Final answer:
The Gram stain technique uses crystal violet as the primary dye, Gram's iodine as the mordant, ethanol or an ethanol/acetone solution as the decolorizing agent, and safranin as the secondary dye to differentiate between Gram-positive and Gram-negative bacteria.
Step-by-step explanation:
The Gram stain is a differential staining technique used in microbiology to differentiate between Gram-positive and Gram-negative bacteria. The process involves several reagents that sequentially apply to a heat-fixed smear:
- Primary dye: Crystal violet is used as the primary stain, which initially gives all cells a purple color.
- Mordant: Gram's iodine acts by binding to the crystal violet dye, forming a complex that clumps within thick peptidoglycan layers in the cell walls, thereby enhancing the dye's retention in Gram-positive cells.
- Decolorizing agent: A solution of ethanol or an ethanol/acetone mix is utilized to decolorize the sample. This agent washes away the dye from Gram-negative cells with thinner peptidoglycan layers, rendering them colorless.
- Secondary dye: Safranin is used as the counterstain, staining decolorized Gram-negative cells pink, while Gram-positive cells retain the purple color from the primary dye.
After the Gram stain procedure, Gram-positive cells remain purple because their thicker cell walls trap the crystal violet-iodine complex, even when exposed to the decolorizing agent. In contrast, Gram-negative cells will appear pink since they absorb the secondary stain after losing the primary dye in the decolorization step.