Figure 1. Electron Micrograph of Mycoplasma Adhering to Host Cells
Background and Hazards of Mycoplasma Contamination
Mycoplasmas are the smallest and simplest known prokaryotic organisms, existing in various forms such as spherical, rod-shaped, and filamentous. Lacking a cell wall, they typically range in diameter from 0.1 to 0.3 μm, allowing them to pass through standard filtration membranes (0.22–0.45 μm).
More than 20 species of mycoplasma have been identified as contaminants in cell cultures, with over 95% of contamination cases attributed to Mycoplasma orale, Mycoplasma arginini, Mycoplasma fermentans, Acholeplasma laidlawii, Mycoplasma hominis, and Mycoplasma hyorhinis. According to literature, the incidence of mycoplasma contamination in stored cell lines ranges from 15% to 35%, and from 0.44% to 6.70% in the biopharmaceutical industry. National and international pharmacopoeias and regulatory guidelines classify mycoplasma contamination as a critical microbiological quality control item due to its harmful effects, including altered cell morphology, inhibited growth, disrupted metabolism, and impaired cell proliferation.
Figure 2. Mycoplasma (in orange) Surrounding and Infecting Host Cells, Potentially Compromising the Validity of Gene Expression Studies
1. What Are the Main Routes of Mycoplasma Contamination in the Laboratory?
Contamination sources can be broadly categorized into five types:
A. Environmental Contamination: e.g., mycoplasma already present in the cell culture room, incubator, or biosafety cabinet.
B. Contaminated Laboratory Equipment: e.g., pipettes, pipette tips, culture dishes, water baths.
C. Contaminated Culture Reagents: e.g., media, trypsin, serum, and cryopreservation solutions.
D. Contaminated Primary Cells.
E. Improper Handling by Laboratory Personnel.
2. What Are the Effects of Mycoplasma Contamination on Cells?
Cells contaminated with mycoplasma may exhibit the following features:
Clear culture supernatant
Slowed growth and proliferation
Abnormal morphology such as filamentous or extensively spread cells
Unresponsive to medium changes or increased FBS concentration
3. What Are the Common Methods for Detecting Mycoplasma in the Laboratory?
Detection methods include:
Culture-based methods
In situ staining
Chemiluminescent assays
Nested PCR
qPCR with specific probes
Isothermal amplification and colorimetric assays
Among these, culture-based detection remains the gold standard in both domestic and international mycoplasma testing.
4. What Are the Methods for Mycoplasma Elimination?
The choice of elimination strategy depends on the sample type:
Biological products: The optimal approach is autoclaving followed by disposal.
Tumor or hybridoma cells: These can be inoculated into syngeneic animal models (e.g., subcutaneously or intraperitoneally in nude mice), leveraging the host immune system to clear the mycoplasma, after which the desired cells are harvested and macrophages are removed.
Valuable cell lines: If autoclaving is not feasible, mycoplasma elimination reagents can be added to the culture (either antibiotic-based or non-antibiotic).
Laboratory environment: Regular disinfection of culture rooms, adherence to standardized SOPs for cell culture, and strict selection of reagents and consumables certified free from mycoplasma are recommended.
5. What Are the Mechanisms of Common Mycoplasma Elimination Reagents?
Mycoplasma elimination strategies typically fall into three categories:
A. DNA Synthesis Inhibitors
Examples: Fluoroquinolone antibiotics
Mechanism: Inhibit DNA replication
Note: Prolonged use of a single antibiotic can induce resistance. If efficacy declines, switch to another antibiotic or use a combination therapy.
B. Protein Synthesis Inhibitors
Examples: Macrolides, tetracyclines, diterpenes
Mechanism: Inhibit protein biosynthesis in mycoplasma
Note: Resistance may also develop with monotherapy; alternating agents or using compound antibiotic formulations is advised.
C. Non-Antibiotic Reagents
Examples: Membrane-active peptides
Mechanism: Specifically bind to and disrupt mycoplasma membranes, leading to complete eradication
Advantages: No risk of resistance development, nearly 100% elimination efficiency, and minimal cytotoxicity to host cells.
Product Overview
|
Product Name |
Specification |
Cat No. |
|
MycAway™ Prophylactic (2000×) - Mycoplasma Prevention Reagent |
1 mL/5 mL |
40608ES03/08 |
|
10 assays/20 assays |
40601ES10/20 |
|
|
100 μL/1 mL/5×1 mL |
40607ES01/03/08 |
|
|
25 T/100 T |
40612ES25/60 |
|
|
25 T/100 T |
40619ES25/60 |