Predict the effect of the following mistake on a Gram stain Image of Staphylococcus aureus (a known Gram-positive cocci bacteria) – reversal of CV and safranin stains A. No effect on results B. Gram-negative result, but false C. Background staining D. No decolorization
The Correct Answer and Explanation is:
The correct answer is B. Gram-negative result, but false.
In a Gram stain procedure, the crystal violet (CV) stain is used to initially stain all cells, and the iodine acts as a mordant to fix the dye inside the cells. The key step that differentiates Gram-positive from Gram-negative bacteria is the decolorization step using alcohol or acetone. Gram-positive bacteria, like Staphylococcus aureus, have a thick peptidoglycan layer in their cell walls that traps the crystal violet dye, making them appear purple. In contrast, Gram-negative bacteria have a thinner peptidoglycan layer, which allows the dye to be washed away during decolorization, and they take up the safranin, appearing pink.
If the stains are reversed—meaning safranin is applied first and crystal violet second—the following will happen:
- The safranin will initially stain all cells, but it won’t effectively bind to the thicker peptidoglycan layer of Gram-positive bacteria.
- After applying the crystal violet stain (which is the incorrect step here), the dye will not get trapped in the Gram-positive bacteria’s peptidoglycan layer as it should during the proper Gram staining process. The dye will be washed out during the decolorization step.
- When you then apply safranin at the end, the cells will appear pink (a characteristic of Gram-negative bacteria).
Since Staphylococcus aureus is a Gram-positive organism, it should appear purple, but due to the reversed staining procedure, it will appear pink, which is characteristic of Gram-negative bacteria. Therefore, the Gram stain results will be falsely interpreted as Gram-negative.
This mistake does not affect background staining, as safranin is the counterstain, and decolorization will still occur, but it will produce misleading results for the bacteria’s Gram classification.
