A.k.a said:

With lc the more nutrient you use the thicker it will grow which sounds good but in practice it can be a pain in the ass cuz it’ll clog your needle up. Really any amount will work though.

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A.k.a said:

If you’re running 18psi for four hours nothing should survive. I guess endospores are possible but I would look into your lid filter or inoculation/sterile technique first.

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dcell said:

Let's assume PC is at 18psi for 4 hours and there is still an occasional bacterial grain jar.. Should the survival of an endospore off of the grain be discounted completely and source of bacteria looked for elsewhere?

Endospores in tap water?
Will bacteria always be visible from LC to agar?

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At what point is the bacteria present? If it's actually bacteria (not mold) and it's showing up in an unopened PC'd jar, there's likely something wrong with your jars (meaning lids, filters, or rings if plastic). With bacteria, I'd be more concerned about moisture content, although 4 hours at 18PSI shouldn't leave tons of water unless your prep is FUCKED.

If it's after inoculation, which it seems to be, it can be any number of things, although it's almost certainly NOT the sterilization run (which is likely overkill). You need to give us more details; everything basically. It could be your inoculant, your technique at introducing this inoculant, etc.

SHIPs should never be replaced. You just shouldn't use them because they fucking suck. They're a shitty crutch for folks to avoid using good technique. Just open the jar and pour in some liquid. It's much easier and involves less tools that can harbor contams. If you're buying LC syringes, just crack the lid in a SAB and squirt into the jar.

What the hell are sticker filters? Is that some new shit you kids are using? The lids only need a single hole for GE, although 3 tiny little holes works just as well. Buy the thick SFDs and you can cover at least 10 jars with one of them. Use RTV silicone around the edges and you'll never have to worry if it's your filter failing.



Post a pictures of your liquid inoculant and record a video of you inoculating jars. If you're using SHIPs for all, I'd stop. You're pushing through any contams on the SHIP itself or around the syringe tip. You want less contact with everything when inoculating. Crack the lid, lift up 1" and move it to the left so the jar is open enough for the syringe squirt. So much easier.

dcell said:

Let's assume PC is at 18psi for 4 hours and there is still an occasional bacterial grain jar.. Should the survival of an endospore off of the grain be discounted completely and source of bacteria looked for elsewhere?

Endospores in tap water?
Will bacteria always be visible from LC to agar?

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ThurmanMurman said:

Im struggling with condensation on my plates after I pour. I used a temp gun to determine the bottle was at around 125f when I poured and they still turned out poorly. Condensation eventually drips and pools on the agar.

1. Is this preventing spore germination? Ive gotten no growth on plates streaked on 7/12 and I've got a little something on 2 plates streaked on 7/20, but I think its contaminates.

2. How do I prevent the condensation here? Ive read that pouring around 120f should have done the trick, but I moved the bottle into the SAB at 125f to give myself some extra time to work. Im only pouring 5 high bc my ceiling is low, and I'm not great with the movements and worried I'll knock them over anyway.

This has been frustrating, to say the least. Thank you for any help.

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ThurmanMurman said:

I saw that pic of yours after someone else mentioned it. Utility AND style. I love it. Sounds like the condensation doesn't totally ruin the plate though? Im struggling to get spores to germ, and wanted to eliminate condensation as a cause.

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