How can I eliminate mycoplasma from infected cell cultures?

The quickest and most reliable way to eliminate mycoplasma is to autoclave the contaminated cultures and any bottles of media or sterile reagents that were used with them (Figure 8a). Autoclave
Figure 8a - Autoclaving is the only guaranteed method for mycoplasma elimination.
Afterwards, replace them with fresh culture stocks which have been certified as mycoplasma-free.  Choosing to proceed with a mycoplasma elimination process may have deleterious effects on the individual culture.  The most common is culture loss from cytotoxicity of the treatment process but there may also be alterations in important culture properties and selection of clonal subpopulations (Uphoff and Drexler, 2002).  Only under exceptional circumstances, when the cultures are very valuable or irreplaceable, should attempts at eliminating the mycoplasma be made.

The first step is to clean and disinfect the hoods, incubators, pipettors and other equipment in the laboratory that may have been in contact with the contaminated cultures. Other cell lines in use within the laboratory should also be tested for mycoplasma to evaluate for possible cross contamination.  It is useless to clean up a contaminated culture if additional sources of mycoplasma are still at large in the laboratory.  Quality control procedures for managing a contamination event will largely be determined by individualized institutional policies. 

There have been many approaches developed for curing cultures of mycoplasma infections. These include use of mycoplasma-specific antisera, passage in nude mice, macrophages, complement, detergents and antibiotics.  To date, there is no universally effective method for eliminating mycoplasma in all cases.  Factors such as antibiotic resistance, cytotoxicity of anti-mycoplasma treatments and reduced viability of chronically infected cells can all hinder success (Fleckenstein and Drexler 1996).

The procedure which has been proven as the most reliable and efficient method for treatment is antibiotic administration (Drexler and Uphoff, 2002).  However, as previously mentioned, development of antibiotic resistance in mycoplasma has occurred.   Research conducted at Bionique® Testing Laboratories evaluated the incidence of antibiotic resistance in mycoplasma isolated from infected cell cultures (Table 8a).  The results demonstrated resistance of 11 to 86% to individual antibiotics which otherwise are considered mycoplasmacidal (Lundin and Lincoln, 1994). Data provided by the DSMZ - (German Collection of Microorganisms and Cell Cultures) also shown mycoplasma resistance within the range of 7 to 24% (Drexler and Uphoff, 2002).   However, despite the presence of resistance, in subsequent studies at their institution evaluating the effectiveness of antibiotic regimens, they were able to cleanse 66 to 85% of the mycoplasma-positive cultures (Drexler and Uphoff, 2002).  Note that these success rates are higher than usual do to the expertise of the authors.

Bionique® Testing Laboratories offers a highly effective method for mycoplasma elimination from cell cultures also based on a chemotherapeutic approach.  This procedure first requires isolating the mycoplasma from the culture and determining its antibiotic sensitivity profile (Del Giudice and Gardella, 1984; Lincoln and Gabridge, 1998). The infected culture is then treated by serial dilution to near clonal levels with a cocktail containing two or three antibiotics that testing has concluded to be most effective.  After consecutive treatments, the culture is expanded and carefully tested to ensure complete eradication. Understandably, the procedure time is lengthy, requiring up to 3 months for treatment and serial follow-up.  Find more information about Bionique's Mycoplasma Testing Services here.

A critical, and frequently over looked, step in the mycoplasma clean up process is continued surveillance of the treated culture to verify complete eradication.  In some instances, the mycoplasma may be simply suppressed and titers lowered to below detection levels of available assays.  Therefore, it is essential that very sensitive mycoplasma testing be performed on any culture that has gone through a mycoplasma clean up procedure to evaluate for re-emergence or reinfection.  Testing should then be done a second time after the culture has grown in antibiotic-free medium for at least four to six subculture passages.

The next section lists suggested readings and technical articles used for these FAQs.

Please call or contact us online to reach our technical experts at Bionique® Testing Laboratories if you have any questions on this material or require additional information.

Table 8a. Antibiotic resistance of mycoplasma from infected cell cultures (Lundin and Lincoln, 1984)
Antibiotic% Resistance
Chloramphenicol 30%
Chlortetracycline 11%
Ciprofloxacin 15%
Erythromycin 98%
Gentamicin 80%
Kanamycin 73%
Lincomycin 28%
Neomycin 86%
Spectinomycin 14%
Streptomycin 88%
Tetracycline 14%
Tylosin 21%
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Shayn Armstrong, Jill Mariano, Cynthia Martino and John Ryan have co-authored the chapter, Identifying Mycoplasma Contamination: Concepts and Tools, in the recently published textbook Microbial Identification: The Keys to a Successful Program, edited by Mary Griffin and Dona Reber and published by PDA and Davis Healthcare International Publishing, 2012