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OfflineFuckUp Eddy
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Agar Issues
    #17266256 - 11/22/12 08:27 PM (12 years, 1 month ago)

Some issues with agar...Im on my fourth transfer and I haven't seen a sector or any rizomorphic mycellium once! It's all cottony all the time. But I believe on my third transfer, I put two pieces on one plate, and they form somewhat of a line in between the growths. Is this a sign of isolate or does it just mean that they are not compatible? With this kind of growth, there is no way in hell Im going to tell when I have a monoculture. All I know is that I started with a drop of spores and now (on fourth transfer) I have about thirty cultures that I dont know what the fuck Im going to do with besides go ahead and try to fruit (man thats a lot of fucking cakes). The agar Im using is MEA Light from a vendor. I use about 5 tablespoons for 750ml water.

Also in the second shitty picture, I put three different pieces of mycellium on agar to see if they will merge or form a seam. One of the pieces kind of looks like mold (wispy/spikey)...not sure. But Ive seen some oyster mycellium that looks just like that. Im not sure what to think. Any feedback on all of this would be great.



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OfflineAmanita virosa
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Re: Agar Issues [Re: FuckUp Eddy]
    #17266374 - 11/22/12 08:59 PM (12 years, 1 month ago)

The first plate looks like two incompatible strains to me. If they were compatible they would have formed dikaryotic hyphae. But it is really hard to see in that shot. Is there a clear line without Myc where they come together?  In other words are they repelling each other?  If so they are incompatible strains. You can always just put a small wedge from say five different plates in each jar to increase likelihood of compatibility

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Offline00funG.I.00
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Re: Agar Issues [Re: FuckUp Eddy]
    #17266403 - 11/22/12 09:04 PM (12 years, 1 month ago)

hey man,
I am looking at your dishes, I see mold on the left one (IMO)... do you have access to a microscope? and at least 400x ? If so ---do a scotch tape mount slide, and look for clamped speta in the hyphae...If it has clamps, then it can not be a mold...

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OfflineRogerRabbitM
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Re: Agar Issues [Re: FuckUp Eddy]
    #17266426 - 11/22/12 09:09 PM (12 years, 1 month ago)

Transfer smaller pieces so you move fewer strains with each transfer.

Don't worry about rhizomorphic mycelium too much.  If you want to see it, reduce the nutrient content of your agar.  Mycelium will often go from cottony to rhizomorphic and back again.
RR


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Offline00funG.I.00
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Re: Agar Issues [Re: 00funG.I.00]
    #17266429 - 11/22/12 09:09 PM (12 years, 1 month ago)

here is one I am working on, this is a piece of grain from a jar that contaminated... If it is a strong strain I think it should be rather quick... (50 hours old @ 81-85 degrees F)..


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OfflineFuckUp Eddy
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Re: Agar Issues [Re: 00funG.I.00]
    #17266449 - 11/22/12 09:14 PM (12 years, 1 month ago)

I really need a flowhood before a microscope. Thats my next move. It would be nice to have a microscope though. I cant get away with it at school right now...lol. Im guessing that if its cobweb then it will spread very fast. If it doesn't do that or change colors then I should be good right?

Also to the first poster. Yes they are rappelling each other especially in the middle. I hope to take better pictures tomorrow. What I need to know is if that is a good thing, because I started off with multispore on agar. Does that indicate that they are new/different strains? I dont want them to combine. I want them to divide. Im trying to get isolates.

@RR...I always take VERY small pieces from different sides of the circle. I just cant tell if I have a monoculture at this point. Im on my fourth transfer if you count the spores going onto agar. Like I said I have about thirty separate cultures now and Im thinking its time to proof. By the way, I tried agar to BRF with good results so far. Problem is that I underestimated the size of the wedge that goes in. I should have put a bigger piece in there. Couldn't find any info on that.


--------------------
If you havent played with this then you are missin out! http://www.albinoblacksheep.com/flash/zoomquilt

Also good video!!! http://www.youtube.com/watch?v=6O6x_m4zvFs

Edited by FuckUp Eddy (11/22/12 09:21 PM)

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Offline00funG.I.00
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Re: Agar Issues [Re: FuckUp Eddy]
    #17266534 - 11/22/12 09:32 PM (12 years, 1 month ago)

yeah, one trick is to leave it in sunlight for a day or 2 (i suppose artificial light will do too), it'll sporylate faster...
I have been using MEA plates, helps mycellium take hold faster, slows contams.

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OfflineAmanita virosa
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Re: Agar Issues [Re: 00funG.I.00]
    #17266585 - 11/22/12 09:42 PM (12 years, 1 month ago)

Quote:

00funG.I.00 said:
hey man,
I am looking at your dishes, I see mold on the left one (IMO)... do you have access to a microscope? and at least 400x ? If so ---do a scotch tape mount slide, and look for clamped speta in the hyphae...If it has clamps, then it can not be a mold...



Yea. That don't look so good for cube Myc now that you mention it. The way it is zonal and not quite pure white. I am betting mold as well

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OfflineFuckUp Eddy
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Re: Agar Issues [Re: Amanita virosa]
    #17266679 - 11/22/12 09:57 PM (12 years, 1 month ago)

Anyone think I should go ahead and proof strains sense I have done four sets of transfers? Should I do all 30? What do you guys normally do?

Also any feedback on what "seams" (incompatible mycellium on agar) actually mean? Different strains?


--------------------
If you havent played with this then you are missin out! http://www.albinoblacksheep.com/flash/zoomquilt

Also good video!!! http://www.youtube.com/watch?v=6O6x_m4zvFs

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OfflinePussyFart
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Re: Agar Issues [Re: FuckUp Eddy]
    #17266798 - 11/22/12 10:17 PM (12 years, 1 month ago)

Quote:

FuckUp Eddy said:
Anyone think I should go ahead and proof strains




What do u mean by this? Do u mean grow each culture out?

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OfflineFuckUp Eddy
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Re: Agar Issues [Re: PussyFart]
    #17266925 - 11/22/12 10:39 PM (12 years, 1 month ago)

yes


--------------------
If you havent played with this then you are missin out! http://www.albinoblacksheep.com/flash/zoomquilt

Also good video!!! http://www.youtube.com/watch?v=6O6x_m4zvFs

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OfflineFuckUp Eddy
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Re: Agar Issues [Re: FuckUp Eddy]
    #17271004 - 11/23/12 08:14 PM (12 years, 1 month ago)

Anyone else...need to know how many of these 30 cultures that I have at the end of four transfers that I should grow out and test...


--------------------
If you havent played with this then you are missin out! http://www.albinoblacksheep.com/flash/zoomquilt

Also good video!!! http://www.youtube.com/watch?v=6O6x_m4zvFs

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Offline00funG.I.00
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Re: Agar Issues [Re: FuckUp Eddy]
    #17271500 - 11/23/12 10:22 PM (12 years, 1 month ago)

I think that is entirely your perogative.. If you have the space and resources "proof" all of them.. and enjoy the bounty..

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OfflineAmanita virosa
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Re: Agar Issues [Re: 00funG.I.00]
    #17272057 - 11/24/12 01:21 AM (12 years, 1 month ago)

Realize that you don't have to grow them all out at once.  You can always do five at a time and just wrap the rest. Parafilm first then stack them and wrap in Saran wrap then foil. Refrigerate. Come back to them in five or six months And they will be fine. Otherwise shit. You could make four grain jars from each plate is 90 jars. That's a lot at one time. Unless you got a very large space and a lot of guts.

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OfflineFuckUp Eddy
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Re: Agar Issues [Re: Amanita virosa]
    #17274809 - 11/24/12 05:26 PM (12 years, 1 month ago)

Thanks guys. No I dont feel like fruiting all those right away...lol. I have too much spawn as it is (G2G's produce so much)! I just started six months or so ago, and I had a hard time with the first WBS to coir grow due to the mean green. This time I pasteurized in jars instead of the bucket tek and I am hoping that will pay off. Now Im experiencing a few problems with mold and such (especially telling the difference between white mold and mycellium sometimes). Its been rough. Right now Im just working on getting an isolate and getting the grains to colonize coir/verm/gypsum...without issues. Got a monotub going and all sorts of other little things Im trying my hand at. I just hate not having a flowhood, as it would make everything so much easier. I will get one soon, thats for sure. I hate failure. Im guessing that encountering contams is the best way to learn what to look for...but the discovery process is a bitch. The website helps but it doesn't beat the experience Im pretty sure.


--------------------
If you havent played with this then you are missin out! http://www.albinoblacksheep.com/flash/zoomquilt

Also good video!!! http://www.youtube.com/watch?v=6O6x_m4zvFs

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OfflinePussyFart
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Re: Agar Issues [Re: FuckUp Eddy]
    #17274821 - 11/24/12 05:31 PM (12 years, 1 month ago)

Quote:

FuckUp Eddy said:
I had a hard time with the first WBS to coir grow due to the mean green. This time I pasteurized in jars instead of the bucket tek and I am hoping that will pay off.




If u had trich before the first flush with coir, even using the bucket tek for semi-pasteurization, I am willing to bet it was from the spawn, not the substrate.

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Offline00funG.I.00
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Re: Agar Issues [Re: PussyFart]
    #17275937 - 11/24/12 09:41 PM (12 years, 1 month ago)

keep in mind, sometimes the colonized grains turn blue-green, blue or purple when damaged.. as long as you are aware what is trich, pennicillium, and bruising......

the best and fastest way to tell If you have a contam is under a microscope.... whick will be over the heads of at least 50% of the people here.... look for (monteic Hyphae -- and clamped septa.. ) it will be the fastest way to Identify...

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InvisibleTheEaglesGift
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Re: Agar Issues [Re: PussyFart]
    #17276544 - 11/24/12 11:47 PM (12 years, 1 month ago)

Quote:

Notahacker420 said:
Quote:

FuckUp Eddy said:
I had a hard time with the first WBS to coir grow due to the mean green. This time I pasteurized in jars instead of the bucket tek and I am hoping that will pay off.




If u had trich before the first flush with coir, even using the bucket tek for semi-pasteurization, I am willing to bet it was from the spawn, not the substrate.




I agree with Notahacker420.

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Offlinetwistedty
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Re: Agar Issues [Re: TheEaglesGift]
    #17276684 - 11/25/12 12:14 AM (12 years, 1 month ago)

i love the bucket tek :shrug:

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OfflineFuckUp Eddy
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Re: Agar Issues [Re: twistedty]
    #17312263 - 11/30/12 10:21 PM (12 years, 1 month ago)

Update from first post...anything fishy with that culture on the left? Note: they are all repelling each other...



--------------------
If you havent played with this then you are missin out! http://www.albinoblacksheep.com/flash/zoomquilt

Also good video!!! http://www.youtube.com/watch?v=6O6x_m4zvFs

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OfflineRogerRabbitM
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Re: Agar Issues [Re: Amanita virosa]
    #17313622 - 12/01/12 08:10 AM (12 years, 1 month ago)

Isolate more if you want single sectors.  You might even start over from scratch, using minimum spores this time.  When you have many thousands of strains on the same dish, it can take a lot of transfers.

Quote:

Amanita virosa said:
The first plate looks like two incompatible strains to me. If they were compatible they would have formed dikaryotic hyphae. But it is really hard to see in that shot. Is there a clear line without Myc where they come together?  In other words are they repelling each other?  If so they are incompatible strains. You can always just put a small wedge from say five different plates in each jar to increase likelihood of compatibility




Just to clarify something, strains already are dikaryotic.  Hyphae are monokaryotic.  If compatible mononucleate hyphae(monokaryotic mycelium) meet, they exchange DNA and become polynucleate (dikaryotic).  You can't have dikaryotic hyphae as that would be like a two wheeled monocycle.
RR


--------------------
Download Let's Grow Mushrooms



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"I've never had a failed experiment.  I've only discovered 10,000 methods which do not work."
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OfflineFuckUp Eddy
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Re: Agar Issues [Re: RogerRabbit]
    #17313836 - 12/01/12 09:22 AM (12 years, 1 month ago)

So what do these seams mean??? I need to start over like you said. Cloning would probably eliminate this many transfers Im thinking?


--------------------
If you havent played with this then you are missin out! http://www.albinoblacksheep.com/flash/zoomquilt

Also good video!!! http://www.youtube.com/watch?v=6O6x_m4zvFs

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OfflineAmanita virosa
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Re: Agar Issues [Re: FuckUp Eddy]
    #17314856 - 12/01/12 01:39 PM (12 years, 1 month ago)

Quote:

RogerRabbit said:
Isolate more if you want single sectors.  You might even start over from scratch, using minimum spores this time.  When you have many thousands of strains on the same dish, it can take a lot of transfers.

Quote:

Amanita virosa said:
The first plate looks like two incompatible strains to me. If they were compatible they would have formed dikaryotic hyphae. But it is really hard to see in that shot. Is there a clear line without Myc where they come together?  In other words are they repelling each other?  If so they are incompatible strains. You can always just put a small wedge from say five different plates in each jar to increase likelihood of compatibility




Just to clarify something, strains already are dikaryotic.  Hyphae are monokaryotic.  If compatible mononucleate hyphae(monokaryotic mycelium) meet, they exchange DNA and become polynucleate (dikaryotic).  You can't have dikaryotic hyphae as that would be like a two wheeled monocycle.
RR



When i first looked at the pict, I was thinking that he may have had two monokaryotic hyphae, which each came from a single spore, on one plate and that they may have not been appropriate mating strains. I guess this is unlikely if you shoot multispore into agar then sub it out cause it only takes two appropriate spores to form a dikaryon and he would have likely shot 1000s onto the plate. I was basing it on some work I did with rhizopus where you start two plus strains (or two minus strains) on opposite sides of an agar plate and where they meet, they will never touch always maintaining a 2 mm clear zone. I.e.like strains repel each other. I realize that the mating system in basidiomycetes is more complex than plus and minus (zygomycota) but could the same thing not happen?

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OfflineFuckUp Eddy
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Re: Agar Issues [Re: Amanita virosa]
    #17315088 - 12/01/12 02:38 PM (12 years, 1 month ago)

I think the one on the left is mold though...compromising the whole dish. Lots of my agar to brf (to proof) has turned out to be mold. In the beginning it looked like the culture on the left. Frustrating. Only three of my seven cultures to brf turned out to be mycellium.


--------------------
If you havent played with this then you are missin out! http://www.albinoblacksheep.com/flash/zoomquilt

Also good video!!! http://www.youtube.com/watch?v=6O6x_m4zvFs

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OfflineFuckUp Eddy
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Re: Agar Issues [Re: FuckUp Eddy]
    #17321843 - 12/02/12 04:42 PM (12 years, 1 month ago)

Is this mold? This is from a clone, 2nd transfer...It doesn't look right to me. Just too whispy. Why are parts of the outside of the circle like that?



--------------------
If you havent played with this then you are missin out! http://www.albinoblacksheep.com/flash/zoomquilt

Also good video!!! http://www.youtube.com/watch?v=6O6x_m4zvFs

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Offline00funG.I.00
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Re: Agar Issues [Re: FuckUp Eddy]
    #17322968 - 12/02/12 08:04 PM (12 years, 1 month ago)

looks like mycellium that has been influenced by fluctuating temps to me...or maybe directional to the heatsource... look under a scope to be certain...

Edited by 00funG.I.00 (12/02/12 08:07 PM)

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OfflineFuckUp Eddy
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Re: Agar Issues [Re: 00funG.I.00]
    #17323023 - 12/02/12 08:12 PM (12 years, 1 month ago)

Dammit I don't have one. You are exactly right though with the fluctuating temps.

It's really hard to make transfers from something like this. Where the hell would I?


--------------------
If you havent played with this then you are missin out! http://www.albinoblacksheep.com/flash/zoomquilt

Also good video!!! http://www.youtube.com/watch?v=6O6x_m4zvFs

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