The History of Gram Staining

History of Gram Stain

• Hans Christian Gram developed the Gram stain in 1884 to visualize cocci in tissue sections of lungs of pneumonia patients.
• Gram's method was based on Robert Koch's staining method for visualizing tuberculosis bacilli.
• The Gram stain uses Crystal Violet as the primary stain, an iodine solution as a mordant, and ethanol as a decolorizer.

Purpose of Gram Stain

• The Gram stain is fundamental to the phenotypic characterization of bacteria.
• It differentiates organisms of the domain Bacteria according to cell wall structure.

Theory of Gram Stain

• The Gram stain is a complex and differential staining procedure.
• Gram-positive cells have a thick peptidoglycan layer and stain blue to purple.
• Gram-negative cells have a thin peptidoglycan layer and stain red to pink.
• The staining procedure is not used for Archeae or Eukaryotes as they lack peptidoglycan.

Gram Stain Procedure

• The Gram stain requires four basic steps: applying a primary stain, adding a mordant, rapid decolorization, and counterstaining.
• The performance of the Gram stain involves the use of crystal violet, Gram's Iodine, and a decolorizing agent.

Chemical Mechanism of Gram Stain

• Crystal violet dissociates into CV+ and Cl– ions that penetrate through the cell wall and membrane.
• The CV+ interacts with negatively charged components of bacterial cells, staining the cells purple.
• Iodine interacts with CV+ to form large CVI complexes within the cytoplasm and outer layers of the cell.
• The decolorizing agent interacts with the lipids of the membranes of both gram-positive and gram-negative bacteria.

Gram-Positive and Gram-Negative Bacteria

• Gram-positive bacteria have cell walls that contain thick layers of peptidoglycan (90% of cell wall) and stain purple.
• Gram-negative bacteria have cell walls with thin layers of peptidoglycan (10% of wall) and high lipid content, and stain pink.
• Gram-negative cells have a slightly different structure of peptidoglycan than gram-positive cells.
• With ethanol treatment, gram-negative cell walls become leaky and allow the large CV-I complexes to be washed from the cell.