Differential Staining of Bacteria: Gram 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 3C
DOI:  10.1002/9780471729259.mca03cs15
Online Posting Date:  November, 2009
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Abstract

In 1884, Hans Christian Gram, a Danish doctor, developed a differential staining technique that is still the cornerstone of bacterial identification and taxonomic division. This multistep, sequential staining protocol separates bacteria into four groups based on cell morphology and cell wall structure: Gram‐positive cocci, Gram‐negative cocci, Gram‐positive rods, and Gram‐negative rods. The Gram stain is useful for assessing bacterial contamination of tissue culture samples or for examining the Gram stain status and morphological features of bacteria isolated from mixed or isolated bacterial cultures. Curr. Protoc. Microbiol. 15:A.3C.1‐A.3C.8. © 2009 by John Wiley & Sons, Inc.

Keywords: Gram stain; differential stain; crystal violet; safranin; morphology; cell wall

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Gram Staining
  • Basic Protocol 2: Potassium Hydroxide String Test
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Gram Staining

  Materials
  • Bacterial sample to be stained and control organisms
  • Crystal violet solution (see recipe)
  • Gram's iodine solution (see recipe)
  • Decolorizing solution (see recipe and Critical Parameters)
  • Safranin counterstain (see recipe)
  • Immersion oil
  • Inoculating loop
  • Clean slides
  • Bunsen burner
  • Microscope slides
  • Clothespin or clamp
  • Staining rack
  • Bibulous paper or paper towels
  • Microscope with oil immersion objective (unit 2.1)
  • Additional reagents and equipment for examining the slide (unit 2.1)
NOTE:Staphylococcus epidermidis is good as a Gram‐positive control and Escherichia coli is a good Gram‐negative control.NOTE: A Gram stain kit (e.g., Fisher) may be used in lieu of the staining reagents described above.NOTE: All staining steps are performed at room temperature.

Basic Protocol 2: Potassium Hydroxide String Test

  Materials
  • 3% KOH
  • Bacterial sample to be stained and control organisms
  • Inoculating loop
  • Clean slides
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Figures

Videos

Literature Cited

   Arthi, K., Appalaraju, B., and Parvathi, S. 2003. Vancomycin sensitivity and KOH string test as an alternative to Gram staining bacteria. Indian J. Med. Microbiol. 21:121‐123.
   Cowan, M.K. and Talaro, K.P. 2009. Microbiology: A Systems Approach, 2nd ed., Chapter 4. McGraw Hill, New York.
   Von Gravenitz, A. and Bucher, C. 1983. Accuracy of the KOH and vancomycin tests in determining the Gram reaction of non‐enterobacterial rods. J. Clin. Microbiol. 16:983‐985.
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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