During tissue culture work, finding unwanted contaminated culture flasks can create many issues, including a question about the data validity from the past and current experiments.

Depending on the source of contaminants, you can detect cell culture contamination by using a light microscope, Gram stain, isothermal amplification, or PCR. While discarding contaminated cell culture flasks is helpful, there are some items in the lab to be discarded (such as ongoing experiments), or decontaminate, including the incubator and tissue culture cabinet.

To prevent contamination, lab personnel must always follow the procedures of aseptic technique. However, when contamination does occur, this article will also cover some approaches to identify cell culture contaminants.


In this article

Why is it important to minimize contamination in cell culture?

What are different types of contaminants in cell culture?

How do you identify bacterial and fungal contamination in cell culture?

How to rescue contaminated cell culture?

How to detect Mycoplasma in cell culture

How viral contamination can be detected in cell culture?

Associated products

References


Why is it important to minimize contamination in cell culture?

Cell contamination leads to all sorts of issues. It doesn’t just impact whether a technique will work or not – it can impact the results and outcomes of your experiment as well. Contamination can also introduce many variables that could go unaccounted for.

Therefore, it is vital to detect, handle and prevent contamination in cell culture, because:

  • Contaminated cultures can affect the growth, morphology, physiology and metabolism of the cells, so the use of these cells in a study can cause doubt in the validity of the results and a retraction of a published work.
  • These cells can cause the loss of time because it’s necessary to repeat the previous and ongoing experiments.
  • Contamination can also cause an almost exponential loss of money, particularly from the costs of media, flasks, and sera.
  • Some microbial contaminants may be potential health hazards for laboratory personnel, particularly viral contaminants.


What are different types of contaminants in cell culture?

There are two types of contaminants in cell culture: chemical contaminants and biological contaminants.

Chemical contaminants usually come from equipment, media, water, or sera.

As for biological contaminants, the most common causes are bacteria, fungi, and yeast. Typically, these types of contaminations are obvious and you can detect this problem visually. However, another source of contaminants, such as viruses, is much harder to detect and deal with.


How do you identify bacterial and fungal contamination in cell culture?

Bacteria and fungi are common contaminants in animal cells in culture and their presence can be detected by observing these following changes (Geraghty et al., 2014):

  • An increase of turbidity of the antibiotic-free medium. In other words, the color of the medium becomes cloudy.
  • A change in pH. For example, the medium can change acidic with bacteria contaminants, whereas the presence of fungi can make the medium to become alkaline. In the media containing phenol red, bacteria are more likely to change the medium’s color to yellow. However, when some fungi contaminate the cell culture, the medium can change its color into pink.
  • A distinct shape under the light microscope. For example, there might be clumps or budding small objects observed for fungal contamination, whereas moving small objects with a different shape from your cell lines might indicate bacterial contamination.

Another way to detect bacteria and fungi is by performing microbiological culture in special media. After growing the culture, test the bacterial cells by using Gram stain (Geraghty et al., 2014). To perform this method, you will need to prepare some solutions, including crystal violet.

If you need a protocol for Gram staining, GoldBio has a really helpful protocol that you can find here: Gram Staining Protocol.


How to rescue contaminated cell culture?

Oftentimes, you can immediately get rid of the contaminated cell culture and start a new culture from your frozen stock. Nevertheless, if you really need to keep it, one way to rescue it is by treating it with some antibiotics and grow the cells in the culture flask in a separate incubator. After the contaminants are completely clear, establish a frozen stock for future work, grow the remaining culture for a month, and retest it.

Below are some antibiotics commonly used for treating contaminated cell culture:

Organism

Antibiotic

Solvent

Working Concentration


Gram Positive Bacteria

Ampicillin

Water

100 mg/liter

Erythromycin

2M HCl or EtOH

100 mg/liter

Gentamicin sulfate

Water

50 mg/liter

Kanamycin sulfate

Water

100 mg/liter

Neomycin sulfate

Water

50 mg/liter

Penicillin-G

(Potassium salt)

Water

100 mg/liter

Streptomycin

sulfate

Water

100 mg/liter

Tetracycline HCl

Water

10 mg/liter


Fungi (molds and yeasts)

Amphotericin B

DMSO; DMF

2.5 mg/liter

Nystatin

DMF

50 mg/liter


Mycoplasma

Gentamicin sulfate

Water

50 mg/liter


To get the full protocol with instructions on how to treat contaminated cell culture, this protocol is extremely helpful. You can find it here:

Antibiotic Treatments of Microbe-Contaminated Cell Cultures


How to detect Mycoplasma in cell culture

Mycoplasma is a group of bacteria that commonly contaminates cell culture, such as in human cells and other mammalian cells. Similar to other microbial contaminants, Mycoplasma contamination can widely affect cell physiology and metabolism (Nikfarjam & Farzaneh, 2012).

There are many methods available to detect Mycoplasma in cell culture, including isolation on selective microbiological growth media, direct or indirect DNA stain by using Hoechst 33258, PCR, Nested PCR, ELISA, autoradiography and immunostaining.

However, one efficient method to detect Mycoplasma is by using isothermal amplification method, such as GoldBio’s kits: certus QC-mycoplasma Detection Kit and certus QC - mycoADVANCED Detection Kit. The advantages of using these kits are high sensitivity, simple, and fast.

To learn more in details about some methods to detect Mycoplasma in cell culture, find GoldBio article below:

Why Test for Mycoplasma in My Cell Culture?


How viral contamination can be detected in cell culture?

Viral contamination is hard to visualize under a regular light microscope, but it can be detected by other methods, such as electron microscopy, immunohistochemistry and ELISA assays (Geraghty et al., 2014). In addition, PCR or RT-PCR with specific primers can also be performed to allow amplification of target viral genes and identify the contaminants.

To get initial information about the possible cause of the contamination, a test by using electron microscopy is useful to find the presence of viral particles. Later on, further testing by using other methods, such as immunohistochemistry or immunofluorescence assays, are also helpful to find out more about the strain of the virus.

Unfortunately, it is hard to recover the cell lines contaminated by viruses, because there are no treatments for it. Therefore, the contaminated flasks must be discarded to prevent cross contaminations of other cell lines.

cell culture contamination


Associated products

Browse our products below:

Dye

Crystal Violet (Cat. No. C-328)

Antibiotics

Ampicillin (Cat. No: A-301)

Erythromycin (Cat. No. E-300)

Gentamicin sulfate (Cat. No. G-400)

Kanamycin sulfate (Cat. No. K-120)

Neomycin sulfate (Cat. No. N-620)

Penicillin-G (Potassium salt) (Cat. No. P-303)

Streptomycin sulfate (Cat. No. S-150)

Tetracycline HCl (Cat. No. T-101)

Amphotericin B (Cat. No. A-560)

Nystatin (Cat. No. N-750)

PCR Kit

Taq DNA Polymerase with Dye plus dNTP (Cat No. T-517)

RT-PCR Kit (Cat No. R-920)

Mycoplasma Detection Kit

certus QC-mycoplasma Detection Kit (Cat No. MD-250)

certus QC - mycoADVANCED Detection Kit (Cat No. MD-500)

Solvent

Sterile Filtered DMSO (Cat. No. D-361)


References

Evanko, D. (2013). A retraction resulting from cell line contamination : Methagora. (n.d.). Blogs.nature.com. Retrieved May 27, 2021, from http://blogs.nature.com/methagora/2013/09/retraction_resulting_from_cell_line_contamination.html.

Geraghty, R. J., Capes-Davis, A., Davis, J. M., Downward, J., Freshney, R. I., Knezevic, I., Lovell-Badge, R., Masters, J. R. W., Meredith, J., Stacey, G. N., Thraves, P., & Vias, M. (2014). Guidelines for the use of cell lines in biomedical research. British Journal of Cancer, 111(6), 1021–1046. https://doi.org/10.1038/bjc.2014.166.

Meet the Culprits of Cell Culture Contamination. (n.d.). Cell Science from Technology Networks. Retrieved May 27, 2021, from https://www.technologynetworks.com/cell-science/articles/meet-the-culprits-of-cell-culture-contamination-329605.

Merten, O.-W. (2002). Virus contaminations of cell cultures -A biotechnological view. Cytotechnology, 39, 91–116. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3463984/pdf/10616_2004_Article_5113486.pdf.

Nikfarjam, L., & Farzaneh, P. (2012). Prevention and detection of Mycoplasma contamination in cell culture. Cell Journal, 13(4), 203–212. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC35844...

Ryan, J. (n.d.). Understanding and Managing Cell Culture Contamination. https://safety.fsu.edu/safety_manual/supporting_docs/Understanding%20and%20Managing%20Cell%20Culture%20Contamination.pdf.

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.