Methods for Reducing Biological Contamination Risks in Your Lab

Probably the most common and annoying problem encountered in a cell culture laboratory is biological contamination.   Contamination can be a minor nuisance or a major catastrophe depending on the type of contamination present and the culture method and volume utilized.  Today reputable media and sera providers do a very good job of filtering their products using specialized filtration systems and therefore most if not all contamination is due to problems with technique, incomplete sterilization of equipment, or some other failure.  Prevention from biological contamination cannot be absolute, biological contamination does sometimes happen especially given that it is recommended that culturists do not use antibiotics.

It is possible to reduce the severity and frequency of biological contamination by following procedures such as the aseptic technique. What are your questions and comments about biological contamination and how can we help you overcome contamination problems in your lab.

Question 1

How can we eradicate the fugal contamination on our lab?

It is difficult to provide an answer unless I have more information. Are you writing about a contamination in a cell culture dish, or the whole lab, or an incubator? Are you having repeated problems with the same contamination? Thanks and I hope to hear back with more details.

Question 2

Is it possible the elimination of contaminated culture from bacterial, yeast, fungal etc.?

Yes, but usually it is difficult and hard on the animal cells. It also depends on the type of contamination. Of course the best solution is to have non-contaminated cells that are frozen away for just that reason. If you are talking about a primary culture, I suggest isolating the cells with a double antibiotic-antimycotic for the first part of the isolation then 1X for the rest. Without knowing more about your culture contamination I would suggest 1 X Pen-strep and fungizone (amphotericin B); (10,000 units/mL of penicillin, 10,000 µg/mL of streptomycin, and 25 µg/mL of Fungizone is the 100 X concentration). The antibiotics penicillin and streptomycin prevent bacterial contamination due to their effective combined action against gram-positive and gram-negative bacteria. Fungizone prevents fungal contamination of cell cultures due to its inhibition of multi-cellular fungus and yeast. It will take multiple passages to eliminate the contamination and normally I would suggest going back and forth between with the antibiotic/antimycotic cocktail and without to see if any contamination continues to grow. In the event that the contamination is not killed over a curing course then switching to another antibiotic and antimycotic will help.

Question 3

How can you ensure that your frozen cells, or cells you obtain through a collaboration aren’t contaminated?

The simple answer is you can not. Normally when you get cells from a repository or a company you might be more assured but you can never be 100 % confident a new line is non-contaminated. This is why it is best to have a quarantine incubator or a way to grow new cells when they arrive, away from your other cells so that you can test for a problem before you freeze back or introduce your new untested cells into your working cell culture room or incubator.

Question 5

After thoroughly cleaning and sterilizing our lab after a bacterial contamination, we are worried about recontamination. Any thoughts about things we can do to ensure sterility after cleaning and any suggestions on how to test to be sure that the contamination is gone.

I am assuming you had a big contamination if you had to go through such extremes but I don't know the particulars or where you think the contamination came from. So, I would first make sure your biological safety cabinets are working and that your incubators are functioning properly. For your incubators, I would have extra sets of shelves and water pans. I would autoclave one set and exchange every week or so. Water pans should be autoclaved and then use sterile water and add a fungal inhibitor. Use disposable pipets and a clean pipette aid for adding media to dishes. I would also recommend using disposable lab coats and gloves along with a sticky mat at the door. These are some suggestions that may help. To test for contamination some some people will use open Trypticase Soy Yeast agar plates (TSY Plates) without antibiotics. Place them in areas of the cell culture room. Leave them open for a hour then incubate at 37C in a bacterial incubator. I hope this helps.

Question 6

Why is it so frowned upon to use antibiotics in cell culture now?

There are times when using antibiotics is justified, for example if you are isolating a primary culture and you can not risk a contaminated sample of tissue. Having mentioned that however, during regular cell culture the use of antibiotics should be avoided. The reasons are many but to list a few….antibiotics can result in persistent low-level contaminations and continued use can allow for resistant organisms to grow. Also antibiotics can be harmful to animal cells depending on both the concentration and media used. Additionally, and I think importantly, the use of antibiotics can mask poor aseptic technique which can become a large problem down the road. It is best to not use antibiotics and learn how to work properly and have good technique which is the best way to reduce contamination.

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