Do/Should you spray/wipe the outside of an ongoing cell culture flask (i.e. with cell+culture media in flask) with any type of disinfectant before place into Biosafety Cabinet (BSC)? What do you use?
I have been in 2 research labs
First lab:
Use Bacdown to spray/wipe all supplies including outside of flask before place into biosafety cabinet, but always treat outside of flask as non-sterile
Was told Bacdown is better than 70% ethanol, but never really explained why it is better or if it is suitable for disinfecting cell culture flask.
Later realized all the research paper/springer methods article used 70% ethanol, never saw Bacdown come up on any paper.
Second lab:
Was told not to spray/wipe outside of flask with anything before placing into biosafety cabinet, but again always treat outside of flask as non-sterile. (other supplies sprayed with 70% ethanol before placing into biosafety cabinet.
Was told Bacdown is bad due to residue build up and difficulty removing residue.
Do you spray serological pipettes (with sterile package on) with 70% ethanol before placing into BSC? One side of the package is paper, I have always wondered if spraying will cause ethanol to leak into the package and potentially cause chemical contamination in cell culture.
I feel that there is a lack of consensus on how to execute sterile techniques in cell culture and really varies from lab to lab, many people I know do certain things certain way only because they were taught to do it that way from someone else.
We should always wipe the exterior of supplies (sterile flasks/serological pipettes/petri-dishes, etc.) with a disinfectant (70% ethanol/isopropanol), particularly containers removed from a water bath. Make sure to spray items with a fine mist of 70% ethanol, this will evaporate quickly and leave no wet mess but still be effective. Segregate items that will remain clean from the ones that may become contaminated.
Dear Yong Ling,
I did some cell culture in a lab that was doing some relatively through drug screening using an immortalized cell line. They just had me spray and wipe everything, and I mean everything sprayed and wiped thoroughly, that went into the biosafety cabinet with 70% EtOH…that was it. This included my gloves and the Pipete-Aids® each and every time I left and entered the biosafety cabinet.
Aside from that, we just irradiated the biosafety cabinets with UV light overnight.
We rarely if ever had pathological problems in our cells (e.g., bacteria, spores, viruses...etc).
I hope this helps.
Best,
Josh
I mostly agree with Joshua. I do cell culture since 1985 and the most important point in my experience is to avoid any contact with the sterile parts of the equipment. This means do never handle above an open flask place everything sterile in the back of the bench, clean the bench before and after manipulations. Excessive use of 70% ethanol or any other cleaning procedure does not help when not sticking to the procedure above. Second, the incubators have to be cleaned regularly. We have two which are intermittendly used and cleaned. I know all this sounds like "everybody" knows, but if you really stick to it, it works.
By the way: Spraying the outside of single sterile packed pipettes is useless or even harmful as Yong Lin suspects.
It's just precaution and may depend on the environment of lab. 70% alcohol may be enough to get rid of accidental contamination. In fact, there are many bacteria and fungi floating around in the air. It is not a protocol, it is better to take precautions. Bacdown seems to be a detergent to wash out some dirt including germs. You can wipe out germs from outside of flasks, but detergent may stay on the surface if you do not wash ( in fact you do not wash with water, right?). In addition, detergents have very strong effects, even if the amount is very small. As long as the detergents on your flasks, it may accidentally get into the inside, but alcohol won't stay on surface. I do not deny to use Bacdown, but you get a risk too using that.
Even if paper wrapped pipets, alcohol would not get into the inside, I guess. I think some water proof treatment is done on it. You can use plastic wrapped pipets instead, if you are worried about it.
Hi Yong,
I've been in similar situations. I've only used 70% ethanol and RNase away (which really just gets rid of RNA) and 10% bleach. The key in using any disinfectant is allowing it the time to work. I believe for EtOH it should be left for two minutes to work. I would often just leave it on until it dried.
A few of the labs I have worked at insisted on using aseptic technique (spraying all non-sterile surfaces before entering the BSC hood as well as gloved hands), but at least one of them sprayed only equipment that stayed in the hoods long-term, or pipette tip boxes, etc., and did not spray cell culture flasks that went into the hood, as long as they came out of sterile packaging outside of the hood. The rationale I heard behind this is that when one is working as quickly one does in the lab, especially handling cells that are not happy outside of the incubator, there is not enough time to let all of the ethanol evaporate before you add the cells to the flask, and even though the EtOH does not get into the flask, the vapors might. Basically, the idea was that the ethanol and additional time outside of the incubator would damage the cells to the point the experiment(s) would not work.
In my experiences, both of these methods work well, as long as you know what you are doing when you bring nonsterile items into the hood. Be mindful of your pipette tips, what they touch, change them if they hit the outside of a nonsterile flask, and always sterilize the hood before someone else comes to use it (and always be mindful of where nonsterile flasks have rested in the hood). Also, I would use aseptic technique when doing something like freezing down a cell line, or establishing a long-term cell culture. I would reserve abandoning aseptic technique for experiments that will quickly end, such as overnight experiments. I hope this helps and good luck!
We use 1% virkon to bleach flasks. The hood, flask etc is sprayed with 70% ethanol. We are cautious about open flasks of bleach as it can damage the surface of the TC hood and there is a slender chance of explosion. Technique is very important and as mentioned above - don't touch anything sterile, tips, pipettes, inside of flasks.
We should always wipe the exterior of supplies (sterile flasks/serological pipettes/petri-dishes, etc.) with a disinfectant (70% ethanol/isopropanol), particularly containers removed from a water bath. Make sure to spray items with a fine mist of 70% ethanol, this will evaporate quickly and leave no wet mess but still be effective. Segregate items that will remain clean from the ones that may become contaminated.
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