Is it true or false that Gram-positive organisms cannot be decolorized after crystal violet staining?

• A. True
• B. False
• C. Sometimes
• D. Not sure

Answer: (B)

Understanding the Gram stain hinges on the decolorization step that separates the two groups. Gram-positive bacteria have a thick peptidoglycan layer that traps the crystal violet-iodine complex, so they typically resist decolorization by alcohol and stay purple after the counterstain. This is why they are expected to appear purple in a properly performed stain.

However, this resistance is not absolute. If the decolorizer is applied too long, if the culture is old, or if the technique is imperfect, Gram-positive cells can lose the crystal violet and take up the counterstain, ending up appearing pink. So the statement that Gram-positive organisms cannot be decolorized is false.