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REFERENCE NO: AHC/1998/3/3.2/1.5



This protocol describes the eradication of mycoplasma from cell cultures by treatment with antibiotics, examples of those used are given below.

Agents Used for Mycoplasma Eradication:

  • Mycoplasma Removal Agent (MRA) - a Fluoroquinolone
    Working concentration: 1 in 100 dilution of stock = 500 ng/ml.
    Supplier: ICN Biomedicals Ltd
  • Ciprofloxacin - a Fluoroquinolone
    Working concentration: 1 in 100 dilution of stock = 20mg/ml
    Supplier: Bayer
  • BM-Cyclin - Antibiotic Combination
    Working concentration: BM - Cyclin 1 - a pleuromutilin derivative 5mg/ml
    BM - Cyclin 2 - a tetracycline derivative 10mg/ml
    Supplier: Boehringer Mannheim


  1. One ampoule is thawed and cells recovered according to laboratory procedure AHC/1998/3/2.5/2 (resuscitation of frozen ampoules of cell lines).
  2. Cells are maintained for at least two cultures in 25 cm2 flasks before treatment with an appropriate antibiotic.
  3. Hybridoma cell lines require antibody testing before treatment with antibiotics and at points 6c and 7. Supply 5ml supernatant from a confluent flask for antibody testing (AHC/1998/3/3.5/2).
  4. Cells are maintained in the presence of antibiotics with specific anti-mycoplasma activity for a period of 14 to 21 days with the appropriate number of passages.
  5. At 14 to 21 days the cells are passaged to three 25 cm2 flasks without antibiotics; flasks are labelled, for example MRA-1.
  6. a) At the second passage without antibiotics (e.g. MRA-2) one of the three flasks is examined for the presence of mycoplasma by Hoechst stain (AHC/998/3/3.2/2.1).
  7. b) If this result is negative, the second flask is expanded for a token freeze of at least seven ampoules.

    c) The remaining flask is subcultured continuously for a total of 10 passages, maintaining three flasks at each passage. One flask is tested by Hoechst stain at each passage and a the results recorded (see herewith). If the culture is Hoechst stain negative after 10 passages the mycoplasma is considered to have been eradicated. A sample is sent for antibody testing at this point (AHC/1998/3/3.5/2).

  8. After ten mycoplasma-free passages an ampoule from the token freeze is resuscitated, grown for two passages in antibiotic-free medium and examined for the presence of mycoplasma by Hoechst stain and isolation (AHC/1998/3/3.2/2.1 and AHC/1998/3/3.2/2.2) and for bacteria and fungi contamination (AHC/1998/3/3.3/2.1). Reserve 5ml supernatant for antibody testing (AHC/1998/3/3.5/2).
  9. If at any stage mycoplasma contamination reappears then the eradication procedure will start with an original culture either with a different antibiotic or combination of antibiotics.

If attempts to eradicate fail, a new deposit may be obtained from the depositor or the cell line has to be taken out of the collection.

Guidelines prepared for CABRI by CERDIC, DSMZ, ECACC, INRC, November 1998
Page layout by CERDIC
Copyright CABRI, 1998

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