Differential Staining of Bacteria: Flagella 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 3G
DOI:  10.1002/9780471729259.mca03gs15
Online Posting Date:  November, 2009
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Abstract

Bacterial flagella are appendages used for motility. Their presence is a useful tool for identification and differentiation of prokaryotes. Since flagella are too thin to be seen by compound light microscopy, staining methods employ the use of a mordant (often tannic acid) to make them thick enough to see using an oil immersion objective. Two protocols are described. Basic Protocol 1 is a modified Leifson method and is the one that many microbiologists have adapted. Basic Protocol 2 is a wet‐mount stain using a Ryu stain and is included because the stain is stable at room temperature. Both of these methods are fairly time‐consuming, taking from 15 to as long as 60 min to perform. Curr. Protoc. Microbiol. 15:A.3G.1‐A.3G.5. © 2009 by John Wiley & Sons, Inc.

Keywords: flagella; bacteria; motility

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Leifson Flagella Stain
  • Basic Protocol 2: Ryu‐Based Wet‐Mount Flagella Stain
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Leifson Flagella Stain

  Materials
  • Bacteria plated on blood agar or tryptic soy agar
  • Sterile water
  • Leifson stain solution (see recipe)
  • Immersion oil
  • Microscope slides (cleaned as described in Critical Parameters)
  • Wax pencil
  • Inoculating loop
  • Bunsen burner
  • Staining rack
  • Microscope
  • Additional reagents and equipment for examining the prepared slides using a compound light microscope (unit 2.1)

Basic Protocol 2: Ryu‐Based Wet‐Mount Flagella Stain

  • Bacterial broth culture or cells from colony suspended in broth
  • Ryu stain working solution (mordant; see recipe)
  • Coverslips
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Figures

Videos

Literature Cited

   Clark, W.A. 1976. A simplified Leifson flagella stain. J. Clin. Microbiol. 3:632‐634.
   Heimbrook, M.E., Wang, W.N.L., and Campbell, G. 1989. Staining bacterial flagella easily. J. Clin. Microbiol. 27:2612‐2615.
   Leifson, E. 1951. Staining, shape, and arrangement of bacterial flagella. J. Bact. 62:377‐389.
Key References
   Heimbrook et al., 1989. See above.
  Contains original protocol. Anticipated results included in figures.
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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