Webinar Highlights: Preventing Contamination in Cell Culture

Dr. Jessica Wagener, Field Application Specialist, Eppendorf

Dr. Jessica Wagener, a Field Application Specialist focusing on cell handling at Eppendorf Headquarters in Hamburg, Germany, discusses tips and procedures to make your daily cell culture routine safer and more reliable in this exclusive webinar, which you can watch on-demand here.

Learn how to recognize and identify different types of contaminants in cultivated cells, plus find out about tips and tricks for aseptic handling. Read on for highlights from the webinar Q&A session.

How can you see the differences between yeast and bacterial contamination?

The first thing that you can distinguish between yeast and bacteria is that bacteria are much smaller. They are usually black and can show different structures such as rod-like structures. Yeast is much bigger than bacteria and if you look through the microscope you can see that bacteria show movement, whereas the yeast cells never move.

How do I identify cross-contamination of different types of cell line in the same culture?

Short tandem repeat (STR) analysis can be used for cross-contamination identification. You can either carry out this analysis in-house or there are a lot of STR services that allow you to simply send in your culture or your cells of interest and they will test them for you. The results will tell you what kind of cross-contamination you might have in your cell lines.

How do I detect / identify mycoplasma in cell lines?

You cannot detect mycoplasma in cell lines with a light microscope; instead you would require an electron microscope. To detect mycoplasma in cell lines, you should perform a test such as a PCR. There are a lot of commercially available test kits that are very straightforward to use, and I would recommend using one of these to identify if you have a mycoplasma contamination. Learn more about the microscopic analysis of cells and how Eppendorf Cell Culture Consumables can help in this application note.

Are there methods other than PCR for detecting mycoplasma?

There are a few different test methods available: microbiological culture, the direct staining of DNA e.g. with DAPI, ELISA tests, and biochemical reactions. They all have their advantages and disadvantages. For instance, when you use direct DNA staining, you have to be experienced with the readout and interpretation of the test. Microbiological culture takes time to cultivate the mycoplasm, and in that time the potential contamination in your cell culture lab has time to spread to other cultures.

Would cells die if grown without antibiotics and infected with mycoplasma?

Most antibiotics are not effective against mycoplasma at all because mycoplasma lack a cell wall. This is the point that most antibiotics, certainly penicillin and streptomycin, target. Cultivating the cells without antibiotics does not kill the cells that have a mycoplasma contamination. The cells might suffer from the mycoplasma contamination because they are competing for the nutrients that are in the medium, but they won’t die immediately.

What would you recommend for aspirating old media from culture dishes: heat-resistant glass pipettes or single wrapped, sterile pipettes changed after contact with medium?

The problem with heat-resistant glass pipettes is that when you have a Bunsen burner in your biological safety cabinet, the flame of the Bunsen burner disturbs the lamina airflow, so it is never recommended to use a Bunsen burner inside the biological safety cabinet. Using disposable single-wrapped serological pipettes is definitely the more convenient and more reliable method of aspiration when it comes to sterility. Learn how Eppendorf Cell Culture Flasks are designed to offer easy pipette access for ergonomic handling in this application note.

Are Eppendorf pipettes autoclavable?

The standard micropipettes, the Reference® 2 and the Research® plus from Eppendorf, are completely autoclavable without any need for disassembly. The Eppendorf automatic pipettes have to be disassembled and only the non-electronic parts of the pipette are autoclavable.

Do you have any recommendations regarding the usage of disinfectants and what is better: spraying or wiping equipment and surfaces?

To give you a direct recommendation regarding disinfectant is quite difficult, because you always have to consider what kind of microbes you want to get rid of and what kind of materials you want to disinfect. For instance, a lot of disinfectants are corrosive so if you want to disinfect a metal surface you have to make sure that you use a disinfectant such as alcohol, which is not corrosive. When it comes to spraying or wiping, I would always recommend wiping any surface or piece of equipment that you want to disinfect, because the use of spray disinfection is a risk for your health, and only by wiping something can you guarantee a complete wetting of the surface. You should always wait a certain time to let the disinfectant do its job so when you use ethanol for instance, wait until it’s dried (at least 30 seconds) before you start your work.

Is it appropriate to use antimicrobial reagents in the water pan of the CO2 incubator to avoid airborne microbial growth?

If you have an incubator which automatically vaporizes the water and distributes it to the incubator chamber, you should not use antimicrobial reagents. If you have an incubator with a water pan, there is usually a recommendation in the manufacturer’s user manual regarding which bactericides or fungicides to use. Eppendorf recommends using copper sulfate in their incubators to avoid microbial growth of any germs in the water.

Microbiological or lab incubators are mostly lined with stainless steel, copper, or sometimes aluminum. Which one is best to prevent contaminants in your experience?

The copper material works against bacterial growth by preventing reproduction.