Abstract
Mycoplasma contamination of cultured cell lines is difficult to detect by routine observation. Infected cells can display normal morphology and the slow growth rate of mycoplasma can delay detection for extended periods of time, compromising experimental results. Positive identification of mycoplasma typically requires cells to be either fixed and stained for DNA or processed with PCR. We present a method to detect mycoplasma using live-cell optical microscopy typically used for routine observation of cell cultures. Images of untreated mycoplasma-infected epithelial cells alongside images of infected cells treated with Plasmocin, a commercially available antibiotic targeted to mycoplasma, are shown. We found that optical imaging is an effective screening tool for detection of mycoplasma contamination. Importantly, we found that cells regained normal function after the contamination was cleared. In conclusion, we present a technique to diagnose probable mycoplasma infections in live cultures without fixation, resulting in faster response times and decreased loss of cell material.
Highlights
Mycoplasmas are small filterable bacteria, 0.1–0.8 microns in size, lack a cell wall, and are resistant to many antibiotics [1]
The aims of this study were (1) to determine if mycoplasma contamination could be detected on living epithelial cell cultures using routine optical microscopy, (2) to identify the mycoplasma strain and determine if treatment with Plasmocin results in elimination of mycoplasma from our cell line, and (3) to determine if clearance of mycoplasma infection restores normal differentiated cell physiology in our cell line
We employed four independent methods to detect the presence of mycoplasma: Optical Microscopy, use of an engineered HEK cell line (PlasmoTest (InvivoGen, San Diego, CA)), DAPI nuclear staining, and PCR
Summary
Mycoplasmas are small filterable bacteria, 0.1–0.8 microns in size, lack a cell wall, and are resistant to many antibiotics [1]. Mycoplasma contamination of cell cultures can progress undiscovered for long periods of time and is a significant problem in experimental research [2]. While these culture methods are considered a “gold standard” of detection [3], these methods are more suitable for large-scale commercial lot release assays and, in the context of laboratory cell culture, may require sacrifice of suspected cells resulting in loss of experimental material and invalid experimental results if undetected contamination occurs during an experiment. Regular electrophysiological measurements of the resistance and current are used to verify that the cell layer is intact and functional
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