Hello Julissa Mejía

There are a number of techniques available that could help identify mycoplasma presence in cell culture.

1) The direct culture method in which an agar plate is inoculated with the culture which is supposed to be mycoplasma contaminated, and incubated for a period of three to four weeks. Positive samples on agar plates will show small colonies similar to fried eggs with 100-400 µm in diameter.

2) The indirect culture method requires culturing the cells under normal growth conditions. Once the cells reach an appropriate confluence, they are stained with Hoechst 33258, a fluorescent dye that binds specifically to DNA. The appearance of filamentous staining in the cytoplasm of the cells in question indicates a positive finding.

3) Mycoplasma contamination can also be detected by polymerase chain reaction (PCR). PCR is easy, sensitive, specific, fast, reliable, efficient and cost effective. The method uses universal PCR primers targeted to the 16S rRNA gene.

4) Bioluminescence assay kits are commercially available which are enzyme-based mycoplasma detection kits. These kits detect mycoplasma enzymes that are not synthesized by eukaryotic cells. First, the kit components rupture the mycoplasma and then specific enzymes are used which trigger cellular mechanisms that generate luminescence. This can then be detected by a luminometer.

5) Non-specific DNA stains can be added in the infected culture medium to detect mycoplasma. When observed under a fluorescent microscope, the mycoplasma DNA appears in the form of small clusters, apart from the cellular DNA. However, some amount of expertise will be required in this process because result interpretation might be difficult.

You may want to refer to the article attached below.

https://www.nature.com/articles/d42473-019-00306-1

Prevention of contamination is always better. This can be done by providing proper training and guidance to the staff working in the tissue culture facility. Proper maintenance of sterility is essential.

In case of mycoplasma contamination of cultures which are really valuable and rare, it is suggested to treat them to eliminate mycoplasma infection. Plasmocin is a widely used drug that can clear most of the mycoplasmas present in the culture media. Also, drugs like BM Cyclin, fluoroquinolone ciprofloxacin, ciprobay, zagam, baytril, tetracycline, etc. are available for mycoplasma removal from the infected culture.

Otherwise, it is recommended to discard mycoplasma contaminated cells since they are considered as a source of contamination in the laboratory.

Best.

Greetings,

There are a few ways though which you can detect mycoplasma in your cultures:

1. PCR amplification.

2. Fluorescent staining with dyes that binds to Mycoplasma spp. DNA (one example is MycoFluor™ here you can check the fundaments https://assets.fishersci.com/TFS-Assets/LSG/manuals/mp07006.pdf).

3. The most recommended is the colony formation on specifically enriched agar plates, however, it includes expensive labile components.

4. A novel approach is to use a procedure that relies on isolation of Mycoplasma spp. on Horse blood Columbia agar directly from exhausted tissue culture supernatants. It is supposedly low-cost and allows the weekly identification of Mycoplasma, with the recommedation of performing a PCR every 2 months.

You can revise the following article for a much extensive explanation: Article A low-cost simple test for weekly detection of Mycoplasma hy...

Check the integrity of your growth media filters before and after filtration? A simple fix, but one that was overlooked by junior lab tech. We lost a whole cell line to mycoplasma

contamination and had to start from ATCC bought stock. Set our research back a month to scale up cell bank back to normal output.

Preventing and detecting mycoplasma contamination in cell lines is crucial to ensure the reliability and accuracy of experimental results. Here are some steps that can be taken to prevent and detect mycoplasma contamination, as well as macro and micro techniques to identify mycoplasma presence in cell culture:

Prevention:

1. Good cell culture practices: Adhering to proper aseptic techniques and following established protocols for cell culture can minimize the risk of mycoplasma contamination.

2. Regular screening: Regularly screen cell lines for mycoplasma contamination to identify any potential issues early on. This can be done using various detection methods (discussed below).

Detection: Macro Techniques:

1. Direct observation: Inspect the cultures visually under a phase-contrast microscope for any morphological changes or the presence of mycoplasma-like structures (e.g., filaments, clusters).

2. Indicator cell culture: Use indicator cell lines that are sensitive to mycoplasma infection, such as Mycoplasma hyorhinis indicator cells. These cells are typically co-cultured with test cells, and the indicator cells show visible changes (e.g., rounding, cytopathic effects) upon mycoplasma contamination.

Micro Techniques:

1. DNA staining: Perform DNA staining methods, such as Hoechst staining or DAPI (4',6-diamidino-2-phenylindole) staining, to visualize mycoplasma DNA within infected cells. Mycoplasma DNA is typically fluorescently stained and can be observed under a fluorescence microscope.

2. PCR (Polymerase Chain Reaction): PCR-based methods, such as nested PCR or real-time PCR, can detect mycoplasma DNA in cell cultures. Specific primers targeting conserved regions of the mycoplasma genome are used to amplify and identify the presence of mycoplasma DNA.

3. ELISA (Enzyme-Linked Immunosorbent Assay): ELISA kits are available that utilize specific antibodies to detect mycoplasma antigens or antibodies produced in response to mycoplasma infection. This technique allows for the detection of mycoplasma proteins or antibodies in cell culture samples.

4. Nucleic acid hybridization: Methods like fluorescent in situ hybridization (FISH) or nucleic acid hybridization assays can be used to detect mycoplasma DNA or RNA within cell cultures. These techniques involve using specific probes that bind to mycoplasma nucleic acids and can be visualized under a fluorescence microscope.

5. MALDI-TOF mass spectrometry: Matrix-Assisted Laser Desorption/Ionization Time-of-Flight mass spectrometry can be used to identify mycoplasma based on their protein profiles. This technique is increasingly being used for rapid and accurate mycoplasma detection.