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LABORATORY PROCEDURES FOR ANIMAL & HUMAN CELL LINES

Appendix

REFERENCE NO: AHC/1998/3/3 Appendix 1


TITLE: GENERAL CONSIDERATIONS ON MYCOPLASMA


INTRODUCTION

Mycoplasma have a cellular-like structure (elementary body) without a true cell wall. They contain protein, DNA, RNA, and enzymes. The elementary bodies vary in shape and size; some are 1/2,500 of an inch long and others are 1/10 that size. Also, mycoplasma are sensitive to heat and some antibiotics. Many plant diseases, previously thought to be caused by viruses, transmitted by leafhoppers and in the "yellows" group, are now known to be caused by mycoplasmas, not viruses. Corn stunt spiroplasma is a mycoplasmal disease.

 

MYCOPLASMA DETECTION AND ELIMINATION

Over the past decades cell culturing has become an indispensable tool for modern research. With the increased use of cell cultures contaminations are increasingly frequent, i.e. cellular contamination (cross-contamination between cell cultures) and contamination with microorganisms (microbial contamination). While bacterial and fungal infections of cultures are relatively easy to detect, to prevent and to treat, contamination with mycoplasmas represents a much bigger problem in terms of incidence, detectability, prevention, eradication and effects. It was estimated that between 5 and 35% of cell cultures in current use are infected with mycoplasmas.

Mycoplasmas belong to the class Mollicutes, which is currently divided into three orders (Mycoplasmataceae, Acholeplasmataceae, Spiroplasmataceae); each order contains different families, and six genera are distinguished by classical taxonomy. For instance, the genus Mycoplasma comprises more than 50 known species which are further subdivided into a multitude of strains. Mycoplasmas are the smallest free-living, self-replicating organisms (0.2-2 Ám in diameter). Mycoplasmas lack a cell wall so that antibiotics interfering with the murein formation of cell walls (such as penicillin which is often added to cell cultures) are not effective at the standard concentrations used. Mycoplasmas in cell cultures are extracellular parasites usually attached to the external surface of the cell membrane.

Mycoplasma contamination is not without serious consequences as virtually every cellular process can be altered by the infection. Mycoplasmas compete effectively with tissue-culture cells for medium nutrients, thus depriving the cells of essential nutrients, resulting in profound effects on cell metabolism and function.*

In the worst scenario, contamination leads to diminished cell growth and eventually to the loss of cultures. Mycoplasmas from human, bovine and porcine sources are the most prevalent groups with Acholeplasma laidlawii, Mycoplasma arginini, Mycoplasma fermentans, Mycoplasma hyorhinis, and Mycoplasma orale being the single most common isolates. Numerous methods for detecting mycoplasma infection have been described. While some tests are rather cumbersome and time-consuming, other less fastidious techniques do not have sufficient sensitivity and specificity. CABRI collections have evaluated and compared the usefulness, practicality and costs of a number of different methods for their daily application in the routine setting of a cell culture bank (1-7). It is agreed that a combination of various tests is the most useful approach for the routine detection of these contaminats, and that the most reliable assays are the following: (direct DNA fluorescence staining, classical broth-agar microbiological colony assay, RNA hybridization in solution and PCR) (2,8). In addition, other assays can be used, which are available in kits, like the Mycotect system (which requires co-cultivation of cells to be tested with 6-methilpurine deoxyriboside (6-MPDR)), the Mycoplasma Detection Kit (an enzyme immunoassay), etc. It has to be kept in mind that these assays present both advantages and disadvantages, and that in no case a single assay is sufficient for exclude the contamination of a culture.

In selected cases, an attempt is made to eliminate mycoplasma contamination by treatment with specific antibiotics: MRA (Mycoplasma Removal Agent) containing a quinolone derivative, Ciprobay containing ciprofloxacin, Baytril containing enrofloxacin, or BM Cyclin containing tiamulin and minocycline. The treatment schedules, concentrations of antibiotics used and other important details have been described extensively elsewhere (1,3,5,7,9). The whole topic of the "mycoplasma Problem" in cell culture, and the detection, elimination and prevention of these contaminations has been summarized and reviewed elsewhere (10).

References:

1. Gignac SM, Brauer S, Häne B, Quentmeier H, Drexler HG: Elimination of mycoplasma from infected leukemia cell lines. Leukemia 5: 162-165 (1991).

2. Uphoff CC, Gignac SM, Drexler HG: Mycoplasma contamination in human leukemia cell lines. I. Comparison of various detection methods. J Immunol Methods 149: 43-53 (1992).

3. Uphoff CC, Gignac SM, Drexler HG: Mycoplasma contamination in human leukemia cell lines. II. Elimination with various antibiotics. J Immunol Methods 149: 55-62 (1992).

4. Uphoff CC, Brauer S, Grunicke D, Gignac SM, MacLeod RAF, Quentmeier H, Steube K, Tümmler M, Voges M, Wagner B, Drexler HG: Sensitivity and specificity of five different mycoplasma detection assays. Leukemia 6: 335-341 (1992).

5. Gignac SM, Uphoff CC, MacLeod RAF, Steube K, Voges M, Drexler HG: Treatment of mycoplasma-contaminated continuous cell lines with Mycoplasma Removal Agent (MRA). Leukemia Res. 16: 815-822 (1992).

6. Hopert A, Uphoff CC, Wirth M, Hauser H, Drexler HG: Specificity and sensitivity of polymerase chain reaction (PCR) in comparison with other methods for the detection of mycoplasma contamination in cell lines. J Immunol Methods 164: 91-100 (1993).

7. Drexler HG, Gignac SM, Hu ZB, Hopert A, Fleckenstein E, Voges M, Uphoff CC: Treatment of mycoplasma contamination in 200 cell cultures. In Vitro Cell Dev Biol. 30A: 344-347 (1994).

8. Hopert A, Uphoff CC, Wirth M, Hauser H, Drexler HG: Mycoplasma detection by PCR analysis. In Vitro Cell Dev Biol 29A: 819-821 (1993).

9. Fleckenstein E, Uphoff CC, Drexler HG: Effective treatment of mycoplasma contamination in cell lines with enrofloxazin (Baytril). Leukemia 8: 1424-1434 (1994).

10. Drexler HG, Uphoff CC: Mycoplasma Contamination of Cell Cultures. Chapter in: The Encyclopedia of Cell Technology, Spier RE (ed). Wiley, New York (in press).

* Rottem S and Barile MF: Beware of Mycoplasmas. TIBTECH 1993, Vol. 11:143-151.

 


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