Cell cultures are extremely prone to contamination, especially when the work area and materials are not maintained in sterility. Some signs of contamination are more obvious than others. The best way to catch contamination early is to routinely check your cell culture via microscopy in order to become familiar with how your cells normally look. The following information will give you some helpful tips to reduce the chances of contamination in your samples.
In this blog post, we'll go over how to identify and handle cells with:
- Bacterial/Fungal/Microbial Contamination
- Mycoplasma Contamination
- Cell Line Cross-contamination
- Virus/Pathogen Contamination
1. Bacterial/Fungal/Microbial Contamination
Bacteria and fungi are all around us; on material surfaces, in the air, and in water. Many species of fungi, in particular, are not only present in non-sterile water, but can also spread easily in air when the fungal fibres form colonies, called molds. In addition, it is easy to introduce contamination when bringing non-sterile equipment, such as micropipettes, pipette tip boxes, falcon tubes, etc., into the BioSafety Cabinet (BSC) space. If you observe signs of microbial contamination in your culture, remove the contaminated cultures immediately to ensure the contamination does not spread to neighbouring cultures and nearby equipment:
a. What should I look out for?
A good practice is to take frequent images of your cell cultures and note detailed observations in your lab notebook. You can then refer to your notes if you notice changes in your cells.
Under a light microscope, you can routinely check for:
- Cloudy appearance in media caused by bacterial colonies/fungi
- Cell morphology changes
- Sudden detachment of adherent cells, or death of cells
b. What are those dark spots?
Oftentimes, you will see dark spots throughout your cultures. These dark spots within your culture can easily be mistaken for contamination, but it may just be cell debris. Certain cell lines will release more debris into the media than others, and a larger amount of debris may be found if your culture contains many dead or unhealthy cells, as they are undergoing apoptosis.
To differentiate between contamination and cell debris, take a look at the cells at a higher magnification and observe how the dark spots move:
- Cell debris: dark spots that move with the motion of the media
- Bacterial/fungal contamination: dark spots that move independently from the motion of the media, sometimes almost with a vibrating or circular motion, as some species of bacteria and fungi have cilia or flagella that allow them to move within the media
c. How can I prevent bacterial/fungal contamination?
To decrease the chances of contamination, it is best to practice aseptic technique. A list of tips for good aseptic technique is listed in this blog post/article. One common practice is to add a low concentration of Penicillin/Streptomycin solution at ~0.5-1.0% into the complete media.
Although most cell lines are not sensitive to these antibiotics, there are some exceptions - it is always a good idea to follow the recommended culturing protocol for your cell line to ensure proper growth conditions.
To test your cell line’s antibiotic sensitivity, compare the growth of your cells in complete media with and without Penicillin/Streptomycin. If there is no difference in cell growth between the two conditions, you should be able to use Penicillin/Streptomycin.
Figure 1: Examples of cell cultures with and without microbial contamination. A. Bacterial contamination at 10X magnification. The dark spots are very prominent throughout the entire culture after 24 hours. B. Bacilli contamination cell culture at 20X magnification. C. Adherent epithelial cells viewed under 10X magnification. The area between the adhered cells have no dark spots alluding to bacterial or fungal contamination. The cells are of proper morphology for their cell type.
abm’s BacAway is a formulation of antibiotics that target gram-positive and gram-negative bacteria.
abm’s Amphotericin B (Fungizone) is an antifungal treatment composed of amphotericin B and deoxycholic acid.
2. Mycoplasma Contamination
There is one genus of bacteria that is an extremely common contaminant in cell culture but not detectable by the naked eye or even by a light microscope - Mycoplasma! Because it is such a small organism (measuring 0.2 - 0.3 μm), it can be present in cell culture media at high concentrations without causing turbidity or pH changes. Filtering your media will not prevent mycoplasma contamination as its size is small enough to pass through the pores of the filter.:
a. Why is Mycoplasma a problem?
Mycoplasma affects virtually every aspect of a cell’s behavior and may cause:
- Inhibition of proliferation due to nutrient withdrawal and secretion of harmful metabolic products
- Immunological reactions
- Effects on virus proliferation and viral infection rate
- Chromosomal aberrations
- Changes to microarray and gene expression profiles
- Decreased cell transfection rate
- Interference in DNA and protein isolation workflows
Figure 2: Absence (A) and presence (B) of mycoplasma in 3T3 cultures.
b. How do I know if I have Mycoplasma contamination?
Mycoplasma can survive in both wet and dry conditions and is therefore easily transferable between one culture to another. It is estimated that contamination rates can be up to 47% in academic labs.
There are numerous ways to detect mycoplasma contamination including:
- Histological staining
- Enzyme activity measurement
- ELISA or fluorescence detection
- DNA staining (DAPI or Hoechst 33258)
- PCR amplification of mycoplasma DNA
Figure 3: Amplification products from cell cultures tested using abm's PCR Mycoplasma Detection Kit were run on a 1% electrophoresis agarose gel. Lane 1 is the negative control, lanes 2-23 are cell culture samples, and lane 24 is the positive control.
Most of the methods above are time-consuming and require specialized equipment - we recommend the PCR method as the quickest and easiest mycoplasma detection method. In this method, mycoplasma DNA in your cell culture sample is amplified and the products are run on an agarose gel for comparison against a positive control.
abm’s PCR Mycoplasma Detection Kit amplifies mycoplasma DNA directly from your cell culture supernatant, enabling detection of 200+ mycoplasma species in only 2 hours.
c. Can I rescue my mycoplasma-contaminated cells?
In the war against mycoplasma, penicillin and streptomycin are ineffective.
For cell lines that are positive for mycoplasma contamination, discard the cells as soon as possible to prevent the spread of the contamination to the rest of your cell cultures. You may also want to clean media and equipment using one or all of the following methods:
- Use of detergents
If your cell lines are too important to discard, the easiest method to decontaminate your cultures is to use a chemical treatment containing antibiotics effective against mycoplasma such as tetracycline, macrolides, and quinolones. In some cases, the mycoplasma strains may be resistant to your antibiotic cocktail, in which case a trial and error process is needed to find a mixture of antibiotics that work.
Depending on the cell type, you may also need to dilute your antibiotic cocktail as its potency can harm certain cell lines. Common dilutions that are often used to resolve mycoplasma are 1:500, 1:1000 and 1:10,000 depending on the intensity of the contamination. Keep in mind that mycoplasma decontamination often takes weeks to months to resolve.
abm’s Mycoplasma Elimination Cocktail is rapid and non-toxic to most mammalian cell lines and can be added directly to cell culture to eliminate mycoplasma in only 4 passages (2 weeks).
3. Cell Line Cross-contamination (STR Profiling and Species Identification)
a. Why is it important to check for cell culture cross-contamination?
Cross-contamination between cell lines has become a big issue in cell culture work as the body of cell culture based research increases. From a publication in 2003, it has been reported that cross-contamination happens 16-35% of the time, with the HeLa cell line being the most common contaminant - having contaminated more than 90 cell lines reported at that time. The HeLa cell line grows much quicker and more robustly than most cell lines, giving it the ability to take over other cell cultures.
Significant consequences to cross-contamination events occur when aseptic techniques are not followed properly and it can lead to inaccurate scientific conclusions. For this reason, researchers submitting publications for journal approvals or grants are required to authenticate the cell line(s) that are part of the publication prior to getting approved. If the authentication shows that there is cross-contamination, the results of the research are invalidated.
b. What is STR Profiling?
STR profiling stands for “short tandem repeats” profiling. This test confirms the identity of a species based on the species’ own distinct STR segment. For example, STR Profiling can be used to confirm a cell line is of human origin by confirming the presence of a segment of repeating 1-6 base pairs in human DNA that is distinct to the human species.
c. When should you do an STR Profile for your cells?
This test should be done for the following reasons:
- If it is the first time you are working with the cell line and you just obtained it from another source (especially for novel cell lines)
- If you are establishing a new cell line from a tissue sample and it will be published in a journal or used to apply for a grant
- Routine checks every few months to ensure that no contamination has occurred during culturing
- If the cells are not looking/functioning as expected
d. Are there ways I can prevent my cell cultures from being cross-contaminated?
In cell culture, it is often easy to cross-contaminate cells across different species if you are working on more than one cell line at a time, or even from errors such as mis-labeling of samples. To reduce the chances of cross-contamination, here are some good techniques to follow:
- Work with one cell line at a time while in the BioSafety Cabinet (BSC) and clean BSC and associated equipment/materials thoroughly in between assays
- Regularly monitor the possibility of changes in cell morphology, growth and function (which means you should be well aware of your cell’s characteristics)
- Aseptic technique is a must to avoid contaminating cell cultures as well as the lab’s shared equipment, reagents, and media
- Pre-label vials/flasks/plates to reduce the risk of mis-labelling samples
If your laboratory does not have a way of identifying cell line species, you can always send your cell line to a laboratory for testing.
abm’s Cell Line Authentication Services can help you identify your human, mouse, rat and canine cell lines.
abm’s Cell Line Species Identification Service can help you identify any cross-contamination between different cell lines.
4. Virus/Pathogen Contamination
a. Are there other kinds of contamination I should be watching out for?
Cell cultures are not only prone to contamination with bacteria, fungi and mycoplasma, but they can also be contaminated with pathogens such as viruses. Host sources can contain endogenous viruses that the cell cultures may inherit and animal-derived reagents may also contain viral contaminants. Common virus contaminants include HPV, HBV, HSV, Cytomegalovirus, and Parvovirus spp. which may not only harm your current cell cultures, but also cause disease outbreaks when these contaminants come into contact with animal hosts. It is therefore a good idea to obtain a profile of possible viral contaminants in your cell cultures to have a better understanding of environmental risks not for laboratory personnel.
abm’s IMPACT Contaminant Detection Service detects pathogens from common animal models used to produce cell lines.
abm’s h-IMPACT Pathogen Testing Service tests for common human pathogens in cell cultures.
Ligasová, A., Vydržalová, M., Buriánová, R., Brůčková, L., Večeřová, R., Janošťáková, A., & Koberna, K. (2019). A New Sensitive Method for the Detection of Mycoplasmas Using Fluorescence Microscopy. Cells, 8(12), 1510. https://doi.org/10.3390/cells8121510
Reid Y, Storts D, Riss T, et al. Authentication of Human Cell Lines by STR DNA Profiling Analysis. 2013 May 1. In: Sittampalam GS, Grossman A, Brimacombe K, et al., editors. Assay Guidance Manual [Internet]. Bethesda (MD): Eli Lilly & Company and the National Center for Advancing Translational Sciences; 2004-. Available from: https://www.ncbi.nlm.nih.gov/books/NBK144066/
Young, L., Sung, J., Stacey, G., & Masters, J. R. (2010). Detection of Mycoplasma in cell cultures. Nature Protocols, 5(5), 929–934. https://doi.org/10.1038/nprot.2010.43