Procedures for the Eradication of

Mycoplasmas from Cell Cultures

 

Before embarking upon eradication, the mycoplasma contaminant is identified and cultured in liquid medium to a high titre. Minimum inhibitory concentrations are established with these cultures against a range of anti-mycoplasma antibiotics suitable for use in cell-cultures. In addition, specific antiserum to the species identified is tested for its ability to inhibit growth of the contaminant.

When these tests are complete, containers of antiserum and anti-mycoplasma antibiotic (at concentrations known to be inhibitory after dilution) are dispatched to the cell culture laboratory with a container of ampicillin. A schedule giving the method of reagent use is included.

Two phases of cell subcultivation are required, one for eradication and one for confirmation that the eradication phase has been successful. Firstly, subculture in medium containing specific antiserum combined with an anti-mycoplasma antibiotic and secondly, subculture in the absence of mycoplasma inhibitors to ensure that any residual mycoplasmas will be detected in the event of an unlikely failure of the eradication phase.

For the first phase, add the contents of the two bijoux labelled Antiserum and Antibiotic to 100 ml. of growth medium, mix and distribute into 5 x 20 ml. amounts for storage at 4C. Use for subcultures A1 to A5 as given in the schedule.

For the second phase, Ampicillin (non-inhibitory for mycoplasmas) is added to growth medium at 1ml./100 ml. Sufficient Ampicillin is provided for 200 ml. of medium to allow culture volume increase prior to storage. Use for subcultures F1 to F5. A mycoplasma cell screen test is conducted at the F1 passage level to confirm satisfactory progress.

Passage F5 can be stored as mycoplasma-free seed and part of this passage is sent for a cell-culture mycoplasma test to confirm the absence of mycoplasmas. Copies of test sheets and media lot validation forms are included with the final report.

Mycoplasma Experience

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