What characteristic of the Ziehl-Neelsen method helps determine acid-fastness?

The characteristic of the Ziehl-Neelsen method that helps determine acid-fastness is the decolorization step. This step is crucial because it differentiates between acid-fast and non-acid-fast organisms based on their ability to retain the primary stain despite the presence of acid-alcohol.

In the Ziehl-Neelsen staining process, carbol fuchsin is used as the primary stain, which penetrates bacteria and binds to the mycolic acids in the cell wall of acid-fast bacteria, such as Mycobacterium tuberculosis. After staining, the slide is subjected to a decolorization process using acid-alcohol. Acid-fast bacteria will not lose the red color of the carbol fuchsin during this step due to the waxy mycolic acids in their cell walls that resist acid-alcohol, thus they remain red.

On the other hand, non-acid-fast bacteria will lose the red stain during the decolorization step and will appear colorless or take up a counterstain, usually methylene blue, during the next step of the staining process. This clear differentiation of retention and loss of stain according to the presence of mycolic acids is what fundamentally allows the identification of acid-fastness. Thus, the decolorization