Diagnosis and Treatment of Mycoplasma‐Contaminated Cell Cultures

Frank W. Fitch1, Thomas F. Gajewski1, Wayne M. Yokoyama2

1 University of Chicago, Chicago, Illinois, 2 University of California School of Medicine, San Francisco, California
Publication Name:  Current Protocols in Immunology
Unit Number:  Appendix 3E
DOI:  10.1002/0471142735.ima03es21
Online Posting Date:  May, 2001
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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.

     
 
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Table of Contents

  • Basic Protocol 1: Testing for Mycoplasma Infection
  • Basic Protocol 2: Treatment of Mycoplasma‐Contaminated Cell Cultures with BM‐Cycline
  • Alternate Protocol 1: Treatment of Mycoplasma‐Contaminted Cell Cultures with Ciprofloxacin
  • Reagents and Solutions
  • Commentary
     
 
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Materials

Basic Protocol 1: Testing for Mycoplasma Infection

  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 BM‐Cycline

  Materials
  • recipeBM‐cycline solutions 1 and 2 (see recipe), sterile
  • Contaminated cells (see protocol 1)

Alternate Protocol 1: Treatment of Mycoplasma‐Contaminted Cell Cultures with Ciprofloxacin

  Additional Materials
  • recipe10 mg/ml ciprofloxacin⋅HCl (see recipe), sterile
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Figures

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Literature Cited

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.
   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.
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.
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