Diagnosis and Treatment of Mycoplasma‐Contaminated Cell Cultures

Helena Harlin1, Thomas F. Gajewski1

1 University of Chicago, Chicago, Illinois
Publication Name:  Current Protocols in Microbiology
Unit Number:  Appendix 3B
DOI:  10.1002/9780471729259.mca03bs00
Online Posting Date:  January, 2006
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

Mycoplasma contamination is a serious and frequent problem in the culture laboratory. Although mycoplasma contamination may be suspected by the failure of cells to thrive, the formal diagnosis rests on the detection of adenosine phosphorylase secretion by infected cell lines. This appendix describes how to test for mycoplasma contamination, and also presents methods for antibiotic treatment of infected cultures.

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Basic Protocol 1: Testing for Mycoplasma Infection by PCR
  • Alternate Protocol 1: Testing for Mycoplasma Infection by Detection of Adenosine Phosphorylase Secretion
  • Basic Protocol 2: Treatment of Mycoplasma‐Contaminated Cell Cultures with Ciprofloxacin
  • Alternate Protocol 2: Treatment of Mycoplasma‐Contaminated Cell Cultures with BM‐Cyclin
  • Reagents and Solutions
  • Commentary
  • Literature Cited
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Testing for Mycoplasma Infection by PCR

  Materials
  • Cell cultures to be tested for contamination
  • Phosphate‐buffered saline (PBS; appendix 2A) or culture medium for cells
  • H 2O, sterile
  • Roche Mycoplasma PCR ELISA kit (cat. no. 1663925) including:
    • Lysis reagent
    • Mycoplasma positive control DNA
    • Neutralization reagent
    • PCR ready‐to‐go mix
  • Taq DNA polymerase
  • Tabletop centrifuge
  • Thin‐walled PCR tubes
  • Thermal cycler
  • Additional reagents and equipment for agarose gel electrophoresis (Sambrook and Russell, ; Voytas, )

Alternate Protocol 1: Testing for Mycoplasma Infection by Detection of Adenosine Phosphorylase Secretion

  Materials
  • SP2/0‐Ag14 hybridoma cells (ATCC #CRL 1581)
  • Cells to be tested in appropriate medium without antibiotics
  • MycoTect (Life Technologies) containing 6‐methylpurine deoxyriboside (6‐MPDR)
  • 6‐methylpurine (6‐MP; Sigma)
  • 96‐well flat‐bottom microtiter plates (Costar, Falcon, or equivalent)

Basic Protocol 2: Treatment of Mycoplasma‐Contaminated Cell Cultures with Ciprofloxacin

  Materials
  • 10 mg/ml ciprofloxacin⋅HCl (see recipe), sterile
  • Contaminated cells (see protocol 1 and protocol 2)

Alternate Protocol 2: Treatment of Mycoplasma‐Contaminated Cell Cultures with BM‐Cyclin

  Materials
  • BM‐cyclin solutions 1 and 2 (see recipe), sterile
  • Contaminated cells (see protocol 1 and protocol 2)
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

   McGarrity, G.J. and Carson, D.A. 1982. Adenosine phosphorylase‐mediated nucleoside toxicity: Application towards the detection of mycoplasmal infection in mammalian cell cultures. Exp. Cell Res. 139:199‐205.
   Phelan, K. 1996. Techniques for mammalian cell tissue culture. In Current Protocols in Molecular Biology (F.M. Ausubel, R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, and K. Struhl, eds.) pp. A.3F.1‐A.3F.14. John Wiley & Sons, Hoboken, N.J.
   Sambrook, J. and Russell, D. 2001. Molecular Cloning: A Laboratory Manual, 3rd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
   Schmitt, K., Däubeaner, W., Bitter‐Suermann, D., and Hadding, U. 1988. A safe and efficient method for elimination of cell culture mycoplasmas using ciprofloxacin. J. Immunol. Methods 109:17‐25.
   Shulman, M., Wilde, C.D., and Köhler, G. 1978. A better cell line for making hybridomas secreting specific antibodies. Nature (Lond.) 276:269‐270.
   Voytas, D. 2000. Agarose gel electrophoresis. In Current Protocols in Molecular Biology (F.M. Ausubel, R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, and K. Struhl, eds.), pp. 2.5A.1‐2.5A.9. John Wiley & Sons, Hoboken, N.J.
   Wirth, M., Grashoff, M., Schumacher, L., and Hauser, H. 1995. Mycoplasma detection by the Mycoplasma PCR ELISA. Biochemica 3:33‐35.
   Yokoyama, W. 1991. Production of monoclonal antibodies. In Current Protocols in Immunology. (J.E. Coligan, A.M. Kruisbeek, D.M. Margulies, E.M. Shevach, and W. Strober, eds.) pp. 2.5.1‐2.5.17. John Wiley & Sons, New York.
Key Reference
   McGarrity, G.J., Sarama, J., and Vanaman, V. 1979. Factors influencing microbiological assay of cell‐culture Mycoplasmas. In Vitro (Rockville) 15:73‐81.
  Classic review of mycoplasma contamination and its evaluation.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library