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Author Topic: Biologists: Aseptic Technique?  (Read 8675 times)
macaroon
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« Reply #15 on: May 03, 2012, 10:24:25 AM »

I agree with others that Tissue Culture is much more stringent than bacterial culture.

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. 




Not really true.

Each microbe smells subtly different.  I rotated in a yeast lab in grad school.  Not only could the members of the group tell the difference between different wild type strains by smell, they could also make a reasonable guess at some of the mutant strains.

If you're ever not sure about your bacterial culture, you can sequence the 16s gene.  It's not difficult or time consuming.

Plus, when you innoculate a flask of bacteria, you're adding a billion cells!   That one Staphylococcus epidermis that flops in doesn't stand a chance of overtaking your culture.   
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eigen
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« Reply #16 on: May 03, 2012, 1:47:30 PM »

Tissue culture background here as well.

No experience with flaming, but for us, all work was in a laminar flow hood. All tips/tubes/glassware was autoclaved and stored sealed, then washed down with Ethanol before going into the laminar flow hood.

Then we did a 30min-1hr UV decontamination on everything (after the Ethanol wash) before doing any work in the hood. And then you cleaned the entire inside of the hood with Ethanol after every use.

And we still got a really bad fungal contamination at one point.
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frogfactory
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« Reply #17 on: May 03, 2012, 1:56:07 PM »

UV is fairly useless, to be fair.

Fungal/yeast contaminations - usual culprit is exposed skin in the hood.  Either someone touching their face/hair with their gloves then putting them in the hood, or gloves that don't stay sealed over the cuffs of the (preferably) Howie-style labcoats.

Or you have an undergrad.
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macaroon
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« Reply #18 on: May 03, 2012, 2:08:37 PM »

UV is fairly useless, to be fair.

Fungal/yeast contaminations - usual culprit is exposed skin in the hood.  Either someone touching their face/hair with their gloves then putting them in the hood, or gloves that don't stay sealed over the cuffs of the (preferably) Howie-style labcoats.

Or you have an undergrad.

About the gloves on the face? I am a tyrant about that.  I embarrass them over and over again if I see the gloves touching anything other than lab equipment. 
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eigen
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« Reply #19 on: May 03, 2012, 2:21:33 PM »

It's true. But every little bit helps.

The problem with these hoods was the huge number of people using them. I don't think we had any undergrads, but it was in a "super lab group" at a medical school- all of the researchers, staff and graduate students from under 7 different PIs used the same two laminar flow hoods and incubators.
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kron3007
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« Reply #20 on: May 03, 2012, 2:25:56 PM »

I agree with others that Tissue Culture is much more stringent than bacterial culture.

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. 




Not really true.

Each microbe smells subtly different.  I rotated in a yeast lab in grad school.  Not only could the members of the group tell the difference between different wild type strains by smell, they could also make a reasonable guess at some of the mutant strains.

If you're ever not sure about your bacterial culture, you can sequence the 16s gene.  It's not difficult or time consuming.

Plus, when you innoculate a flask of bacteria, you're adding a billion cells!   That one Staphylococcus epidermis that flops in doesn't stand a chance of overtaking your culture.   

Not all microbes smell that strong, and if you have them growing together I doubt your nose would know.  Sequencing the 16s gene may be a good approach, I dont really know, but the better approach is good sterile technique to avoid the problem in the first place.  That one contaminating microbe may seem insignificant among the millions you add, but that dosnt mean that they cant co-exist, and you would no longer have a pure culture.  Further, you are assuming that the million cells you add are pure to start with...  

Contamination is not always obvious, and even if you think you have a pure culture you may not.  Even with plant tissue culture, contamination is not always easy to see and can spring up months after you think it is clean.  Why microbiologists gamble like this when the solution is relatively simple I will never know.





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greyscale
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« Reply #21 on: May 03, 2012, 3:18:19 PM »

I don't usually go all-out with sterile technique for yeast or bacteria, but I just started working with RNA and I'm practically having nightmares about RNAses lurking everywhere. (I'm not much of a bench scientist. I'm sure once I've done it a while, I'll have a system that works for me without having it on my mind so much.)
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macaroon
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« Reply #22 on: May 03, 2012, 3:20:51 PM »

 Why microbiologists gamble like this when the solution is relatively simple I will never know.


Speed.

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frogfactory
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« Reply #23 on: May 03, 2012, 5:02:02 PM »

I don't usually go all-out with sterile technique for yeast or bacteria, but I just started working with RNA and I'm practically having nightmares about RNAses lurking everywhere. (I'm not much of a bench scientist. I'm sure once I've done it a while, I'll have a system that works for me without having it on my mind so much.)

This is one of those things that often goes wrong in the hands of novices, but the solution is generally not DEPC treating everything, but rather to learn proper handling.
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cyano
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« Reply #24 on: May 05, 2012, 11:50:42 PM »

I do bacterial culture. I'm at a PUI so I only have undergrads working in my research lab. The students work in a laminar flow hood, autoclave everything possible then sterilize everything possible with alcohol then flame everything possible. It slows things down, but with undergrads, I like to ensure that multiple failures are necessary for contamination.
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