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Rizzle4Drizzle

The fact that the bacterial growth is not only near the tissue suggests that it's not coming from the tissue. Either your agar is not fully sterilized or possibly the plates themselves aren't sterile - which seems likely. It looks like condensation has dragged some bacteria onto the surface of the plates.


DirtyHarry133

Oh that rly makes sense. It seemes to me that condesensation kind of brought the plague but i didn't make the connection. Thx! I'll check the other sleeves for any punctures


enonesohc

Are those tissue cultures?


DirtyHarry133

It's an isolate from a tissue culture from some time ago, it went through multiple plates till now. Inside is a piece of agar from an earlier batch


enonesohc

I’ve had a lot of bacterial contam associated with t.c. I’ve had some limited success in taking a sample of the mycelium as far as possible from the contamination and placing it in a ketchup cup with peroxide until the bubbles stop the placing the sample on clean agar it’ll take a bit for the myc to recover but works well about half the time


DirtyHarry133

I don't think that's the issue though because i've had more than 10 transfers since the initial tc and they all looked great (besides the first 2).not saying it's impossible there weren't hiddem contams still but it s pbb highly unlikely


enonesohc

Yeah I agree after that many transfers it’s probably environmental. Do you incubate you plates for a few days at room temperature before you inoculate to make sure they’re clean


bullish88

Have you been spraying your area with lysol?


DirtyHarry133

Yes. Just the general area 15-30 minutes before starting work in the sab. Might that be a problem? It shouldn't have touched the inside of the plates theoretically


bullish88

Spray room, spray sab. Have clean tissue samples, and the agar don’t look good. Which agar method?


DirtyHarry133

Yes, i spray everuthing with iso, the inside of the sab etc. I'm using pda agar with powdered potato


bullish88

Dont use potato it has mad endospores. And two thats whats making the white stuff its potato starch.


ben_roxx

Could it be from your dishes not being sterile? I had this kind of issues lately!


DirtyHarry133

I guess it miight but it's the same provider and the plates are from the same order from a while ago. I guess the plastic in which they're packaged might have been broken, i should check for that


[deleted]

I've gotten plastic petri dishes with pinholes in the plastic wrapping. If there is a hole than they will be contaminated. Usually when I get a box of them at least one sleeve is compromised. Gotta check them before use


TundraSpice

How are you sterilizing your agar?


johnnyhitch1

Condensation problems. Do you poly film them after transfer? Try storing upside down after PC