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Reliability / reproducibility What is inter‐ and intra‐laboratory reproducibility? Are results consistent across batches, labs, or days? What is the variation or “noise floor”?
Inter-laboratory and inter-operator differences can yield highly inconsistent—even opposite—results across the three methods. The principal cause is human operation. SOPs should be detailed yet not overcomplicated. Focus on critical checkpoints and avoid nonessential items (e.g., routine growth curves). A simple, structured approach can reduce operator burden and errors: always include a negative control; check every 12 hours; if slight color change appears within 12 h, extend culture by 12 h; if around 2 days, extend by 24 h. This standardization improves reproducibility.
Background noise / assay robustness Could background DNA (from cells, reagents) or residual reagent signals interfere? How to design good negative and positive controls?
Yes, these limitations can lead to both false negatives and false positives. The emphasis should be on SOP execution and primer design. With time, more suitable commercial kits are expected to appear.
Applicability / coverage scope Can the method detect all mycoplasma species, including non‐cultivable ones? Can it work in different sample types (culture supernatant, cells, biologics, viral preparations)?
No single method detects all Mycoplasma species; medium formulations are continuously being developed and updated. Culturing feline mycoplasmas remains notably unsatisfactory. For each material, first identify likely contaminating species, then decide whether culture alone is sufficient.
Cost / instrumentation / resource constraints What is the cost difference among methods and reagents (kits)? Are specialized instruments required? What are budget‐friendly / mid‐cost solutions?
Cost ranking is roughly: culture < cell-based assays < PCR (which varies greatly by instrument and kit brand). Under regulatory requirements, validate multiple methods and vendors before finalizing a routine approach.
Removal / decontamination strategies If a culture tests mycoplasma‐positive, how can one eliminate it? Use anti‐mycoplasma antibiotics? Discard the cell line? Quarantine? Prevent recontamination?
Antibiotic resistance is severe worldwide; quinolone-resistant, uncommon field mycoplasmas (e.g., wild boar, some plant mycoplasmas) have been reported. Removal strategies depend on sample type: serum → filtration; reagents → high-temperature/pressure sterilization; cells → consider antimicrobial peptides. Prevention hinges on testing incoming materials, lab disinfection, and, most importantly, personnel training.
Interpretation / result judgment How to read colony growth, color changes, fluorescent dots, amplification curves, etc.? What thresholds or criteria define positive / negative? When to retest or confirm?
At low magnification, Mycoplasma colony morphology is consistent; to confirm species, increase magnification to 100×. A positive should be based on agar colonies; liquid color change is only supportive and error-prone. Growth curves are of limited qualitative value because color-change timing varies widely:
— ATCC 15531: red → yellow in ~3–5 days
— ATCC 23714: red → dark red/orange, then deeper red in ~2–4 days
— M. synoviae: orange to yellow within ~1 day.
Observe at least daily and subculture promptly to plates to avoid missing transiently viable cells.
Sample processing / sample preparation How should the sample be pre‐treated (centrifugation, filtration, concentration, dilution) to be compatible with detection? How to mitigate sample matrix effects or inhibitors?
Preservation is paramount: use CDC-recommended viral transport tubes or compatible buffers for Mycoplasma, keep samples cold, and test within 3 days to avoid death-related false negatives. In fermenters, dead zones and poor mixing make distribution uneven; therefore, multi-site sampling (e.g., ≥10 mL as many regulations require) is necessary. Begin Mycoplasma testing only after sterility has been confirmed. If antibiotics or inhibitors are suspected, filter to collect Mycoplasma and reculture rather than testing the raw liquid directly.
Validation / regulatory compliance How to validate the method (sensitivity, specificity, robustness)? Does it meet USP / EP / GMP / FDA requirements when used for biologic or cell therapy product quality control?
Each of the three regulatory methods (culture, cell culture, PCR) has gaps for comprehensive Mycoplasma detection. Culture media are often outdated and inadequate for many fastidious species, hence the use of cell culture. Cell-based methods are difficult to read and some species cannot be grown in eukaryotic cells, making medium-based culture necessary. PCR suffers from false negatives/positives due to insufficient primer coverage. Regulations define a baseline, but in practice one must optimize media and, especially, SOP.
How can a mycoplasma-contaminated sample affected by bacterial overgrowth be salvaged?
Because of their strong suppressive effects, bacterial contamination must be excluded before attempting mycoplasma detection in pharmaceutical testing.
Bacteria suppress mycoplasma via nutrient competition, space occupation, pH shifts, and by secreting antibiotics or lytic enzymes.
Therefore, early detection is critical. If bacterial contamination is suspected or confirmed and previous storage conditions were acceptable, proceed by first filtering out the bacteria and then diluting the sample to reduce concentrations of inhibitory substances.
Even if the mycoplasma concentration becomes very low, repeated passaging and dilution can reduce the impact of inhibitors and potentially allow recovery of viable mycoplasma.
How can mycoplasma be collected from a sample?
For biopharmaceutical samples that are confirmed to be bacteria-free, you can simply filter through a 0.1 µm membrane to collect mycoplasma. The membrane can then be placed onto mycoplasma culture medium.
For clinical research samples, it is recommended to first filter out bacteria using a 0.45 µm membrane, followed by capturing mycoplasma with a 0.1 µm filter.
Which regulations are essential for understanding mycoplasma detection?
The following are key regulatory references:
USP <63>: Mycoplasma Tests
EP 2.6.7: Mycoplasmas
JP (17th Edition or latest): Mycoplasma Testing section
ICH Q5D: Derivation and Characterization of Cell Substrates Used for Production of Biotechnological/Biological Products
FDA CMC Biologics: Applicable to biologics, cell-based products, and vaccines, outlining detection requirements for mycoplasma
Are there differences in mycoplasma medium evaluation standards among JP, EP, and USP?
Yes. There are slight differences in the species required for validation, due to variations in traditional classification and growth behavior. The required fold increase for confirming effectiveness also varies—USP tends to be slightly stricter.
However, in practical application, these differences rarely result in significant outcome variations. Similar discrepancies exist in incubation temperature standards.
The number and type of mycoplasma strains required for detection also differ. The listed strains represent a minimum requirement. It is recommended to enhance internal SOPs by including additional species based on local epidemiological reports, industry standards, and operational experience.
How to Prevent Mycoplasma Contamination in Pharmaceutical Manufacturing
A. Raw material and cell bank testing: Ensure all materials and cell stocks are mycoplasma-free before use.
B. Personnel entry and operations: Enforce strict access control and comprehensive aseptic operation training.
C. Equipment sterilization and routine sanitation: Validate cleaning protocols and schedule full-environment sanitization regularly.
D. Physical and procedural separation: Implement physical barriers and process separation to minimize contamination risk.
E. Contamination response SOPs: Develop and enforce emergency SOPs to contain and eliminate contamination swiftly before it spreads.
How to Eliminate Mycoplasma
A. Sterilization of instruments and equipment using autoclaving (steam sterilization) or peracetic acid-based agents.
B. Environmental decontamination: Use hydrogen peroxide (H₂O₂) vapor for space decontamination; after exposure, leave the area unoccupied and then perform follow-up mycoplasma testing to confirm decontamination.
C. Limitations of alcohol and UV light: These are only partially effective; also, don’t overlook the importance of eliminating mycoplasma spores if present.
Can KUSADA products be used in clinical settings?
We’re sorry, but KUSADA products are not certified for clinical use at this time. They are intended for research purposes only, including use in pharmaceutical mycoplasma testing.
How to download a product COA
COAs (Certificates of Analysis) and SDSs (Safety Data Sheets) are available for download in the “Quality Documents” section or on each product’s detail page.
Simply enter the product code and lot number to access and download the COA directly.