Fluorescent Staining of Bacteria: Viability and Antibody Labeling

Rita B. Moyes1

1 Texas A&M University, College Station, Texas
Publication Name:  Current Protocols in Microbiology
Unit Number:  Appendix 3K
DOI:  10.1002/9780471729259.mca03ks15
Online Posting Date:  November, 2009
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Abstract

This appendix presents several methods for using fluorescence to evaluate bacterial viability and to explore the cell surface for the presence of various antigens for diagnostic and taxonomic purposes. The use of fluorescent labeling will allow fast and accurate analysis and monitoring of microbial populations in ecological and clinical environments. Curr. Protoc. Microbiol. 15:A.3K.1‐A.3K.13. © 2009 by John Wiley & Sons, Inc.

Keywords: fluorescent staining; bacteria; direct fluorescent antibody; indirect fluorescent antibody; propidium iodide

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Viability Staining of Cell Suspension Using One Dye
  • Basic Protocol 2: Viability Staining of Cell Suspension Using Two Dyes—SYTO9/Propidium Iodide
  • Alternate Protocol 1: Viability Staining of Cell Suspension Using Two Dyes—Fluorescein Diacetate/Propidium Iodide
  • Basic Protocol 3: Direct Immunofluorescent Labeling of Cell Surface Components of Bacterial Cells
  • Basic Protocol 4: Indirect Immunofluorescent Labeling of Cell Surface Components of Bacterial Cells
  • Alternate Protocol 2: Immunofluorescent Amplification Using Streptavidin‐Biotin Antibody Conjugates
  • Alternate Protocol 3: Dual Immunofluorescent Labeling
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Viability Staining of Cell Suspension Using One Dye

  Materials
  • Cultures to be tested
  • Phosphate‐buffered saline (PBS; see appendix 2A)
  • Propidium iodide (1 mg/ml in water; or can be purchased from Invitrogen, cat. no. P3566)
  • Immersion oil
  • Centrifuge
  • Microcentrifuge tubes
  • Slides (cleaned with 95% ethanol followed by water rinse and dried with laboratory wipes)
  • Coverslips
  • Fluorescent microscope

Basic Protocol 2: Viability Staining of Cell Suspension Using Two Dyes—SYTO9/Propidium Iodide

  Materials
  • Cultures to be tested
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • SYTO9‐green fluorescent nucleic acid stain (see recipe)
  • Propidium iodide (1 mg/ml in water; or can be purchased from Invitrogen, cat. no. P3566)
  • SYTO9/PI‐Live/dead Baclight bacteria viability kit (Invitrogen, cat. no. L7012), optional
  • Immersion oil
  • Centrifuge
  • Microcentrifuge tubes
  • Slides (cleaned with 95% ethanol followed by water rinse and dried with laboratory wipes)
  • Coverslips
  • Fluorescent microscope
  • 0.2‐µm pore polycarbonate filter, optional.

Alternate Protocol 1: Viability Staining of Cell Suspension Using Two Dyes—Fluorescein Diacetate/Propidium Iodide

  • Propidium iodide (PI)‐for dual‐label viability (see recipe)
  • Fluorescein diacetate (FDA; see recipe)
  • Ice

Basic Protocol 3: Direct Immunofluorescent Labeling of Cell Surface Components of Bacterial Cells

  Materials
  • Cultures (test organism and positive and negative control cultures)
  • 95% (v/v) ethanol
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • Fluorochrome‐labeled primary (antigen specific) antibody (can be purchased from Pierce, R&D Systems, or Accurate Chemicals)
  • Buffered glycerol (see recipe) or commercial mounting media (ProLong Antifade Kit; Invitrogen, cat. no. P7481)
  • Clear fingernail polish (for sealing coverslip)
  • Low‐fluorescing immersion oil
  • Slides (cleaned with 95% ethanol followed by water rinse and dried with laboratory wipes)
  • Inoculation loops
  • Coplin jars
  • Humidified chamber (e.g. petri dishes with moist filter paper)
  • Bibulous paper
  • Coverslips
  • Fluorescent microscope

Basic Protocol 4: Indirect Immunofluorescent Labeling of Cell Surface Components of Bacterial Cells

  • Fluorochrome‐labeled secondary (anti‐IgG) Ab (directed against primary Ab species; can be purchased from Sigma, Pierce, R&D Systems, or Accurate Chemicals)
  • 37° and 55°C incubators

Alternate Protocol 2: Immunofluorescent Amplification Using Streptavidin‐Biotin Antibody Conjugates

  • Biotinylated secondary antibody
  • Fluorochrome‐streptavidin conjugate (Invitrogen, cat. no. S869 for the FITC‐labeled conjugate or S11223 for Alexa Fluor 44—labeled conjugate)
NOTE: Alexa Fluor conjugates fluoresce brighter and are more pH stable and photobleaching resistant.
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Figures

Videos

Literature Cited

   Jones, K.H. and Senft, J.A. 1985. An improved method to determine cell viability by simultaneous staining with fluorescein diacetate‐propidium iodide. J. Histochem. Cytochem. 33:77‐79.
   Ono, M., Murakami, T., Kudo, A., Isshiki, M., Sawada, H., and Segawa, A. 2001. Quantitative comparison of anti‐fading mounting media for confocal laser scanning microscopy. J. Histochem. Cytochem. 49:305‐311
   Xu, H.‐S., Roberts, N.C., Adams, L.B., West, P.A., Siebeling, R.J., Huq, A., Huq, M.I., Rahman, R., and Colwell, R.R. 1984. An indirect fluorescent antibody staining procedure for detection of Vibrio cholerae serovar 01 cells in aquatic environmental samples. J. Microbiol. Methods 2:221‐231
Internet Resources
  http://www.accuratechemical.com
  Supplier with a huge inventory of polyclonal and monoclonal antibodies.
  http://www.invitrogen.com
  Supplier of fluorescently labeled probes and staining kits (formerly Molecular Probes).
  http://www.microbelibrary.org
  Select the visual collection icon and search the free image bank for your desired organism and stain.
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