Mycoplasma Detection Methods: DNA Stain
To detect Mycoplasma infection within a cell line, cell culture samples are co-cultivated with an indicator cell line, typically Vero cells which have large cytoplasm area around the nucleus, for two to three days before staining. This involves the use of a fluorochrome dye (Hoechst 33258) which binds specifically to DNA, causing fluorescence when viewed with an epiflorescent microscope, with Mycoplasmas appear as granules surrounding the nucleus. Samples are compared with known positive and negative control samples.
This method is useful for detecting contamination with the non-cultivable strains of M.hyorhinis. It is recommended that all samples be tested using two methods of detection.
Stained Mycoplasma infected cells viewed under epifluorescence
(x 1000 magnification)
ECACC offer the Hoechst DNA stain method to both regulatory protocols and in-house variations of pharmacoepial methods.
Mycoplasma Detection Methods:
| Assay type | Assay Characteristics | ||||
|---|---|---|---|---|---|
| Sensitivity | Speed | Species Range | ECACC In-house Protocol |
US FDA Approved Protocol | |
| PCR | Low/Medium | 1 day | Majority | Yes | No |
| DNA Stain | Medium | 2-3 days | All | Yes | Yes* |
| Culture Isolation | High | 3-4 weeks | Majority | Yes | Yes |
*European Pharmacopoeia protocol also available by arrangement
