Waste bottle - (Sep/18/2009 )

Hi everybody,

I've recently been put in charge of the cell culture in my lab, and I want to have everything running as smooth as possible. I noticed a few things that I'd like to change, but I want to be sure that it's not going to affect the sterility of my cell culture hood before changing them:

- where do you keep your liquid waste bottle? Currently, ours is outside the hood and the way it is now, there's a tube running from the inside of the hood to the outside waste bottle, and the glass pane of the hood can't be completely closed. The tube is also touching the air vents. I want to put the waste bottle inside, and bleach it regularly, but the guy that was taking care of the cell culture before me doesn't agree. What would be the best, inside or out?

- also, he's keeping flasks and tubes in their original plastic bags in the hood, to be "more sterile". I don't like the idea of having plastic bags that were in a drawer or a box in the lab for a few months being put in the hood, even after washing them with alcohol. I also don't think having a half-open bag around flasks will avoid contamination. Is there any risk in leaving the flasks by themselves in the hood? (They have filter caps for gas exchange, but I checked the company's website and the pore size prevent common contaminations).

Thank you!

Dear OK13,

these are very interesting questions and concerns. With regards to the waste bottle, there is no problem with it being outside the hood, and with the tubing stopping the hood from being sealed. Just make sure you dust the area under the work surface every now and again.

If you are concerned about the suction tube being a source of contamination, many labs store the tubing outside attached to the side of the hood. They rinse the outside of the tubing with 70% ethanol before placing back into the hood and using and there are no problems with contamination. I have also seen laminar flow units with an outlet on the side that you can attach "internal" tubing and then there is an outlet for attaching the tubing that connects to the waste bottle. Does your hood have this?

Keeping things inside the hood (waste bottle, partially opened tube bags etc) is a semi-personal thing. In my experience, labs where this is done have more contaminations than labs where everything is removed, allowing complete disinfection of the work surface. In labs where you need GMP, you are not allowed to store anything in the hood. I have also seen disgustingly messy cell culture hoods where people never see contaminations and then I have seen exceptionally clean hoods where staff get all sorts of problems. This could be related more to antibiotic use than "good" asceptic technique.

Let me know what you do and how you succeed in changing things.

AussieUSA.

Thanks for your advice! I decluttered the hood as much as possible, kept the waste bottle outside and hooked up the aspiration tube on the side panel. So far so good, no contamination.
Thanks again.

We keep our liquid waste bottle outside too, no problem. In my opinion the main function of the hood is to produce sterile air. So any objects in the hood (bottles and plastic bags) could interfere with airflow and therefore the sterileness (is that a word?) of the hood. Besides, working in a clean and empty hood is preferable above working in a cluttered, full hood.

I think the waste bottle position is just a personal preference, i worked in labs which the waste bottle inside the BSC and outside the BSC, it doesn't make any difference, however, I do found if the waste bottle is outside the hood, you got more space to work with especially you are working with a 4' BSC.

Also, if you working in an accredited lab, the accessors would prefer you have nothing inside the hood, so, just my experience.

agree...

i think the practice in cell culture sometimes is a very personal "ritual", as long as you feel comfortable with it and it doesn't cause contamination problem.
In the lab i am working now, we too keep the waste bottle outside the hood. All the flasks etc. etc were also kept in drawer outside hood. None of them were kept inside hood.
However, in the lab i used to work in, we use to keep flasks/ spinning tubes inside hood. It never cause us any problem and there is no contamination issue.

I guess the main point is, having a good aseptic technique.
If the user of tissue culture hood is not too many, you can consider to keep things inside. In the lab i used to work in, only 2 person working in tissue culture room. We all have a very good understanding of how things work in the room.
But if the laminar hood was shared by many many people (just like the lab i work in now), you better don't keep things inside the hood. It'll be very messy.

OK13 on Sep 18 2009, 06:36 PM said:

Depending on the design, a LAF bench can serve to 1) protect the work, 2) protect the operator or 3) protect both. If you work in a LAF bench to protect both you and what you are working with, the waste flask should ideally be in the cabinet.

The waste flask should be within the "sterile" cabinet with the connection between the flask and the vacuum source protected by a filter capsule. The flask should also contain a suitable amount/type of disinfectant commensurate to the work you are undertaking.

In this way, any infectious agent remains the cabinet, or is disinfected in the waste flask before it is removed from the protective environment of the sterile cabinet. It is good practice to work with such a setup if you are not sure the cells/solutions you are working with are safe. It is preferable to handle all human-derived material (blood, bone marrow etc) in this way. As far as transformed human cell lines (eg Hela, lymphoblastoid etc) it is probably best to be on the careful side.

Hi OK13,

When I worked in a diagnostic lab, the laminar was completely emptied and disinfected every time we finished work. (Our waste container was a beaker, and not a vacuum bottle, so that doesn't really answer your question.)

In the research labs, we left the peristaltic pump or vacuum bottle outside, but autoclaved the tubing every time we used it. We would then open the sterile package inside the hood, anchor one end of the tube inside, and then extend the tubing outside to the pump. We completely emptied and disinfected the hood every time we finished work.

In one lab, we tried leaving flasks inside the hood, but they invariably got moldy (one person had some mold issues). We decided to abandon the flasks entirely, and used beakers inside or a vacuum bottle outside instead.

regards,

lab rat

lab rat on Nov 6 2009, 11:01 PM said:

With GLP and appropriate infection control practice a "moldy flask" never occurs. The flask plus disinfectant is simply cleaned/replaced at the end of every day/work shift. I work in a routine diagnostic lab mostly with HepABC etc/hiv bloods. The rule is that all aspirations result in aerosol generation and that these should remain in the LAF bench and hence so should the waste flask. I agree that a "waste beaker" is often a good idea but sometimes it is necessary to aspirate a solution.

I agree with you Klinmed, but at the time I was the new lab manager arguing with a 30-year veteran who didn't believe wearing gloves or a lab coat in the LAF was necessary. This person also didn't know how to tear down a hood for complete cleaning, and thanked me for my "suggestions" (explanations of GLP and aseptic technique) but firmly insisted that no changes would be made to their routine because they had "been doing it this way for years, and it has worked just fine" for them. Since this person also objected to being "watched" while working, I have no idea whether disinfectant was ever added to the flask.