Acid-fast stains may be negative if the tissue was fixed in which fixative?

• A. Carnoy solution
• B. Formalin
• C. Alcohol
• D. Bouin's solution

Answer: (A)

Acid-fast staining depends on the lipid-rich, waxy cell wall of Mycobacteria to resist decolorization by acid alcohol; if the lipid components are removed or leached out during fixation, the stain can be washed away and yield a negative result. Carnoy's fixative (ethanol, chloroform, acetic acid) aggressively dissolves lipids in tissues, including the mycobacterial cell wall lipids, which disrupts the waxy barrier and makes it easier for the acid-fast stain to be decolorized. Because of this lipid extraction, acid-fast organisms may not retain the red dye and appear negative after processing. Formalin fixes by crosslinking proteins and generally preserves the wall structure, Bouin's preserves tissues with some lipid content but is less disruptive than Carnoy’s, and pure alcohol can also distort lipids, but Carnoy's combination of solvents is particularly prone to causing false negatives in acid-fast staining.