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Offlineoculodextro
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Registered: 04/10/13
Posts: 1,205
Loc: Space is the Place
Last seen: 1 year, 10 months
Clean agar to bacteria city
    #26642189 - 05/01/20 08:56 PM (3 years, 9 months ago)






I'm losing my fucking mind here. Went from 90% success rate to failure consistently. I haven't changed any procedures, or even agar recipe and this is how every plate is turning out.

I am pressure cooking my agar for 40 minutes in a 500 mL flask, using grain soaked water, in a still air box. As you can see this really nice looking lions mane culture is a bacterial soup.

What the fuck happened and I am consistently seeing P. aeruginosa but now there's new bacteria arising. I'm almost out of my clean Lions Mane agar and really don't wish to buy another syringe.


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InvisibleStrainsfordaze


Registered: 05/10/18
Posts: 669
Re: Clean agar to bacteria city [Re: oculodextro]
    #26642256 - 05/01/20 09:30 PM (3 years, 9 months ago)

I would get your experience on potato agar or malt extract agar before doing grain water.
Are you venting off your pc?
What is the dilution of your grain water?


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Offlineoculodextro
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Re: Clean agar to bacteria city [Re: Strainsfordaze]
    #26642294 - 05/01/20 10:05 PM (3 years, 9 months ago)

I mean I've done 200-300 grain plates with no issues. I let the PC cool completely before opening (thinking I need to sit it in front of a laminar flow as it cools).

It doesn't matter what media I use, MEA, PDA, Grain, my lions mane contaminates consistently.

The grain water I've played with pure grain water, 75%, 50%, 25% and all show bacteria. The original Lions Mane plates are clean.

I'm wondering my scalpel is contaminating and needs a new blade.


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Invisiblec10h12n2o
serial dilutor
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Registered: 01/21/15
Posts: 3,200
Loc: the abyss
Re: Clean agar to bacteria city [Re: oculodextro]
    #26642414 - 05/01/20 11:30 PM (3 years, 9 months ago)

are test plates contaming, or just the LM inoculated ones?

i know it aint kosher but i pop my pressure valve with a tiny bit of positive pressure with agar, to prevent it sucking in contams. works flawlessly every time and i do a ridiculous amount of agar

how long you PC and at what pressure?

the bacteria has to come from somewhere

are you pressure cooking your scalpel? i wrap my handles in foil and pc, then use either a fresh blade or one that got red hot in a bacticinerator

what kind of agar are the original plates on?


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

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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Offlineoculodextro
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Re: Clean agar to bacteria city [Re: c10h12n2o]
    #26642506 - 05/02/20 12:37 AM (3 years, 9 months ago)

Test plates are not contaming. I have done oyster, cubes and pans at the same time with same agar. Which leads me to believe i am missing contams on the initial LM plates.

I may need to start PCing my scalpel, never done that before. Just iso and flame.

PCing 40mins at 15psi. Increased from 20m @15psi.

Agar has been just grain water. I have MEA plates that show same contam as well as PDA.

I'm almost suspecting my ISO is bunk. Took me forever to find some recently ordered from a medical supply company and have never smelt ISO  like this.


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Invisiblec10h12n2o
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Registered: 01/21/15
Posts: 3,200
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Re: Clean agar to bacteria city [Re: oculodextro]
    #26642618 - 05/02/20 01:53 AM (3 years, 9 months ago)

Sounds like a dirty culture

Few things to try:

Antibiotics (gentamicin and chloramphenicol)

You could try basically streaking the myc, like we would typically streak spores (as in my sig). Grab clean colony as soon as its visible and transfer it

Start over


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

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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Offlineoculodextro
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Re: Clean agar to bacteria city [Re: c10h12n2o]
    #26642962 - 05/02/20 06:47 AM (3 years, 9 months ago)

I have years of microbiology and virology work. I've def. gotten lazy with this hobby, time to bust out my innoc loops and do the classic streaking like your sig.

Yeah it was a syringe from spore works, and it started just with P. aerguonisa and now it's moved on to that clusterfuck.

Do scalpel blades get replaced that often? I figured alcohol/red hot would be more then enough.


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Invisiblerickyswamps
Bad Apple
I'm a teapot


Registered: 11/08/18
Posts: 1,192
Re: Clean agar to bacteria city [Re: oculodextro]
    #26642976 - 05/02/20 06:55 AM (3 years, 9 months ago)

Those plates are not being contaminated by your LM transfer.  The contams are everywhere.  Its either agar that wasn't sterilized or plates left open/improperly stored etc...


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OfflineDomno
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Registered: 01/30/20
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Re: Clean agar to bacteria city [Re: oculodextro]
    #26642977 - 05/02/20 06:56 AM (3 years, 9 months ago)

I dont personally use a scalpel, but I use exacto blades. Flaming them makes the layers of metal separate over time as the blades are cheap. I replace them every once in a while because I worry about a contam in a crack that may not get hot enough. I'm not paranoid about it, but if a blade starts looking crappy I just toss it out and get a new one.


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Offlineoculodextro
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Re: Clean agar to bacteria city [Re: Domno]
    #26642984 - 05/02/20 07:00 AM (3 years, 9 months ago)

I guess i should say xacto and not scalpel, yeah I see that the blade isn't red hot everywhere and the blade has defintely lost it's original length/sharpness.

Time for a new blade. Been so hungry for these LMs and they are teasing me. Few of my bags are contaming out now, that looked great just a few days ago. My oyster is perfect and the cubes have never been an issue.

Oh well, love problem solving. Just running out of clean multi spore plates.


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InvisibleStrainsfordaze


Registered: 05/10/18
Posts: 669
Re: Clean agar to bacteria city [Re: oculodextro]
    #26643087 - 05/02/20 07:58 AM (3 years, 9 months ago)

I replace my scalpel blade every session or 40 plates. It starts bending or becoming frail in areas. It’s all preference though.


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Offlinepoisoned
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I'm a teapot User Gallery


Registered: 04/17/13
Posts: 1,738
Loc: Yurop
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Re: Clean agar to bacteria city [Re: Strainsfordaze]
    #26643478 - 05/02/20 11:16 AM (3 years, 9 months ago)

I replace my blade if I'm taking clones or transferring reishi. Otherwise, it's fine.


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