Question

Place the steps of the Gram stain in the correct order.

1-Alcohol-acetone
2-Crystal violet
3-Safranin
4-Iodine

A. 1-3-2-4
B. 4-3-2-1
C. 1-2-3-4
D. 2-4-1-3
E. 2-1-4-3

Answer 1

The correct sequence of steps for the Gram stain is crystal violet (2), iodine (4), alcohol-acetone (1), and safranin (3), which corresponds to the answer choice D. 2-4-1-3. Gram-positive cells remain purple, and gram-negative cells turn pink.

Step-by-step explanation:

The correct order of the steps for a Gram stain is as follows:

1. Begin with the application of crystal violet, which serves as the primary stain.
2. Add iodine, which acts as a mordant to form a complex with the crystal violet, making it more difficult to wash out from certain types of cells.
3. Apply an alcohol-acetone solution to decolorize the cells, removing the primary stain from those with thinner cell walls.
4. Finally, counterstain with safranin, making the previously decolorized cells visible with a pink color.

Therefore, the correct order is D. 2-4-1-3.

After the Gram stain procedure, gram-positive cells appear purple because they retain the crystal violet-iodine complex due to their thick peptidoglycan layers. In contrast, gram-negative cells appear pink, because the dye is washed out during the decolorization step and they take up the pink counterstain safranin.