Question to Consider: Which step of the Gram stain is most critical with respect

Question

Question to Consider: Which step of the Gram stain is most critical with respect to timing?

1- The smear is first flooded with the primary stain, crystal violet in this case. The primary stain is the first dye applied in differential staining and generally stains all cells.

2-The smear is rinsed to remove excess dye and then flooded with a solution called Gram's iodine. The iodine combines with the crystal violet to form a dye-iodine complex, thereby decreasing the solubility of the dye within the cell.

3-The stained smear is rinsed, and then a decolorizing agent—95% alcohol or a mixture of alcohol and acetone—is briefly added. These chemicals quickly remove the dye-iodine complex from Gram-negative, but not Gram-positive, bacteria.

4-A counterstain is then applied to give a different color to the now colorless Gram-negative bacteria. For this purpose, the red dye safranin is used. This dye enters Gram-positive cells as well, but because they are already purple, it makes little difference to their color.

Explanation / Answer

Step 3 is the most crucial step of the Gram staining with repect to timing.

The stained smear is rinsed, and then a decolorizing agent—95% alcohol or a mixture of alcohol and acetone—is briefly added. These chemicals quickly remove the dye-iodine complex from Gram-negative, but not Gram-positive, bacteria. If this step is done for too long, even Gram positive bacteria will lose their color giving false gram negatives and if this step is completed too quickly, gram negative bacteria also will not loose their color thereby giving false gram positives.Also, if too much of a decolorizer is used then , the gram positive cells will lose the primary stain and be counterstained pink. if too little is used, gram negative cells will not lose the primary stain and will remain purple

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