differentially stain acid-fast incrustations in the cuticular hyphae of certain species of fungi in the genus Russula. Some free endospores can be confused with small yeasts, so staining is used to identify the unknown fungi. It is also useful in the identification of some protozoa, namely Cryptosporidium and Isospora. The Ziehl–Neelsen stain can also hinder diagnosis in the case of paragonimiasis because the eggs in sputum sample for ovum and parasite (O&P) can be dissolved by the stain, and is often used in this clinical setting because signs and symptoms of paragonimiasis closely resemble those of TB. History In 1882 Robert Koch discovered the etiology of tuberculosis. Soon after Koch's discovery, Paul Ehrlich developed a stain for mycobacterium tuberculosis, called the alum hematoxylin stain. Franz Ziehl then altered Ehrlich's staining technique by using carbolic acid as the mordant. Friedrich Neelsen kept Ziehl's choice of mordant but changed the primary stain to carbol fuchsin. Ziehl and Neelsen's modifications together have developed the Ziehl–Neelsen stain. Another acid-fast stain was developed by Joseph Kinyoun by using the Ziehl–Neelsen staining technique but removing the heating step from the procedure. This new stain from Kinyoun was named the Kinyoun stain. Procedure A typical AFB stain procedure involves dropping the cells in suspension onto a slide, then air drying the liquid and heat fixing the cells. Studies have shown that an AFB stain without a culture has a poor negative predictive value. An AFB culture should be performed along with an AFB stain; this has a much higher negative predictive value. Mechanism explanation The mechanism of action of the Ziehl-Neelsen stain is not completely understood, but it is thought to involve a chemical reaction between the acidic dyes and the cell walls of the bacteria. The acidity of the dyes causes them to bind more strongly to the cell walls of the bacteria than to other cells or tissues. This results in the selective staining of only those cells that have a high density of cell wall material, such as acid-fast bacteria. The Ziehl-Neelsen stain is a two step staining process. In the first step, the tissue is stained with a basic fuchsin solution, which stains all cells pink. In the second step, the tissue is incubated in an acid alcohol solution, which decolorizes all cells except for acid-fast cells, which retain the color and appeared as red. The mechanisms by which this color is produced