Differential Staining of Bacteria: Acid Fast Stain

Jackie Reynolds1, Rita B. Moyes2, Donald P. Breakwell3

1 Richland College, Dallas, Texas, 2 Texas A&M University, College Station, Texas, 3 Brigham Young University, Provo, Utah
Publication Name:  Current Protocols in Microbiology
Unit Number:  Appendix 3H
DOI:  10.1002/9780471729259.mca03hs15
Online Posting Date:  November, 2009
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Abstract

Acid‐fastness is an uncommon characteristic shared by the genera Mycobacterium (Section 10A) and Nocardia. Because of this feature, this stain is extremely helpful in identification of these bacteria. Although Gram positive, acid‐fast bacteria do not take the crystal violet into the wall well, appearing very light purple rather than the deep purple of normal Gram‐positive bacteria. Curr. Protoc. Microbiol. 15:A.3H.1‐A.3H.5. © 2009 by John Wiley & Sons, Inc.

Keywords: differential stain; bacteria; acid‐fast stain; Mycobacterium; carbol fuchsin

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Ziehl‐Neelsen Acid‐Fast Stain
  • Alternate Protocol 1: Acid‐Fast Stain Cold (Kinyoun) Method
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Ziehl‐Neelsen Acid‐Fast Stain

  Materials
  • Beaker (500 ml or 1 liter) of boiling water
  • Distilled or deionized water
  • Specimens to be stained (Mycobacterium smegmatis is good as a + control)
  • Carbol fuchsin solution (see recipe)
  • Acid alcohol solution (see recipe)
  • Methylene blue solution (see recipe)
  • Immersion oil
  • Hot plate
  • Wire overlay for the top of the beaker (or another heat‐resistant structure that will hold the slide over the beaker rim while steaming)
  • Slides
  • Bunsen burner or electric burner
  • Paper towel (cut the size of the slide)
  • Bibulous paper
  • Additional reagents and equipment for examining the slides using a compound light microscope (unit 2.1)
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Figures

  •   FigureFigure a0.3H.1 Shows acid‐fast staining of Mycobacterium smegmatis.

Videos

Literature Cited

   Brock, T. 1999. Milestones in Microbiology 1546 to 1940: 1546 to 1940. ASM Press. Washington, D.C.
   Hussey, M. and Zayaitz, A. September 8, 2008. MicrobeLibrary: Acid‐fast protocol. Retrieved from http://www.microbelibrary.org/Edzine/details.asp?id=2552&Lang=
   Root, R.K., Waldvogel, F., Corey, L., and Stamm, W.E. 1999. Clinical Infectious Diseases: A Practical Approach, Oxford University Press, New York.
Internet Resources
  http://delrio.dcccd.edu/MFarinha/lab_manual/TOC
  Online lab manual of this section's author (J. Reynolds), with information on other stains and tests.
  http://www.urmc.rochester.edu/Path/zqu/StainsManual/index.html?MicrowaveZiehl‐NeelsenAcid‐fast
  Detailed information on the acid‐fast stain, from a clinical perspective.
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