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Offlinesinubrius
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AGAR | Please help me identify what's on my plates!
    #26733904 - 06/10/20 09:27 AM (3 years, 8 months ago)

Hi there,

Extremely new to agar and have been trying to get a clean culture out of some spore swabs I bought but have yet to do so (by my reckoning)

I got all excited when I saw these plates and did some transfers, but once opening one of them I realised it doesn't really smell mushroomy, and it definitely doesn't seem rhizo, at least to my untrained eye.

Is this tomentose mycelium? Or is it white mold?

All help greatly appreciated.



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InvisibleLadysKnight
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Re: AGAR | Please help me identify what's on my plates! [Re: sinubrius]
    #26733916 - 06/10/20 09:36 AM (3 years, 8 months ago)

Healthy mold.


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Offlinesinubrius
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Re: AGAR | Please help me identify what's on my plates! [Re: LadysKnight]
    #26733927 - 06/10/20 09:41 AM (3 years, 8 months ago)

Any advice?

I'm using my SAB, I'm flame heating my scalpel, I'm holding the plates upside down while swabbing, sterilizing my space, I'm pressure cooking the agar, I'm blasting my room with disinfectant and also myself...

I just can't seem to get anything but mold out of these swabs. Is there a better method than just swabbing? I've only got two of these things left and I really don't want to waste them, I've used two already over probably about 15+ plates and this is the first thing I've gotten that even vaguely resembles healthy mycelium to me.

I swear to christ up and down that I've put endless hours of research into this but I'm at my wit's end. Agar is not as easy as everyone says it is, but I've no mind to quit.


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InvisibleAntigov
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Re: AGAR | Please help me identify what's on my plates! [Re: sinubrius]
    #26734027 - 06/10/20 10:31 AM (3 years, 8 months ago)

It’s moldy but don’t give up, you’re on the right track, keep on keeping on, success will come.


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


Edited by Antigov (06/10/20 10:32 AM)


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Offlineredhandmat
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Re: AGAR | Please help me identify what's on my plates! [Re: sinubrius]
    #26734056 - 06/10/20 10:46 AM (3 years, 8 months ago)

Quote:

sinubrius said:
Any advice?

I'm using my SAB, I'm flame heating my scalpel, I'm holding the plates upside down while swabbing, sterilizing my space, I'm pressure cooking the agar, I'm blasting my room with disinfectant and also myself...

I just can't seem to get anything but mold out of these swabs. Is there a better method than just swabbing? I've only got two of these things left and I really don't want to waste them, I've used two already over probably about 15+ plates and this is the first thing I've gotten that even vaguely resembles healthy mycelium to me.

I swear to christ up and down that I've put endless hours of research into this but I'm at my wit's end. Agar is not as easy as everyone says it is, but I've no mind to quit.




You could always try to do no pour agar, decreasing vectors of contamination. Either by PCing small pp5 containers or glass petri dish. Add an additional 5 minute to your PC time. You could also use an inoculation loop that you flame sterilize instead of using cotton swabs.


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Offlinesinubrius
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Re: AGAR | Please help me identify what's on my plates! [Re: redhandmat]
    #26734242 - 06/10/20 12:10 PM (3 years, 8 months ago)

Thanks for your reply and the other replies in the thread!

I am actually using pasty plates tek, I've just got a bunch of different jars and stuff too, including a bunch of pp5 containers and some squatty jars, and also some old PF Tek jars I repurposed after dedicated sterilization.

This is an MEA mix, 500ml, 10g/10g ME/Agar

I've also made some grain boil water agar (yes, I transferred this mold to some grain jars :/ ) that I'll be using too - 500ml undiluted to 10g agar.

My agar is clean from additional contam follwing my transfer of this mold to other plates, but I can never get any mycelium growth. I've thought about using antibiotics in the agar to try and get an initial clean culture, but I can't find anything available in Australia that would work specifically - it's all ambiguous and I can't be sure - any confirmed info here would be greatly appreciated.

I just feel like I'm missing something, and I don't wanna consider the possibility that maybe these spore swabs are bunk.. :/


EDIT**
Innoc. loop is my next test from a scalpel - do I literally just scrape the wire against the swab and then against the agar? Those parts have always been unclear to me. I know about the Z pattern and such, just innoc. loops confuse me.

The swabs themselves came from a vendor, so I'm trying to get something to grow in the first place, so I can then start cloning and isolating fruits and mucking around with that.


Edited by sinubrius (06/10/20 12:16 PM)


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Offlineredhandmat
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Re: AGAR | Please help me identify what's on my plates! [Re: sinubrius]
    #26734297 - 06/10/20 12:32 PM (3 years, 8 months ago)

Quote:

sinubrius said:
Thanks for your reply and the other replies in the thread!

I am actually using pasty plates tek, I've just got a bunch of different jars and stuff too, including a bunch of pp5 containers and some squatty jars, and also some old PF Tek jars I repurposed after dedicated sterilization.

This is an MEA mix, 500ml, 10g/10g ME/Agar

I've also made some grain boil water agar (yes, I transferred this mold to some grain jars :/ ) that I'll be using too - 500ml undiluted to 10g agar.

My agar is clean from additional contam follwing my transfer of this mold to other plates, but I can never get any mycelium growth. I've thought about using antibiotics in the agar to try and get an initial clean culture, but I can't find anything available in Australia that would work specifically - it's all ambiguous and I can't be sure - any confirmed info here would be greatly appreciated.

I just feel like I'm missing something, and I don't wanna consider the possibility that maybe these spore swabs are bunk.. :/


EDIT**
Innoc. loop is my next test from a scalpel - do I literally just scrape the wire against the swab and then against the agar? Those parts have always been unclear to me. I know about the Z pattern and such, just innoc. loops confuse me.

The swabs themselves came from a vendor, so I'm trying to get something to grow in the first place, so I can then start cloning and isolating fruits and mucking around with that.




Alright I see., you dont have a spore print, but actual swabs with spores. The loop wont do anything in that case, just ignore that. Also, antibiotics don't do anything against mold, they only help against bacterial contaminations. IT could be that you got dirty swabs. You can always contact your vendor again and send them pic of all the failed plates together with your order id. Should send you new ones.

Otherwise, you dont actually have any other choices than to start from 0. Clean the entire room you are working in,use loads of soap should be enough but a bit of chlorine never hurts. vacuum, wipe the floor, benches, all flat areas, wash textiles etc. Prepare clean clothes, turn off ac, close windows and everything that may move the air around. Take a shower, put on clean clothes, wash mouth, put on a hat to get hair out of the way. prepare sab, put clean towel on the bottom of SAB. put all the stuff you need inside of SAB. Was hands one last time, thoroughly. Put hands in SAB, prepare everything so your hand movement will be as limited as precise and limited as possible. wait for air inside SAB to settle for a few moment, then GO.


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Offlinesinubrius
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Re: AGAR | Please help me identify what's on my plates! [Re: redhandmat]
    #26734328 - 06/10/20 12:44 PM (3 years, 8 months ago)

I'll give that a go with this new agar I've made. I'll also try and be quicker, the technique you've outlined seems quicker than mine - maybe I'm waiting a little too long for the SAB to settle with everything inside. I'm also going to deep clean my room.

Thanks for your advice!! Appreciate being able to troubleshoot with someone experienced. I'll probably post again when/if I think I've been successful again. :smile:


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InvisibleLadysKnight
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Re: AGAR | Please help me identify what's on my plates! [Re: redhandmat]
    #26734341 - 06/10/20 12:47 PM (3 years, 8 months ago)

From your description, the swabs are highly suspect. I'd try to get a couple prints from different sources and use a loop.

I don't know Australian law, but in the US, spores are only legal to look at, not grow, so if it's the same there, I'd tell the vendor all I see is mold, and swap for prints.


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Offlinesinubrius
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Re: AGAR | Please help me identify what's on my plates! [Re: LadysKnight]
    #26734366 - 06/10/20 12:55 PM (3 years, 8 months ago)

Yeah I think it's a similar circumstance. These are from sporeswap on reddit tho, so not an actual technical commercial vendor. Getting anything Down Under is out of bounds for most commercial vendors so it had to be there. Can't really go back to them with agar plates, as they're all for 'microscopy only' - any talk of cult. is against the rules. Not sure how I should approach it really. :/

The spores themselves are supposed to be APE - the tips of the swabs look more blue than purple, though I thought maybe this was a trait of the albinism. Kinda look like they've been hit with a blue marker tho.



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Offlineredhandmat
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Re: AGAR | Please help me identify what's on my plates! [Re: sinubrius]
    #26734408 - 06/10/20 01:11 PM (3 years, 8 months ago)

Quote:

sinubrius said:
Yeah I think it's a similar circumstance. These are from sporeswap on reddit tho, so not an actual technical commercial vendor. Getting anything Down Under is out of bounds for most commercial vendors so it had to be there. Can't really go back to them with agar plates, as they're all for 'microscopy only' - any talk of cult. is against the rules. Not sure how I should approach it really. :/

The spores themselves are supposed to be APE - the tips of the swabs look more blue than purple, though I thought maybe this was a trait of the albinism. Kinda look like they've been hit with a blue marker tho.






Spores can look blue-ish. I dont think someone would bother to send you sawbs of nothing bruv. I have two ideas.
First, cut a tiny bit of the cotton on a plate (or just swab the surface well and only on one spot) while being as cautious as you possibly can be of keeping everything clean. As soon as you see growth, pour hot (kinda, not boiling hot) agar o top of the plate. Wait. As soon as you see a growth break through the new agar on top, transfer it to a new plate. Maybe, just maybe, you can beat the mold or bacterial issue you have to the punch.

My second idea, is that in the mean time (as you are doing the above), keep scanning the trade forum on shroomery. Sooner or later someone will be posting free prints internationally. Im sure that within a month max you will be sent a free spore print.


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Offlinesinubrius
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Re: AGAR | Please help me identify what's on my plates! [Re: redhandmat]
    #26734454 - 06/10/20 01:32 PM (3 years, 8 months ago)

Dude thanks heaps for this! Had no idea there was a trade forum on here, I'll look around for it and keep my eyes peeled. This website truly is Pandora's Labyrinth, haha!

I'll also try what you've said about cutting the swab and adding the hot agar! I have a media bottle that I can P/C then microwave so that won't be an issue. I'll only microwave it til it's liquid so it's not too hot, since it's already sterile.

Legend! :smile:


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InvisibleRoger Clemency
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Re: AGAR | Please help me identify what's on my plates! [Re: sinubrius]
    #26734481 - 06/10/20 01:43 PM (3 years, 8 months ago)

I’ve heard of people just leaving the swab in the agar. Those swabs are probably the toughest thing to start with since they were rubbed against a mushroom that fruited in open air. You can get lucky and have it go well but probably you’ll need to work to get it clean. Using taller jars and stuff will make that more difficult as well, petris are where it’s at.

APE would be about the only time I’d advise getting a spore syringe to go to agar. Just some thoughts.


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Offlinedeniskov
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Re: AGAR | Please help me identify what's on my plates! [Re: Roger Clemency]
    #26734928 - 06/10/20 05:17 PM (3 years, 8 months ago)

By trade forum do you mean the marketplace or is there another trade forum?


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Offlinesinubrius
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Re: AGAR | Please help me identify what's on my plates! [Re: Roger Clemency]
    #26735563 - 06/10/20 09:29 PM (3 years, 8 months ago)

I've done that with the previous two swabs, but the growth that emerged was just as questionable and very quickly turned green.

Say I were to take one of these swabs and quirt some water from a sterile syringe on it and into a shot glass - could I then get spores in the syringe to use on the Agar?

Also, is there a specific reason for this in terms of their genetics?

Appreciate your advice. :smile:


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InvisibleSmartattack
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Re: AGAR | Please help me identify what's on my plates! [Re: sinubrius]
    #26735615 - 06/10/20 09:55 PM (3 years, 8 months ago)

I've had good luck with all swabs ive worked with. However I have never had a single plate germinate other than the plate where I cut off the top and just jammed it into the agar. Works every time so far.


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InvisibleAlkeides
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Re: AGAR | Please help me identify what's on my plates! [Re: sinubrius]
    #26735621 - 06/10/20 10:02 PM (3 years, 8 months ago)

So you're having issues with no-pours, are you PCing your agar for at least 20 minutes? Are any of your prepared (but unused) no-pours showing contam growth?

It sounds like you've done your research and are using good practices from what you've said. If your plates are clean otherwise, it would have to be those swabs.

Out of everything I've tried I've had best success going from spore print > streak plate > isolations or MS plate. It sucks that you spent money on those swabs but maybe you should """trade""" a different vendor for an actual print.

Also you won't be able to access the shroomery trade forum until you have 50 posts and have been a member for 90 days here.


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OfflineAbilor
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Re: AGAR | Please help me identify what's on my plates! [Re: Alkeides]
    #26735825 - 06/11/20 12:19 AM (3 years, 8 months ago)

Your Tek sounds legit. Likely dirty swabs. It's the weakest link in your sterile Kung Fu.


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InvisibleMateja
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Re: AGAR | Please help me identify what's on my plates! [Re: sinubrius]
    #26735911 - 06/11/20 02:10 AM (3 years, 8 months ago)

First of all you probably don't need to wash anything down with soap or with chlorine, the problem isn't dirty air and the problem isn't sterilization of agar, you're obviously not getting satellites on the outer edges of the agar. Instead this 'questionable' culture is growing from the transferred wedge so everything is as its supposed to be. What is relevant here is how your germination plates look, do you have pics of those? Also what's worked for me in the past is to cut off a portion of the swab with clean scissors and then wipe it onto the germination plate in a zig zag pattern as is recommended when germinating spores.


Also what you could do with this plate before throwing it away (if you choose to discard it) is to open it and let it dry out to see if it bruises or press the culture your fingertip all over to see if it responds similarly to bruising cube mycelium. Be aware that mold colonies often grow in circular fashion, I wouldn't transfer things from germination plats that look perfectly circular, that to me is a warning sign. Gl


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Offlinesinubrius
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Re: AGAR | Please help me identify what's on my plates! [Re: Alkeides]
    #26735960 - 06/11/20 03:37 AM (3 years, 8 months ago)

There's so much constructive info here, thanks heaps for your help!!

For reference, I pc my plates at 15psi for at least 25 minutes at at most 35 minutes using a presto 23qt, plates are above water line on multiple trivets and I use around 2qts of water, with no problems with water loss.

I leave the plates in the pc over night once it's off the heat without opening it to help combat the condensation. Majority of my pasty plates (all the ones you see here, incl. plastic ones) are fitted with SFDs that are siliconed on with RTV over a single 1/4" hole.

Other plates I've done with these swabs have shown something similar, or have just bacced out entirely after showing no growth for an extended period of time. I have a bunch of clean cultures of this mold and some of them I opened and had a poke around, no bluing so far as I can tell. It also doesn't feel like any mycelium I've touched in the past (old PF cakes, things like that).

Plates that I've left aside as control plates to spec out any potential contam never show any signs of growth before I either use them or turf them to use the container again for a different recipe, but I keep the control plate around for a week at least, even if I've already used the other plates beforehand, so that if something pops up on the control I can keep an eye out for it in the others.

Quote:

Lerman said:
Also you won't be able to access the shroomery trade forum until you have 50 posts and have been a member for 90 days here.




Do you reckon there's any forum mods I could message for potential inclusion before this threshold? I've had this account for a while now, 2 years atleast. I just try to lurk because I'm not that experienced yet so I can't render much help, and I definitely can't just spam the forums. If you don't think it's possible, fair enough, just figured I'd ask if something like that can happen in anyone's experience here on the forums. :smile:

Really appreciate the help!!!!!!

**EDIT - Also, no photos of the original culture, but it looked identical to above photos. I've got no doubt that there was never any mycelia there in the first place, unfortunately.

- Not quite sure what you mean by germination plates - do you mean the plate with the original culture that I took the mold from, or any plate I have that shows mycelia? (No plates so far have shown what I can determine as mycelia). Sorry if this is nooby!!


Edited by sinubrius (06/11/20 03:46 AM)


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