be formulated. It is agreed that disruption of the cell membrane by chemical carbol-fuchsin and for the artifacts that have been observed.

Transcription

1 THE NATURE OF THE ACID-FAST STAIN CARL LAMANNA Camp Detrick, Frederick, Maryland, Received for publication April 1, 1946 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, ; 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 made 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 the Louisiana State University School of Medicine, Department of Pathology and Bacteriology, New Orleans, Louisiana, and continued at Camp Detrick. 99

2 100 CARL LAMANNA [VOL. 52 consolute or critical temperature in the presence of these substances. Some of such data are recorded in table 1. As indicated by the rise of the consolute temperature, these data prove that sodium chloride decreases the solubility of phenol in water. The effect of basic fuchsin is more complex. A small amount causes a lowering of the consolute temperature, whereas larger quantities cause a rise. The exact concentration of dye at which this reversal takes place varies for each batch of dye as prepared for commercial use. By adding sufficient dye the solubility of phenol in water can be decreased to less than is normal for a mixture of phenol and water alone. The addition of NaCl, which is soluble in water and not in phenol, accentuates this loss of solubility by phenol. It is also possible to "salt out" the dye in the water phase by adding high enough concentrations of sodium chloride. The end effect on the solubility of phenol in carbol fuchsin would seem to depend on the relative concentrations of inorganic salts, alcohol, and dye, and the exact composition of the batch of basic fuchsin utilized. A generalization which can be made from the work of Yegian and Baisden (1942), Yegian and Budd (1943), and Porter and Yegian (1945) is that factors which decrease the 8olubility of phenol in water are those leading to the occurrence of artifacts in the Ziehl-Neelsen procedure. Thus these investigators add sodium chloride to carbol-fuchsin to get staining solutions which will regularly yield a large percentage of stained "beaded" cells. They have noted that reduction of the concentration of the stain is accompanied by a decrease in beading. Yegian and Budd (1943) have found that carbol-fuchsin made up with the acetate salts of rosaniline and para-rosaniline gives more beading than when the chloride dye salts are used. It is significant that the acetate salts are more soluble in alcohol than the chloride salts. Therefore, when equal quantities of saturated alcoholic solutions of these dye salts are used to make up the carbol-fuchsin, it is more likely that with the acetate dye salts high enough concentrations of dye will be present to cause a loss in solubility of the phenol than in the case of the chloride. In agreement with this statement is the reported observation (Yegian and Baisden, 1942) that employment of the acetate dye salts to make up carbolfuchsin gave a colloidal suspension instead of a clear solution as in the case when the chloride dye salts were used. The colloidal suspension that is noted for some batches of carbol-fuchsin and for all batches to which inorganic electrolytes are added is the result of the separation of some of the phenol present into a separate phase. When such a solution is centrifuged, two layers are isolated. The more intensely colored layer is at the bottom and consists in part of a viscous liquid with a boiling range of 180 to 185 C. This is within the range of boiling for phenol. In accordance with the work of Yegian and Baisden (1942) and Porter and Yegian (1945) it has been confirmed that beading occurs in the step of the Ziehl- Neelsen procedure in which the smears are washed with water. The action is sudden and independent of the species of acid-fast organism being studied. But it is dependent on the employment of a suitable batch of carbol-fuchsin and regularly occurs when sodium chloride has been added to the staining solution.

3 1946] NATURE OF ACID-FAST STAIN 101 Upon the basis of the foregoing discussion, the beads or artifacts are postulated to be the separation of phenol into a separate liquid phase upon the penetration of water into the cell. In this wise the instantaneous formation, the more or less spherical shape, the concentration of color, and the solubility in alcohol and phenol of the beads can be accounted for. This explanation presumes that the use of carbol-fuchsin in which phenol is present to the extent of saturation or supersaturation with respect to water permits the accumulation of the phenol in a similar state in the bacterial cell. The presence of water-insoluble cellular lipids in which phenol is soluble, or of salts insoluble in phenol, would accentuate TABLE 1 Consolute temperature of phenol-water in the presence of components of carbol-fuchsin ITEMS ADDED CONSOLUTE TEMPERATURE (C) None (control: phenol-water) Ethyl alcohol, 10 per cent NaCl, 0.5 per cent Ethyl alcohol, 10 per cent, and NaCl, 0.5 per cent 58 Coleman and Bell Co.: Basic fuchsin, CF-25, 0.5 per cent Basic fuchsin , 0.5 per cent Basic fuchsin , 0.5 per cent and NaCl, 0.5 per cent Allied Chemical and Dye Corp.: Para-rosaniline base, 9753, 0.5 per cent Basic fuchsin, NF 40, 0.5 per cent Basic fuchsin, NF 45, 0.5 per cent Basic fuchsin, NF 43, 0.5 per cent Basic fuchsin, NF 43, 1.0 per cent Basic fuchsin, NF 43, 1.0 per cent, and NaCl, 0.5 per cent Basic fuchsin, NF 43, 2.0 per cent Basic fuchsin, NF 43, 2.0 per cent, and NaCl, 0.5 per cent Difco bacto basic fuchsin, per cent per cent, and NaCl, 0.5 per cent per cent per cent, and NaCl, 0.5 per cent per cent per cent, and NaCl, 0.5 per cent the loss of solubility of phenol in water. The role of basic fuchsin is not specific, since malachite green has been substituted for the basic fuchsin in the preparation of staining solutions that favor beading. This proposed explanation of beading is further supported by evidence that the phenomenon is not limited to acidfast organisms. Four unlike non-acid-fast bacterial species have been studied: Bacillus megatherium, Corynebacterium diptheriae, Corynebacterium hofmanni, and an unidentified species of Vibrio. Cells of these species when stained with carbol-fuchsin are rapidly decolorized by alcohol. But when washed with cold water they are decolorized more slowly and at varying rates. Most important of all is the fact that, with the exception of Bacillus megatherium, decolorization

4 102 CARL LAMANNA [VOL. 52 with water is accompanie(i by the instantaneouis appearance of highly colored "beads" when a batch of carbol fuchsin is tused to which soditum chloriide has been added. Figuire 1, nos. 1, 2, and 3 illtustrate the situation that obtains with the vibrio. With (1orynebacterium diphtheriac decolorization with water was least rapid, and the beads were evident for the greatest length of time before being completely wn-ashed out of the cells. If the properties of acid-fast cells stained wtith carbol-fuchsin are depen(lent on the relative state of solubility of the phenol in water an(d cell constituents, it should be possible to (lecolorize these stained cells merely by immersing them in water at the consolute temperature. The presence of cell constituients in the acid-fast cells that decrease the solubility of phenol in wn-ater would not abolish Abv I\ ': AV,) efi" ; - \j {J E X~ ill t ~.. X t*. SIb I./~~~~~~~~~"I4 t, $ vt i 1 e XI-,\X.,,. ''i s,!.}.... f e.,..1 Z Z bbb 4* *f f ^, >.t i, - 3!? &... wss e e. sqaf: F a \,: ġr t *,. ^,. _)tis. IN:A. FIG. 1. BEADING OF VIBRIO SP. WHEN STAINEI) BY ZIEHM-NEELSEN'S CARBOL-FUCHSIN, WITH NaCl ADDED, AND WAShED wvith COLD WATER No. 1. (Left) Cells stained solidly with carbol-fuchsin. Stain ap)pliedl an(l drained off without, washing with wat,er. X 1,040 No. 2. (Center) Stained cells washed for a few secon(ls. Cells lose stain an(l beading becomes evident. X 1,040 No. 3. (Right) Continue(d washing in water results in practi(ally all cells becomiing beaded. Washing beyon(d t,his stage would result in complete loss of stain. X 1,040. the consolute temperature; they wvould only raise it. Yegian and Baisden (1942) report that at 90 C Mycobacterium tuberculosis (strain H37) can be decolorized wxith water. l'his observation has been confirmed. A strain of Mllycobactcriutm phici available in our laboratory can be in(duced to lose the stain when heated in water at 80 C for several minutes. A strain of the avian tubercle organism was decolorized in water at 70 to 75 C. On the other hand, heating at the same temperature and time with 95 per cent ethyl alcohol does not result in a loss of color. The alcohol-fastness is explained if lipids are present in which a mixture of alcohol and phenol is soluble, and if the lipids do not dissolve out of the cell by the method used for staining w-hen the membrane is intact. Thus the role of the membrane in maintaining the acid-fast properties of cells is harmonious with the suggested thesis. On the other hand, if the cytoplasmic membrane is the principal substrate of the acid-fast property as claimed by

5 1946] NATURE OF ACID-FAST STAIN 103 Knaysi (1946), the hypothesis offered would still be applicable. In this case the mutual solubility of the dye solution and lipids of the cytoplasmic membrane would be chiefly concerned. Added significance of the explanation offered for the beading phenomenon lies in the indication that differences of solubility of phenol in cell constituents (chiefly lipid) may account for the various types of acid-, alkaline-, and alcoholfastness recognized by the bacteriologist. Since the qualitative and quantitative chemical constitution of different species are dissimilar, the situation within the cell would be complex, and different for each kind of cell. The response to the presence of decolorizing agents would be the summation of all the factors influencing the solubility of phenol and dye within the bacterial cell. The essential difference between the acid-fast and non-acid-fast cell would rest on the greater solubility of the phenol and dye in cell constitutents of acid-fast cells than in the decolorizing solution bathing the cell. In the case of non-acid-fast organisms, the phenol and dye, being more soluble in the decolorizing agent than in the cell constituents, would leave the cells in the presence of the decolorizing agent. An in vitro test of the hypothesis suggested would require a phase rule study of the solubility of carbol-fuchsin and its constituents in cell lipids, and other cellular material, and in combinations of these materials in the proportions they occur in cells. SUMMARY The beading of cells when stained with Ziehl-Neelsen's carbol-fuchsin is postulated to be the result of phenol and dye separating out as a liquid phase. Evidence for this point of view is presented. It can be considered that the basis for the acid-fast property lies in the greater solubility of the phenol and dye in the cell constituents than in the decolorizing agent. REFERENCES ANDERSON, R. J The chemistry of the lipoids of tubercle bacilli. Concerning the so-called tubercle bacilli wax. Analysis of the purified wax. J. Biol. Chem., 83, KNAYSI, G On the existence, morphology, nature, and functions of the cytoplasmic membrane in the bacterial cell. J. Bact., 51, LONG, E. R Lipin-protein in relation to the acid-fastness of bacteria. Am. Rev. Tuberc., 6, PORTER, K. R., AND YEGIAN, D Some artifacts encountered in stained preparations of tubercle bacilli. II. Much granules and beads. J. Bact., 50, TAMURA, S Zur Chemie der Bakterien. Hoppe-Seyler's Z. physiol. Chem., 87, WELLS, H. G., AND LONG, E. R The chemistry of tuberculosis. 2d ed. Williams and Wilkins Co., Baltimore. YEGIAN, D., AND BAISDEN, L Factors affecting the beading of the tubercle bacillus stained by the Ziehl-Neelsen technique. J. Bact., 44, YEGIAN, D., AND BUDD, VERA Ziehl-Neelsen technique: Staining properties modified by different preparations of basic fuchsin. Am. Rev. Tuberc., 48, YEGIAN, D., AND PORTER, K. R Some artifacts encountered in stained preparations of tubercle bacilli. I. Non-acid-fast forms arising from mechanical treatment. J. Bact., 48, 83-91: