Unless otherwise specified, this method is used to confirm the absence of detectable mycoplasma in a given sample. The procedures are conducted in accordance with each section below. For viral suspensions, samples must be taken prior to filtration for live virus vaccines, and prior to both filtration and inactivation for inactivated virus vaccines.
If the test is performed within 24 hours of sample collection, samples must be stored at 2–8°C. If not, they must be stored at ≠−60°C. Generally, culture methods are used, and may be supplemented by nuclear staining using indicator cells or nucleic acid amplification techniques.
A. Culture Method
Media
Unless otherwise specified, use Mycoplasma Agar Medium ("agar plates") and Mycoplasma Liquid Media I and II. Other media are acceptable if they meet the growth promotion criteria below.
Growth Promotion Test
For Liquid Medium I: Inoculate with ≤100 CFU of a glucose-fermenting Mycoplasma species (e.g., Mycoplasma pneumoniae ATCC 15531 or equivalent).
For Liquid Medium II: Inoculate with ≤100 CFU of an arginine-metabolizing species (e.g., Mycoplasma orale ATCC 23714 or equivalent).
The medium must clearly change color within 7 days at 35–37°C.
For agar plates: Colonies of mycoplasma must be observable within 14 days after inoculation with <100 CFU of either test strain.
Growth Inhibition Test and Neutralization
Test whether the sample inhibits mycoplasma growth. For formulations with the same process, this test does not need to be repeated for each batch. Use Acholeplasma laidlawii unless another strain is known to be more sensitive.
Inoculate ∼100 CFU of the test strain into the sample mixed with appropriate liquid media (I for glucose-fermenters, II for arginine metabolizers) and incubate for 7 days. If no growth or delayed growth is observed compared to control, the sample has inhibitory activity. In such cases, take measures (e.g., dilution, neutralization, or passage) to remove inhibitory substances, and retest to confirm removal.
Culture Test Methods
If no inhibition is observed, perform both:
4.1 Direct Agar Culture: Inoculate 0.2 mL of sample per agar plate, using at least 10 plates per sample. Incubate at 35–37°C under 5–10% CO2 or nitrogen gas with appropriate humidity.
4.2 Enrichment Culture: Inoculate 0.2 mL of sample into each of at least 10 tubes of both Liquid Media I and II (10 mL/tube). Incubate sealed tubes for ≥14 days. On days 5–7 and the final day, transfer 0.2 mL into fresh medium and continue culture. If turbidity or color change occurs, dilute 10-fold and subculture 0.1 mL onto at least 2 agar plates per dilution.
4.3 Membrane Filtration: For samples with strong inhibitory activity, filter through 0.1 μm membrane, wash, and culture the membrane in 100 mL of both Media I and II. Follow the same enrichment culture procedure as above.
Observation
Monitor liquid media for color change. For agar plates, observe for colony formation on days 7 and 14. Use Diene's stain for questionable colonies.
Judgement
If no mycoplasma growth is observed in any of the above tests, the sample passes.
B. Indicator Cell Nuclear Staining Method Inoculate the sample into indicator cells. After culturing, stain the cells with a fluorescent nucleic acid dye and examine under a microscope. Detect presence of mycoplasma nucleic acid outside host nuclei.
If the sample or virus suspension is cytotoxic, neutralize it first. Test for any mycoplasma inhibitory activity, including from added neutralizing agents.
Validation of Method: Before testing, verify with Mycoplasma hyorhinis (ATCC 29052/17981 or equivalent) and Mycoplasma orale (ATCC 23714 or equivalent) at ≤100 CFU using the same indicator cells (e.g., Vero) under antimicrobial-free conditions. Detection of both indicates suitability.
Testing Procedure:
2.1 Seed indicator cells (e.g., Vero or equivalent) at 1×10⁴ cells/mL on coverslips in dishes and culture at 35–38°C with 5% CO2 for 1 day.
2.2 Inoculate culture supernatant as the test sample. Include negative control and positive controls (as above). Culture for 3–6 days until cells reach 50% confluency.
2.3 Fix coverslips with 3:1 methanol/acetic acid, dry, stain with a fluorescent dye (e.g., bisbenzamide), rinse, dry, and mount for observation under a fluorescence microscope (400–600x or greater).
Observation and Evaluation:
In negative control: only host nuclei fluoresce.
In positive control: scattered fluorescent dots indicate mycoplasma nucleic acid.
In test: if no such dots are observed, the sample passes.
C. Nucleic Acid Amplification Test (NAT) This method amplifies and detects nucleic acid specific to the class Mollicutes, including Mycoplasma, Ureaplasma, Spiroplasma, and Acholeplasma. Primers and probes must be specific and conserved across multiple species of Mollicutes, and must not cross-react with other bacteria (e.g., Clostridium, Lactobacillus) or host cell DNA.
Evaluate detection limit by testing serial 10-fold dilutions of known CFU or gene copy numbers. Use multiple Mollicutes species or strains (e.g., Mycoplasma hyorhinis ATCC 29052/17981 or equivalent, and Acholeplasma) as positive controls. Test should also evaluate matrix effects of sample on NAT. If no amplification is detected, the sample passes.