Assessing and Controlling Microbial Contamination in Cell Cultures

Rosalie Coté1

1 Becton Dickinson Microbiology Systems, Sparks, Maryland
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 1.5
DOI:  10.1002/0471143030.cb0105s01
Online Posting Date:  May, 2001
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Abstract

This unit describes how to detect and recognize bacterial, fungal, or mycoplasma contamination and how to verify the presence of mycoplasma. Strategies are described for dealing with culture contamination, if necessary.

     
 
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Table of Contents

  • Basic Protocol 1: Testing for Bacterial and Fungal Contaminants
  • Basic Protocol 2: Testing for Mycoplasma Contamination by Direct Culture
  • Alternate Protocol 1: Indirect Testing for Mycoplasma by Staining for DNA
  • Alternate Protocol 2: Indirect Testing for Mycoplasma by PCR
  • Support Protocol 1: Agarose Gel Electrophoresis of PCR Products
  • Basic Protocol 3: Use of Antibiotics to Control Microbial Contamination
  • Reagents and Solutions
  • Commentary
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Testing for Bacterial and Fungal Contaminants

  Materials
  • Medium for bacterial detection: e.g., brain heart infusion (BBL, Difco), fluid thioglycollate medium (BBL, Difco), HTYE broth (see recipe), soybean/casein digest broth USP (e.g., trypticase soy broth, BBL; tryptic soy broth, Difco), or trypticase soy agar (BBL)
  • Medium for mycelial and yeast fungal detection: e.g., Sabouraud's dextrose agar (Emmon's modified; BBL, Difco), or YM agar (Difco)
  • Sterile, defibrinated sheep blood (e.g., Colorado Serum, Waltz Farm)
  • Cell culture test samples
  • Antibiotic‐free culture medium (optional)
  • Conductivity meter (Corning model 162 or equivalent), if not integrated with the laboratory water purification system
  • 50°C water bath
  • 16 × 125–mm borosilicate screw‐cap test tubes with rubber‐lined caps
  • 100 × 15–mm sterile plastic disposable petri dishes
  • Semiautomated repeat‐volume filling unit to accurately dispense 5‐ to 24‐ml aliquots (optional)
  • Incubators: 26°C, 35° to 37°C, and 37°C with 5% (v/v) CO 2
NOTE: To avoid inadvertent contamination of clean cell lines, bacterial and fungal testing should be segregated to a laboratory not used for general cell culture work.

Basic Protocol 2: Testing for Mycoplasma Contamination by Direct Culture

  Materials
  • Cell line for testing
  • Mycoplasma broth medium (see recipe): 6 ml medium in 16 × 125–mm screw‐cap test tubes
  • Mycoplasma agar plates (see recipe): 10 ml solidified medium in 60 × 15–mm petri dishes
  • 37°C incubators: one without CO 2 and one humidified with 5% (v/v) CO 2
  • Inverted microscope with 100 to 300× magnification
NOTE: To avoid inadvertent contamination of clean cell lines, mycoplasma testing should be segregated to a laboratory not used for general cell culture work.

Alternate Protocol 1: Indirect Testing for Mycoplasma by Staining for DNA

  Materials
  • Complete EMEM‐10: Eagle's minimum essential medium (EMEM) with Earle's salts (Life Technologies), 100 U/ml penicillin, 100 µg/ml streptomycin, and 10% (v/v) bovine calf serum (see unit 1.2 for media preparation methods)
  • Indicator cell line: e.g., African green monkey cell line Vero (ATCC #CCL81) or 3T6 murine cell line (ATCC #CCL96)
  • Cell culture for testing
  • Mycoplasma hyorhinis (ATCC #29052) or a known mycoplasma‐infected cell line to use as a positive control, actively growing
  • Fixative: 3:1 (v/v) absolute methanol/glacial acetic acid
  • Hoechst stain (see recipe)
  • Mounting medium (see recipe)
  • 60 × 15–mm culture dishes, sterile
  • No. 1 or no. 1 ½ coverslips, sterilized by autoclaving (unit 1.4)
  • 37°C, 5% (v/v) CO 2/95% air incubator
NOTE: To avoid inadvertent contamination of clean cell lines, mycoplasma testing should be segregated to a laboratory not used for general cell culture work.

Alternate Protocol 2: Indirect Testing for Mycoplasma by PCR

  Materials
  • Cells for testing
  • 10× PCR buffer (usually provided with Taq polymerase)
  • 2.5 mM 4dNTP mix: 2.5 mM each dGTP, dCTP, dTTP, and dATP
  • 25 mM MgCl 2
  • 5 U/µl Taq DNA polymerase
  • Mineral oil (if needed for thermal cycler)
  • Mycoplasma detection kit (ATCC), containing first‐ and second‐stage primer mixtures (total 7 primers), as well as two positive control mycoplasma DNAs (Mycoplasma pirum, Acholeplasms laidlawii)
  • Thin‐wall microcentrifuge tubes
  • Aerosol‐preventive micropipettor tips, sterile
  • Positive‐displacement micropipettors
  • Picofuge
  • Thermal cycler
  • Additional reagents and equipment for agarose gel electrophoresis (see protocol 5)
NOTE: To avoid inadvertent contamination of clean cell lines, mycoplasma testing should be segregated to a laboratory not used for general cell culture work.NOTE: To avoid amplification of contaminating DNA from laboratory workers, room contaminants, or previous mycoplasma DNA amplifications, all PCR should be performed using aseptic technique (also see special considerations for PCR experiments in appendix 2A).

Support Protocol 1: Agarose Gel Electrophoresis of PCR Products

  Materials
  • Agarose (e.g., NuSieve, FMC Bioproducts)
  • 1× TBE electrophoresis buffer ( appendix 2A)
  • 10 mg/ml ethidium bromide solution
  • Second‐stage PCR products from test samples and controls (see protocol 4)
  • 6× electrophoresis sample buffer (see recipe)
  • Molecular weight marker (100‐bp DNA ladder)
  • Electrophoresis apparatus with a 10 × 14–in. gel tray and a 1‐mm, 10‐tooth comb
  • Power supply
  • UV light box

Basic Protocol 3: Use of Antibiotics to Control Microbial Contamination

  Materials
  • Contaminated cell culture
  • Sterile antibiotic stock solution(s) as appropriate (Table 1.5.1)
  • Additional reagents and equipment for identifying microbial contamination (see Basic Protocols protocol 11 and protocol 22; see Alternate Protocols protocol 31 and protocol 42)
    Table 1.5.1   Materials   Antibiotics a   Antibiotics

    Organism Antibiotic Solvent Stability (days at 37°C) Working concentration
    Bacteria (grem‐positive only) Ampicillin Water 3 100 mg/liter
    Erythromycin 2 M HCI 3 100 mg/liter
    Gentamicin sulfate Water 5 50 mg/liter
    Kanamycin sulfate Water 5 100 mg/liter
    Neomycin sulfate Water 5 50 mg/liter
    Penicillin‐G, K+ salt Water 3 105 U/liter
    Streptomycin sulfate Water 3 100 mg/liter
    Tetracycline HCI Water 4 10 mg/liter
    Fungi (molds and yeasts) Amphotericin B DMSO DMF b 3 2.5 mg/liter
    Nystatin DMF 3 2.5×106 U/liter
    Mycoplasma Gentamicin sulfate Water 5 50.0 mg/liter

     aAll antibiotics must be filter sterilized in the slovent noted. Antibiotics are meant only for short‐term use; if contamination is not cleared after 14 days of treatment or two subcultures, discard culture.
     bAbbreviations: DMF; dimethylformamide; DMSO, dimethyl sulfoxide.
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Figures

Videos

Literature Cited

Literature Cited
   Sambrook, J., Fritsch, E.F., and Maniatis, T. 1989. Molecular Cloning: A Laboratory Manual 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
Key References
   Barnhart, E.R. 1990. Physicians' Desk Reference. Medical Economics Company, Oradell, N.J.
  Provides invaluable information on the mode of action of, for instance, antibiotics and antagonisms.
   Budavari, S., O'Neil, M.J., Smith, A., Heckelman, P.E., and Kinneary, J.F. (eds.) 1996. The Merck Index. Merck Research Laboratories Division of Merck & Co., Whitehouse Station, N.J.
  A technical reference providing information on physical characteristics, solubilities, and stabilities of most chemicals (including antibiotics) used in a tissue culture laboratory.
   Freshney, R.I. 1994. Contamination. In Culture of Animal Cells: A Manual of Basic Technique, 3rd ed., pp 243‐252. Wiley‐Liss, New York.
  This chapter of the classic text on cell culture discusses various types of contamination and methods for detection and eradication.
   Hay, R.J., Caputo, J., and Macy, M.L. 1992. ATCC Quality Control Methods for Cell Lines, 2nded. American Type Culture Collection, Rockville, Md.
  A manual of procedures used in this cell culture collection's quality control program.
   Sigma‐Aldrich Co. 1998. Cell Culture Catalogue. Sigma‐Aldrich Co., St. Louis, Mo.
  Most specialty chemical companies feature a wealth of useful, technical information with their catalogues. This particular edition can be most helpful to the novice user of cell cultures.
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