The nature of the acid-fast stain

1946 Journal of Bacteriology  
A satisfactory explanation of the nature of the Ziehl-Neelsen stain remains to be formulated. It is agreed that disruption of the cell membrane by chemical or physical means is accompanied by a loss of acid-fastness (Long, 1922-23; Yegian and Porter, 1944) , and that acid-fast lipids peculiar to the mycobacteria exist (Tamura, 1913; Anderson, 1929) . A critical review of the literature on the subject has been presented by Wells and Long (1932) . Since then a new and major observation has been
more » ... de by Yegian and Baisden (1942) and Porter and Yegian (1945) , who have demonstrated that beading and Much's granules are artifacts dependent upon the staining procedure. No theory of the acid-fast stain offered to date can satisfactorily explain the origin of these artifacts. The present paper develops a hypothesis which, if it can stand the test of time, presumably can account for the properties of acid-fast cells when stained with carbol-fuchsin and for the artifacts that have been observed. Carbol-fuchsin is a mixture including phenol, water, and a dye (basic fuchsin) much less soluble in water than in phenol. When carbol-fuchsin penetrates into a cell, the distribution of phenol and dye within the cell should be a function of their concentration in the reagent and their solubility in the lipids and water phases present within the cell, assuming the absence of chemical reactions. Factors which influence the mutual solubility of the phenol and dye, or phenol, dye, and lipids, should affect the appearance of the stained cell. When an aqueous solution of phenol is added to a lipid (water-insoluble) two phases will result, a water and a lipid phase. The phenol will be distributed among the phases depending upon its relative solubility in the phases. For example, with oleic acid the phenol would tend to concentrate in the oleic acid phase. Basic fuchsin and a great variety of other dyes are more soluble in phenol and some other organic solvents than in water. Thus when oleic acid is added to carbol-fuchsin and vigorously mixed, and then centrifuged, two phases separate out, a top layer of oleic acid and a more lightly colored bottom water layer. The dye and phenol tend to be concentrated in the oleic acid. If a solvent is added, such as ethyl alcohol, in which both water and oleic acid are infinitely soluble, the two phases disappear and a homogeneous colored solution results. A simple analysis of the solubility of phenol in water as influenced by the presence of other substances, such as basic fuchsin, inorganic salts, and ethyl alcohol, which all occur in carbol-fuchsin, is provided by the determination of the 1 The work reported was initiated at
pmid:20994874 fatcat:vdvs2nl6yvblpmo75wuusulzke