Isolation, Culture, and Characterization of Bacteriophages

Adam J. Pelzek1, Raymond Schuch2, Jonathan E. Schmitz3, Vincent A. Fischetti4

1 New York University School of Medicine, New York, New York, 2 ContraFect Corporation, Yonkers, New York, 3 Vanderbilt University Medical Center, Nashville, Tennessee, 4 The Rockefeller University, New York, New York
Publication Name:  Current Protocols Essential Laboratory Techniques
Unit Number:  Unit 4.4
DOI:  10.1002/9780470089941.et0404s07
Online Posting Date:  October, 2013
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Abstract

Bacteriophages (phages) are viruses that infect bacteria. Phages are the most abundant biological entities on Earth, and have influential effects on every ecosystem. Phage genomes represent a vast gene pool from which bacteria can draw upon for rapid evolution. Since the co‐discovery of phages in 1915 by Frederick Twort and 1917 by Felix d'Herelle, their potential as subjects of laboratory research has been exploited to great effect. Phage research has resulted in important strides in biology, from the elucidation of the function of DNA and the discovery of messenger RNA to the study of basic molecular interactions and genetic regulation. These fascinatingly simple and diverse entities are also valuable as diagnostic tools, genetic screening vectors, and potential therapeutics. This article provides an overview of techniques essential for entering the world of bacteriophage study, and contains protocols for the propagation, maintenance, storage, and basic characterization of phages. Curr. Protoc. Essential Lab. Tech. 7:4.4.1‐4.4.33. © 2013 by John Wiley & Sons, Inc.

Keywords: bacteriophage; phage; phage isolation; phage culture; metagenomics; phage titer; phage characterization; phage DNA; single step growth curve; DNA library; lysin screen

     
 
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Table of Contents

  • Overview and Principles
  • Strategic Planning (Host‐Bacterium Considerations)
  • Safety Considerations
  • Protocols
  • Basic Protocol 1: Phage Titering and Enumeration Using a Bacterial Reporter Strain
  • Basic Protocol 2: Phage Isolation and Expansion from a Single Plaque
  • Basic Protocol 3: Phage Expansion to Obtain a High‐Titer Stock
  • Basic Protocol 4: Storage and Handling of Bacteriophage Stocks
  • Basic Protocol 5: Bacterial Host‐Range Analysis
  • Basic Protocol 6: Single‐Step Growth Curves
  • Basic Protocol 7: Characterization of Bacteriophages
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Phage Titering and Enumeration Using a Bacterial Reporter Strain

  Materials
  • Bacterial host strain(s)
  • Bacterial growth medium (liquid and plates), and supplementary components (autoclave or filter‐sterilize prior to use, add non‐autoclavable supplements after cooling); medium type will vary depending on host strain(s); use BHI, LB, THY, Mueller‐Hinton, SOC, tryptic soy broth, minimal medium, etc. (refer to unit 4.2)
  • Bacto Agar (BD Difco) or agarose for solid medium preparation
  • Bacteriophage liquid stock (preferably high‐titer, 106 to 109 phages/ml; protocol 3)
  • 1× phosphate‐buffered saline (PBS; see recipe) or 5 mM CaCl 2
  • Erlenmeyer flasks (autoclaved) for bacterial growth
  • 15‐ and 50‐ml conical polypropylene centrifuge tubes (e.g., BD Falcon)
  • Sterile cotton or cheesecloth
  • Incubator (temperature‐regulated, rotational shaking model) with inserts for 125‐ml, 250‐ml, 500‐ml, 1‐liter, and 2‐liter Erlenmeyer flasks (some bacteria may require a 5% CO 2 atmosphere for growth)
  • Incubator (temperature‐regulated, stationary model) for growing organisms in liquid culture or on plates (some bacteria may require a 5% CO 2 atmosphere for growth)
  • Anaerobic cultivation packets for liquid culture, and nitrogen‐purging jars (for anaerobic hosts only; e.g., Remel AnaeroPack and AnaeroPouch system; Thermo Scientific)
  • Water bath (capable of heating from 30° to 70°C)
  • Petri dishes, sterile plastic: small size (100 × 15 mm) and large size (150 × 15 mm)
  • Petri dishes (glass 150 × 15 mm): wash, dry, wrap in aluminum foil, autoclave (can be used as an alternative to disposable plastic dishes)
  • 13‐ml round‐bottom polypropylene tubes, sterile (Sarstedt)
  • 1.5‐ml snap‐cap microcentrifuge tubes (e.g., dry‐cycle autoclaved before use; e.g., Posi‐Click tubes from Denville; http://www.denvillescientific.com/)
  • Additional reagents and equipment for bacterial medium preparation (unit 4.2)

Basic Protocol 2: Phage Isolation and Expansion from a Single Plaque

  Materials
  • Bacterial lawn containing phage plaques ( protocol 1)
  • Petri dishes containing bottom agar with top (soft) agar overlay containing bacteria ( protocol 1, step 6a)
  • Bacterial growth medium (liquid and plates), and supplementary components (autoclave or filter‐sterilize prior to use, add non‐autoclavable supplements after cooling); medium type will vary depending on host strain(s); use BHI, LB, THY, Mueller‐Hinton, SOC, tryptic soy broth, minimal medium, etc. (refer to unit 4.2)
  • 1× phosphate‐buffered saline (PBS; see recipe) or 5 mM CaCl 2
  • Chloroform (CHCl 3; optional)
  • Toothpicks (autoclaved)
  • Syringes (sterile, variable sizes: 1, 5, 10, 20, 30, 60 ml) and syringe filters (Millipore, Millex‐GP 0.22 µm, Green): contains Millipore Express (PES) membrane for low protein binding and fast‐flow
  • 50‐ml conical polypropylene tubes (e.g., BD Falcon)
  • Centrifuge (tabletop, swinging‐bucket model with 15‐ml and 50‐ml tube adapters)

Basic Protocol 3: Phage Expansion to Obtain a High‐Titer Stock

  Materials
  • Phage stock ( protocol 2)
  • DNase I (optional, create a 1 mg/ml stock, then deliver 1 µg/ml final concentration)
  • Chloroform
  • LB (unit 4.2) or BHI (BD, Difco) plates
  • Cuvettes (plastic, disposable) for OD 600 measurement of bacterial cultures
  • Nephelo culture flask (Wheaton, 100 ml or 300 ml, for turbidity measurement using spectrometer, an optional alternative to cuvettes)
  • Spectrophotometer for measuring DNA concentrations (use quartz cuvette with small aperture) and taking OD 600 turbidity measurements
  • 1.5‐ml snap‐cap tubes, dry‐cycle autoclaved to sterilize before use: Posi‐Click tubes from Denville (http://www.denvillescientific.com/) are recommended , easy to open/close, prevents splash‐back upon opening)
  • 50‐ml conical polypropylene centrifuge tubes (e.g., BD Falcon)
  • Tabletop centrifuge
  • Centrifuge (floor model, Beckman or Sorvall) with angle rotors GS3, GSA, SS‐34
  • Centrifuge bottles [Sorvall rotor‐dependent: GS‐3 rotor (500‐ml bottles), GSA rotor (250‐ml bottles), SS‐34 rotor (30‐ml Oak Ridge tubes)]; wash, wrap with lid (unsealed) in aluminum foil, and autoclave
  • Syringe filters (Millipore, Millex‐GP, 1.0 µm)
  • Sterile filtration apparatuses for bottle tops (Millipore, 0.22‐µm membrane, 150 ml and 500 ml, fast PVDF or nitrocellulose filter units; order correct neck size for your bottles)
  • Cell scrapers
  • End‐over‐end mixer
  • Additional reagents and equipment for titering phage ( protocol 1) and bacterial cell growth including cell counting (unit 4.2)

Basic Protocol 4: Storage and Handling of Bacteriophage Stocks

  Materials
  • Phage stock (high‐titer; protocol 3)
  • Chloroform
  • Skim milk (pasteurized, powdered)
  • Glycerol (or other cryoprotectant like sucrose or peptone)
  • Sterile filtration apparatuses for bottle tops (Millipore, 0.22‐µm membrane, 150 ml and 500 ml, fast PVDF or nitrocellulose filter units; order correct neck size for your bottles)
  • Cryovials (if using liquid nitrogen freezer)
  • Dry ice/ethanol bath
  • Lyophilizer or freeze‐drying apparatus
  • 2.0‐ml screw‐cap microcentrifuge tubes (Sarstedt or Denville, http://www.denvillescientific.com, for freeze‐drying or frozen storage of bacterial or phage stocks at −80°C)

Basic Protocol 5: Bacterial Host‐Range Analysis

  Materials
  • Bacterial strains (related and unrelated to host used for propagation)
  • 5 mM CaCl 2
  • Additional reagents and equipment for titering of bacteriophage ( protocol 1)

Basic Protocol 6: Single‐Step Growth Curves

  Materials
  • Bacterial host (for this protocol, Bacillus sp.)
  • Brain‐Heart Infusion (BHI) broth and plates (BD Difco; BHI medium is typically used for Bacillus strains)
  • Diluent: medium, PBS (see recipe), or 5 mM CaCl 2
  • 37° and 46°C water baths
  • Centrifuge
  • 15‐ml conical polypropylene centrifuge tubes (e.g., BD Falcon)
  • Timer
  • Graphing software (e.g., GraphPad Prism or Microsoft Excel)
  • Additional reagents and equipment for titering bacteriophage ( protocol 1)
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Figures

Videos

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  Westwater, C., Kasman, L.M., Schofield, D.A., Werner, P.A., Dolan, J.W., Schmidt, M.G., and Norris, J.S. 2003. Use of genetically engineered phage to deliver antimicrobial agents to bacteria: an alternative therapy for treatment of bacterial infections. Antimicrob. Agents Chemother.47:1301‐1307.
  Wilhelm, S.W. and Suttle, C.A. 1999. Viruses and nutrient cycles in the sea. BioScience49:781‐788.
  Willner, D., Furlan, M., Schmieder, R., Rohwer, F., and Haynes, M. 2011. Metagenomic detection of phage‐encoded platelet‐binding factors in the human oral cavity. PNAS108:4547‐4553.
  Wright, A., Hawkins, C.H., Anggard, E.E., and Harper, D.R. 2009. A controlled clinical trial of a therapeutic bacteriophage preparation in chronic otitis due to antibiotic‐resistant Pseudomonas aeruginosa: A preliminary report of efficacy. Clin. Otolaryngol.34:349‐357.
Key References
  Ackermann, 2009. See above.
  Resource for molecular biology protocols concerning phage characterization.
  Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G., Smith, J.A., and Struhl, K. 2013. Current Protocols in Molecular Biology. John Wiley & Sons, Hoboken, N.J.
  Resource for general molecular biology methods.
  Kutter and Sulakvelidze, 2005. See above.
  This book was used as a general reference throughout this unit. Great written history on phage research.
  Bykowski, T. and Stevenson, B. 2008. Aseptic technique. Curr. Protoc. Essential Lab. Tech.0:4.1.1‐4.1.11.
  Unit on aseptic technique in this manual.
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