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CplCauliflower
shroomyboi


Registered: 02/21/15
Posts: 135
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Multispore Agar Inoculation Help
#26465017 - 02/02/20 10:10 AM (3 years, 11 months ago) |
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Hey all, needing some assistance as I dive into agar work. Last year I had great success pouring my own plates and taking clones of the grows I already had going, I was able to get another round out before senescence kicked in and the mycelium stopped spreading as well as it should have. I purchased a multispore syringe from a sponsored source I've used for a long time, I used it to inoculate 10 plates by dropping a drop or two on different areas of the plates. They were quickly overwhelmed with bacteria and I don't think I even waited two weeks before throwing them out.

I initially suspected dirty syringe so I purchased several more MS syringes from a different sponsored source I've also used and the same thing happened on 20 more plates - bacteria everywhere except for one plate had this growth on it I suspect to be psilocybe cubensis. I have another plate that grew very clear cobweb mold so I've used it for comparison.

I did some research and learned some lessons pertaining to inoculation of agar with MS syringes - only one drop or less and drag the needle across the surface as you drop it. I also learned I should dilute the agar a little bit more as bacteria is more likely to overwhelm a culture on a nutrient-rich medium. I inoculated 10 more plates this way and took many many cuts of the culture I suspect (hope) to be P.cubensis. Probably 30+ plates were done in an attempt to extract something here.
This is the culture I took all of my cuts from that I'm keeping in case it sporulates or reveals itself to be something else... I think it looks like p.cubensis.

On the left you can see my failed attempts at inoculation. Back left is cobweb, back middle is my mother culture. The stacks in the middle are cuts from that culture, and the two rows in the back are culture I incoculated with more spore solution.

This is the growth I'm seeing on 15+ plates as I hope to get some kind of culture out of these attempts... any advice on what this is? Looking around on Shroomery and the internet in general I'm seeing conflicting images as to what I'm actually cultivating here. Any help is appreciated on this. It might be pin mold or p.cubensis I'm still very new to MS agar work. I'm more used to MS -> grain jars and cloning into LC or master grain jars.




I think I have a clear example of satellite colonies of Trichoderma and the other colonies are dusty white on top - obviously not p.cubensis. I'm wondering if the transfer in the middle is my huckleberry because I almost feel like it's too fuzzy or it might just be tomentose growth.. All of my transfered plates look like this except most don't have contaminates growing in them, I pulled this one out because I felt ok handling it for pictures since it was the dirtiest transfer. I can take many more pictures of my plates at request.
Help me Shroomery-Wan Kenobi, you're my only hope.
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A.k.a
Stranger



Registered: 10/27/19
Posts: 16,782
Loc: Gaming the system
Last seen: 17 minutes, 34 seconds
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Man in those close ups I thought you had plates full of the craziest contam ever, I was like wtf is that it looks like carpet...
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CplCauliflower
shroomyboi


Registered: 02/21/15
Posts: 135
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Re: Multispore Agar Inoculation Help [Re: A.k.a]
#26465108 - 02/02/20 11:13 AM (3 years, 11 months ago) |
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A bath towel I use to line my table, soaks up my isopropyl spray while I work. Mostly a temporary setup while I work inside of my tub, if I'm letting the plates sit for long term colonization I saran wrap them and put them in my closet under my light. Can't remember where I picked the idea up from, no idea if it's even still applicable in today's methodology.
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A.k.a
Stranger



Registered: 10/27/19
Posts: 16,782
Loc: Gaming the system
Last seen: 17 minutes, 34 seconds
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Well that one piece you diced up looks like cube to me. I’d hope with 30 transfers you’ll have more than enough.
I had some problems with bacteria until I started making sure the whole blade got red hot before I used it, not just the tip half.
And make sure to not get your hands over the plate, or the scalpel handle for longer than needed. Wiping the whole handle down with 70 iso should help too.
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LAGM2020     
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Sockadin



Registered: 01/03/10
Posts: 7,244
Last seen: 2 months, 20 days
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Re: Multispore Agar Inoculation Help [Re: A.k.a]
#26465177 - 02/02/20 12:15 PM (3 years, 11 months ago) |
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So I am suspecting the bath towel is your problem. There is so much bacteria on sponges and towels. If you had a way to PC the towel for like 45 mins you might have better luck. With that much contamination. I don't think it's the syringes as much as the air circulating off the bottom of the SAB
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A.k.a
Stranger



Registered: 10/27/19
Posts: 16,782
Loc: Gaming the system
Last seen: 17 minutes, 34 seconds
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Re: Multispore Agar Inoculation Help [Re: Sockadin]
#26465204 - 02/02/20 12:37 PM (3 years, 11 months ago) |
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As long as you’re not putting everything right on a dirty towel that shouldn’t matter right?
The whole point is there’s no air movement
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CplCauliflower
shroomyboi


Registered: 02/21/15
Posts: 135
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Re: Multispore Agar Inoculation Help [Re: A.k.a]
#26465212 - 02/02/20 12:39 PM (3 years, 11 months ago) |
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I don't know if that makes much sense considering the contamination only happens in areas where I dropped the spore solution, and I've been using the towel method for years and have not had these kinds of issues.
I do change and wash the towel out, it doesn't just sit there endlessly.
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Sockadin



Registered: 01/03/10
Posts: 7,244
Last seen: 2 months, 20 days
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Have you swabbed the towel and put it to agar? SAB will limit air movement, but it isn't gonna stop it. The air inside the SAB moves when you move your hands and unless you have it perfectly sterile, which you cant. I think your picking up contams from the towel. But even if you solutions are dirty, that is why we do agar, just takes longer to clean it up. I will post some pictures later of a transfer I did from a spore discharge on my fruiting tray that has been sitting in my drafty garage from 3 years ago. You should still be able to transfer away from the bacteria and mold. I do think lowering your nutrients will help with that.
Edited by Sockadin (02/02/20 01:05 PM)
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CplCauliflower
shroomyboi


Registered: 02/21/15
Posts: 135
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Re: Multispore Agar Inoculation Help [Re: Sockadin]
#26465304 - 02/02/20 01:27 PM (3 years, 11 months ago) |
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Fantastic, I'll probably end up experimenting with removing the towel. Any idea if the culture in the pictures above is p.cubensis?
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Sockadin



Registered: 01/03/10
Posts: 7,244
Last seen: 2 months, 20 days
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3rd picture looks like cubes with mold growing at 4 o clock. 4th looks promising.
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CplCauliflower
shroomyboi


Registered: 02/21/15
Posts: 135
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Re: Multispore Agar Inoculation Help [Re: Sockadin]
#26466346 - 02/03/20 06:50 AM (3 years, 11 months ago) |
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Thank you, I'm glad I can get some supporting opinions on this culture. I'll probably update in a day or two with more pictures of how these plates are turning out.
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jbgtaa
extraterrestrial



Registered: 06/09/19
Posts: 1,785
Last seen: 3 years, 3 months
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Re: Multispore Agar Inoculation Help [Re: Sockadin]
#26466356 - 02/03/20 06:56 AM (3 years, 11 months ago) |
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Quote:
Sockadin said: So I am suspecting the bath towel is your problem. There is so much bacteria on sponges and towels. If you had a way to PC the towel for like 45 mins you might have better luck. With that much contamination. I don't think it's the syringes as much as the air circulating off the bottom of the SAB
Nah. The towel isn’t your problem, but if it was, you can just put a piece of foil down and work on top of that. You’re supposed to raise your work off of the towel and not work directly on it, but I usually do if it was the towel, a piece of foil will fix the problem because it’s sterile on the roll.
My question is: are you wearing gloves?
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CplCauliflower
shroomyboi


Registered: 02/21/15
Posts: 135
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Re: Multispore Agar Inoculation Help [Re: jbgtaa]
#26466526 - 02/03/20 09:38 AM (3 years, 11 months ago) |
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When I am working inside of the SAB and actively performing transfers/inoculations I wear gloves and use IPA on my hands and arms, as well as spraying the inside of the tub and the environment immediately outside where I work.
In these pictures I only washed my hands and arms because I removed these plates to take pictures and compare - I have plenty more plates with no contamination to choose from. I'm just really hoping that the cultures I have currently are in fact p.cubensis.
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