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Aseptic Technique for Cell Culture

Rosalie J. Coté1

1Becton Dickinson Microbiology Systems, Sparks, Maryland

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

  • Unit Introduction
  • Basic Protocol 1: Aseptic Technique
  • Basic Protocol 2: Use of the Horizontal Laminar-flow Clean Bench
  • Alternate Protocol: Use of the Vertical Laminar-flow Biosafety Cabinet
  • Commentary
  • Bibliography
  • 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: Use of the Vertical Laminar-flow Biosafety Cabinet

 Materials (also see Basic Protocols 1 and 2)
  • 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

  • Figure 1.3.1
    Horizontal laminar-flow clean cabinet. Solid arrows, dirty room air; open arrows, clean HEPA-filtered air; circled +, positive pressure with respect to room air.

  • Figure 1.3.2
    Biological safety cabinet, Class II, Type A. Note that filtered air is contaminated after passing through the work space, and is filtered again whether it is recycled to the workspace(70%) or exhausted (30%). Solid arrows, dirty (room/contaminated) air; open arrows, filtered air.

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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