Testing for mycoplasma

Observation of a monolayer of contaminated animal cells sometimes gives early warning of the presence of mycoplasma. Thus cells 

---

The presence of mycoplasmal DNA in the cell cytoplasm or attached to the cell membrane may be detected by staining with the fluoro-chrome Hoechst 33258 (Appendix 3). This intercalating dye fluoresces under ultraviolet light and this forms the basis of a very rapid test for mycoplasmas. 

Ohno T Takeuchi M (1990) Test for mycoplasma contamination. Standardized protocols for quality control of animal cell lines. Report from JTCA Cell Bank Committee, Tissue Culture Research Communications 9 Suppl. 9-11. 

The qualification programs involve extensive testing of the master, working, and post-production ceU banks, including tests for Mycoplasma, steriUty, and viruses [8]. These cell lines are subjected to an extensive program of in vivo- and in vitro tests for known and unknown vimses ... 

She was treated with oxamniquine and transported to the USA, where evaluation showed flaccid paralysis and decreased sensation of touch and of temperature over the skin of the legs. Cerebrospinal fluid examination showed pleocytosis and protein elevation. Serologic tests for mycoplasma and viral pathogens were negative. A myelogram. showed no masses amenable to surgical removal. 

Mycoplasma contamination of cell culture systems continues to present major problems for monoclonal antibody production. Mycoplasma-positive cell cultures are themselves the major source of infection. It is recommended that all myeloma cell lines be tested for mycoplasma prior to fusion. If a hybridoma culture is considered irreplaceable, it is possible to eliminate effectively the mycoplasma contamination by injecting the hybrids into mice. The animal s immune system will destroy the infection and effectively clean up the cells. The mycoplasma-free cells are then recovered by draining the ascites fluid. Drug treatment is also an effective method to decontaminate hybrid cells from mycoplasma infection. 

GL34 Biologicals Mycoplasma Test for the detection of Mycoplasma contamination... 

Prior to further processing of the bulk harvest, it should be tested for sterility, mycoplasma, and viral contamination. In the case of CHO cells, an in... 

There are various methods for detecting Mycoplasma contamination of cell culture. A sensitive polymerase chain reaction test with broad specificity for Mycoplasma species is our method of choice (8). There are several products available for the eradication of Mycoplasma species from cell lines. The effectiveness of the treatment will depend on the cells and involves trail and error. This is because some cell lines are very sensitive to the chemicals used to eradicate Mycoplasma and may become static or die during treatment. 

Mycoplasma. The test for the presence of mycoplasma relies on expansion of cells in antibiotic-free conditions and detection of the organism using dye or PCR, as well as growth of mycoplasma on appropriate agar media. 

As mycoplasmal contamination of cell cultures is not always so obvious as bacterial contamination, it is important to 1) be aware of the effects of mycoplasmas on cell cultures, and 2) carry out routine tests for their presence. This is especially important as a contaminated culture may have 108 mycoplasma per ml, i.e. there may be 100 mycoplasma per cell. The mycoplasma often grow attached to the surface of the cell providing it with a prokaryotic coat. 

Is there a better PCR technique Over the past few years different authors have described other 16S rDNA-based PCR methods. Spaepen et ah (1992) used a nested PCR system with great sensitivity, but the use of a second amplified cycle dramatically increased the risk of DNA carryover contaminations, van Kuppeveld et al. (1994) reported a single PCR system that seems to be very suitable to detect cell culture contamination but it requires a DNA extraction stage, which is very time consuming. Moreover, a new marked PCR method is available (Stratagene, CA). The primers used make it possible rapidly to (4-5 h) test eukaryotic cells for mycoplasma infection but this method seems to be less sensitive than our PCR technique. 

This incident emphasizes the critical importance of diligent testing of cell cultures for contaminant microorganisms. By combining procedures such as those described here with procedures included elsewhere in this volume (e.g. fluorescent or nucleic acid probes for mycoplasma and viruses) one can be more certain that clean cell cultures are available for experimentation. 

During the course of antibiotic treatment and also 5-7 days after treatment in the case of bacteria and fungi, and 25-30 days after treatment for mycoplasma-contaminated lines, re-test the culture by an appropriate method. Regular routine testing should, however, occur after this period. This is particularly important in the case of mycoplasmas, where low levels of infection may persist after antibiotic treatment. 

A more general example from virus vaccine production is the rigorous examination of tissue cultures to exclude contamination with infectious agents from the source animal or, in the cases of human diploid cells or cells from continuous cell lines, to detect cells with abnormal characteristics. Monkey kidney cell cultures are tested for simian herpes B virus, simian virus 40, mycoplasma and tubercle bacilli. Cultures of human diploid cells and continuous line cells are subjected to detailed kary-ological examination (examination of chromosomes by microscopy) to ensure that the cells have not undergone any changes likely to impair the quality of a vaccine or lead to undesirable side-effects. 

Latex agglutination immunoassays are easily formatted into simple kits which can provide yes/no and semiquantitative estimates of antigen (or antibody) in a sample. The first such assay was developed in 1957 for rheumatoid factor (15) and assays are on the market for the deterrnination of many species of bacteria, fungi. Mycoplasma, parasites, ckettsia, and vimses, as well as for the deterrnination of autoimmune disease, hormones (qv), dmgs (see Pharmaceuticals), and blood proteins (16). Latex agglutination is also the basis of many home pregnancy tests. 

Contamination (including mycoplasma) and differences in serum lots for cultures can affect bioassay performance. Consequently, it is important to maintain cell lines adequately and use consistent sources of material to ensure reproducibility of test results. Bioassay data generally follow a sigmoidal curve, which can complicate the interpretation of results. 

Difficulty is encountered in detecting mycoplasma in certain cell lines and, for this reason, indicator cells e.g. mouse 3T3 cells are often used. 24 hours after establishing a cover slip culture of 3T3 cells a sample of the supernatant of the cells under test is added to the culture. Positive and negative controls should also be included. Three to four days later cultures are tested using fluorescence staining and another technique. 

After 24 h add 50 jal 6-MPDR (final concentration 30 juM) to duplicate test and control wells and continue the incubation for a further 3-4 days when the negative control wells should be confluent. The positive controls and those wells containing mycoplasma contamination will show no cells, or a reduced number of cells. 

---