The Nature of Acid-Fastness

Abstract

Many theories have been proposed to explain the acid-fast property of the tubercle bacillus, but no satisfactory explanation has yet been given. It is suggested that the property is dependent upon the permeability of the cytoplasmic membrane. Evidence will be presented in support of this concept. It will be shown that when the Ziehl-Neelsen technique is employed the dye exists within the cell in two distinct portions: a small portion is bound to the cytoplasm and the remainder is free. The characteristic color of the stained bacillus is due to the free dye which can be removed without altering the acidfast believed that this phenomenon of acidfastness might be based upon the properties of chemicals isolated from the tubercle bacillus or of complexes of these chemicals. At one time there was a general belief that the presence of a wax sheath around the cell was responsible for its peculiar staining characteristics, but this is no longer tenable after the study made by Knaysi (1929). Some authors including Ehrlich thought that acid-fastness was related to the permeability of the cell membrane, but this structure was not clearly defined and experimental evidence was not given. The one point on which there is complete agreement is that the integrity of the cellular structure must be maintained to preserve the acid-fast property.Since our proposed explanation of the mechanism of acid-fastness is based on the function of a cellular structure, a brief review of pertinent information on the cytology of the tubercle bacillus is indicated.The cell wall of the tubercle bacillus has been observed directly with the electron microscope by Mudd and Anderson (1944). Mudd and Mudd (1927) demonstrated the hydrophobic property of the surface of the bacillus. The apparent functions of the cell wall are to protect the cell from mechanical injury and to impart to the cell its characteristic shape. The electron microscopic studies of Mudd, Polevitsky, and Anderson (1942) and Mudd and Anderson (1944) give direct evidence of the cytoplasmic membrane in bacteria. According to Knaysil (1929Knaysil ( , 1938Knaysil ( , 1944Knaysil ( , 1946, who refers frequently to this structure, the membrane of the tubercle bacillus probably consists of lipids and protein. With ordinary technique it appears to be the external boundary of the cytoplasm rather than a separate strtucture. EXPERIMENTAL RESULTSAlthough there is some evidence that the cell wall of the tubercle bacillus retains small amounts of certain other dyes, our observations suggest that this 1 A recent personal communication from Knaysi states that what he referred to as the cell "membrane" in his paper (1929) is now usually known as the cytoplamic membran. 777

Mycobacterium

“…Since the time of Stodola et al (1103), it has been held that mycolic acids per se were acid-fast. However, Richards (975) and Rich (972), as well as Yegian and Vanderlinde (1294), have regarded that as untrue, perhaps because thin films of mycolic acid were only faintly pink following exposure to acidic ethanol. The faintness of color was due, no doubt, (i) to the low degree of binding of dye when the lipoidal receptors for that dye were dispersed rather than ordered in the ultrastructure of the Mycobacterium and (ii) to the absence of the background color provided by intracellular dye (see above).…”

“…Such materials contain dye complexes that are not decolorized following exposure to acidic ethanol or mineral acids. This general property exists in a variety of entities, including spores of a number of fungi (1294), the spores ofBacillus cereus (1297), human sperm (125), the embryophores of Taenia saginata (914), the hooklets of Taenia echinococcus (194), corynebacteria and/or certain of their inclusions (929a, and our unpublished data), tubercle bacilli (623), leprosy bacilli (1151), keratin (C. A. Fisher, unpublished data), nuclear DNA (as in the Feulgen reaction), and chitin following exposure, in situ, to mild oxidation (916). In each case the biological poduct responsible for stably combining with the dye is apparently different: for example, the capacity for acid-fastness of the spores of B. cereus is associated with f3-hydroxybutyrate and can be removed by extracting the spores with chloroform (1297); that of leprosy bacilli can be extracted with pyridine whereas that of tubercle bacilli cannot (377).…”

The Use of Spectroscopic Techniques in the Characterization of Mycobacterial Metabolites

“…7 The chemical nature of this interaction has been widely speculated but with contradicting evidence pointing in different directions, it remains a debatable research point. [8][9][10] Historically, several classifications of Mycobacteria have been used and most have relied on a specific morphological feature or a biochemical reaction. The most acceptable nowadays is the traditional classification of Mycobacteria into slow growers and fast growers subgroups 1 (excluding the uncultivable M. leprae).…”