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Mushrooms, Mycology and Psychedelics > Mushroom Cultivation
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Re: Agar culture question [Re: mushpunx]
    #25808890 -

Nah I poured, and I’m glad I did bc I made a lot of mistakes and am already much better. The syringes cumming all over my plate was the main issue, but also my pour technique could use work.

I also had mad bubbles (agar was shaken, not swirled before pour which I know better about now lol) and issues with condensation. Which btw, anyone know how to correct that/prevent for future reference?

The recipe was premade MEA from amazon. When I sterilized in pc, I noticed by smell and color the agar leaked into the water in the cooker during the cycle. Could that have thrown it off?


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“Time flies. It’s up to you to be the navigator.”

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Re: Agar culture question [Re: Funnguiy]
    #25809267 -

You can't always prevent leaking, a little bit is OK. I've used liquor bottles, I use Erlenmyer flasks with threaded caps right now but the media bottles meant for agar would be the best to go with. The best way to reduce/prevent it I know is to raise the water level to the level of the agar in the bottle, stops boil over.

If you are getting condensation you are pouring too hot. If you have an infrared thermometer it can help you with consistency, but I do it by feel. When the agar is cool to the point it might start to solidify any minute, that is when I pour.
Hot water baths are another way to get it to temp, Bodhisatta mentions a way to do that in his agar teks. The lab water baths are super nice but expensive - with those you can just pull sterilized (solidified) agar out of the fridge and bring it right up to pouring temp!

If you do still get condensation on top plates, set a mug (I use a small mason jar with lid) of hot water on top of the plate as they cool. But if you pour at a low temp, bone dry- since I started doing that I haven't had a drop of condensation again.

Did your plates contaminate before inoculation?


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Amateur Mycologists United
AMU Q&A

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Re: Agar culture question [Re: mushpunx]
    #25809543 -

Not all plates contamed, but most did. I tried the water bath, but the agar wasn’t even close to budging so I nuked it in the microwave, about 2-3ish mins, but had to loosen the cap so you know it wouldn’t explode. But that could’ve been a potential contam problem too. Or a mix of all this stuff.

What I don’t get is this, how did I get both the too hot agar and too cool agar issues with my plates? I nuked and shook the crap out of it but it still poured a lot of clumps:/ but then I get condensation on more than half my plates? What gives?


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“Time flies. It’s up to you to be the navigator.”

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Re: Agar culture question [Re: Funnguiy]
    #25809769 -

when microwaving agar, some of it will boil while some of it has yet to reach the melting temp. It's best to nuke it until some of it melts, stop and let it rest, give it a short blast until more melts, stop and let it rest, blast again..... and carry on until it's all melted. at this point it'll be way too hot to pour and you'll have to wait for it to cool before pouring to avoid condensation.

I'd not recommend loosening the lid to nuke. some people do it but it makes me way too nervous. I put an sfd on my captain morgan handle lid just so i could nuke it without loosening the lid. Watch out though, nuking agar can get rather explosive. I've had it literally explode in my hand and shoot burning hot agar all over me and the entire kitchen (ceiling included). that was a painful mess.


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Masters and slaves hunting clones        How not to isolate (it worked, but)


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Re: Agar culture question [Re: verum subsequentis]
    #25810335 -

Yes, the stuff is freakin atomic. They weren’t lying when they said if you take your eye off for even a second it will boil over fast in the microwave. Shit made me super nervous too, hell I risked contams by loosening the lid just to be sure it wouldn’t blow  :psychsplit:


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“Time flies. It’s up to you to be the navigator.”

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Re: Agar culture question [Re: Funnguiy]
    #25813341 -

Here’s an update for other people to learn from:

As stated previously, most of my plates had a contamination, surprisingly not too bad on the nocced ones, but almost every other agar plate had some spot on it. After I made the transfers, I isolated and cut out the contam spot with like idk 1/4” or so perimeter around it. I used xacto over an alc lamp, and once the contam was cut out with that, it was quickly discarded into a cup with iso in it.

That was done on 2/12, and my sterile technique was MUCH better. I got it done fast and relatively smoothly. Now it is 2/14 (happy Valentine’s Day!) and there is no sign of any contam on the plates yet, at least that I can tell, just unsightly holes from where I cut lol. And my transfers are slowly taking hold of their new agar plates. I guess the real test will be growing cultures but so far it looks good and clean.

Follow your instincts! :rockon:

Also, thank you all for your inputs and advice! Helps a lot!


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“Time flies. It’s up to you to be the navigator.”

Edited by Funnguiy (02/14/19 09:41 PM)

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Re: Agar culture question [Re: Funnguiy]
    #25813426 -

congrats and good luck man. what kind of contams did you cut out? if you can't name them, would you describe them for me


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Masters and slaves hunting clones        How not to isolate (it worked, but)


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Re: Agar culture question [Re: verum subsequentis]
    #25814886 -

Yeah I actually was too lazy to throw out the cup so I have a picture of it. The one shown was the only one still in tact after the iso soak, but all the spots I cut more or less looked like that. Small yellow/orange dots, usually just one per plate, however one had two spots, and I removed both, seems ok tho.

The only one that looked different from the rest was grey and a little smaller than dime sized on the plate. For that one I cut it out in a big wedge, and the plate seems ok.


(Ignore the black areas, that was just from the tool I used to poke it to pick up, not a rogue contam)




Also, the mycelium I have going is growing pretty well, but it seems a little too fuzzy and whispy, not as ropey and solid. My temps are around 70-76F and they are stored in a small 6qt tote in mostly dark. Is it fine or do you think it looks off too. I’m judging by what it looked liked in a brf jar I did a while ago. Here’s what a couple of the original ms syringe plates look like:





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“Time flies. It’s up to you to be the navigator.”

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Re: Agar culture question [Re: Funnguiy]
    #25815191 -

looks like you cut out a little bacteria. Who knows if it's all over the plate and just hasn't shown itself yet. I'd guess it'd have shown by now but the fact that you said there was a ton of solution rolling around the plate makes me nervous. or was that the first plate? damn, sometimes i forget what thread i'm in.

any who, I can't tell you much about the first culture because the reflection makes it almost impossible to see. Doesn't look great but I'd hesitate to say without a better photo. the second plate looks potentially fine but may have bacteria spots all over it. i can't tell. I'd take some tiny transfers from the leading edge of the culture just above the light reflection.


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Masters and slaves hunting clones        How not to isolate (it worked, but)


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Re: Agar culture question [Re: verum subsequentis]
    #25815277 -

Yeah unfortunately without the flash you really couldn’t see anything. The solution was an issue for about 3-4 plates which I didn’t try to fix as they’re definitely beyond repair. The others just had those spots. Yeah I was wondering too if the contam is just microscopic and waiting to grow but it’s been a few days now so.

Those two plates pictured both had transfers taken pretty early you can see from the black singe mark the xacto made when I cut out the culture. It just looks weak and thin, but still keeps growing, I’m not sure if that’s just how they grow on agar or not. I’ll try to get a better pic.


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“Time flies. It’s up to you to be the navigator.”

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Re: Agar culture question [Re: Funnguiy]
    #25815311 -


(If you look to the right you can see the hole I cut out in this plate where a contam was)


(You can see the fuzzy better in this picture on the left culture growing out, it’s pretty fuzzy lol)


My transfers like this look more promising, they all are growing like this one more or less.

P.S. the strains I’m using are:
C- Cubensis Chitwan
PE- Penis Envy
Pan- Panaeolus Cyan Hawaii


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“Time flies. It’s up to you to be the navigator.”

Edited by Funnguiy (02/15/19 08:40 PM)

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Re: Agar culture question [Re: Funnguiy]
    #25821356 -

Quick update: most of the plates I cut contam out have sprouted new contams and with a vengeance. Oh well, I’m glad I tried. A couple still seem ok tho, but at this point I think I’m just gonna make new plates, I’m running out anyway:/


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“Time flies. It’s up to you to be the navigator.”

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Re: Agar culture question [Re: verum subsequentis]
    #25821389 -

i wise man once said,


verum subsequentis said:
theoretically sure. Chances of success. .00000000000000006%



:douchewink:


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Masters and slaves hunting clones        How not to isolate (it worked, but)


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Re: Agar culture question [Re: verum subsequentis]
    #25821437 -

Lmao I know. I just figured if they would go in the trash anyways what’s the harm in trying it out? Was good practice working with the plates too, so to me not a failure!


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“Time flies. It’s up to you to be the navigator.”

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Re: Agar culture question [Re: Funnguiy]
    #25821489 -

Absolutely! "The master never fails, he only learns to succeed." That's the spirit, mate.


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Masters and slaves hunting clones        How not to isolate (it worked, but)


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Re: Agar culture question [Re: verum subsequentis]
    #25821638 -

Yes. You can’t expect everything to go perfectly, besides, what’s the fun in that! But I think I have my overall agar technique down better bc of this so next batch should be lit, hopefully for clonesssss.


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“Time flies. It’s up to you to be the navigator.”

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Re: Agar culture question [Re: Funnguiy]
    #25821689 -

you got it man.


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Masters and slaves hunting clones        How not to isolate (it worked, but)


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Re: Agar culture question [Re: verum subsequentis]
    #25828535 -

Instead of making a new thread I’m gonna post a quick question I have here. I have rye jars sterilized and ready to rock, but my agar plates are sloooowwwww. I still only have some dime-quarter sized cultures growing atm. I need to start the process and start moving forward, but I’m worried taking agar partially uncolonized will jeapordize my rye jars. Is this a big issue? Should I just try to keep my pieces all white and avoid plain agar pieces? All the plates are clean as far as I can tell. I just don’t have a freaking month to wait for them to grow on the plates unfortunatelyyyy.

If it’s that big a risk, my plan b would be use what I have left in the ms syringe, which id REALLY rather not, specially considering all this work I did to clean up the culture first. Any thoughts?


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“Time flies. It’s up to you to be the navigator.”

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Re: Agar culture question [Re: Funnguiy]
    #25828915 -

You can use a culture that size, although I usually wait until it colonizes about half the plate, a little larger than a quarter, I guess. Using more inoculant (your agar in this case) just means that your jars will finish faster and have a head start on any contaminants which they may have to compete against (since we do not sterilize 100% completely). It really shouldn't take more than a few days more to expand to that size, not a month, at least. If it's taking that long, there might be something else up, or it's just a very slow culture :shrug:


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To avoid all evil, to cultivate good, and to cleanse one's mind - this is the teaching of the Buddhas

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Re: Agar culture question [Re: juniperus]
    #25829010 -

It might be genes, or maybe it’s just that slow and I’m impatient. Idk, but thanks for the reply. I’ll probably cut a grid ish shape into the circle and get a few pieces for each jar either tonight or tomorrow. Hopefully they grow a little faster on the grain, lol.


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“Time flies. It’s up to you to be the navigator.”

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Shop: North Spore Injection Grain Bag   Original Sensible Seeds Bulk Cannabis Seeds   Sporeworks.EU Spores for European Microscopy   Mushroom-Hut Liquid Cultures   MagicBag.co All-In-One Bags That Don't Suck   Myyco.com Isolated Cubensis Liquid Culture For Sale

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