We recently finished our Ask the Expert discussion on Methods for Reducing Biological Contamination Risks in Your Lab. The result was a great discussion of real examples of lab contaminations and solutions to common biological contamination risks. Topics included fungal contamination, microbial contamination, avoiding recontamination after clean up, eliminating contamination in cultures, getting rid of white flocks contamination, problems with sharing cells and the use of antibiotics.

Unfortunately, biological contamination is one of the most common cell culture lab problems. Levels of contamination vary greatly and can be minor problems or major catastrophes. Prevention of biological contamination cannot be absolute, biological contamination does sometimes happen especially given that it is recommended that culturists do not use antibiotics. So how do we best avoid the risk of contamination and what to do after contamination occurs? These are just some of the issues discussed during our Ask the Expert discussion.

This Ask the Expert Session was Sponsored by Life Technologies and hosted by Timothy Fawcett, Ph.D.  Dr. Fawcett has been in the biotechnology business for over 30 years.  Trained as a biochemist he has held senior positions in both academics and industry and has been a mentor to many young scientists throughout his career.  For the last 12 years, Dr. Fawcett has been the Director of the BioTechnical Institute of Maryland (BTI) a non-profit institute located in Baltimore, Maryland.  He is also the Founder and Director of BioSciConcepts, a social venture of BTI that provides hands-on training for professional scientists in cell culture, baculovirus based expression, as well as topics such as molecular biology, PCR and real-time PCR. BioSciConcepts is an internationally recognized provider of expertise in the biological sciences and has provided consultation services to several small and large biotechnology companies.

Below is a sneak peek of the discussion. For a full transcript of the discussion, please see – Ask the Expert Methods for Reducing Biological Contamination Risks in Your Lab.

Question:

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.

The Answer:

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 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 an hour then incubate at 37C in a bacterial incubator. I hope this helps.

Question:

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

The Answer:

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 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:

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

The Answer:

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.