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Invisiblemicro
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Tissue culturing skills
    #2041278 - 10/25/03 02:10 AM (13 years, 1 month ago)

I figure I'll post my view on this. People with whom I work (in a lab) still can't figure it out, but there are a lot of tricks-of-the-trade, and if you get your TC skills down you'll be a lot better at this stuff. People seem to be somewhat meticulous when it comes to cleaning, but it seems like people don't have the basic lab skills down, in a lot of cases. Honestley, I use non-antibiotic media without a flow hood or glove box, but nothing gets contaminated for that reason. I'm a bit drunk right now, so I'll probably miss stuff, and I'll include it later/

Loostening caps:

One of the most important things, IME. You want to keep your lids to your sterile media only open a crack. If the lids are just loosened and not opened you shouldn't get contamination because you should only be cracking the lids for a second or two. Let's say you want to transfer something from jar 1 to jar 2 sterilly. Loosen both the lids and then take what you need from your sterile stock solution by cracking the lid with one hand and pulling the sterile media into a sterile pipette or syringe or whatever with the other hand, so you keep the time the lid was cracked open to a minimum.

DO NOT put the extra media back into your stock solution. If you pulled up too much, throw it out, into a jar or whatever. Even with cotton-plugged pipettes this can contaminate the media. I always assumed since there was a liquid barrier, the bottom should still be sterile, but it isn't. I've contaminated my LB media many a time this way. If you pull up too much, throw it out. Don't ask me how it contaminates; it just does.

If working with petri dishes, it's a lot better to work with them upside-down. You just have to keep condensation at a minimum, so after you pour them let them sit for 4-6 hours.

Also, working behind a flame from a bunson burner, or whatever creates the flame, helps with airborne contamination a lot. If you are pouring petri dishes, heat the top of the jar from which you're pouring every other dish or so with a flame to reduce contamination. The petri dishes should be sterile and closed in the first place, but the tip of the jar remains open to the air, and flaming it will kill everything that might land on it.

Just my take; to be continued....

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OfflineSuntzu
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Re: Tissue culturing skills [Re: micro]
    #2044676 - 10/26/03 02:37 PM (13 years, 1 month ago)

Great post, micro. I agree that good technique is key to success. Another is addressing the general air quality of the working space; labs usually are in a building with HVAC systems that [at least] somewhat filter the air. Bedrooms and such can be notoriously full of airborne particles; gloveboxes are effective when used properly, but even more so the cleaner the outside air. Even a decent, inexpensive air filter running in a room can make a huge difference in some contamination situations.
One addition to the actual technique. . .Plan all moves in advance. Have the tools in position, the jars/plates in order, so that all inoculations occur with a smooth transition and minimum open-time.
Again, great post, micro.


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Invisiblemicro
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Re: Tissue culturing skills [Re: Suntzu]
    #2044986 - 10/26/03 04:45 PM (13 years, 1 month ago)

Yeah -- I completely agree. It really, really helps if you have a seperate room to do this stuff in with an HEPA filter running, or at least a clean room without carpet.

If you have carpet get a really good vacuum cleaner.

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Re: Tissue culturing skills [Re: micro]
    #2045220 - 10/26/03 06:18 PM (13 years, 1 month ago)

Oh -- one more thing -- when dealing with petris you can just put them back into the original sleeve (they should be upside-down when storing them to prevent condensation.) I think this is a good idea, in general.

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