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Nutritional effects of culture media on mycoplasma cell size and removal by filtration
Martha Folmsbee1, Glenn Howard, Morven McAlister
1Pall Corporation, 25 Harbor Park Dr., Port Washington, NY 11050, USA. martha_folmsbee@pall.com
Abstract:
Careful media filtration prior to use is an important part of a mycoplasma contamination prevention program. This study was conducted to increase our knowledge of factors that influence efficient filtration of mycoplasma. The cell size of Acholeplasma laidlawii was measured after culture in various nutritional conditions using scanning electron microscopy. The maximum cell size changed, but the minimum cell size remained virtually unchanged and all tested nutritional conditions resulted in a population of cells smaller than 0.2 microm. Culture in Tryptic Soy Broth (TSB) resulted in an apparent increase in the percentage of very small cells which was not reflected in increased penetration of non-retentive 0.2 microm rated filters. A. laidlawii cultured in selected media formulations was used to challenge 0.2 microm rated filters using mycoplasma broth base as the carrier fluid. We used 0.2 microm rated filters as an analytical tool because A. laidlawii is known to penetrate 0.2 microm filters and the degrees of penetration can be compared. Culture of A. laidlawii in TSB resulted in cells that did not penetrate 0.2 microm rated filters to the same degree as cells cultured in other media such as mycoplasma broth or in TSB supplemented with 10% horse serum.
Insights
Media filtration is key for mycoplasma contamination control. Acholeplasma laidlawii cell size varies with media, but even small cells don't always penetrate 0.2 micrometer filters, especially when cultured in Tryptic Soy Broth (TSB).
Area of Science:
- Microbiology
- Cell Biology
- Filtration Technology
Background:
- Mycoplasma contamination poses a significant threat to cell cultures and biopharmaceutical production.
- Effective media filtration is a critical component of mycoplasma contamination prevention strategies.
- Understanding factors influencing mycoplasma cell size and filter penetration is essential for optimizing filtration efficiency.
Purpose of the Study:
- To investigate the impact of nutritional conditions on the cell size of Acholeplasma laidlawii.
- To evaluate the penetration of A. laidlawii through 0.2 micrometer rated filters under various culture conditions.
- To identify media formulations that may influence mycoplasma filterability.
Main Methods:
- Scanning electron microscopy was employed to measure Acholeplasma laidlawii cell dimensions after culture in diverse nutritional media.
- 0.2 micrometer rated filters were challenged with A. laidlawii cultured in different media, using mycoplasma broth base as the carrier fluid.
- Filter penetration levels were quantified to assess the efficacy of filtration under varying conditions.
Main Results:
- While maximum cell size varied with nutritional conditions, minimum cell size remained consistent, with all cells measuring below 0.2 micrometers.
- Culture in Tryptic Soy Broth (TSB) led to an apparent increase in very small A. laidlawii cells, but this did not correlate with increased filter penetration.
- A. laidlawii cultured in TSB exhibited reduced penetration through 0.2 micrometer filters compared to cells grown in mycoplasma broth or TSB with horse serum.
Conclusions:
- Nutritional media significantly influences Acholeplasma laidlawii cell size distribution.
- Tryptic Soy Broth (TSB) appears to reduce the filterability of A. laidlawii through 0.2 micrometer filters, despite not altering the minimum cell size.
- These findings suggest that media composition is a crucial factor to consider in mycoplasma filtration strategies for contamination control.
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