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Author Topic: Biologists: Aseptic Technique?  (Read 8677 times)
chaosbydesign
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« on: May 02, 2012, 3:07:01 PM »

When did it become acceptable/common to not use aseptic technique/sterile tips, microfuge tubes etc. when working with bacteria in the lab? Is this just something people stop doing when they're not being forced to do things aseptically in class labs? I don't understand how you can be sure you're not getting contamination if you have everything open on the bench and non-sterile tips etc.

(I'm not snarking, I'm genuinely curious -- nobody uses aseptic technique here in any of the labs I've been in. People look at me like I have three heads if I ask where the Bunsen burner is.)

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« Reply #1 on: May 02, 2012, 3:15:14 PM »

The bunsen burner is not necessary, and it involves the bottle being open longer. 

Also, if you're working with antibiotic resistant bacteria, that's going to be robust and you won't get contamination on your amp or kan plates very easily.  Especially not on the kan plates. 
Tips used for PCR with specific primers?  Yeah, no need to keep those very clean. 

Are you talking about in an undergraduate laboratory class?  Have you tried to teach aseptic technique to students?  It's not a 5 minute job.  It's a three lab session job.
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pathogen
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« Reply #2 on: May 02, 2012, 3:20:22 PM »

I  teach aseptic technique, and I insist my students use it. Everything we use is either sterilized by us or comes sterile in package. But I don't flame bottles usually. I tend to spray them down with ethanol before I use them.
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frogfactory
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« Reply #3 on: May 02, 2012, 3:26:39 PM »

I've definitely noticed that health and safety is generally much laxer in the US (not just at my current place, and obviously not in GLP labs).  I don't know if that reflects the heavy regulation culture in the UK more than laxity over here.

But I'm still a bit icked out at not having a handwash sink in the room I use for tissue culture - a dedicated handwash sink in every room is mandated in UK labs, as I understand it.
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chaosbydesign
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« Reply #4 on: May 02, 2012, 4:05:05 PM »

The bunsen burner is not necessary, and it involves the bottle being open longer. 

Also, if you're working with antibiotic resistant bacteria, that's going to be robust and you won't get contamination on your amp or kan plates very easily.  Especially not on the kan plates. 
Tips used for PCR with specific primers?  Yeah, no need to keep those very clean. 

Are you talking about in an undergraduate laboratory class?  Have you tried to teach aseptic technique to students?  It's not a 5 minute job.  It's a three lab session job.

No, I'm not teaching. I'm just working in a lab. We were taught aseptic technique in undergrad and if I remember correctly it was done in half a lab session.

Re the bunsen burner, I was taught to flame everything when working with bacteria, including the top of the bottle of media. We used glass universals and flamed them too. This is also what people did in research labs.
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« Reply #5 on: May 02, 2012, 4:14:08 PM »

The bunsen burner is not necessary, and it involves the bottle being open longer. 

Also, if you're working with antibiotic resistant bacteria, that's going to be robust and you won't get contamination on your amp or kan plates very easily.  Especially not on the kan plates. 
Tips used for PCR with specific primers?  Yeah, no need to keep those very clean. 

Are you talking about in an undergraduate laboratory class?  Have you tried to teach aseptic technique to students?  It's not a 5 minute job.  It's a three lab session job.

No, I'm not teaching. I'm just working in a lab. We were taught aseptic technique in undergrad and if I remember correctly it was done in half a lab session.

I can promise you that it was not learned in one lab session.  There is a lot of muscle memory involved in learning aseptic technique. 


Re the bunsen burner, I was taught to flame everything when working with bacteria, including the top of the bottle of media. We used glass universals and flamed them too. This is also what people did in research labs.

Yeah, not necessary.  I'm a non-flamer and I don't contaminate my LB or TSB, really, ever. 
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slinger
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« Reply #6 on: May 02, 2012, 5:34:18 PM »

The bunsen burner is not necessary, and it involves the bottle being open longer. 

Also, if you're working with antibiotic resistant bacteria, that's going to be robust and you won't get contamination on your amp or kan plates very easily.  Especially not on the kan plates. 
Tips used for PCR with specific primers?  Yeah, no need to keep those very clean. 

Are you talking about in an undergraduate laboratory class?  Have you tried to teach aseptic technique to students?  It's not a 5 minute job.  It's a three lab session job.

No, I'm not teaching. I'm just working in a lab. We were taught aseptic technique in undergrad and if I remember correctly it was done in half a lab session.

Re the bunsen burner, I was taught to flame everything when working with bacteria, including the top of the bottle of media. We used glass universals and flamed them too. This is also what people did in research labs.

I took general microbiology 10 years ago, have never used any of it since, until now, when I do it regularly in my job. I prepare cultures and media for the labs and research. I do know that I'm not as careful and particular as they try to get undergraduates to be. 

Perhaps it's the throw everything at the wall and hope something sticks routine?
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frogfactory
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« Reply #7 on: May 02, 2012, 11:01:14 PM »

I never really learned micro, but I did train and work extensively in tissue culture.  Apart from the setting fire to everything, I'd say that's probably a more rigourous background in aseptic technique.  Seems to work for me, at least.  I don't flame flask necks (unless I'm pouring for some reason), but I always flame the sponge bungs since there's generally no choice but to set them down on the bench after removing them.  Contact with surfaces = bad.
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bioteacher
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« Reply #8 on: May 02, 2012, 11:07:06 PM »

When I was at the bench in grad school, the lab I was in still used aseptic technique. I never new any other way than the super-careful approach.
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chaosbydesign
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« Reply #9 on: May 02, 2012, 11:18:08 PM »

I never really learned micro, but I did train and work extensively in tissue culture.  Apart from the setting fire to everything, I'd say that's probably a more rigourous background in aseptic technique.  Seems to work for me, at least.  I don't flame flask necks (unless I'm pouring for some reason), but I always flame the sponge bungs since there's generally no choice but to set them down on the bench after removing them.  Contact with surfaces = bad.

The good thing about not using a Bunsen with flasks is that you know for sure that you're not going to end up with a flaming flask. We used cotton wool wrapped in foil in ours with tape holding the foil, and every so often someone in the lab would set fire to one of their flasks.

Tissue culture practice is a little different here from what I learned previously as well. For example, I would not normally store anything in the laminar flow, and I would usually drench everything in 70% ethanol before letting it anywhere near the hood. That seems to be pretty unnecessary (though I still ethanol-spray pipettors even when other people don't) as I haven't had any significant contamination issues.

The person who taught me a lot of this stuff was obsessively obsessive about things being sterile (and pretty much everything else, actually). There was a bit of a contamination problem in that lab, though. Some people were not as careful as they could have been, and we had some weird (and extensive) cross-contamination from another groups' experiments at one point. Also, unlabeled plates of Serratia marcescens would occasionally appear in my fridge.
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frogfactory
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« Reply #10 on: May 03, 2012, 8:25:53 AM »

That seems to be pretty unnecessary (though I still ethanol-spray pipettors even when other people don't) as I haven't had any significant contamination issues.

No no no.  Good tissue culture practice depends on a) paranoia and b) redundancy of technique.

Eg. All surfaces must be sterilized before use.  But if you put anything down on them, or touch them with a pipette tip, you must assume they are contaminated.  You can get away with not doing this for decent periods of time, but you will eventually have an expensive and time consuming crash.

The number and nature of people using the facility does matter - for instance, if you have undergraduates in TC (I would *never* let an undergrad work in TC, even in a dedicated TC lab.  Their projects should be taking cells grown by other people on the side and, say, doing ICC on them or RT-PCR), you will get frequent contamination events.  If you have a dozen people sharing a hood, you need to strip and clean it every two weeks instead of every two months.
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kron3007
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« Reply #11 on: May 03, 2012, 8:42:52 AM »

Coming from more of a plant tissue culture background, microbiologists blow my mind.  I would never consider working outside of a flow bench being sufficiently sterile, but microbiologists do it all the time.  We sterilize every piece of equipment and do all of our work in the flow bench, if we didn't it would be a mess.

I have always doubted that bench work is good enough unless you are using highly selective media.  The worst part about bacterial cultures is that you really dont know for sure if it is contaminated with another bacteria, especially if the other bacteria is similar.  Personally I dont get why more precautions are not taken.

Just for the record, when I worked in a microbiology lab we used a flow bench and flamed everything. 



 
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frogfactory
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« Reply #12 on: May 03, 2012, 8:49:11 AM »

I know, right?  There has to have been an event in micro similar to the HeLa crisis by now (in which, on testing, large number of cell lines around the world believed to be specific cell types actually turned out to be highly invasive, robust cancer cells of the HeLa line, invalidating gods know how many papers and theses).
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chaosbydesign
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« Reply #13 on: May 03, 2012, 9:15:19 AM »

That seems to be pretty unnecessary (though I still ethanol-spray pipettors even when other people don't) as I haven't had any significant contamination issues.

No no no.  Good tissue culture practice depends on a) paranoia and b) redundancy of technique.

Eg. All surfaces must be sterilized before use.  But if you put anything down on them, or touch them with a pipette tip, you must assume they are contaminated.  You can get away with not doing this for decent periods of time, but you will eventually have an expensive and time consuming crash.



Yes, I do all of that. Nobody else does, though, (at least they haven't in previous labs -- just sprayed gloves and nothing else) and I don't go in the hood and sterilize all of their stuff they have in there before doing anything.
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frogfactory
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« Reply #14 on: May 03, 2012, 9:22:21 AM »

That seems to be pretty unnecessary (though I still ethanol-spray pipettors even when other people don't) as I haven't had any significant contamination issues.

No no no.  Good tissue culture practice depends on a) paranoia and b) redundancy of technique.

Eg. All surfaces must be sterilized before use.  But if you put anything down on them, or touch them with a pipette tip, you must assume they are contaminated.  You can get away with not doing this for decent periods of time, but you will eventually have an expensive and time consuming crash.



Yes, I do all of that. Nobody else does, though, (at least they haven't in previous labs -- just sprayed gloves and nothing else) and I don't go in the hood and sterilize all of their stuff they have in there before doing anything.

*twitches*
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