| Bionique® Testing
Laboratories, Inc., offers Antigen Production, consulting, validation and other contractual services. |
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These very common contaminants are: Mycoplasma hyorhinis, Mycoplasma arginini, Mycoplasma salivarium, Mycoplasma orale, Mycoplasma fermentans and Acholephasma laidlawii.
Mycoplasmas have been shown to alter virtually every property and parameter measured in cell cultures, including hybridoma selection rates, protein and nucleic acid synthesis, immunogenicity, chromosomal breakage and production of virus and biologicals such as interferon and monoclonal antibodies. As a result of this widespread problem, bio production and research is often unknowingly done using mycoplasmal contaminated cell cultures. The validity and significance of research and the safety of the biologicals produced from contaminated cell cultures must be questioned.
1. The absence of visible signs of mycoplasmal contamination including changes in turbidity, cytopathic effect and pH, even in heavily contaminated cultures, leads to a false sense of security. Once present in the laboratory, mycoplasmally infected cultures often cross-contaminate the other cell lines being used. 2. The continuous use of antibiotics which are not usually mycoplasmacidal in long-term cell cultures can mask poor aseptic technique. This leads to accidental introduction of mycoplasmas along with other microorganisms. 3. The lack of simple, easy-to-use and reliable detection methods in the past has resulted in many cell lines being untested. Because of this, many cell lines brought into a lab from outside sources although appearing normal and healthy, are in fact already infected with mycoplasma. 4. Because of their small size and lack of cell wall, mycoplasmas occasionally penetrate the filters used to "sterilize" cell culture media and sera, resulting in low levels of these organisms being accidentally introduced into cultures during routine feeding.
Direct culture requires the use of one or more complex nutritionally enriched mycoplasmal media and carefully controlled environmental conditions. Even then, some mycoplasmal strains are difficult to grow in culture without cells. Properly done, with appropriate positive and negative controls, direct culture testing offers the greatest security, but is rather slow, usually requiring up to 28 days for completion. There are many indirect tests of varying sensitivity and convenience, including DNA fluorochrome staining, DNA probes, PCR, ELISA, autoradiography, immunofluorescence and specific biochemical assays. While faster than direct culture methods, indirect tests are not yet as sensitive and usually require higher levels of contamination (i.e. 104+ organisms/mL) for detection. Indirect tests offer two advantages over direct culture methods: first, they can detect so-called "non-cultivable" mycoplasmal strains that direct culture may miss; second, they are faster, usually taking only 1-5 days to complete. What Does Bionique® Testing
Laboratories Recommend? 1. What is the intrinsic value of your cell lines and the work performed with them? 2. What are the consequences of infection of these cell lines by mycoplasmas, or the subsequent loss of the cell cultures? 3. What resources are or can be made available to implement the necessary program? This will help you to determine whether to do it yourself in-house or to contract with an outside reference laboratory. Once these three questions have been fully answered, a mycoplasmal testing program can be implemented. Always select the best test you can afford and commit enough resources to do an effective job. An inadequate testing program results in a false sense of security that can lead to serious problems later on. There are 5 basic steps when setting up a good mycoplasmal testing program: 1. Test all of your cell lines that are currently in use to eliminate these cultures as potential sources of contamination. 2. Quarantine and then test all cell lines coming into the lab from any outside sources. Do the same for any cultures currently stored in your cell repository that were not tested at the time of storage. 3. Test continuously maintained cell cultures at defined intervals (most experts recommend monthly to quarterly). 4. Make sure all new additions to your cell repository are thoroughly tested at the time of freezing. This saves both time and money by eliminating the needs for testing these cultures each time they are thawed. 5. Test new lots of sera whenever possible, and always buy from a reputable source that tests each lot. If serum testing is not feasible, be sure to carefully retest cells after they have been cultured for 4-6 weeks in media containing the serum.
ECONOMY - We provide the expensive equipment and all the special media and chemicals; you use your valuable time and resources for research and/or production. SAFETY - Bionique® Testing has all the necessary live mycoplasmal control cultures required for valid testing; you avoid the risks of accidentally contaminating your own cultures. Cell Culture User's Guide to Mycoplasma Detection &
Control
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