Differential Staining of Bacteria: Capsule Stain

Donald P. Breakwell1, Rita B. Moyes2, Jackie Reynolds3

1 Brigham Young University, Provo, Utah, 2 Texas A&M University, College Station, Texas, 3 Richland College, Dallas, Texas
Publication Name:  Current Protocols in Microbiology
Unit Number:  Appendix 3I
DOI:  10.1002/9780471729259.mca03is15
Online Posting Date:  November, 2009
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Abstract

Bacterial capsules are composed of high‐molecular‐weight polysaccharides and/or polypeptides, and are associated with virulence and biofilm formation. Unfortunately, capsules do not stain well with crystal violet, methylene blue, or other simple stains. This unit describes two methods of capsule staining. The first is a wet‐mount method using india ink; the capsule is visualized as a refractile zone surrounding a cell. The second is a direct‐staining dry‐mount method that precipitates copper sulfate and leaves the capsule as a pale blue zone. Both methods are easily performed within ∼5 min. Curr. Protoc. Microbiol. 15:A.3I.1‐A.3I.4. © 2009 by John Wiley & Sons, Inc.

Keywords: differential stain; capsule stain; india ink; nigrosin; negative stain; wet mount; dry mount

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Duguid Wet‐Mount Method
  • Basic Protocol 2: Anthony Direct–Dry Staining Method
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Duguid Wet‐Mount Method

  Materials
  • Nigrosin ampules (Becton Dickinson)
  • Bacterial culture
  • Clean glass microscope slides
  • Coverslips
  • Filter paper
  • Compound light microscope
  • Additional reagents and equipment for examining the slides using a light microscope (unit 2.1)

Basic Protocol 2: Anthony Direct–Dry Staining Method

  Materials
  • Bacterial culture
  • 1% (w/v) crystal violet solution (see recipe)
  • 20% (w/v) copper (II) sulfate (see recipe)
  • Microscope slide
  • Paper towel
  • Compound light microscope
  • Additional reagents and equipment for examining the slides using a light microscope (unit 2.1)
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Figures

Videos

Literature Cited

   Anthony, E.E. Jr. 1931. A note on capsule staining. Science 73:319‐320.
   Bailey, H.D. 1930. A practical flagella and capsule stain for bacteria. Science 72:95‐96.
   Duguid, J.P. 1951. The demonstration of bacterial capsules and slime. J. Pathol. Bacteriol. 63:673.
   Hiss, P.H. Jr. 1905. A contribution to the physiological differentiation of Pneumococcus and Streptococcus. J. Exp. Med. 6:317‐345.
Key References
   Hiss, 1905. See above.
  One of the original capsule staining protocols.
   Moat, A.G., Foster, J.W., and Spector, M.P. 2002. In Microbial Physiology, 4th ed. pp. 315‐323. Wiley‐Liss, Inc. New York.
  A detailed description of capsule structure and physiology, together with micrographs comparing staining techniques.
Internet Resources
   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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