You’re doing a transfection experiment. The efficiency is far too low, so you call technical support to help troubleshoot the problem. One of the first things they ask is whether you’ve screened for Mycoplasma contamination.

Mycoplasma is the genus of a group of hardy bacteria that are common laboratory contaminants. These cells are very small—less than 1 μm in diameter—and come in a variety of shapes. Additionally, they have no cell wall, making them resistant to common antibiotics that target cell wall synthesis. They are difficult to detect, challenging to get rid of, and can have a negative impact on your experiments—one example of which is the transfection frequency.

“There can be various possible reasons for low transfection efficiency, but in many cases, it is because of Mycoplasma contamination,” says Claudia Schwartz, Ph.D., product manager of Lonza Bioscience Solutions. “Once you eliminate the contamination (e.g., by starting a fresh culture), transfection efficiency improves significantly. Therefore, Mycoplasma testing is one of our standard troubleshooting approaches.”

What’s the big deal?

According to Kevin Grady, senior product line business manager of the American Type Culture Collection (ATCC), an estimated 15–35% of cell cultures used in laboratories across the world are contaminated with Mycoplasma. But what does this mean for research?

“Mycoplasma contamination can result in a number of deleterious effects,” Grady explains, “for example, chromosomal aberrations, disruption of nucleic acid synthesis, changes in membrane antigenicity, inhibition of cell proliferation and metabolism, decreased transfection rates, changes in gene-expression profiles, and cell death.”

Schwartz adds that the reliability, reproducibility, and consistency of results are all impacted by Mycoplasma contamination. “This represents a major problem for basic research as well as for the manufacturing of bio products,” she says. “Standard testing for Mycoplasma is an important quality control method.”

Where does it come from?

The most common sources of Mycoplasma contamination are these:

1. Infected cell cultures sent from another lab

2. Contaminated cell culture reagents

3. Laboratory personnel

“The best practice is to adopt appropriate handling and testing procedures to minimize the risk of contamination from these sources,” Grady says. “Always use certified or authenticated cell lines. Use effective quality control testing to detect contamination in media and reagents used in cell culture.”

When you receive new cells—especially if they come from another lab—make sure to quarantine them while you screen for Mycoplasma. Only move them into the incubator with the rest of your cells when you are sure they are clean.

“Working with only one cell line at a time in the hood, cleaning the hood between cell lines, and using a dedicated media bottle for each cell line can significantly reduce the danger of cross-contamination from other cultures,” Schwartz suggests. She also recommends that you purchase “high-quality cell culture media and sera from reputable manufacturers.”

To avoid contaminating your cell cultures yourself, make sure to follow good cell culture practice: “Wearing appropriate protective equipment (e.g., sterile gloves, lab coat, safety glasses) as well as washing gloved hands with 70% ethanol and allowing to air dry for 30 seconds helps to reduce the contamination risk,” Schwartz says. “Also, directing verbal communication away from the biosafety cabinets is an important aspect that is often disregarded.”

However, despite your best efforts, it’s possible that someday you’ll find that you’ve got a Mycoplasma infection in one of your cell cultures. Modify your cell handling practices now so that, if the time comes, you can prevent Mycoplasma from spreading to the rest of your cultures.

“Aerosols and operator error are the most common means of transferring Mycoplasma contamination from one culture to another. The safest way to limit your risk of Mycoplasma contamination is to use good aseptic technique,” Grady explains. “It is prudent to use a vertical laminar flow hood that is isolated from other standard cell culture facilities. Inner surfaces of the hood should be disinfected with 70% ethanol or 10% bleach solutions between each use. Gloves and lab coats should always be worn. Incubators should be routinely cleaned. Cell cultures should be regularly assayed by standard methods for detection of Mycoplasma.”

He also recommends that you avoid the “indiscriminate use of antibiotics,” as this practice “permits repeated lapses in aseptic technique, masks low-grade contaminations, and leads to resistant organisms.”

Take-Home Points: Avoiding Contamination
  • Use certified or authenticated cell lines. If not possible, quarantine new cells until you can verify that they do not have Mycoplasma.
  • Freeze a back-up copy of each of your cultures in case the cells you’re working with become infected.
  • Work with one culture at a time in the hood, clean the hood between uses, and don’t use the same media for different cultures.
  • Wear sterile gloves and a lab coat at all times.
  • Purchase your cell culture reagents from reputable manufacturers.
  • Assay your cultures regularly to screen for Mycoplasma contamination.

Finally, ATCC recommends a “monthly surveillance program” in which you clean and disinfect all working areas and screen your cell cultures for Mycoplasma contamination. They also recommend you maintain a “rigorous cell-banking program” so that you have clean backups for each culture in case contamination is found.

How do I detect it?

“Mycoplasma contamination is not easily detected,” Grady says. “It does not cause media turbidity like bacterial or fungal contaminations, produces few metabolic by-products, does not alter the pH of the culture media, and is too small to be detected by routine microscopy.”

Basically, you’re not going to find Mycoplasma unless you screen for it. There are a number of methods used for this purpose, each with its own pluses and minuses. The most common are the direct culture, DNA staining, PCR, and enzyme-based methods.

“When testing for Mycoplasma contamination, the highest sensitivity is achieved by the direct culture and Hoechst stain methods,” explains Schwartz. “However, for both methods, Mycoplasma positive controls need to be cultured, which is not advisable in a regular cell lab. These assays are usually outsourced and are therefore costly. Additionally, they take around one week (DNA stain) or four weeks (direct culture) to complete.”

Grady adds that “as far as methodologies go, the FDA’s ‘Points to Consider’ (direct agar method) is still regarded as the gold standard for Mycoplasma testing.” However, he also says that there are several PCR-based assays available that can give reliable results in a much shorter timeframe.

“Biochemical and PCR methods are performed with a noninfectious positive control and can be executed in a normal cell culture lab,” Schwartz explains. “Both methods detect all Mycoplasma that are usually faced as contaminants in cell cultures.”

Lonza’s MycoAlert™ Mycoplasma Detection Assay and the Universal Mycoplasma Detection Kit (ATCC® 30-1012K™) are examples of biochemical- and PCR-based methods, respectively, of screening for Mycoplasma. Both are safe to use in the lab and can give results in a number of hours.

“The advantage of a biochemical assay such as Lonza’s MycoAlert Mycoplasma Detection Assay is the speed and convenience of the test. It can be easily incorporated in the regular splitting routine in a cell culture lab,” Schwartz notes.

The Universal Mycoplasma Detection Kit, Grady says, “detects over 60 species of Mycoplasma, Acholeplasma, Spiroplasma, and Ureaplasma, including the eight species that are most likely to afflict cell cultures: M. arginini, M. fermentans, M. hominis, M. hyorhinis, M. orale, M. pirum, M. salivarium, and A. laidlawii.”

According to Grady, samples that are positive will give a distinct PCR product ranging in size from 434 to 468 base pairs, and the band is easily recognizable on an agarose gel. “The assay is sensitive to as few as 20 genome copies with results available in about 3 hours,” he adds.

So I’ve got contamination… what do I do now?

Both Grady and Schwartz agree that the best solution to Mycoplasma contamination is to throw out the affected cells. While there are reagents that can help save your culture, most are toxic, and the resulting cell population may no longer exhibit qualities of the parental line. The process is time consuming, and it’s not guaranteed to work.

“You should dispose of contaminated cultures and thoroughly disinfect your work area—including biosafety cabinets, incubators, and water baths—and begin with a new freeze of cells,” Grady recommends.

This is another reason why it’s important to have clean back-up freezes for each of your cultures—but if you don’t have any, you’ve still got some options.

“When curing a line of Mycoplasma, the cells are cultured for one to two weeks in the presence of an antibiotic such as Plasmocin, BM Cyclin, or Ciprofloxacin and then cultured without the antibiotic for one to two months,” Grady explains. “At this point, the line is retested to make sure that the culture is clean.” He also recommends periodic retesting to make sure that the contaminant doesn’t come back.

You may also consider trying Lonza’s MycoZap Elimination™ Reagent, which Schwartz tells us is a specially formulated reagent that combines antibiotic and antimetabolic agents.