In the movie “My Big Fat Greek Wedding,” the father of the bride believes that the glass cleaner Windex^® is a cure-all for any and all ailments. As ridiculous as this sounds, some researchers use antibiotics and cell selection agents in a similar way: “Contamination? Throw some antibiotics on it, it will be fine!”

Antibiotics and cell selection agents are powerful tools used for a variety of research applications. But they’re not miraculous cure-alls. Careful and appropriate usage of these tools is essential for obtaining accurate and reproducible results.

Common mistakes when working with antibiotics in cell culture include improper aseptic technique or aliquot storage, using wrong antibiotic concentrations, ignoring mycoplasma contamination, or the side effects of antibiotics and not checking for combined antibiotic interactions.  

In this article, we’ll discuss 6 common mistakes associated with antibiotics and cell selection agents in cell culture so that you can avoid making these same missteps in your own research.

 

Article Table of Contents

Using antibiotics as a replacement for aseptic technique

Using the wrong concentration

Improper preparation or storage of antibiotic stocks

Not controlling for the side effects of antibiotics

Ignoring mycoplasma contamination

Using multiple antibiotics without checking for interactions

References

 

Using antibiotics as a replacement for aseptic technique

Antibiotics are great for preventing contamination in cell cultures. The best way to prevent contamination, however, is to do your best to make sure your cultures don’t get infected in the first place.

While antibiotics can cover up for some lapses in proper aseptic technique, you usually don’t want to count on them in this way.

Aseptic technique refers to practices used to prevent microbial contamination: using sterile personal protective equipment (gloves, goggles, lab coats, and sometimes masks), establishing and maintaining sterile working areas, and using sterile lab equipment.

The exact standards for proper aseptic technique will differ a bit depending on what exactly you’re doing. However, the constant is that you will want to use aseptic technique to prevent your cultures from getting infected in the first place.

As we’ll cover in a few sections below, antibiotics can have nuanced impacts on the cells that they don’t kill such as influencing their gene expression, metabolism, and more. So, if you can bypass using antibiotics in your culture then you won’t have to worry about these consequences. However, there are several types of cell culture where it is critical to use antibiotics. Knowing when to use antibiotics and when you can skip them is quite important.

In either case, it is critical to use proper aseptic laboratory techniques to keep your cultures sterile. When your technique gets sloppy your cultures will get infected eventually, but you’ll just find out much sooner if you’ve been working without antibiotics.

 

Using the wrong concentration

Different antibiotics and cell selection agents have different working concentrations. This is even more complicated since the ideal working concentration for each antibiotic varies in different types of cells (Delrue et al., 2018).

If you’re working with too little antibiotic, then you run the risk that cells without the resistance gene will still grow. If you’re using too much antibiotic, then even the cells with the resistance gene will still die or have their growth stalled. This is one of those situations where knowing the just-the-right concentration for your experimental setup can really save you time and effort in the long run.

If you need to define the ideal concentration of antibiotic or cell selection agents for your cells, then you will want to run a kill curve, ideally against both resistant and sensitive cells. Resistant cells are those that express the resistance gene, whereas sensitive cells lack the resistance gene. By titrating the antibiotic into both cell types, you can find the concentration that maximizes the selectivity between resistant and sensitive cells (Figure 1).

Figure 1. Kill curves against resistant (purple) and sensitive (orange) cells. Usually, you will want to use the lowest antibiotic concentration that reliably eliminates sensitive cells while maintaining acceptable survival and growth of resistant cells, as indicated by the selectivity arrow. 

 

Improper preparation or storage of antibiotic stocks

Let’s be honest, making stock solutions is not the part of research that gets you out of bed in the morning! But it is really crucial for getting consistent and reproducible results. When it comes to making antibiotic stock solutions there are quite a few ways to flub the preparation, storage, or use:

• Miscalculating the concentration
• Using the wrong solvent
• Improper storage of antibiotic powder or stock solutions
• No filter sterilization
• Adding antibiotics to the media while it’s still hot

It’s important to accurately calculate the antibiotic stock and final concentrations. Mistakes in the calculation will lead to complications with having the wrong antibiotic concentration, as we just discussed in the previous section.

Most antibiotics are readily soluble in water, so water is a great choice when preparing stock solutions. However, a few antibiotics are poorly soluble in aqueous solutions. Macrolides such as azithromycin, clarithromycin, and erythromycin are more soluble in organic solvents such as ethanol and DMSO. If you try using water for these antibiotics, the stock solution concentrations will be much lower than expected.

When antibiotic powders are received from GoldBio, it is important to store them properly until you’re ready to use them or to make stock aliquots. The exact storage condition varies by antibiotic, but many should be stored in a refrigerator at 4 °C.

Some antibiotics require additional precautions such as storing them desiccated and limiting light exposure.

Make sure to check, and follow, the storage specifications for the antibiotics that you’re using.

After solubilizing your antibiotic, it is important to filter sterilize it to make sure that there are not any microorganisms in your stock solution. Otherwise, you may contaminate your cultures when you add antibiotics.

If you’re making antibiotic aliquots for future use, follow the recommended storage conditions for your specific antibiotic. Many antibiotic stock solutions can be stored frozen in a -20 °C freezer.

When you’re adding one of your aliquots to your cell growth media, make sure that media isn’t still boiling hot from the autoclave. Hot media will degrade the antibiotic, ruining its potency. Allow the media to return to room temperature, or at least the growth temperature for your cultures, before adding antibiotics in.

If you have questions about your antibiotic stock solutions, the easiest way to check if they are still good is to test them. By making sure that your stock is killing sensitive cells, and not killing resistant cells, you can be confident that your stock solutions are still good (Figure 2).

Figure 2. If your antibiotics are still working, sensitive cells will have very few colonies, whereas resistant cells will have a lot (left). In contrast, if your antibiotics have lost activity then there will be a lot of colonies for both sensitive and resistant cells (right).

 

Not controlling for the side effects of antibiotics

For certain experiments it is important to have a control sample to make sure that antibiotics are not impacting the biological factor that you’re measuring. Ideally, antibiotics would kill sensitive cells while not impacting resistant cells at all.

However, antibiotics can have more subtle impacts on resistant cells, even if they don’t kill them, such as by changing the cells’ gene expression, metabolism, and differentiation (Figure 3) (Cohen et al., 2006; Elliot and Jiang, 2019; Nygaard et al., 2015; Ryu et al., 2017; Varghese et al., 2017).

Figure 3. Common side effects from antibiotics on surviving cells include changes in metabolism, gene expression, and differentiation.

By having a control where you independently assess any effect the antibiotic is having on your experimental readout, you can make sure that you’re not confounding results due to cellular stress responses to antibiotics. See this article for more details about these controls and why they’re so important. 

In mammalian cell culture you don’t always have to use antibiotics, and in some situations, it is actually advisable to skip them. Knowing when to skip antibiotics will simplify your experimental approach in terms of not needing an antibiotic-only control.

 

Ignoring mycoplasma contamination

If you’ve ever wondered what the nightmare scenario is for cell culture (and let’s be honest, who hasn’t), well meet Mycoplasma.

Mycoplasma are tiny bacteria that lack a rigid cell wall, and their contamination of cell lines is pervasive; upwards of 10% of cell lines are infected with this annoying microorganism (Olarerin-George & Hogenesch, 2015).

Working with contaminated cell lines can impact a wide range of experiments because whichever measurement you are reading out will come from both the Mycoplasma cells and your intended cells in the culture.

Since Mycoplasma lack a cell wall, penicillins (ampicillin, carbenicillin, etc.) and vancomycin that target the cell wall are ineffective at targeting them (Gautier-Bouchardon, 2018). Other antibiotics such as gentamicin and kanamycin do work on Mycoplasma, but effective concentrations may be higher than those typically used in cell culture, and susceptibility varies among Mycoplasma species (UNC Lineberger Comprehensive Cancer Center, 2019).  

If you suspect you might have Mycoplasma infection, definitely don’t ignore it! There are methods for identifying Mycoplasma infection and treating it including a simple PCR test you could do yourself.

Using contaminated cells runs the risk of providing inaccurate and hard to reproduce results because Mycoplasma confounds your experimental measurements either directly or indirectly.

Often the best path forward when dealing with Mycoplasma contamination is to throw away the infected cell line and start over with a fresh, uninfected aliquot. 

 

Using multiple antibiotics without checking for interactions

When using multiple antibiotics at the same time, one antibiotic can sometimes impact the potency of the other. In most cases they don’t, and both antibiotics combine as you would expect, which is called additive.

However, antibiotics can work more potently as a pair (synergistic) or less potently as a pair (antagonistic). When antibiotics are synergistic or antagonistic this is called an interaction.

The tricky thing about how antibiotics combine is that it is also cell-type specific (Brochado et al., 2018). What this means is that a pair of antibiotics could be additive in one type of cell, synergistic in another, and antagonistic in a third cell type.

If antibiotics have an interaction, then combining them at the same concentration as they would be used as single agents will be either too potent or too weak at killing or stalling cell growth.

So, if you’re using antibiotics and cells that haven’t been combined before in your lab, then it’s worth testing to see if those antibiotics have an interaction.

Checking for interactions is especially important for experiments with longer selection protocols, which are more frequently done in mammalian cells. By getting the antibiotic concentration just right, you can make sure that you’re selecting for the desired cells and minimizing side effects.

Shorter experiments, such as expressing proteins in bacteria, tend to be less sensitive to antibiotic interactions, though you could unnecessarily slow down culture growth with synergistic antibiotics.

 

GoldBio is your go-to source for a wide selection of affordable and reliable antibiotics. Following this guidance and using GoldBio’s antibiotics is a sure-fire recipe for experimental success!

 

References

Brochado, A. R., Telzerow, A., Bobonis, J., Banzhaf, M., Mateus, A., Selkrig, J., Huth, E., Bassler, S., Zamarreño Beas, J., Zietek, M., Ng, N., Foerster, S., Ezraty, B., Py, B., Barras, F., Savitski, M. M., Bork, P., Göttig, S., & Typas, A. (2018). Species-specific activity of antibacterial drug combinations. Nature, 559(7713), 259–263. https://doi.org/10.1038/s41586-018-0278-9

Delrue, I., Pan, Q., Baczmanska, A. K., Callens, B. W., & Verdoodt, L. L. M. (2018). Determination of the Selection Capacity of Antibiotics for Gene Selection. Biotechnology journal, 13(8), e1700747. https://doi.org/10.1002/biot.201700747

Elliott, R. L., & Jiang, X. P. (2019). The adverse effect of gentamicin on cell metabolism in three cultured mammary cell lines: "Are cell culture data skewed?". PloS one, 14(4), e0214586. https://doi.org/10.1371/journal.pone.0214586

Gautier-Bouchardon A. V. (2018). Antimicrobial Resistance in Mycoplasmaspp. Microbiology spectrum, 6(4), 10.1128/microbiolspec.arba-0030-2018. https://doi.org/10.1128/microbiolspec.ARBA-0030-2018

Nygaard, U. H., Niehues, H., Rikken, G., Rodijk-Olthuis, D., Schalkwijk, J., & van den Bogaard, E. H. (2015). Antibiotics in cell culture: friend or foe? Suppression of keratinocyte growth and differentiation in monolayer cultures and 3D skin models. Experimental dermatology, 24(12), 964–965. https://doi.org/10.1111/exd.12834

Olarerin-George, A. O., & Hogenesch, J. B. (2015). Assessing the prevalence of mycoplasma contamination in cell culture via a survey of NCBI's RNA-seq archive. Nucleic acids research, 43(5), 2535–2542. https://doi.org/10.1093/nar/gkv136

Ryu, A. H., Eckalbar, W. L., Kreimer, A., Yosef, N., & Ahituv, N. (2017). Use antibiotics in cell culture with caution: genome-wide identification of antibiotic-induced changes in gene expression and regulation. Scientific reports, 7(1), 7533. https://doi.org/10.1038/s41598-017-07757-w

UNC Lineberger Comprehensive Cancer Center. (2019, March 1). Mycoplasma. https://unclineberger.org/tissueculture/contaminant/mycoplasmacontam/

Varghese, D. S., Parween, S., Ardah, M. T., Emerald, B. S., & Ansari, S. A. (2017). Effects of Aminoglycoside Antibiotics on Human Embryonic Stem Cell Viability during Differentiation In Vitro. Stem cells international, 2017, 2451927. https://doi.org/10.1155/2017/2451927

 

 

Tags

antibiotics cell culture Simon Currie

• Share :
• Deel
• X
• LinkedIn

---