Preliminary Staining of Bacteria: Negative Stain

Rita B. Moyes1, Jackie Reynolds2, Donald P. Breakwell3

1 Texas A&M University, College Station, Texas, 2 Richland College, Dallas, Texas, 3 Brigham Young University, Provo, Utah
Publication Name:  Current Protocols in Microbiology
Unit Number:  Appendix 3F
DOI:  10.1002/9780471729259.mca03fs15
Online Posting Date:  November, 2009
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Abstract

Negative staining is one of the many staining techniques that can be employed for viewing of bacterial cell morphology and size. The advantages of the negative stain include the use of only one stain and the absence of heat fixation of the sample. Negative staining employs the use of an acidic stain and, due to repulsion between the negative charges of the stain and the bacterial surface, the dye will not penetrate the cell. In negative staining, the results yield a clear cell with a dark background. Curr. Protoc. Microbiol. 15:A.3F.1‐A.3F.5. © 2009 by John Wiley & Sons, Inc.

Keywords: negative stain; bacteria; nigrosin; indirect stain

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Bacterial Negative Stain
  • Alternate Protocol 1: Negative Staining with a Black Marking Pen
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Bacterial Negative Stain

  Materials
  • Bacterial culture to be tested
  • Nigrosin or other negatively charged dye (e.g., Congo red or India ink; see reciperecipes)
  • Immersion oil
  • Clean microscope slides
  • Inoculating loops
  • Bunsen burner
  • Microscope with oil immersion objective
  • Biohazard container
  • Additional reagents and equipment for examining the slides using a compound light microscope (unit 2.1)
NOTE: Commercially prepared nigrosin solutions can be purchased from various chemical companies.

Alternate Protocol 1: Negative Staining with a Black Marking Pen

  • Water‐resistant, black blunt‐tipped marking pen (e.g., Marks‐a‐lot by Avery)
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Figures

Videos

Literature Cited

   Woeste, S. and Demchick, P. 1991. New version of negative stain. Appl. Envir. Microbiol. 57:1858‐1859.
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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