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Offlinegabbk
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Alternatives to gentamicin for bacterial contamination * 1
    #26624238 - 04/24/20 09:24 AM (3 years, 9 months ago)

Hi all.
I've been dealing with bacterial contamination on the mycelium from a long time now and discovered it came from the syringe. It's not the typical bacteria that leaves stains on the agar. Instead, it rides along with the mycelium kind of 'hitch-hiking' on it, impersonating 'healthy' fungal growth.


I've already done around 5 transfers and still persists, so now I'm looking to add an antibiotic to the agar and try doing hot pour. I know the hot pour tek by RR doesnt need any antibiotic but I would like to add it, as some other users did, and see how it goes. Here at my nearest pet store they don't have gentamicin. Instead they do have available cephalexin, doxycycline, ampicillin and phenoxymethylpenicillin.
Which one is autoclavable or stands the highest temperature possible? (If not autoclavable I could add it after sterilizing the agar while hot).


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InvisiblebodhisattaMDiscordReddit
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Re: Alternatives to gentamicin for bacterial contamination [Re: gabbk] * 1
    #26625155 - 04/24/20 04:36 PM (3 years, 9 months ago)

Doesn't appear to be contaminated at all :shrug:


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Offlinegabbk
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Re: Alternatives to gentamicin for bacterial contamination [Re: bodhisatta]
    #26625812 - 04/24/20 10:20 PM (3 years, 8 months ago)

Sorry if this is a long post. But I think you need to catch up what I've done to get here.
Every tub I make seemed to be overrid with bacteria. I tried already eliminating every vector I could and straightening every technique possible. Every grain jar seemed to look fine, such as this one:

(At that moment the jar was just took out from the fridge, but anyways those spidery growths weirded me out, making me think it COULD be bacterial too).
None of these jars has areas uncolonized, although two of them took two more days to colonize completely (one more red flag to point out).
Made 3 tubs, the first one in April 2nd, the second one in April 15th and the third
one in April 19th.
The first one was made with both or your teks (shoebox tek and bucket), but I made the mistake of doing it with already hydrated coir, so it was waaay over hydrated, so then I had to get rid of lot of water to reach field capacity, and then finally mix it. I sprayed it with mineral water and let it sit covered with a plastic bag instead of using the lid. Within just two days this showed up on the surface:

Bacterial blobbing that at the time I didn't know; it impersonated "aerial mycelium".
That first tub seemed stalled. Just within the couple first days it had pretty acidic/fermented odor, clearly from bacterial colonization (the walls smelled just as when you leave a bottle of water two weeks without opening it and then peak to smell it, horrifying). It went 14 days straight and it was getting worse, so I searched a lot and decided to give a try to the hippie3 bleach dunk tek. At first the substrate seemed to be dead, but after 5 days it gave pins, so I consider it succesful. Today, at day 22 it isn't even full colonized yet, it's around 80% but despite of it it's giving fruits and really has no trace of that horrible smell. Now it has the beautiful mushroomy smell.
With this experimentation done I decided to make a second attemp on a new tub, this time doing it 'correctly'. This time I decided to SFF shoebox tek and do a proper pasteurization because I thought the issue of the first tub was improper pasteurization. To pasteurize I did a tek on PC by RR: 5 mins on, 30 off, then again 5 mins on and then leave it for 2 hours. And then again, the same thing, sloooow growth and same smell.
Later then I remembered something pretty unorthodox that I saw GordoTek did in a post and everyone bashed him, spraying H2O2 (hydrogen peroxide) to the casing. So I thought this case of mine is not so common, so it maybe could work out. So  19th April decided to make a third tub, doing exactly the same as the second tube (SSF + proper pasteurization) but this time spraying H2O2 on the casing. And just within a few days, this particular tub with H2O2 colonized waaaaaaay quicker than the second tub.
The picture I took today is self explanatory (the one above -the third one, that was made six days ago- is the one with hydrogen peroxide, while the one below -the second one, made ten days ago-is the one with mineral water, just like the first tub that contaminated):

So that now leaves me pointing out that the original issue was in the syringe. That syringe carried some bacteria in it that mixed in with the spores. It looked and looks okay for you, so I did G2G and carried away to all these tubs. It really might seem okay, but one thing that doesn't allow me to be in peace is this behavior that has that fuzzy little """mycelium""" that grows riding over the real mycelium. I've noticed that if I make a transfer and allow the dish to get some air (just the time needed to do the transfers), in a matter of one or two days that mycelium-looking structure (which I suspect it is bacteria) completely takes over all the reproduction work that did the mycelium, difficulting a lot its visibility. So it isn't really a coincidence that in a matter of two days, the previously mentioned first tub that I made it developed those big aerial blobs, isn't it? It seems that when that organism gets some fresh air it skyrocket it's reproduction rate. That bacteria really makes it impossible to get clean transfers, because it grows so fast that I can't ever just transfer pure mycelium, it would always be attached in some extent. And if it there at least in a minimal extent, it will always be there dominating the mycelium, just because of the fact bacteria grows immensely faster than fungi. So today I bought amoxicillin-clavulanate and already made some agars (I added the amoxicillin after sterilizing the agar, before pouring it) and already made some transfers. These days I'm going to check these agars closely and look how it evolves.


Edited by gabbk (04/24/20 11:09 PM)


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InvisibleAndyHinton


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Re: Alternatives to gentamicin for bacterial contamination [Re: gabbk]
    #26625846 - 04/24/20 10:39 PM (3 years, 8 months ago)

See Addgene's table of antibiotics doses. I've used chloramphenicol with fungi to good effect, and my colleagues usually use carbenicillin and kanamycin for plasmid work.


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Edited by AndyHinton (04/24/20 10:40 PM)


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InvisiblebodhisattaMDiscordReddit
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Re: Alternatives to gentamicin for bacterial contamination [Re: AndyHinton] * 1
    #26626239 - 04/25/20 04:44 AM (3 years, 8 months ago)

Agar culture looks fine. Your contamination gets in during sterile technique moving from agar to grain. Or improper grain prep/sterilization.


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Invisiblepanne cyanne
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Re: Alternatives to gentamicin for bacterial contamination [Re: bodhisatta]
    #26627861 - 04/25/20 06:21 PM (3 years, 8 months ago)

bod has it right on this.
id spare the antibiotics and tighten up the lab cleaning.


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