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Are these clean cultures?
    #27255647 -

So I've done about 30-40 plates now in total, but always ended up with contamination when innoculating rye. Havent had any success yet, although spore syringe worked really well for me I'd really like to get a hang of agar. So this time I want to really make sure before innoculating grain that it looks good on agar. I ordered new spore print and switched to MEA from PDA, recipe I'm using is 10g malt extract and 10g agar per 500ml.

They don't look as fluffy as before but still no rhyzo anywhere to be seen, yet they don't look contaminated either, at least to my untrained eye. Till this day I've only gotten rhyzo growth once. So right now my plan is to make less nutrient MEA, about 5g ME per 500ml to see what happens then, and maybe just to be safe hot pour onto that and take one final transfer that I'll use to inoculate grain, but that would be 6-7th transfer, I'm wondering if it'll still be enough strains to get at least one good performer and get a clone. Not too concerned about rhyzo just to be sure there aren't any contaminations.

So any input on how these look and how to proceed?





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Re: Are these clean cultures? [Re: otsilla]
    #27255679 -

Those look nice and clean to me. I see some odd dots in the underside pics but I'm assuming those are just some sort of reflection or something? They are at 12:00 on the 4th pic and 9:00 on the 3rd pic. Maybe a divet in the plastic plate from when it was molded by the factory?

Don't worry about fluffy vs rhizo off a spore print. In my experience, spore inoculation usually produced fluffy mycelium - likely because you have so many different spore germinations all climbing on top of one another. I wouldn't worry about changing your agar formula either. I tried both regular and half strength and noticed no difference, personally.

You don't need to do so many transfers unless you are really having trouble getting away from contams. If your plate that you inoculate with spores is clean, I'd put that to grain. Gives you the widest genetics possible and best chances of catching a good one to clone from. If my spore plate isn't clean, I'll take as many samples of as many individual mycelium growths as I can, and transfer each of those to a different clean plate, so I'll go from one spore plate, to like 5-10 T1 plates. If those are clean, I put each one to grain separately and grow it out to see how it does in hopes I get a good one. You can also use the POD tek to find good genetics even faster and with less effort.

I can't diagnose your grain issue, but if it's anything like mine, I have found that over-wet grains are usually the contamination cause, assuming you have your sterile SAB procedure down and you are PCing your grains long enough (90-120 mins), and assuming you are not leaving your grain jars sitting for more than 12 hours before knocking them up. I also find it best not to use more than 1/4 plate of agar into the grain jar.... I've found in my own experience that having a ton of agar in the jar invites trouble.


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* How eliminate condensation from no-pour agar plates
* Build a steam pasteurizer
* Learn about trich


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Re: Are these clean cultures? [Re: Hindsight] * 1
    #27255684 -

Hindsight said:
Those look nice and clean to me. I see some odd dots in the underside pics but I'm assuming those are just some sort of reflection or something? They are at 12:00 on the 4th pic and 9:00 on the 3rd pic. Maybe a divet in the plastic plate from when it was molded by the factory?

Don't worry about fluffy vs rhizo off a spore print. In my experience, spore inoculation usually produced fluffy mycelium - likely because you have so many different spore germinations all climbing on top of one another. I wouldn't worry about changing your agar formula either. I tried both regular and half strength and noticed no difference, personally.

You don't need to do so many transfers unless you are really having trouble getting away from contams. If your plate that you inoculate with spores is clean, I'd put that to grain. Gives you the widest genetics possible and best chances of catching a good one to clone from. If my spore plate isn't clean, I'll take as many samples of as many individual mycelium growths as I can, and transfer each of those to a different clean plate, so I'll go from one spore plate, to like 5-10 T1 plates. If those are clean, I put each one to grain separately and grow it out to see how it does in hopes I get a good one. You can also use the POD tek to find good genetics even faster and with less effort.

I can't diagnose your grain issue, but if it's anything like mine, I have found that over-wet grains are usually the contamination cause, assuming you have your sterile SAB procedure down and you are PCing your grains long enough (90-120 mins), and assuming you are not leaving your grain jars sitting for more than 12 hours before knocking them up. I also find it best not to use more than 1/4 plate of agar into the grain jar.... I've found in my own experience that having a ton of agar in the jar invites trouble.


This is all good advice. Especially the part about too much agar in the grain jar. If I put a little sliver in the grain jar it might be slow, but contams are way down. Every single time I try to put more agar in the jar it contams out.

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Re: Are these clean cultures? [Re: Blazer Man]
    #27255782 -

I don't like the uneven growth. I transfer until I get even growth before using. I have always had problems using cultures that split like that.


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My no pour Petri TEK - https://www.shroomery.org/forums/showflat.php/Number/27252059/page/1

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Re: Are these clean cultures? [Re: Professor X]
    #27255865 -

No not clean. The wedge peices missing make me think bacteria. Take another transfer until you get round even growth

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Re: Are these clean cultures? [Re: FrugalFungi]
    #27255896 -

I'm really curious why everyone always says uneven growth is bacteria. Who started this trend and what's the logic? I've seen plenty of people on here running similar agar plates with no issues though. Maybe it just comes down to selection bias and people tend to not run agar plates unless they're immaculate so they have less experience with odd growth patterns.

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Re: Are these clean cultures? [Re: AntsInYourPants]
    #27255897 -

The mycelium is avoiding something on that plate. Period. They aren't picky about the agar they won't colonize something that is already colonized.


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My no pour Petri TEK - https://www.shroomery.org/forums/showflat.php/Number/27252059/page/1

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Re: Are these clean cultures? [Re: Professor X] * 2
    #27255900 -

That is just genetic sectoring. I don't see any bacteria on those plates. Some mycelium will act funny on plates if you transfer a large section with alot of genetic diversity.
Amazon Cross with lots of diversity



Large Wedge Transfer can also cause this.


Perfect uniform growth culture.


Edited by Sockadin (03/16/21 05:16 PM)

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Re: Are these clean cultures? [Re: Sockadin]
    #27255904 -

In his pics the top plate has a ring around it when he shines the light through. The other has lighter growth in the gaps...


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My no pour Petri TEK - https://www.shroomery.org/forums/showflat.php/Number/27252059/page/1

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Re: Are these clean cultures? [Re: Professor X] * 1
    #27255915 -

In the last picture of the OP I count 31 sectors that are all viable genetic variations from the others. This is sectoring out a plate. So he can basically take 31 different transfers and have 31 plates that may have different pheno characteristics, or they can all look the same, but some may have heavier spore deposits or lighter spore deposits or some sectors might not fruit at all. Im just saying that I do not see mold or bacteria in those plates and the reason mycelium sectors out like that is not because it is running from a contamination or competitor in this case.

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Re: Are these clean cultures? [Re: Sockadin]
    #27255939 -

Sockadin said:
That is just genetic sectoring. I don't see any bacteria on those plates. Some mycelium will act funny on plates if you transfer a large section with alot of genetic diversity.



Yes that is my understanding as well.

OP, most mycelium growth will not be compatible with other mycelium growth on the same plate and so when they reach eachother, there will be a line that separates them, unless they are compatible and can fuse. I read all the technical terms for this phenomenon (I believe there is a scientific term for that line of segmentation, and also the fusion) but have forgotten them both. The reason it's wide is that most of the visible mycelium has growth out in front of it that you can't see and when those touch, that's where they stop.

I try to get the most genetics I can into a grain jar and then a tub. No point isolating things that you don't know will perform. The fewer transfers the better.


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* How eliminate condensation from no-pour agar plates
* Build a steam pasteurizer
* Learn about trich


Edited by Hindsight (03/16/21 05:35 PM)

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Re: Are these clean cultures? [Re: Hindsight]
    #27255968 -

I never thought about it that way. Geez I’ve been getting way ahead of myself


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Re: Are these clean cultures? [Re: NOS4A2]
    #27256060 -

I just keep gping until it looks nice. It's much better imo to do a few more transfers than throw out moldy/bacterial jars. Even tho it might be clean, I like to be positive. If I have a question, it's not good enough for grains that's just how I do it.

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Re: Are these clean cultures? [Re: Blazer Man]
    #27257578 -

Yes those reflections are the rivets from the mold.

And yeah, not too anal about rhyzo growth, just I'd be more confident to rule out possible contams. Getting the most genetic material to fruit and then clone seems the best option to me too, just want to be really sure it's clean before moving to spawn due to the previous failures.

But for about too wet grains, I've noticed the last times I have failed they started out nicely but pooled some water on the bottom after a while and thats when they stalled and started to have contam issues, so my problems may just lay there.

So there are a lot of mixed answers here as always, I've prepared 5 grain jars now which I dried more carefully, I guess I'll knock them up once again and see how it works out, hope for the best, but I think I'll also prepare some lighter MEA and make some transfers to there and see how it plays out just for the plan B and report back how it turns out.

Thank you all for your input.

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Re: Are these clean cultures? [Re: otsilla]
    #27257637 -

I wouldn't put any of that to grain. Im fairly sure there is something intertwined in that colony besides cube myc

Might work fine, but too much of a risk imo

What i would do in this case is find some sectors to transfer that have the least cloudiness on the leading edge. Id do several more transfers at least

I do have a strong bias for organized growth though. But even more so, i dont like when the leading edge (and edges between sectors) look so drastically different than the rest of the colony


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C10's Agar Guide + Tips and Tricks | c10's Flow Hood Build Guide


"Partial knowledge is more triumphant than complete knowledge; it takes things to be simpler than they are, and so makes its theory more popular and convincing."

"Convictions are more dangerous enemies of truth than lies"
― Friedrich Nietzsche

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Re: Are these clean cultures? [Re: c10h12n2o]
    #27259060 -

Too bad, but as I already made and sterilized 5 grain jars I'm gonna put them to grain, otherwise I'd waste the grain anyways. I will also take transfers to continue from there, as I'm used to grain failing.

One thing I cant wrap my head around is these interwined contams, how to get rid of them? I've never tried antibacterial agar but as I've read not many recommend it, and since I'm in europe I can't find antibacterial fish medicine that has antibiotics. I've also tried hot pour, transfer and put to grain, but still got contam problems.

Since my agar plates sit for weeks and no contams found mostly(few here and there). And I've had uncolonized grain jars sit for weeks with no visible contam so I think I have my sterile tek down. I ordered new spores thought that maybe that was the issue, my previously sloppy taken spores, but as the results seem to be the same, I don't get where do these hidden contams come and how to get rid of them. So my only thought was maybe too wet grains, not much left.

Anyways here are boths ancestors for funs:

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Re: Are these clean cultures? [Re: otsilla]
    #27259083 -

Antibacterial agar wouldn't fix mold/trich/yeast contamination issues. And in fact, I have experimented with fish antibiotics mixed in agar and it didn't work - the spores wouldn't germinate and while the antibiotics slowed the bacteria down, it didn't stop them. They still grew. The only thing that worked for me when working with a dirty source genetics was germinating on BRF cakes in a jar. Works EVERY time for me. You said "spore print" so I assume you are not working with a syringe, but if you were, BRF cakes work great. Every dirty syringe I use to knock up a BRF cake works perfectly, and I even end up successfully fruiting the BRF cake in the end (though that's not my goal - my goal with the BRF cake is to get clean mycelium growing that I can then take a little sample of to put on agar and grow out).

And for the record, I'm not saying I personally think your plates are contaminated. Just answering your hypothetical question about how to deal with bacterial contamination if and when you have it.


--------------------
* How eliminate condensation from no-pour agar plates
* Build a steam pasteurizer
* Learn about trich


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Re: Are these clean cultures? [Re: Hindsight]
    #27259193 -

I think that the point with antibiotics is to slow down bacterial growth to make the transfer from healthy myc ASAP not to completely eliminate it.

Never tried BRF cakes before, seems too much of an hassle, but if this also fails I just might try it to get some fruits to clone.

Anyways appreciate your input, guess we'll see whether something comes out of it or not.

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Re: Are these clean cultures? [Re: otsilla]
    #27259271 -

U don't need anti bacterial agar. Work on your clean work,by transferring through antibacterial agar you cultivate some mold/bacteria that is even stronger and antibacteria resistance ! It is used for some special uses.
In the past I transferred some varieties up to the point where I got to some monocultures on agar, I was so happy and excited to grow them but one ended up being a very slimy genetic one was a short fruiter and on was gonna take so long to fruit another one I wasn't content with the potency ! So.. I started from spores again, germinated on agar and that time didn't go more that T2 just to make sure I'm having a clean and diverse culture and now I can clone from fruits I like and have the desired monoculture.

Edited by windyroom (03/18/21 05:11 PM)

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Re: Are these clean cultures? [Re: otsilla] * 1
    #27259306 -

otsilla said:
Never tried BRF cakes before, seems too much of an hassle, but if this also fails I just might try it to get some fruits to clone.



If you have a contaminated syringe, it is literally the only way you'll get mycelium out of it. At least, that's been my experience. BRF cakes are easier than agar. Mix up 2 parts vermiculite, 1 part water, and 1 part brown rice flour, spoon it into a couple pint jars until it's a couple inches deep, put a lid on it with a filter and PC it for 90 mins. Cool, then inoculate. About a week later you'll have some nice mycelium growing on the top that you can pull from to inoculate as many agar dishes as you want.


--------------------
* How eliminate condensation from no-pour agar plates
* Build a steam pasteurizer
* Learn about trich


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