Mycoplasma Testing

Genetica DNA Laboratories now offers the “Gold Standard” in Mycoplasma detection assays for cell culture – contact Genetica DNA Laboratories to find out which Mycoplasma Detection assay is right for your cell culture.

Appropriate positive and negative controls are included in every assay to ensure the validity of the test results.

Call for pricing and sample preparation/shipping requirements.

• DNA Fluorochrome Staining with Indicator Cell Line - Conventional method for mycoplasma detection. This assay has the capacity of detecting non-cultivable mycoplasma species or strains. This assay provides reliable and timely results.
 
• Lower Limit of Detection (LLOD) - 100 CFU/ml
• Recommended For - Continuous cell cultures including cell repositories
• Cell line Species Accepted - All species
• Advantages - Enhancement of sensitivity and specificity by reducing background signal from cellular debris; amplification of mycoplasma titers and detection of non-cultivable organisms
• Test Turnaround Time - Three (3) to five (5) days
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• DNA fluorochrome staining of normal (left image) versus mycoplasma infected cells (right image)
• Source: Bionique Testing Laboratories®, Inc.

• Multi-Media Direct Culture Method PLUS DNA Fluorochrome Staining with Indicator Cell Line (GMP service) - Comprehensive culture-based tests consisting of both a multi-media direct culture procedure and indicator cell culture with DNA fluorochrome staining assay. This option is the optimum assay and is considered the “Gold Standard” for detection of Mycoplasma contamination in cell cultures. The submitted cell culture specimen is inoculated in both the broth and agar systems for three individual mycoplasma media formulations. The addition of the DNA Fluorochrome Staining Assay adds the ability to detect potential non-cultivable mycoplasma contaminants.
• Lower Limit of Detection (LLOD) - 10 CFU/ml
• Recommended For - Master cell banks and working cell banks
• Cell line Species Accepted - All species
• Advantages - GMP service; enhancement of sensitivity and specificity by reducing background signal from cellular debris; amplification of mycoplasma titers and detection of non-cultivable organisms
• Test Turnaround Time - 28 days; DNA staining results in three (3) to five (5) days

NOTE: Mycoplasma detection services are FOR RESEARCH ONLY; not for clinical diagnostic purposes.

MYCOPLASMA INFORMATION

Mycoplasma, considered the smallest free-living organism capable of self-replication, is a bacterium which lacks a cell wall; they can be as small as 0.2-0.3 µm in diameter and are known to freely pass through some filters used to remove bacteria from cell culture. Their genome sizes range from about 600 to 2200kb. Mycoplasma has become one of the most widespread and severe microbial contaminants within cell culture systems in research institutions.

Mycoplasma can attain high densities in cell cultures (i.e. 10⁷ - 10⁸ mycoplasma/mL) without ANY noticeable changes to the pH of the medium and without causing increased turbidity. Typically there is an absence of visible morphological changes. Mycoplasma contamination cannot be detected using bright-field or phase microscopy, even at high magnification. In other words, you do NOT know mycoplasma is contaminating the cell culture without testing.

Estimates vary but research studies performed by different groups have shown anywhere from 5-35% of all cell cultures are contaminated with mycoplasma; the real percentage may be even higher because many researchers don’t know that their cultures are contaminated. If bacterial contaminations are a common occurrence in your lab, you most likely have mycoplasma contamination as well and just don’t know it.

Due to the typically high concentrations of mycoplasma in infected cultures, mycoplasma often out-compete the host cells for essential nutrients resulting in altered growth and protein production. Mycoplasma contamination has been shown to alter almost every cell culture property and characteristic measured including altered DNA, RNA and protein synthesis, lowered ATP levels, altered enzyme expression and activity, variable growth rates and viability, nucleic acid synthesis which leads to aneuploidy and other chromosomal irregularities, cytotoxicity, decreased transfection efficiency and culture starvation. As with using a misidentified cell line, using cell cultures contaminated with mycoplasma calls into question the validity of the experiments using that cell line.

No! Actually, mycoplasma contamination rates are much higher than average for cell lines that are routinely grown in medium containing antibiotics (72% were contaminated with mycoplasma) versus no antibiotics in the medium (7% contamination) (Barile, et al.1973). This is due to the mycoplasma developing partial or complete antibiotic-resistance to antibiotics such as penicillin and streptomycin. In medium containing antibiotics, other microbial contaminants disappear allowing plenty of room for antibiotic resistant mycoplasmas to grow and spread through the culture undetected.

Cell cultures used for pharmaceutical manufacturing MUST be mycoplasma-free, as required by the U.S. Pharmacopoeia and FDA regulatory requirements (CFR Title 21, Sec. 610.30). Due to the negative effects mycoplasma contamination has on a cell cultures (see “Why is Mycoplasma contamination bad for cell culture?” above), it is not out of the question that in the near future, scientific journals and funding agencies would start requiring Mycoplasma contamination testing PRIOR to submitting a research article that contains experiments using any cell line. Again, the validity of the scientific data is called into question when a cell culture becomes infected with Mycoplasma.

Just like cell line authentication, testing for mycoplasma should occur at a regular basis while the cell culture is actively growing. It is recommended that one of the above mycoplasma detection assays be performed, as applicable:

• When creating a master cell bank OR working cell bank
• Before beginning a new series of experiments
• When the cell line is not behaving as expected
• Every 2 months that the cell culture is actively growing
• Before publication of experimental data

These steps are crucial to the success and repeatability of the research and to the acceptance of the research in the scientific community.

• Armstrong, S.E., Mariano, J.A. and D.J. Lundin. “The scope of mycoplasma contamination within the biopharmaceutical industry”, Biologicals 38 (2010) 211-213.

• Barile, et al. “The identification and sources of mycoplasmas isolated from contaminated cell cultures”. Science 255 (1973) 251-254.

• Bionique Testing Laboratories®, Inc.

• U.S. Food and Drug Administration – www.fda.gov