A New Sensitive Method for the Detection of Mycoplasmas Using Fluorescence Microscopy

Abstract

Contamination of cell cultures by mycoplasmas is a very common phenomenon. As they can substantially alter cell metabolism and potentially spread to all cell cultures in laboratory, their early detection is necessary. One of the fastest and cheapest methods of mycoplasma detection relies on the direct staining of mycoplasmas’ DNA by DAPI or Hoechst dyes. Although this method is easy and fast to perform, it suffers from the low signal provided by these dyes compared to the nuclear DNA. Therefore, the reporter cell lines are used for cultivation of mycoplasmas before DAPI or the Hoechst staining step. In the study presented, we have developed and tested a new immunofluorescence assay for the detection of mycoplasmas. The method is based on the enzymatic labeling using DNA polymerase I and modified nucleotides utilizing nicks in the mycoplasmas’ DNA. Modified nucleotides are incorporated into mycoplasmas’ DNA and subsequently visualized by immunofluorescence microscopy. The developed approach is independent of the mycoplasma strain, does not intensely stain nuclear DNA, does not stain other bacteria, and provides higher sensitivity than the approach based on the direct labeling using DAPI or Hoechst dyes.

Aptamer Cocktail to Detect Multiple Species of Mycoplasma in Cell Culture

“…Most make use of DNA amplification techniques that are characterized as stable, specific, and rapid, such as general and real-time PCR assays [6,[19][20][21], Cycleave PCR [22], and loop-mediated isothermal amplification [23]. Some are based on immunological principles, such as enzyme-linked immunosorbent assays [24,25], and some are based on microscopic techniques, including electron microscopy and fluorescence microscopy [26][27][28]. Other detection methods include Fourier transform infrared microspectroscopy [29], biochemical tests [30], and Gaussia luciferase-based assays [31].…”

Anchored linear oligonucleotides: the effective tool for the real-time measurement of uracil DNA glycosylase activity

“…The cells were cultivated at 37°C in a humidified atmosphere containing 5% CO 2 . The cell line was regularly tested for mycoplasma contamination by PCR and enzymatic detection [ 41 ].…”

“…The obtained data were analysed using CellProfiler [ 43 , 44 ], ImageJ and Microsoft Excel software and the final graphs were plotted in GraphPad Prism 6 [ 41 ]. The graphs were constructed using the following functions: one-phase decay was used in the case of the analysis of data in figure 2 a–c , one-phase association was used for the analysis of data in electronic supplementary material, figure S3, second-order polynomial (quadratic) regression was used for the analysis of the data in figures 1 b and 8 a – c (except m-sensors with T in 8 a , b ), and electronic supplementary material, figures S1, S2 (6-FAM/Cy5) and S5 (probe B 30FAM C UQ ).…”

High ATF4 Expression Is Associated With Poor Prognosis, Amino Acid Metabolism, and Autophagy in Gastric Cancer

“…The method is as follows: the nucleus was stained with DAPI and observed under an oil microscope. Mycoplasma contamination is characterized by radiating or satellite-shaped spots around the nucleus (22). All cell lines were cultured in RPMI-1640 medium (Corning, Corning, NY, USA), supplemented with 10% fetal bovine serum (FBS) (Clark Bioscience, Richmond, VA, USA), penicillin (100 U/ ml), and streptomycin (100 mg/ml) (HyClone, Logan, UT, USA) in a humidified atmosphere at 37°C with 5% CO 2 .…”