11 replies to this topic

[GeneBee]

Hi, i am new to cell culture.

I would like to know, actually, what's the reason of using fire in the hood?

I was told that the hood should maintain laminar flow and we should not put too much stuff in hood.

So, will the fire change the laminar flow of the hood?

Thanks!

[Bomber]

using fire in the hood is pretty much old fashioned.
there is no good reason for that. in fact, as you said, it might even disturb the flow.
things going into the hood should be sterilized anyways before and it is no extra safety to flame things

[pcrman]

For sterilization. We still use it.

[Stephan]

The flame should not really disturb the laminar flow (depends how big it is).  I think that the benefits of a flame outweigh the possibility of this.

The flame is used particularly for the lids and openings of tubes or bottle as they are the things that are being handled most.  The most important part of flaming is flaming a tube/bottle just before closing it as it creates a rush of air out the bottle and thus carries any bugs out the air above the liquid.  

Flaming only really helps if your methodical but quick with it.  I have heard some people say that they never flame anymore because their reagents always get contaminated when they are flaming everything.  I believe this is just bad technique and/or reliance on only the flame for sterilization.

My flamer has a motion sensor and thus does not require any touching of the flamer.  I think if you can only get an alcohol burner it may not be worth it but if you can get a fireboy then I would suggest it.

[bob1]

Flames do disturb the laminar flow in hoods and can make your barrier ineffective.  Most modern tissue/cell culture (not bacterial/fungal) labs do not flame, as all the media and equipment going into the hood should be sterile and the outside sprayed and wiped down with 70% ethanol to inactivate live bacteria and fungi.  With good technique you should not be getting more than an occasional contamination, even without antibiotics.

If you do use a flame, you need to get one of the ones with a pilot flame, that will go to a full flame when touched.  Be aware that spraying ethanol near these is not a good idea also!

I have just remembered that if you are using loops and things like that, there are flameless ones available, that are essentially a heating element inside a cylinder that you poke your implement into.  Not much use if you want to flame the neck of a bottle, which you shouldn't be doing for cell culture anyway.

Also remember that the air flow barrier in a class II hood is there to protect YOU, not just your cells.

Edited by bob1, 01 June 2009 - 04:44 PM.

[GeorgeWolff]

There are plenty of claims that flames DO disrupt the laminar flow
(an example from Auburn for example: www.auburn.edu/administration/rms/pdf/biosafetycabinet-faq.pdf ).
What are the data that this does not occur?

I'd also take with a grain of salt the legend that flaming the mouth of a flask causes the "air to rush out."

[bob1]

GeorgeWolff, on Jun 2 2009, 02:32 PM, said:

There are plenty of claims that flames DO disrupt the laminar flow
(an example from Auburn for example: www.auburn.edu/administration/rms/pdf/biosafetycabinet-faq.pdf ).
What are the data that this does not occur?

I'd also take with a grain of salt the legend that flaming the mouth of a flask causes the "air to rush out."

I am pretty sure I have seen footage of a bunsen inside a biosafety cabinet, taken with an IR camera showing the air flow with it on and off (may be on Youtube, which I can't search seeing as I am at work).  A quick google search also indicates that there is a significant risk of a fire starting from the behaviour of the flame in the down-draft.

[GeorgeWolff]

The air in a flask or tube is not heated - only the neck - and the IR observartion doesn't track air - it tracks heat.  Even if there were such a video it hardly endorses the claim and aseptic technique itself is more than enough in a laminar flow hood to maintain sterile in a sterile vessel.

Yup - saw the cautions re flames.

[Matt105]

If the hot air is rushing out of the bottle, then I would imagine that cooler air would be replacing it at the same time.

[bob1]

GeorgeWolff, on Jun 3 2009, 12:34 PM, said:

The air in a flask or tube is not heated - only the neck - and the IR observartion doesn't track air - it tracks heat.  Even if there were such a video it hardly endorses the claim and aseptic technique itself is more than enough in a laminar flow hood to maintain sterile in a sterile vessel.

I know the IR doesn't track air, but the heat in the video isn't travelling in straight lines, as you would expect electro-magnetic waves to.  Therefore it must be interacting with something, which in this case is air, so you can track the air with an IR camera.  I totally agree that aseptic technique should be enough (even on an open bench), I never use a flame in a hood.

[Stephan]

This seems to be a typical argument of personal opinion and experience more than actual evidence.  I do think that not using a flame may work but this does not mean it is the best way or that it works better than when using a flame.  The type of flame used is so important, if using a simple alcohol burner or even bunsen burner - this may have less possibility for improving sterility but if a hands-free, non-continuous, high heat, low flame burner is available and used correctly, I think sterility can only be improved.  Also, about the air rushing out the bottle, this is not a 100% extraction of the air but physics would prove at least a good portion of the air is removed which can only improve sterility.  This air will be replaced with other colder air but hopefully cleaner than the air previously above the fluid.
Sterility is all about improving the odds, I'm sure u can get perfect sterility on the bench top, but not consistently and should this be what is taught to new comers to culture, who have not such refined techniques to start out with?

[Dominic]

i'm the other way round - gadgets are a bonus but sterility is always reliant on your aseptic technique - if your technique is not good enough seek out someone who's is better and ask for help.
you can use the best hoods, flame everything, spray your nitrile gloves in 70% ethanol, deep fry your lab coat - its all for naught if you touch the tip of a pippette

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