Staphylococcus aureus Gram Stain: Introduction, Principle, Procedure and Result Interpretation
Staphylococcus aureus in single, pairs and clusters in Gram stain as shown above picture. Gram stain is a differential stain and therefore it uses to differentiate Gram-positive and Gram-negative bacteria. It was devised originally by a Danish bacteriologist, Hans Christian Joachim Gram (1884) as a method of staining bacteria in his laboratory.
Principle of Gram stain
Principle of Gram stain on the various basis, like permeability of cell wall of bacteria and formation of dye-iodine complex, affinity, pH of the cytoplasm of organisms andpresence of magnesium ribonucleate as follows:
Permeability of cell wall of bacteria and formation of the dye-iodine complex (lake): The permeability of the cell wall of Gram-negative bacteria is more porous compared to the Gram-positive bacterial cell wall. The formation of the lake takes place in the cytoplasm of bacteria. The decolorizer ( alcohol) can not pass through the cell wall of Gram-positive bacteria and hence these cells become purple or violet whereas the Gram-negative bacterial cell wall permits the above decolorizer into the cell. Since the complex is soluble in organic solvent and insoluble in water, the dye-iodine complex will be washed away from the cell. As a result, these cells, now, are colorless and will take the color of counterstain, i.e. safranin or neutral red or dilute carbol fuchsin and appear pink/ red as shown in negative control as shown above picture.
Acidic substances have an affinity to basic dye and basic substances have an affinity to acidic dye.
The pH of the Cytoplasm of 0organisms
pH could be another factor that researchers have shown to describe the principle of Gram stain. The pH of the cytoplasm of Gram-positive bacteria is 2-3 whereas that of Gram-negative bacteria is 4-5. The acidic nature of the cytoplasm is further increased after the addition of iodine. Thus, it is the acidity of the cytoplasm of Gram-positive bacteria, which has an affinity to basic dye.
Presence of Magnesium ribonucleate
Magnesium ribonucleate in the Gram-positive bacteria has an affinity to basic due. As a result, Gram-positive bacteria take the color of crystal violet. It has also been proved that these bacteria become Gram-negative after removal of Magnesium ribonucleate.
Requirements for Staining Staphylococcus aureus
a) Compound light microscope
b) Reagents and glasswares
Clean grease-free slides
Crystal violet (Basic dye)
95% ethanol (decolorizing agent)
1% safranin or dilute carbol fuchsin or neutral red