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Mycoplasma Detection

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BACKGROUND:

Mycoplasmas are very small free-living organisms similar to bacteria but lacking a cell wall. They are ubiquitous in nature and are potential contaminants in biotechnology and cell-based products. Biological products thus require examination for presence/absence of Mycoplasmas before release for sale/use.  Since Mycoplasmas have been commonly found to contaminate cell cultures, it is also wise to check these periodically to ensure they remain free from infection.  This is especially important where these cells may be developed as Master Cell stock for product development purposes. There are several detection methods available, including conventional culture enrichment and growth on agar, cell culture substrate with Hoechst staining and more rapid Polymerase Chain Reaction ( PCR) methods.  Eurofins | ams is able to offer this detection service.

PRINCIPLES:

Mycoplasma Detection by Conventional Test

Two test methods are followed to detect Mycoplasma contamination:

  1. Enrichment in broth and detection by colony formation on selective agar plates.  Samples are set up as per pharmacopoeia method whereby product is inoculated into Mycoplasma enrichment broth and onto selective medium agar plates.  Broths are sub-cultured onto further agar plates for up to two weeks.  Cultures are incubated aerobically and microaerophilically.  Inoculated media are examined for evidence of Mycoplasma growth.

  2. Enrichment in cell culture and characteristic fluorescent staining of DNA.  Samples are inoculated onto susceptible Mycoplasma-free host cell cultures and incubated for 4 days.  Cells are then passaged to fresh cultures and further incubated.  After incubation, cells are stained with Hoechst stain and examined under a fluorescent microscope for signs of Mycoplasma infection. 

Mycoplasma Detection by PCR

 Two test methods are available:

  1. PCR Mycoplasma Screening Test: A more rapid detection method for screening Mycoplasma is available through the use of PCR technique.

  2. Rapid PCR test as an alternative to conventional methods: Unlike the conventional procedure, enrichment in cell culture for four days and then the cell suspension is analyzed immediately using PCR  technique. The entire process takes seven days to complete. This has recently been approved by BP/EP as a suitable alternative to the conventional method.   

METHOD REFERENCE:

  1. Current BP, Appendix XVI B Microbiological Examination of Non-sterile Product, Section 3 – Test for absence of Mycoplasmas.

SAMPLE REQUIREMENTS

 Please contact the laboratory to discuss your project requirements 

Test Protocols
Method Code Test Description Turnaround Time (TAT)
TMCV-240 Mycoplasma - Culture Method 4-5 weeks
TMCV-242 Mycoplasma - PCR 3-5 days