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Which of the following lists the correct order of the four reagents used in the Gram stain procedure?
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9. Microscopes
Gram Stain
Scelta multipla
In the Gram stain procedure, which reagent is used as the decolorizer to remove the crystal violet–iodine complex from Gram-negative cells?
A
95% ethanol (or an acetone-alcohol mixture)
B
Gram’s iodine (mordant)
C
Safranin (counterstain)
D
Crystal violet (primary stain)
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Understand the purpose of each reagent in the Gram stain procedure: Crystal violet is the primary stain that colors all cells purple initially.
Gram's iodine acts as a mordant, forming a crystal violet–iodine complex that helps retain the dye in Gram-positive cells.
The decolorizer, typically 95% ethanol or an acetone-alcohol mixture, is applied next to selectively remove the crystal violet–iodine complex from Gram-negative cells due to their thinner peptidoglycan layer.
After decolorization, Gram-negative cells become colorless, while Gram-positive cells remain purple because the complex is retained.
Finally, safranin is used as a counterstain to color the now colorless Gram-negative cells pink/red, allowing differentiation under the microscope.
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