Canadian Society for Medical Laboratory Science (CSMLS) Practice Exam

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Which stain is NOT used in the gram stain procedure?

Methylene blue
Safranin
Acetone
Crystal violet

The correct answer is: Methylene blue

In the Gram stain procedure, methylene blue is not utilized as one of the primary stains. The primary stain used in this technique is crystal violet, which stains all of the cells, followed by iodine, which helps fix the dye inside the cells. After that, a decolorizing agent, typically acetone or ethyl alcohol, is applied, which differentiates between Gram-positive and Gram-negative bacteria by washing out the dye from Gram-negative cells. Finally, safranin is used as a counterstain, which provides a contrasting color for the decolorized Gram-negative bacteria. Methylene blue is more commonly associated with different staining procedures for specific types of techniques, such as identifying certain cell types in cytology, rather than being part of the classical Gram stain procedure.