Fixation: The sample (e.g., sputum, gastric lavage) is smeared on a slide and heat-fixed. Primary Stain: The slide is flooded with carbol fuchsin dye and heated to allow the dye to penetrate the bacterial cell wall. Decolorization: The slide is then treated with a decolorizing agent (usually acid-alcohol) to remove the stain from non-acid-fast cells. Counterstaining: Methylene blue or malachite green is applied to provide a contrasting background.
Acid-fast organisms retain the red color of carbol fuchsin, while non-acid-fast organisms take up the blue or green counterstain.
