Spotting Culture Contaminants

Researchers have a new tool for detecting ruinous mycoplasma infections in cell cultures. A glowing enzyme can quickly assess whether mycoplasmas have invaded (Anal. Chem., DOI: 10.1021/ac2033112).

Many microbes can contaminate mammalian cell cultures, but some of the most worrisome are mycoplasmas. The family of tiny bacteria, estimated to contaminate 15 to 70% of cell cultures, threatens cell viability and alters cell behavior.

Currently, the most reliable way to detect mycoplasmas involves extracting and amplifying their DNA with polymerase chain reaction (PCR) techniques. “But PCR is time-consuming, and you can have false positives and false negatives,” says neuroscientist Bakhos A. Tannous of Harvard Medical School.

He and his colleagues realized they had developed a faster, luminescence-based technique when they noticed unusual behavior in a biological assay. They had genetically engineered Gaussia luciferase, a luminescent protein from a small marine crustacean, into brain tumor cells to monitor the expression of specific genes.

Tumor cells synthesize and secrete the protein into the cell medium, where the researchers can sample it. Normally, the protein has a half-life of about six days in the medium.

But when the researchers performed the assay on several cell lines, they found that the half-life of the luminescence varied greatly. “Sometimes it was seven days, which was expected; sometimes it wasn’t even 24 hours,” Tannous says.

While Tannous’ lab members were scratching their heads one day about the half-lives, Tannous asked them whether they had performed their routine screening for mycoplasmas that month. They hadn’t. They soon discovered that only contaminated cultures showed the drop in luminescence. To investigate further, they infected other brain tumor cells with three common species of mycoplasma and measured Gaussia luciferase luminescence. After three days, the contaminated cultures showed a 14 to 81% decline in luminescence.

The team’s method could detect low concentrations of the bacteria that commercial tests could not. The team hypothesizes that mycoplasmas produce a protease that breaks down the luciferase protein.

Tannous thinks the simple technique could be of use to any laboratory working with mammalian cells already engineered to express Gaussia luciferase. For other cells, researchers could just add the protein to the cell medium, he says.

“This looks like a very interesting new test,” says Charles J. Rosser, of M. D. Anderson Cancer Center, Orlando. In earlier work, he and his colleagues found that mycoplasma infection causes benign prostate cells to look cancerous.

Rosser says the new technique could make it easier for a lab to screen many cultures at once: “To do 30 cultures the old way would take a substantial amount of time. To do 30 cultures this way, you could do it all in one morning and have the results by lunchtime.”