Sterilization and Filtration

Rosalie J. Coté1

1 Becton Dickinson Microbiology Systems, Sparks, Maryland
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 1.4
DOI:  10.1002/0471143030.cb0104s01
Online Posting Date:  May, 2001
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Abstract

This unit on sterility in the tissue culture environment describes methods for sterilization of liquid and dry goods used for tissue culture and filtration of liquids to prevent contamination of cultures.

     
 
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Table of Contents

  • Basic Protocol 1: Autoclaving Liquids
  • Alternate Protocol 1: Autoclaving Dry Goods
  • Alternate Protocol 2: Autoclaving for Decontamination of Biological Waste
  • Support Protocol 1: Use of Biological Indicators for Monitoring Autoclave Processes
  • Basic Protocol 2: Dry‐Heat Sterilization and Depyrogenation
  • Support Protocol 2: Use of Biological Indicators for Monitoring Dry‐Heat Sterilization
  • Basic Protocol 3: Use of Disinfectants: 70% Ethanol
  • Alternate Protocol 3: Use of Disinfectants: Quaternary Ammonium Compounds
  • Alternate Protocol 4: Use of Disinfectants: Sodium Hypochlorite
  • Basic Protocol 4: Vacuum Filtration
  • Basic Protocol 5: Small‐Volume Positive‐Pressure Filtration of Nonaqueous Solutions
  • Alternate Protocol 5: Large‐Volume Positive‐Pressure Filtration of Nonaqueous Solutions
  • Alternate Protocol 6: Large‐Volume Positive‐Pressure Filtration Using Pressurized Nitrogen
  • Reagents and Solutions
  • Commentary
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Autoclaving Liquids

  Materials
  • Heat‐resistant containers and vessels (e.g., borosilicate glass, high‐grade stainless steel, noncytotoxic plastic)
  • Liquid to be autoclaved
  • Moisture‐resistant labels
  • Paper or aluminum foil
  • Autoclave indicator tape
  • Autoclave
  • Autoclavable discard pans

Alternate Protocol 1: Autoclaving Dry Goods

  • Items to be autoclaved
  • Shallow heat‐resistant container

Alternate Protocol 2: Autoclaving for Decontamination of Biological Waste

  • Items for decontamination
  • Plastic (polyethylene or polypropylene) autoclavable bags for biohazardous waste

Support Protocol 1: Use of Biological Indicators for Monitoring Autoclave Processes

  • Biological indicator ampules: standardized concentration of Bacillus stearothermophilus (ATCC #7953) spores suspended in growth medium containing bromcresol purple as a pH indicator (e.g., Prospore from Raven Biological Laboratories)
  • 55° to 60°C incubator

Basic Protocol 2: Dry‐Heat Sterilization and Depyrogenation

  Materials
  • Items to be sterilized
  • Heat‐resistant outer containers (borosilicate glass or stainless steel) for small items
  • Aluminum foil
  • Heat‐resistant labels or tape
  • Dry‐heat indicator tape
  • Laboratory oven (operating temperature of 140° to 180°C for sterilization; 220° to 350°C for depyrogenation)

Support Protocol 2: Use of Biological Indicators for Monitoring Dry‐Heat Sterilization

  • Biological indicator strips containing standardized concentrations of Bacills subtilis (ATCC #9372) spores (e.g., Spore‐O‐Chex; PyMaH, or VWR)
  • Trypticase soy broth (see recipe)
  • 30°C incubator

Basic Protocol 3: Use of Disinfectants: 70% Ethanol

  Materials
  • 100% denatured ethanol
  • Ethanol‐resistant spray‐type storage container

Alternate Protocol 3: Use of Disinfectants: Quaternary Ammonium Compounds

  Materials
  • Quaternary ammonium compound disinfectant of choice: e.g., Roccal (Sterling Winthrop), Micro‐Quat (Ecolab), Zephirol (Bayer)
  • Tightly closed containers
  • Spray bottles
  • 1‐gallon jugs

Alternate Protocol 4: Use of Disinfectants: Sodium Hypochlorite

  Materials
  • Household liquid bleach (e.g., Clorox, Dazzle)
  • 5% (w/v) sodium thiosulfate solution

Basic Protocol 4: Vacuum Filtration

  Materials
  • Solution to be filtered
  • 47‐mm funnel/support assembly (optional; e.g., Kontes, Millipore) attached to a 1‐ to 2‐liter vacuum filtration flask (Fig. )
  • 47‐mm glass fiber depth filters (optional; Gelman, Millipore)
  • 47‐mm membrane filters (optional; 0.45‐µm and 0.2‐µm pore sizes)
  • Disposable, sterile filter unit (e.g., Corning, Nalgene) including:
  •  Filter funnel, housing an integrally sealed 0.2‐µm filter membrane
  •  Funnel dust cover
  •  Removable receiver bottle and cap
  •  Barbed tubing adapter
  •  Nonsterile depth prefilters (included by most manufacturers)
  • Vacuum source

Basic Protocol 5: Small‐Volume Positive‐Pressure Filtration of Nonaqueous Solutions

  Materials
  • Dimethyl sulfoxide (DMSO)
  • Glass 25‐ml syringe with Luer‐lok tip
  • Sterile syringe filter: 25‐mm‐diameter nylon membrane, 0.2‐µm pore size, polypropylene housing (Nalgene or equivalent)
  • Laminar‐flow cabinet
  • Sterile amber glass storage vessels with polytetrafluoroethylene (Teflon, PTFE)–lined screw‐cap closure

Alternate Protocol 5: Large‐Volume Positive‐Pressure Filtration of Nonaqueous Solutions

  Materials
  • Dimethyl sulfoxide (DMSO)
  • Sterile filter capsule: 400‐cm2‐surface‐area nylon membrane, 0.2‐µm pore size, polypropylene housing (Whatman Polycap 36AS or equivalent)
  • Glass 25‐ml syringe with Luer‐lok tip
  • Worm drive clamps
  • PTFE tubing: polytetrafluoroethylene (PTFE or Teflon) with 0.25‐in. (6.4‐mm) i.d., 0.06‐in. (1.6‐mm) wall thickness, 0.38‐in. (9.5‐mm) o.d. (Norton or equivalent)
  • Peristaltic pump assembly capable of providing an operating pressure of 15 to 20 lb/in.2
  • Sterile amber glass storage vessels with PTFE‐lined screw caps

Alternate Protocol 6: Large‐Volume Positive‐Pressure Filtration Using Pressurized Nitrogen

  • 5‐liter pressure vessel (or size to fit application; Gelman or Millipore)
  • Pressurized nitrogen tank
  • Additional tubing to fit pressure vessel and nitrogen tank
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Figures

  •   FigureFigure 1.4.1 Funnel/support assembly for vacuum prefiltration.
  •   FigureFigure 1.4.2 Positive‐pressure filtration assembly for use with a peristaltic pump.
  •   FigureFigure 1.4.3 Positive‐pressure filtration assembly for use with pressurized nitrogen.

Videos

Literature Cited

Key References
   Barkley, W.E. and Richardson, J.H. 1994. Laboratory safety. In Methods for General and Molecular Bacteriology (P. Gerhardt, R.G.E. Murray, W.A. Wood, and N.R. Krieg, eds.) pp. 715‐734. American Society for Microbiology, Washington, D.C.
  A well‐written chapter covering all aspects of safety in any laboratory dealing with biological agents.
   Block, S.S. 1983. Disinfection, Sterilization, and Preservation, 3rd ed. Lea & Febiger, Philadelphia.
  A substantial text covering all aspects of the discipline. Contributors provide a great deal of information to the individual chapters.
   Brock, T.D. 1983. Membrane Filtration: A User's Guide and Reference Manual. Science Tech, Inc., Madison, Wis.
  Includes detailed information on the principles of membrane filtration, selection of filtration systems, and use of membranes for specific applications. Lacks information on the newer membrane types developed since its publication date, but still a worthwhile reference.
   Perkins, J.J. 1976. Principles and Methods of Sterilization in Health Sciences. Thomas, Springfield, Ill.
  A classic reference text that covers all aspects of dry‐heat and steam sterilization.
   U.S. Pharmacopeial Convention. 1995. The U.S. Pharmacopeia XXIII/The National Formulary XVIII. U.S. Pharmacopeial Convention, Rockville, MD.
  The U.S. official standard for sterilization criteria and sterility testing. Describes methods for using biological indicators.
   Vesley, D. and Lauer, J. 1986. Decontamination, sterilization, disinfection, and antisepsis in the microbiology laboratory. 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. 182‐198. American Society for Microbiology, Washington, D.C.
  Presents substantial information on all aspects of decontamination concerns for the laboratory, including choice of disinfectants, spill containment and cleanup, and routine cleaning.
   Millipore. 1993. Millipore Direct. Millipore, Bedford, MA.
  This filter manufacturer's catalog and reference guide provides a wealth of background, practical, and technical information to assist a user in appropriate choices for filters and filtration equipment.
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