The History of Gram Staining

Study Notes

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.

Description

Learn about the origin of the Gram stain, a method used to visualize bacteria in tissue samples, and its developer Hans Christian Gram. Discover how the staining method was created and its importance in microbiology.