Gram positive bacteria retain the primary stain because of the:
high content of the peptidoglycan in the cell wall
Gram-positive bacteria retain the primary stain (crystal violet) because of the high amount of peptidoglycan present in their cell walls. Peptidoglycan is a polymer of sugar and amino acids that provides rigidity and structural support to the cell wall. The crystal violet binds to the peptidoglycan layer, which is thick in Gram-positive bacteria, leading to the retention of the stain. After staining with crystal violet, the bacteria are treated with iodine, which forms a complex with the crystal violet in the peptidoglycan layer, making it difficult to wash away. Subsequent treatment with alcohol or acetone-based decolorizer dissolves the outer membrane of Gram-negative bacteria leading to loss of the stain, while Gram-positive bacteria retain it due to their thicker peptidoglycan layer. Finally, a counterstain (usually safranin) is used to stain the decolorized Gram-negative bacteria, helping to differentiate between Gram-positive and Gram-negative bacteria under a microscope.
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