Topic 10: Gram Staining

• The gram stain is a differential stain that allows you to classify bacteria as either gram positive or gram negative.
• Gram staining technique discovered by Hans Christian Gram in 1884.
• Steps
  • Application of primary stain (crystal violet) - all bacteria are stained purple by this basic dye
  • Application of mordant (gram’s iodine) - iodine combines with the crystal violet in the cell to form a crystal violet-iodine complex (CV-I)
  • Application of decolorizing agent (ehtyl alcohol or ethyl alcohol-acetone) - primary is washed out of some bacteria while others are unaffected
  • Application of secondary stain or counterstain (safranin) - basic dye stains the decolorized bacteria red
• The most important determining factor in the procedure is that bacteria differ in their rate of decolarization.
  • Those that decolorize easily are gram-negative
  • Those that decolorize slowly, and retain primary stain, are gram-positive
• CV-I complex cannot be washed out of gram-positive cells
• Decolorizing agent dissolves the outer lipopolysachharide layer and CV-I complex washes out through thin layer of peptidoglycan in gram-negative cells
• Gram stain is most consistent when done with young cultures of bacteria (less than 24 hours old)
  • When bacteria die, their cell walls degrade and may not retain the primary stain → gives inaccurate results
• Gram staining is usually first step in identifying bacteria

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