It's one of my culture. The mycoplasma test is positive and I saw those within what looks like a cell.
Can they be explained by mycoplama or they're something else?
Hello Yongjie Lin
An easy way to test and confirm the presence of mycoplasma in your culture is by carrying out DAPI staining on your cells. Contaminated culture will show a typical "starry sky" appearance. You may refer to the protocol as well as the images provided in the attached link.
https://projects.iq.harvard.edu/files/hlalab/files/mycoplasm-test_hla.pdf
If the culture is negative for Mycoplasma, then it will show only nuclear fluorescence. Occasionally micronuclei or nuclear fragments from dead and disrupted cells will appear as spherical bodies. Their large size and brighter fluorescence will distinguish them from Mycoplasma.
However, if the culture is positive for Mycoplasma, then along with nuclear fluorescence it will also show extra-nuclear fluorescence. Mycoplasma can be identified by small pinpoint dots of fluorescence, either aggregated in clusters or scattered uniformly over the cytoplasm and sometimes in the intercellular spaces as the dye will also label the DNA of the intracellular mycoplasma.
Best.
Hi;
The two reference methods are the culture and fluorescence methods on indicator cells. The Pharmacopoeia (Ph. Eur. 2.6.7 & USP) also presents the FDA recognized use of nucleic acid amplification techniques as a real alternative. Fluorescence on indicatory cells allows the detection of certain strains of mycoplasma not detectable by the culture method.
One of the fastest and most economical methods of mycoplasma detection is based on direct staining of mycoplasma DNA with DAPI or Hoechst stains. Although this method is easy and quick to perform, it suffers from the weak signal provided by these dyes compared to nuclear DNA. Therefore, reporter cell lines are used for mycoplasma culture before the DAPI or Hoechst staining step. In the attached paper, the authors have developed and tested a new immunofluorescence assay for the detection of mycoplasma. The method is based on enzymatic labeling with DNA polymerase I and modified nucleotides using notches in the mycoplasma DNA. The modified nucleotides are incorporated into the mycoplasma DNA and then visualized by immunofluorescence microscopy.
My regards
- Badges
- Science topic