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OfflineSoulButter
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Agar trouble :/
    #23537315 - 08/13/16 08:49 AM (7 years, 5 months ago)

no matter what i do with this agar it seems to always get contaminated and NEVER host the proper mycelium. Below i have a picture of some inner stipe tissue transferred to MEA. It appears to have significant bacterial contamination, and half a millimeter or so of a mycelial ring around the tissue sample. I submerged in the 10:1 diluted 3% H2O2 before transfer, as well as doing it inside a SAB and sterilizing utensils. I have yet to produce anything viable with agar and ive done many runs. this was the first attempt at cloning however.



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InvisibleSupalemonhaze
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Re: Agar trouble :/ [Re: SoulButter]
    #23537544 - 08/13/16 10:43 AM (7 years, 5 months ago)

Bacteria from clones is pretty damn common. I took 7 clones a few days ago and 5 of them went bacterial. As long as there is mycelium, you can always clean it up.

Sandwich tek that bitch and it will be clean as a whistle. Take a transfer of mycelium, put on new plate, flame scalpel, cut a square of agar from new plate, cover your transfer with it. Once it breaks the surface of the square on top of it, scrape some mycelium and transfer to another new plate. Bang, clean culture.


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Invisibleblindingleaf
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Re: Agar trouble :/ [Re: SoulButter]
    #23537547 - 08/13/16 10:45 AM (7 years, 5 months ago)

You will get better at cloning and working with inner tissue the more u do it.

There is no saving that plate.

How many times have you tried spores to agar?  What's the story there, same deal with bacteria or does it get mold or just nothing grows?

Spores can have competitors intermingled just like fruit bodies can, but you need to give us some more info about what u do and some pictures of other plates that have gone bad. 

Don't get discouraged


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A few thoughts on cultivation
MICROBIAL HUSBANDRY!!!!

The whole is greater than the sum of its parts


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Invisibleblindingleaf
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Re: Agar trouble :/ [Re: blindingleaf]
    #23537549 - 08/13/16 10:46 AM (7 years, 5 months ago)

Oh...the sandwich, That's true.  That's a lot of bacteria tho


--------------------
A few thoughts on cultivation
MICROBIAL HUSBANDRY!!!!

The whole is greater than the sum of its parts


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InvisibleSupalemonhaze
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Re: Agar trouble :/ [Re: blindingleaf]
    #23537552 - 08/13/16 10:47 AM (7 years, 5 months ago)

Oh and also, diluting 3% h202 is for cuts and bruises. Forgetting the fact that h202 is only ever used on cobweb mold, it's always used undiluted when you have the 3% one.


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InvisibleSupalemonhaze
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Re: Agar trouble :/ [Re: blindingleaf]
    #23537567 - 08/13/16 10:52 AM (7 years, 5 months ago)

Quote:

blindingleaf said:
Oh...the sandwich, That's true.  That's a lot of bacteria tho




Generally I give up with clones if mycelium won't even grow through the bacteria. If it manages to crawl through it, even if ever so slowly, I try sandwiching it. Sometimes I notice after sandwiching, the bacteria seems to effect the mycelium more, to the point where it won't even grow but IME, this happens less often given that the mycelium was able to recover through the bacteria from the original clone tissue though.


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Invisibleblindingleaf
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Re: Agar trouble :/ [Re: Supalemonhaze]
    #23537583 - 08/13/16 11:00 AM (7 years, 5 months ago)

Do u hot pour it or cut from another plate?  I've done the hot pour when I had spores that always carried bacteria but never tried it with clones.  Maybe u have to let it cool longer cause its live tissue?  Pasty does it a lot I think


--------------------
A few thoughts on cultivation
MICROBIAL HUSBANDRY!!!!

The whole is greater than the sum of its parts


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OfflineSoulButter
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Re: Agar trouble :/ [Re: blindingleaf]
    #23537589 - 08/13/16 11:02 AM (7 years, 5 months ago)

Quote:

blindingleaf said:

How many times have you tried spores to agar?  What's the story there, same deal with bacteria or does it get mold or just nothing grows?



Ive used homemade spore syringes that worked for WBS, but led to bacteria and cobweb on agar. Ive even used a syringe straight from sprworks that produced nothing but bacteria and cobweb. no mycelial development. Is it possible to cook the agar for too long, making it less hospitable for fungi? I thought the contam was because of my dusty old mold house, but now im having contam issues in a cleaner area. Its been different contam types here than at the old house which makes me think it would be my sterile technique letting in some native orgs during the making of the syringe or inoculation, but why then does good mycelium NEVER grow??

ive tried it probably less than 10 times with 5-10 jars each time. no luck. i am the dactylium master though haha


also, what should i surface sterilize with if not h2o2?


Edited by SoulButter (08/13/16 11:06 AM)


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OfflineNDStepp84
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Re: Agar trouble :/ [Re: SoulButter]
    #23537605 - 08/13/16 11:07 AM (7 years, 5 months ago)

Quote:

SoulButter said:
no matter what i do with this agar it seems to always get contaminated and NEVER host the proper mycelium. Below i have a picture of some inner stipe tissue transferred to MEA. It appears to have significant bacterial contamination, and half a millimeter or so of a mycelial ring around the tissue sample. I submerged in the 10:1 diluted 3% H2O2 before transfer, as well as doing it inside a SAB and sterilizing utensils. I have yet to produce anything viable with agar and ive done many runs. this was the first attempt at cloning however.





You have a decent SAB with large armholes? Also make sure you are splitting the stipe by hand instead of cutting in half, cutting drags nasties from the outside to the center. Bacteria usually spreads by direct contact hitching rides. Flame the blade before taking sample. As said don't give up or get discouraged, you'll get it :goodluck:


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

"I am free, no matter what rules surround me. If I find them tolerable, I tolerate them; if I find them too obnoxious, I break them. I am free because I know that I alone am morally responsible for everything I do.
-Robert A. Heinlein 

:takingnotes: Links and teks:takingnotes:
ND's grow log and discussion
Plant thread


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InvisibleSupalemonhaze
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Re: Agar trouble :/ [Re: blindingleaf] * 1
    #23537618 - 08/13/16 11:11 AM (7 years, 5 months ago)

Probably because the contams are growing too much by the time the spores are getting close to germinating. The few times I used spore solutions, they always took way longer than a print to germinate. Prints are also cleaner while syringes are always said to have at least some bacteria in them. If you are using agar, better to invest in prints than syringes.

Quote:

blindingleaf said:
Do u hot pour it or cut from another plate?  I've done the hot pour when I had spores that always carried bacteria but never tried it with clones.  Maybe u have to let it cool longer cause its live tissue?  Pasty does it a lot I think




I never hot poured contams. I do warm pours for spores though to give them a push. This is something that OP can most definitely try.

I usually cut the square from the same plate I transferred to. I like to cut the square close to where I put my transfer so all I have to do is flip the square on top of it.

Oh, and something to add to ND's post. I notice that hollow fruits often have bacteria on the inside as well. Even those fruits which are not entirely hollow but have that fuzzy mycelium like tissue on the inside. I suspect this is why most of my clones have bacteria. I have been trying to take clones from caps to counter this but I haven't done it enough to see if it's any better than stems in terms of contamination so far.


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OfflineSoulButter
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Re: Agar trouble :/ [Re: Supalemonhaze]
    #23537647 - 08/13/16 11:25 AM (7 years, 5 months ago)

Quote:

Supalemonhaze said:
Prints are also cleaner while syringes are always said to have at least some bacteria in them. If you are using agar, better to invest in prints than syringes.




No shit! what about homemade prints? Is bacteria unable to persist on aluminum? I always assumed prints would be more ridden with contam because they are less protected from the world than a closed up syringe. Thanks for the tip.


Edited by SoulButter (08/13/16 11:33 AM)


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InvisibleSupalemonhaze
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Re: Agar trouble :/ [Re: SoulButter]
    #23537655 - 08/13/16 11:27 AM (7 years, 5 months ago)

Bacteria on prints are less common than bacteria in syringes. Homemade prints would be more prone to molds since bacteria needs moisture to survive. A good print would be bone dry, not a good place for bacteria to grow/survive.

I would take a homemade print over a vendor syringe any day of the week but my title probably gives that away.


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OfflineSoulButter
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Re: Agar trouble :/ [Re: Supalemonhaze]
    #23537664 - 08/13/16 11:30 AM (7 years, 5 months ago)

Well i trust that you know better than I do! now just to wait for something to print.....


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InvisibleSupalemonhaze
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Re: Agar trouble :/ [Re: SoulButter]
    #23537689 - 08/13/16 11:35 AM (7 years, 5 months ago)

You should definitely try what BL said in the meantime. Do a warm pour over your spore inoculated plate, it can stunt contaminants enough to give a window of opportunity for your mycelium to grow. That and warm pours generally help a lot when it comes to germinating spores faster. Had this vial which wouldn't germinate at all on agar, until I warm poured on it.


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InvisiblebodhisattaMDiscordReddit
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Re: Agar trouble :/ [Re: Supalemonhaze]
    #23537702 - 08/13/16 11:39 AM (7 years, 5 months ago)

I've only ever had trouble with trade prints and syringes. Vendor syringe has never gave me much of any trouble even if it wasn't perfectly clean.


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InvisibleSupalemonhaze
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Re: Agar trouble :/ [Re: bodhisatta]
    #23537717 - 08/13/16 11:43 AM (7 years, 5 months ago)

I have a couple of prints that never germinated that I got from the same guy but apart from that, every print I recieved was great. Much better than the 2 vendor vials I bought. Fucking spore solutions. :melt:


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OfflineSoulButter
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Re: Agar trouble :/ *DELETED* [Re: SoulButter]
    #23537729 - 08/13/16 11:46 AM (7 years, 5 months ago)

would something like this work?



Edited by SoulButter (08/14/16 11:16 AM)


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InvisiblebodhisattaMDiscordReddit
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Re: Agar trouble :/ [Re: SoulButter]
    #23537757 - 08/13/16 11:55 AM (7 years, 5 months ago)

I would act like you never saw that garbage info.

That's not even middle school level. That's just plain wrong


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Invisibleblindingleaf
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Re: Agar trouble :/ [Re: bodhisatta]
    #23537775 - 08/13/16 11:59 AM (7 years, 5 months ago)

:whathesaid: 

Everyone knows that technique only works with a microwave anyway.


--------------------
A few thoughts on cultivation
MICROBIAL HUSBANDRY!!!!

The whole is greater than the sum of its parts


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InvisibleSupalemonhaze
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Re: Agar trouble :/ [Re: blindingleaf]
    #23537975 - 08/13/16 01:19 PM (7 years, 5 months ago)

I hate it when someone says something silly and then deletes the comment. Makes me curious for one and if everyone did that newbies can't learn.


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InvisiblebodhisattaMDiscordReddit
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Re: Agar trouble :/ [Re: Supalemonhaze]
    #23537996 - 08/13/16 01:25 PM (7 years, 5 months ago)

Something about using hydrogen peroxide to get a clean clone lol


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InvisibleSupalemonhaze
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Re: Agar trouble :/ [Re: bodhisatta]
    #23538005 - 08/13/16 01:28 PM (7 years, 5 months ago)

Oh. That's not too bad. Here I was thinking it was gonna be another one of those "mushroom cap for a lid on an LC" or some shit. :rofl:


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OfflineSoulButter
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Re: Agar trouble :/ [Re: Supalemonhaze]
    #23540526 - 08/14/16 11:18 AM (7 years, 5 months ago)

my bad, i edited it back. also helped a friend last night and took back some agar inoculated with b+ and golden teacher spore syringe. 2 of each, with about a ml left of golden teacher. used pretty good technique, fingers crossed.


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InvisiblePastywhyteMDiscord
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Re: Agar trouble :/ [Re: Supalemonhaze]
    #23540592 - 08/14/16 11:46 AM (7 years, 5 months ago)

Quote:

Supalemonhaze said:
Oh. That's not too bad. Here I was thinking it was gonna be another one of those "mushroom cap for a lid on an LC" or some shit. :rofl:




Damn that one just disappeared didn't it, I want an update so we know how it turned out :rolleyes: :lol:


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InvisibleSupalemonhaze
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Re: Agar trouble :/ [Re: Pastywhyte]
    #23542845 - 08/15/16 03:02 AM (7 years, 5 months ago)

It did? Damn. Here I was thinking he was on the verge of a breakthrough. :lol:


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Offlinesandy_vag
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Re: Agar trouble :/ [Re: Supalemonhaze]
    #23543059 - 08/15/16 06:51 AM (7 years, 5 months ago)

Non standard agar work will be hit or miss, lots of miss. Are you using a flowhood? Real agar plates poured in a flowhood are very reliable, anything else is just a hope and a prayer. Don't dip a clone in h202. Just pluck, take to hood, rip down the stem to expose the inside, cut a little slice by going against the grain for 2 cuts about 3mm apart and lift out between the cuts to get a easy slice and lay it on your properly prepared agar and that's it.


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InvisiblePastywhyteMDiscord
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Re: Agar trouble :/ [Re: sandy_vag]
    #23543063 - 08/15/16 06:54 AM (7 years, 5 months ago)

Quote:

sandy_vag said:
Non standard agar work will be hit or miss, lots of miss. Are you using a flowhood? Real agar plates poured in a flowhood are very reliable, anything else is just a hope and a prayer.




I do just fine with no pour and a SAB :shrug:


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Offlinesandy_vag
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Re: Agar trouble :/ [Re: Pastywhyte]
    #23543072 - 08/15/16 07:03 AM (7 years, 5 months ago)

But ur special :crazy2:

Of course it can work, but if it doesn't then there's your problem. I had fairly good luck with no pours in tupperwares back in the day.


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