Aseptic Technique for Cell Culture

Rosalie J. Coté1

1 Becton Dickinson Microbiology Systems, Sparks, Maryland
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 1.3
DOI:  10.1002/0471143030.cb0103s00
Online Posting Date:  May, 2001
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Abstract

This unit describes some of the ways that a laboratory can deal with the constant threat of microbial contamination in cell cultures. A protocol on aseptic technique is described first. This catch‐all term universally appears in any set of instructions pertaining to procedures in which noncontaminating conditions must be maintained. In reality, aseptic technique encompasses all aspects of environmental control, personal hygiene, equipment and media sterilization, and associated quality control procedures needed to ensure that a procedure is, indeed, performed with aseptic, noncontaminating technique. Although cell culture can theoretically be carried out on an open bench in a low‐traffic area, most cell culture work is carried out using a horizontal laminar‐flow clean bench or a vertical laminar‐flow biosafety cabinet. Both are described here.

     
 
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Table of Contents

  • Basic Protocol 1: Aseptic Technique
  • Basic Protocol 2: Use of the Horizontal Laminar‐flow Clean Bench
  • Alternate Protocol 1: Use of the Vertical Laminar‐flow Biosafety Cabinet
  • Commentary
  • Figures
     
 
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Materials

Basic Protocol 1: Aseptic Technique

  Materials
  • Antibacterial soap
  • 70% ethanol or other appropriate disinfectant
  • 95% ethanol
  • Clean, cuffed laboratory coats or gowns
  • Latex surgical gloves
  • Clean, quiet work area
  • Shallow discard pans containing disinfectant
  • Bunsen burner or pilot‐activated burner (e.g., Touch‐o‐Matic, VWR)

Basic Protocol 2: Use of the Horizontal Laminar‐flow Clean Bench

  Materials
  • 70% ethanol or other disinfectant
  • Horizontal laminar‐flow hood, certified for use
  • Swabs (e.g., cheesecloth, paper towels)
  • Pilot light–activated Bunsen burner (e.g., Touch‐o‐Matic, VWR)

Alternate Protocol 1: Use of the Vertical Laminar‐flow Biosafety Cabinet

  • Class II, Type A Biosafety Cabinet (BSC), certified for use
  • Pilot light–activated Bunsen burner (e.g., Touch‐o‐Matic, VWR) or electronic incinerator (e.g., Bacti‐Cinerator III, VWR)
  • Closed‐front laboratory gowns (for personnel working with biological agents)
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Figures

Videos

Literature Cited

Literature Cited
   Richmond, J.Y. and McKinney, R.W. (eds.) 1993. Biosafety in microbiological and biomedical laboratories, 3rd ed. U.S. Government Printing Office, Washington, D.C.
   NSF (National Sanitation Foundation) International. 1992. Class II (laminar flow) biohazard cabinetry (NSF 49‐1992). NSF International, Ann Arbor, Mich.
Key References
   Barkley, W.E. and Richardson, J.H. 1994. Laboratory safety. In Methods for General and Molecular Bacteriology, 2nd ed. (P.E. Gerhardt, R.G.E. Murray, W.A. Wood, and N.R. Krieg, eds.) pp. 715‐734. American Society for Microbiology, Washington, D.C.
  Chapter provides an overview of general concerns for working with biological agents, from a classic publication on general methods in bacteriology that often overlaps to satisfy the technical needs of cell biologists.
   Chatigny, M.A. 1986. Primary barriers. In Laboratory Safety: Principles and Practices (B.M. Miller, D.H.M. Gröschel, J.H. Richardson, D. Vesley, J.R. Songer, R.D. Housewright, and W.E. Barkley, eds.) pp.144‐163. American Society for Microbiology, Washington, D.C.
  Offers detailed considerations on the types and uses of laminar‐flow barrier technology. The main publication is well worth its price for anyone (staff, supervisors, administrators) responsible for safety in a biological laboratory.
   Freshney, R.I. 1994. Culture of Animal Cells: A Manual of Basic Technique, 3rd ed. pp. 51‐69. Wiley‐Liss, New York.
  Offers suggestions for maintaining aseptic conditions while working with cell cultures. A classic cell culture publication that surveys the field while providing enough detail for an individual with intermediate knowledge of microbiology and cell biology.
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