agar has less mass and is more reactive to temperature fluctuations.
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solumvita
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Re: Mycelium won't colonize my agar plate [Re: Cyber]
#16663938 - 08/08/12 12:16 AM (11 years, 8 months ago) |
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you can see the mycelium is trying to grow, like mentioned above, isolate the good growth away, onto new plates, the thickness of the agar has nothing to do with growth, except that thinner paltes dry out quicker. Never put water onto the agar, if there are bacteria present you are giving them a way to spread and get onto the mycelium.
the mycelium might not like the agar that you are giving them try a simple PDA without any special additives.
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joyless
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Re: Mycelium won't colonize my agar plate [Re: solumvita]
#16666662 - 08/08/12 04:02 PM (11 years, 8 months ago) |
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i've had similar situation few years back. i mixed up some agar and malt extract...and nothing, no growth, no germination, mycelium that i transfered from PDA plates would grow upwards, like it was trying to avoid the MEA. i even left some plates open for 3 weeks, agar dried out, but there was no sign of bacteria. tossed agar and malt away, bought different brands and it was all back to normal again.
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Mykes logos
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Re: Mycelium won't colonize my agar plate [Re: solumvita]
#16668259 - 08/08/12 09:56 PM (11 years, 8 months ago) |
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Quote:
Terry M said:
Looks to me like you've got a plateful of contaminants.
Quote:
solumvita said:
you can see the mycelium is trying to grow, like mentioned above, isolate the good growth away, onto new plates, the thickness of the agar has nothing to do with growth, except that thinner paltes dry out quicker. Never put water onto the agar, if there are bacteria present you are giving them a way to spread and get onto the mycelium.
the mycelium might not like the agar that you are giving them try a simple PDA without any special additives.
I agree... but perhaps an antibiotic MEA would work much better in OPs situation? OP stated that this has been growing for "several weeks"... that seems like way too many weeks- maybe the plate dried out a while back? There's only one way to find out tho... but the timeframe doesn't seem right to me
TBH I'm with Terry M on this one... OP should start a new batch of plates w/ a different recipe and thicker wedges w/ the original print.
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IndigoSky
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Re: Mycelium won't colonize my agar plate [Re: Mykes logos]
#16696460 - 08/13/12 11:10 PM (11 years, 8 months ago) |
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I'm surprised no one has said anything about those clumps of spores. I guess that's the way it goes when flaking them onto agar though?
 I've only used a loop for spores to agar.
Maybe the hundreds of strains are inhibiting each other? I'd go with solumvitas adivce and start isolating the mycelium.
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donrjuan
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Re: Mycelium won't colonize my agar plate [Re: LastBreath]
#16702984 - 08/15/12 06:21 AM (11 years, 7 months ago) |
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Great point Indigo. Try streaking the spores next time. I use a section on thin stainless steal wire twisted careful into a loop. If you are making your own loop, be sure not to use pliers with teeth to twist the wire as it will leave sharp ridges that will damage the spores, trust me. I flame the loop, touch the agar to cool (also moistens the loop) and touch to the sore print. Streak directly on the agar making an S. Another option is to make very watered down spore syringe and just use a drop or two per plate. Remember LastBreath, less is more when it comes to agar work. Less spores/spore solution also equals less contaminants from the spore print. Good Luck!
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Terry M
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Re: Mycelium won't colonize my agar plate [Re: donrjuan]
#16703047 - 08/15/12 06:51 AM (11 years, 7 months ago) |
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I'm wondering how you get flakes of spores to begin with. Even oyster mushrooms spore prints aren't heavy enough to make flakes.
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Liberté, égalité, humidité.
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donrjuan
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Re: Mycelium won't colonize my agar plate [Re: Terry M]
#16703060 - 08/15/12 06:55 AM (11 years, 7 months ago) |
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In the begging I used Stamets method of simply folding a prints and rubbing it together to get the spores to release. Huge clumps every time that would show little signs of growth. It is like they are to dense or crowded to take off. I personally use the weak syringe method and streak one small drop per plate.
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Amanita virosa
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Re: Mycelium won't colonize my agar plate [Re: LastBreath]
#16756751 - 08/29/12 08:39 PM (11 years, 7 months ago) |
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Quote:
LastBreath said:
Well now it is since I dripped some sterile water onto it and shook it up a bit.
never a good idea. high likely hood for contam doing that....is it a premixed PDA or did you make it urself? looks awefully clear for pda. is it possible that is is just agar without any nutrients? that would explain it. just enuff nutrient in the agar itself to get the spores to germinate but thats about all they are gonna do.
redo...or sub out to new plates
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tahoe
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Re: Mycelium won't colonize my agar plate [Re: Amanita virosa]
#16758087 - 08/30/12 03:36 AM (11 years, 7 months ago) |
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I am claiming the mycelium does not like the spuds. Or its too dry but it doesnt look it
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Stop experimenting half way through your first grow. Grow it to maturity, watch it, learn from it. Do this a few times then experiment with different ideas and figure out what works best for you.
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Teh=The
I need to proofread
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Amanita virosa
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Re: Mycelium won't colonize my agar plate [Re: tahoe]
#16760461 - 08/30/12 03:04 PM (11 years, 7 months ago) |
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Quote:
tahoe said:
I am claiming the mycelium does not like the spuds. Or its too dry but it doesnt look it
Never met a mycelium that didn't like spuds. Guess theres a first for everything.
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tahoe
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Re: Mycelium won't colonize my agar plate [Re: Amanita virosa]
#16760597 - 08/30/12 03:35 PM (11 years, 7 months ago) |
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I have met some that doesn't like wheat. I hate wheat agar.
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Stop experimenting half way through your first grow. Grow it to maturity, watch it, learn from it. Do this a few times then experiment with different ideas and figure out what works best for you.
My Legacy
https://www.shroomery.org/forums/showflat.php/Number/22140987#22140987
Teh=The
I need to proofread
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LastBreath
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Re: Mycelium won't colonize my agar plate [Re: tahoe]
#16763295 - 08/30/12 11:02 PM (11 years, 7 months ago) |
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I abandoned my agar project, I don't know what to do with it. I don't even know why I tried to do it in the first place, I guess I was just curious.
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IndigoSky
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Re: Mycelium won't colonize my agar plate [Re: LastBreath]
#16764199 - 08/31/12 05:32 AM (11 years, 7 months ago) |
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Try again with a clone rather than spores, it's worth it.
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pablokabute
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Re: Mycelium won't colonize my agar plate [Re: IndigoSky]
#16764222 - 08/31/12 05:47 AM (11 years, 7 months ago) |
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tissue clone direct to the recieving sub! hehe.
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Fermented Mushrooms!!
--- https://www.shroomery.org/forums/showflat.php/Number/23378638/fpart/1/vc/1
'The second seal: “All CONTAMINATED things and events are unsatisfactory.”'
"I envy you. You North Americans are very lucky. You are fighting the most important fight of all - you live in THE HEART OF THE BEAST."
--Anonymous Guerilla, or is he..
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LastBreath
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Re: Mycelium won't colonize my agar plate [Re: IndigoSky]
#16766442 - 08/31/12 03:21 PM (11 years, 7 months ago) |
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Quote:
IndigoSky said:
Try again with a clone rather than spores, it's worth it.
So I would just carve out a piece of mushroom tissue and put it in an agar dish? How long will it keep? I have some good fruits that are worth cloning. There's also the cardboard method which I read a while ago, I might try that too.
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Amanita virosa
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Re: Mycelium won't colonize my agar plate [Re: LastBreath]
#16766757 - 08/31/12 04:18 PM (11 years, 7 months ago) |
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cube? at least six months parafilmed, wrapped in foil in the fridge
edibles even longer
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IndigoSky
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Re: Mycelium won't colonize my agar plate [Re: Amanita virosa]
#16766805 - 08/31/12 04:28 PM (11 years, 7 months ago) |
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And that doesn't mean you lose your culture, it just means it's time to make new petris with the old ones. Slants should last even longer.
In a nutshell yeah, transfer a tissue sample from the stem to agar. Watch RR's video on cloning if you can, there's also a lot of in depth teks about it here. I wouldn't use the cardboard method.
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LastBreath
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Re: Mycelium won't colonize my agar plate [Re: IndigoSky]
#16766844 - 08/31/12 04:33 PM (11 years, 7 months ago) |
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Quote:
IndigoSky said:
I wouldn't use the cardboard method.
Why?
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IndigoSky
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Re: Mycelium won't colonize my agar plate [Re: LastBreath]
#16766916 - 08/31/12 04:46 PM (11 years, 7 months ago) |
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I don't see the point other than money. A plate of agar cost about $1 if you make it yourself. It seems like a PITA (I haven't tried it though) to cut out pieces to transfer to new dishes and inoculate grain. It seems like it'd also be harder to isolate sectors too. I think you would also have to sterilize the cardboard in the petris, you'd need glass sealed in bags so you don't screw with the moisture content or half pint jars.
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donrjuan
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Re: Mycelium won't colonize my agar plate [Re: LastBreath]
#16780210 - 09/03/12 06:38 AM (11 years, 7 months ago) |
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The cardboard method was really intended for isolating from contams or "cleaning up" a culture, not inoculating. One samples from a specimen and then sandwich that small piece between moist cardboard. You then wait until you see mycelium peeking at the edge, at which point you take a sample and plate that. This process will not be used once you already have it isolated on a plate.
No disrespect to others but my agar plates only cost $0.25 to produce so cost should not be anyone's issue. Just do not throw your hands up and quit. Try it a few times! Good Luck.
Edited by donrjuan (09/03/12 06:48 AM)
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LastBreath
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Re: Mycelium won't colonize my agar plate [Re: donrjuan]
#16784049 - 09/03/12 09:16 PM (11 years, 7 months ago) |
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So if I clone a mushroom and redo the growing process, will the fruits all be very similar to the one I took the clone from?
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donrjuan
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Re: Mycelium won't colonize my agar plate [Re: LastBreath]
#16785429 - 09/04/12 06:41 AM (11 years, 7 months ago) |
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If you have a monoculture from the clone then yes. The subsequent grows will be genetically identical. Be sure to pay careful attention to the mycelium running from the sample you take. Be sure it is not sectoring indicating the presence of another strain in the mushroom. If it does sector, just collect a sample of each and grow them out to see which produces. It can be daunting sometimes as I have had four sectors from a single clone and when grown out three of the four fruited well. Mycology is a strange science. Good luck!
Edited by donrjuan (09/04/12 06:50 AM)
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pablokabute
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Re: Mycelium won't colonize my agar plate [Re: donrjuan]
#16786087 - 09/04/12 10:49 AM (11 years, 7 months ago) |
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Quote:
donrjuan said:
If you have a monoculture from the clone then yes. The subsequent grows will be genetically identical. Be sure to pay careful attention to the mycelium running from the sample you take. Be sure it is not sectoring indicating the presence of another strain in the mushroom. If it does sector, just collect a sample of each and grow them out to see which produces. It can be daunting sometimes as I have had four sectors from a single clone and when grown out three of the four fruited well. Mycology is a strange science. Good luck!
never ever thought this was possible, i thought a clone would be the very clone you cloned it from.. got a detailed explanation of this(page referral and whatnot)
thanks.
--------------------
Fermented Mushrooms!!
--- https://www.shroomery.org/forums/showflat.php/Number/23378638/fpart/1/vc/1
'The second seal: “All CONTAMINATED things and events are unsatisfactory.”'
"I envy you. You North Americans are very lucky. You are fighting the most important fight of all - you live in THE HEART OF THE BEAST."
--Anonymous Guerilla, or is he..
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LastBreath
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Re: Mycelium won't colonize my agar plate [Re: pablokabute]
#16786398 - 09/04/12 11:58 AM (11 years, 7 months ago) |
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BTW the sterile water I dripped into my plates didn't contaminate at all, it was microwaved for only 2 minutes or so.
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Amanita virosa
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Re: Mycelium won't colonize my agar plate [Re: LastBreath]
#16788346 - 09/04/12 05:49 PM (11 years, 7 months ago) |
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Quote:
LastBreath said:
BTW the sterile water I dripped into my plates didn't contaminate at all, it was microwaved for only 2 minutes or so.
you got lucky
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LastBreath
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Re: Mycelium won't colonize my agar plate [Re: Amanita virosa]
#16789453 - 09/04/12 09:16 PM (11 years, 7 months ago) |
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Quote:
Amanita virosa said:
Quote:
LastBreath said:
BTW the sterile water I dripped into my plates didn't contaminate at all, it was microwaved for only 2 minutes or so.
you got lucky
The water got so hot it exploded inside the microwave, then I slurped some up into a syringe while it was still boiling hot and let it cool. Worked for me.
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lipa
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Posts: 2,684
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Re: Mycelium won't colonize my agar plate [Re: LastBreath]
#16790043 - 09/04/12 10:37 PM (11 years, 7 months ago) |
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Personally I like spore dilutions for germinating spores. Gives you a chance to hydrate the spores first and spreads things out making it easier to isolate away from contamination. I usually do 3-4 dilutions. If you have antibiotic agar you can just scratch the print ever so lightly and just plunge it strait down into the middle of the agar so the spores get sandwiched in between the agar giving them a nice humid place to germinate. you can also plunge into the edge of the agar and slide it around the perimeter of the dish (between the dish and agar) spreading them out more.
Other things that work are smearing the spores on the agar and place a glass slide slightly over the smear of spores. they germinate under the slide and crawl up and over the slide sometimes making it easy to isolate the mycelium on top of the glass. Most other contaminants wont follow.
Lipa
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