Already available was a staining method designed by Robert Koch for visualizing turbercle bacilli. Gram devised his method that used Crystal Violet as the primary stain, an iodine solution as a mordant followed by treatment with ethanol as a decolorizer. The basic principle of gram staining involves the ability of the bacterial cell wall to retain the crystal violet dye during solvent treatment.
This staining procedure left the nuclei of eukaryotic cells in tissue samples unstained while the cocci found in the lungs of those who had succumbed to pneumonia were stained blue/violet.
The term for organisms that retain the primary color and appear purple-brown under a microscope is Gram-positive organisms. Gram-positive bacteria have cell walls that contain thick layers of peptidoglycan (90% of cell wall).
The organisms that do not take up primary stain appear red under a microscope and are Gram-negative organisms. Gram-negative bacteria have walls with thin layers of peptidoglycan (10% of wall), and high lipid content.
Gram-negative cells have thin layers of peptidoglycan, one to three layers deep with a slightly different structure than the peptidoglycan of 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.
Principle of gram staining
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| Gram-positive staining |
