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OfflineBismillah
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Registered: 07/11/22
Posts: 231
Last seen: 3 days, 7 hours
Working with a contaminated swab
    #28185063 - 02/13/23 07:24 AM (1 year, 10 months ago)

I've been trying to get clean myc from a APE-revert swab for the past 4 months. Here's what I've tried:
  • Transfering healthy looking myc to new plates
  • Josex' poke and transfer to BRF
  • Transfer of myc from BRF to agar
  • Cloning from BRF fruit
  • Cloning plate pins


TL/DR: The plate pins seem more promising than the other techniques, especially when I chop them off instead of plucking them. Anyone else have experience with this? BRF fruits were big enough for sampling internal tissue but I still have contams. Is it possible that bacteria enter the fruits?

Story so far:

Two weeks after streaking the swab and chopping it up, there's visible bacteria growing on all plates:


I transferred some rhizo sections of the streak plates, and obviously some bacteria came along:


I used a syringe with distilled water, poked the plates and squirted into 5 BRF pucks.

I kept the shitty looking plates, and got some pins in late January this year. Also the BRF pucks started fruiting:


Notice the blue surface, not sure if that's myc or contam. The fruits I cloned showed extreme blueing. Only two pucks produced fruits, the others just shriveled up.



I transferred the plins to new plates. I noticed they would lift off easily (potentially bringing in bacteria from the agar), so later I started chopping them off using two scalpels. The procedure looks like this:


Here's a plin ready for cloning next to the stem of a plin I cloned before:



I did a few transfers from BRF pucks to agar. A challenge here was that the myc grew downwards, with little myc on the surface. I would guess the chance of contam is higher near the syringe squirt, so I sampled on the myc rim. I also sampled from the fully colonized pucks.


The early transfers I did from BRF were bacterial. Later transfers (when pucks were fully colonized) so far looks better, but maybe early to say. I find it harder to judge MS plates with regard to contams. The first two (top left) are from BRF, the rest are plin clones:



A lot of these transfers have like a fuzzy edge, where the agar is slightly opaque near the myc. Closeup of the above BRF transfer (top left, left half of plate):


The clones look more promising. These were chopped off, not plucked.

I had high hopes for this one:


but it started growing all werid after the first transfer. Notice the pic above, there is a small speck of bacteria: I cut that out earlier but didn't get it all. The transfer also doesn't look great.


Maybe there's something I can work with here:



Clone material from the BRF fruits also seem to be contaminated. Top row is from the big fuit, "aper jcl" is from the small fruit. The rest are plins.


This fella looked pretty good, so I transferred to both LC and grain.


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Edited by Bismillah (02/13/23 07:26 AM)

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Offlinephenyl
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Registered: 09/03/22
Posts: 544
Loc: One toke over the line
Last seen: 33 minutes, 48 seconds
Re: Working with a contaminated swab [Re: Bismillah]
    #28185100 - 02/13/23 08:00 AM (1 year, 10 months ago)

You have so much patience. I would have nuked everything with tetracycline by now.


--------------------
The fool who persists in his folly will become wise.

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Offlinedyel
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Registered: 10/15/21
Posts: 920
Last seen: 3 months, 4 days
Re: Working with a contaminated swab [Re: phenyl]
    #28185177 - 02/13/23 09:22 AM (1 year, 10 months ago)

Quote:

BRF fruits were big enough for sampling internal tissue but I still have contams




my suspicion is something with the process, don't know if I missed it but are using a SAB or flow hood?
breathing on the plates by any chance?

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OfflineBismillah
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Registered: 07/11/22
Posts: 231
Last seen: 3 days, 7 hours
Re: Working with a contaminated swab [Re: dyel]
    #28185227 - 02/13/23 10:02 AM (1 year, 10 months ago)

I have a wild pan cyan swab as well, figured I need the practice to clean myc. Also, I'd like to limit international post to the bare minimum.

I use a SAB, clear face shield, latex sleeves, nitrile gloves. I started this hobby in August last year, so yeah maybe I'm missing something. Still, I've had 4 shoebox grows and one mini mono, grown from spore to agar and LC without issues. Also quite a few ziplocks for testing strains. Ok, I had one grain jar go green on me in January, but my plates have been clean.


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