Detection of Mycoplasma Contamination in Cell Cultures

Cord C. Uphoff1, Hans G. Drexler1

1 Leibniz‐Institute DSMZ—German Collection of Microorganisms and Cell Cultures, Braunschweig
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 28.4
DOI:  10.1002/0471142727.mb2804s106
Online Posting Date:  April, 2014
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Abstract

Mycoplasma contamination of cell lines is a major problem in cell culture technology. This unit presents protocols involving either the polymerase chain reaction (PCR) or fluorescent in situ hybridization (FISH) to provide independent, fast, and sensitive techniques to monitor mycoplasma contamination in laboratory cultures. Special emphasis is placed on the integration of control reactions to prevent false‐negative as well as false‐positive results due to reaction inhibition or contamination and background staining, respectively. Curr. Protoc. Mol. Biol. 106:28.4.1‐28.4.14. © 2014 by John Wiley & Sons, Inc.

Keywords: contamination detection; FISH; mycoplasmas; PCR

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Detection of Mycoplasma by PCR
  • Alternate Protocol 1: Fluorescent in Situ Hybridization for Detection of Mycoplasma
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Detection of Mycoplasma by PCR

  Materials
  • Adherently growing cells or suspension cell cultures, and appropriate culture medium
  • Phosphate‐buffered saline (PBS; ), autoclaved
  • 5 mM dNTP mix (see recipe)
  • 5 µM each of the forward primers as mixture (Myco‐5′; see recipe for primers)
  • 5 µM each of the reverse primers as mixture (Myco‐3′; see recipe for primers)
  • 5 U/µl Taq DNA polymerase and appropriate 10× buffer without MgCl 2 (Platinum hot‐start Taq polymerase; Life Technologies)
  • 50 mM magnesium chloride (MgCl 2)
  • Internal control DNA (see recipe; also see http://www.cell‐lines.de)
  • Positive control DNA (see recipe; also see http://www.cell‐lines.de)
  • 1.3% agarose‐TAE gel containing 0.3 µg/ml ethidium bromide (unit )
  • TAE buffer ( )
  • 6× loading buffer (see recipe)
  • DNA extraction and purification system with columns (RTP DNA/RNA Virus Mini Kit from Stratec, or Wizard DNA Clean‐Up System from Promega, or apply a method described in unit or another commercially available kit; also see Commentary)
  • 0.2‐ml PCR tubes
  • Thermal cycler (we used Applied Biosystems GeneAmp PCR System 9700)
  • Additional reagents and equipment for extraction and purification of DNA (unit ), PCR (unit ), and agarose gel electrophoresis (unit )

Alternate Protocol 1: Fluorescent in Situ Hybridization for Detection of Mycoplasma

  Materials
  • Adherently growing cells or suspension cell cultures, and appropriate culture medium
  • Phosphate‐buffered saline (PBS; ), autoclaved
  • 4% paraformaldehyde (see recipe)
  • 1:1 (v/v) PBS/ethanol (store and use at –20°C)
  • Washing buffer (see recipe)
  • 50%, 80%, and 100% (absolute) ethanol
  • Hybridization solution (see recipe)
  • 10 mg/ml denatured fish sperm DNA
  • Fluorescence primer: 5 µg/ml EUB16S‐338, 5′‐AlexaFluor 488–gct gcc tcc cgt agg agt‐3′ (Sigma‐Aldrich)
  • Background control primer: 5 µg/ml Anti‐EUB16S‐338, 5′‐AlexaFluor 488–cga cgg agg gca tcc tca‐3′ (Sigma‐Aldrich)
  • Mounting medium (see recipe)
  • Immersion oil
  • Hybridization oven
  • Humidified chamber (see step 7 annotation)
  • Coplin jars
  • Cytocentrifuge (Shandon Cytospin 3)
  • Slides: soak slides in 1% HCl overnight, wash slides twice in a slide washer without detergent, soak slides overnight in 70% ethanol, and wipe with lint‐free cloth. Store the washed slides in a dry place.
  • Pap pen
  • Coverslips
  • Fluorescence microscope equipped with filters for green fluorescence and a UV lamp for DAPI stain
  • x63 Plan Apochromat (oil) objective (or equivalent)
  • Additional reagents and equipment for cell culture techniques including trypsinization ( )
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Figures

Videos

Literature Cited

Literature Cited
   Amann, R.I. , Ludwig, W. , and Schleifer, K.H. 1995. Phylogenetic identification and in situ detection of individual microbial cells without cultivation. Microbial. Rev. 59:143‐169.
   Anonymous. 2013. Mycoplasmas 2.6.7. In European Pharmacopoeia, 8th ed., Vol. 1, pp. 178‐182. Council of Europe, Strasbourg, France.
   Coecke, S. , Balls, M. , Bowe, G. , Davis, J. , Gstraunthaler, G. , Hartung, T. , Hay, R. , Merten, O.W. , Price, A. , Schechtman, L. , Stacey, G. , and Stokes, W. 2005. Guidance on good cell culture practice: A report of the second ECVAM task force on good cell culture practice. Altern. Lab. Anim. 33:261‐287.
   Drexler, H.G. and Uphoff, C.C. 2002. Mycoplasma contamination of cell cultures: Incidence, sources, effects, detection, elimination, prevention. Cytotechnol. 39:23‐38.
   Kong, F. , James, G. , Gordon, S. , Zelynski, A. , and Gilbert, G.L. 2001. Species‐specific PCR for identification of common contaminant Mollicutes in cell culture. Appl. Environ. Microbiol. 67:3195‐3200.
   Razin, S. 2006. The genus Mycoplasma and related genera (class Mollicutes). In The Prokaryotes. Volume 4 ( M. Dworkin , S. Falkow , E. Rosenberg , K.‐H. Schleifer , and E. Stackebrandt , eds.), pp. 836‐904. Springer, New York.
   Uphoff, C.C. and Drexler, H.G. 1999. Detection of mycoplasma contaminations in cell cultures by PCR analysis. Human Cell 12:229‐236.
   Uphoff, C.C. and Drexler, H.G. 2001. Prevention of mycoplasma contamination in leukemia‐lymphoma cell lines. Human Cell 14:244‐247.
   Uphoff, C.C. and Drexler, H.G. 2002. Comparative PCR analysis for detection of mycoplasma infections in continuous cell lines. In Vitro Cell. Dev. Biol. Anim. 38:79‐85.
   Uphoff, C.C. and Drexler, H.G. 2010. Contamination of cell cultures, mycoplasma. In The Encyclopedia of Industrial Biotechnology. Volume 5 ( M. Flickinger , ed.) pp. 3611‐3630. John Wiley & Sons, Hoboken, New Jersey.
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