Detection of mycoplasma contamination in cell culture

Importance of Mycoplasma Detection in Cell Culture

It is estimated that about 5 to 30% of the world's cell lines are contaminated with mycoplasmas. However, the actual rates are probably higher in a significant number of laboratories. It was reported that the level of mycoplasma contamination of the cell cultures evaluated in Mexico was 88.7%. Mycoplasmas cannot be seen by visual examination or light microscopy. Accordingly, experiments in which cell culture is used are at risk and their results cannot be reliable without checking for mycoplasma contamination. Moreover, nutrient competition or toxic metabolites due to mycoplasma can affect bioproducts which are produced by mycoplasma contaminated cell cultures.

Detection Methods of Mycoplasma Contamination in Cell Culture

A large number of simple, reliable, sensitive and specific tests are now available to detect mycoplasma contamination in cell culture as the below table shows.

Microbiological Culture for Mycoplasma Contamination Detection

It is an officially approved method to detect mycoplasma contamination in cell culture. In this test, a sample of cell culture supernatant is added to a liquid medium for mycoplasma culture. After few days, a sample of cell culture supernatant is cultivated on a mycoplasma agar medium and will incubate in an aerobic condition at 37ºC for two weeks. Positive samples on agar plates will show small colonies similar to fried eggs with 100-400 μm in diameter.

Advantages: The microbiological culture method is a gold standard to detect every kind of mycoplasma contamination in cell culture without considering the origin and species of mycoplasma. This test is theoretically highly sensitive.

Disadvantages: It requires a lot of time (up to 4 weeks) and expensive media. Furthermore, many types of mycoplasmas poorly grow on cell-free media, whereas some of them are impossible to grow in vitro. In this test, the medium can also become infected from the outside: either from a researcher, medium components, or laboratory facilities. Thus, this detection method includes the risk of obtaining false-positive and false-negative results.

DNA Staining for Mycoplasma Contamination Detection

(1)Direct DNA stain by fluorochromes [such as 4, 6-diamidino-2-phenylindoledihydrochloride (DAPI) and Hoechst 33258 stain]. It is also an approved method.

Advantages: It is practically an easy and rapid test. The result can be obtained in as early as 2–3 hours.

Disadvantages: Sometimes interpreting the results is difficult and some experience is definitely necessary. If the condition of cell culture is not good, the DNA staining results will be misinterpreted.

(2)Indirect DNA stain using indicator cells e.g. 3T3 or 3T6. In this analysis, the test culture supernatant is added to a mycoplasma-free indicator cell culture (lines Vero B4, NIH 3T3 or 3T6). Cells are grown in flasks containing sterile slips, which are washed and stained with fluorescents after several days of cell culture growth.

Advantages: Improved sensitivity and specificity compared with direct DNA staining method.

Disadvantages: Slower and more time consuming than direct stain. Prolonged duration of the test poses a risk that contaminants would spread in the laboratory.

Molecular Biology Tests for Mycoplasma Contamination Detection

(1)Hybridization analysis

(2) Dot-blot hybridization with specific probes

(3)PCR based (TransDetect^® PCR Mycoplasma Detection Kit-Cat. No. FM311)

These methods are based on the development of gene or DNA probes, which were first introduced in the 1980s. The principle is simple as genes or genomic sequences, that are specific for a single species or a particular group of mycoplasma or universally for nearly all mycoplasmas, have been identified and synthesized; these probes are used for DNA or RNA hybridization (Hybridization analysis). The development of PCR enables the amplification of the target DNA in the specimen using specific synthetic oligonucleotides complementary to conserved rRNA sequences and increases the sensitivity by several orders of magnitude. Positive hybridization signals as dots (Dot-blot hybridization) on filters or scintillation counts and visual demonstration of the PCR amplicon in gels indicate the presence of the infectious agent.

PCR based method. PCR, as an approved method, is nowadays the most effective way to detect mycoplasma. It has been suggested that they have the potential superiority over conventional microbiological methods for mycoplasma testing in terms of analytical sensitivity, simplicity and turnaround time. The PCR test is based on the detection of 16S rRNA molecules of the most common species of mycoplasma contaminating cell cultures. The specificity of primers in this method should be broad enough to recognize Acholeplasma as well, but narrow enough to prevent amplification of common bacteria which might exist in the PCR reagents.

Advantages: Rapid and very sensitive. Several good commercial kits are available.

Disadvantages: Both false positives and false negatives may occur. False positives are due to carry over contamination from positive controls and samples. False negatives are due to PCR inhibitors in sample.

Biochemical Assays for Mycoplasma Contamination Detection

(1) Detection of adenosine phosphorylase activity (6-MPDR)

(2) Enzymatic conversion АТР→АDP detected by luciferase(TransDetect^® Luciferase Mycoplasma Detection Kit-Cat. No. FM301)

(3)Chromatographic detection of the transformation of radioactively labeled uridine to uracil with the uridine phosphorylase of mycoplasma

The method of Enzymatic conversion АТР→АDP detected by luciferase (or Bioluminescent assay) provides a new, sensitive and rapid biochemical methodology based on a bioluminescent assay which can detect mycoplasma carbamate kinase and/or acetate kinase within 30 minutes.

Advantages: This is a very rapid and specific method, which can only be used to detect biologically active mycoplasmas. It is good for urgent testing of small sample numbers.

Disadvantages: It requires access to a luminometer.

Immunoassays for Mycoplasma Contamination Detection

(1) Immunofluorescence

(2) ELISA

Immunoassays are about using specific antibodies to detect mycoplasma.

Advantages: Rapid and cheap. It is useful and simple for screening large numbers of samples.

Disadvantages: It will not detect all Mycoplasma species, and requires access to ELISA spectrophotometer.

References for Detection of Mycoplasma Contamination in Cell Culture

·   Drexler H G, Uphoff C C. Mycoplasma contamination of cell cultures: Incidence, sources, effects, detection, elimination, prevention[J]. Cytotechnology, 2002, 39(2): 75-90.

·   Nikfarjam L, Farzaneh P. Prevention and detection of Mycoplasma contamination in cell culture[J]. Cell Journal (Yakhteh), 2012, 13(4): 203.

·   Falagan-Lotsch P, Lopes T S, Ferreira N, et al. Performance of PCR-based and bioluminescent assays for mycoplasma detection[J]. Journal of microbiological methods, 2015, 118: 31-36.

·   Harlin H, Gajewski T F. Diagnosis and treatment of mycoplasma‐contaminated cell cultures[J]. Current protocols in cytometry, 2008, 43(1): A. 3C. 1-A. 3C. 7.

·   Geraghty R J, Capes-Davis A, Davis J M, et al. Guidelines for the use of cell lines in biomedical research[J]. British journal of cancer, 2014, 111(6): 1021-1046.

·   Chernov V M, Chernova O A, Sanchez-Vega J T. Mycoplasma contamination of cell cultures: vesicular traffic in bacteria and control over infectious agents[J]. Acta Naturae (англоязычная версия), 2014, 6(3 (22)).

Related Pages for Mycoplasma Contamination Detection

Mycoplasma Solutions-Detection & Elimination & Prevention

Mycoplasma Contamination in Cell Culture