6 replies to this topic

[J Bardo]

Will sealed agar tubes stay sterile forever?
I am trying to use screw-capped tubes to store 12 mL aliquots of agar. I will be melting these down as needed to pour into plates but I'd just like to store them for now. My problem is that, while making room in my fridge over the last week, almost all the screw-capped tubes with solid agar have growth on the top surface while sitting at room temp. I had previously autoclaved them for 30 min (tubes with the screw caps on) so the contents should be sterile indefinitely, but why aren't they?
Cleanliness of the tube initially?
Shouldn't autoclaved screw tubes more than anything stay sterile indefinitely? With slants I have made in the past (that I have left out for a few weeks), I still see growth showing up. I use the sterility tape in the autoclave to confirm sterility but still I end up with growth eventually.
Is this normal?

[phage434]

Were the tops loose on the tubes while being autoclaved? Steam must be able to reach the inside of the tube during autoclaving for it to be effective.

[microgirl]

It is pretty hard to pour completely sterile plates/tubes. What you've got growing is probably fungus/mold - there's so much of that in the air that it gets into your tubes. You don't usually notice it because it grows much more slowly than o/n bacteria. If you keep them at 4C that should inhibit the growth so that you can keep them for a couple of months. If your still seeing a lot of growth then maybe your media or your tubes is/are not completely sterile, or your aseptic technique is having a glitch!

[microgirl]

Also the autoclave tape does not confirm sterility - just that steam touched the tape. You need to be sure you're autoclaving at 121 for 15 minutes.

[J Bardo]

thank you for the replies. my problem is basically with autoclaving. i am under the assumption that anything that goes in the autoclave, in relatively clean glassware, will be sterile after 45 minutes. i am making liquid agar, pipetting it into tubes, then autoclaving them with the lids slightly loosened for 45 minutes (15 min warmup, 30 minute sterilize).
i put them in the incubator and they show growth within a few days and i dont know why.

[phage434]

I think your autoclave needs to validated. Is it reaching pressure? It should be about 15 psi, or 1 atmosphere.

[Joneslab]

J Bardo, on Feb 16 2010, 12:23 AM, said:

thank you for the replies. my problem is basically with autoclaving. i am under the assumption that anything that goes in the autoclave, in relatively clean glassware, will be sterile after 45 minutes. i am making liquid agar, pipetting it into tubes, then autoclaving them with the lids slightly loosened for 45 minutes (15 min warmup, 30 minute sterilize).
i put them in the incubator and they show growth within a few days and i dont know why.

Slight overkill, but have you thought about running the empty glass tubes through the autoclave first to sterilise them BEFORE you pipette in the liquid agar, then autoclaving to sterilise the agar?