Mollicutes: Wall-Less Wonders & How ATCC Helps Them Thrive

Mollicute culture essentials: Media, pH, and serum supplements

Media matters
Mollicutes typically require rich broth media, as many species do not grow on agar. Even in broth, growth is often barely visible, so media are commonly supplemented with pH indicators to monitor metabolic activity. Because colony formation is minimal or absent, visual confirmation of growth is unreliable. Biochemical markers and regular monitoring are essential for assessing culture viability.

pH is critical
Careful pH monitoring is vital as mollicutes are extremely sensitive to even minor fluctuations. These shifts can signal cellular stress or impaired growth. Maintaining a stable pH between 7.2 and 7.6 is generally optimal; deviations can disrupt membrane function, enzyme activity, and nutrient uptake. Regular subculturing helps preserve viability and prevents the buildup of toxic metabolic byproducts. Buffering reagents can help maintain pH stability within the ideal range.  

Serum supplements
To support membrane integrity, media should be supplemented with 10% fetal bovine serum (FBS) or horse serum. These components stabilize membranes, buffer pH, and inactivate proteolytic enzymes. Serum also provides essential lipids and growth factors that enhance cellular viability and proliferation.

Growth on Mollicute time: A lesson in patience

Mollicutes grow slowly—some species may take several weeks to show signs of activity. Cultivating mollicutes requires patience and precision, but with the right strategies, success is achievable. Common challenges include:  

• No visible growth: Extend incubation time, use pH indicators, and confirm media enrichment. Also, a 10% inoculum is recommended when scaling up cultures to ensure robust growth.
• Atmospheric sensitivity: Some species require microaerophilic or CO₂-enriched conditions (e.g., 5–10% CO₂ or GasPak systems (BD)).
• Poor growth: Avoid freeze-thaw cycles and ensure serum supplementation.
• Incorrect pH or osmolarity: Consider HEPES-buffered media, which helps maintain a stable and physiologically favorable pH environment—crucial for supporting mollicute growth and minimizing cell stress. 
• Growth timeline: Mollicute growth may take several days to weeks, depending on species and conditions—especially on solid media. Be patient and monitor cultures for up to 14 days. 

ATCC: Your Mollicute resource

ATCC maintains a scientifically valuable repository of mollicute strains that facilitate advanced research across a wide range of disciplines. Notable examples include: 

• Mycoplasma pneumoniae: A key model for studying respiratory infections, immune evasion, and antibiotic resistance.
• Spiroplasma citri: Widely used in agricultural research, particularly for plant-pathogen interactions and membrane protein studies.
• Ureaplasma urealyticum: Important for reproductive health research, including infertility, pregnancy complications, and neonatal infections.
• Mycoplasmoides genitalium: With its minimal genome, it serves as a powerful system for investigating essential gene functions and sexually transmitted infections.

Together, these strains form a robust platform for advancing mollicute biology and driving innovation in diagnostics, therapeutics, and systems biology.

Streamlining Mollicute research with ATCC’s proven resources

From troubleshooting stubborn cultures to scaling up for genomic analysis, ATCC equips researchers with validated strains, optimized media, and detailed protocols. These resources simplify cultivation even for fastidious species and ensure consistency for downstream applications such as microbial quality control testing, sequencing, transcriptomics, and vaccine development.

Whether you're advancing clinical microbiology, innovating in synthetic biology, or exploring agricultural applications, ATCC’s mollicute resources are designed to streamline your workflow and accelerate discovery. For more insights, watch the webinar: ATCC’s Best Practices for Optimal Growth and Propagation of Bacteria and Bacteriophages, where I explore effective strategies for cultivating mollicutes. 

Did you know?

ATCC offers a Mycoplasma growth supplement that can be used in broth and agar media to help you isolate and grow various Mycoplasma species.

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