Factors Which Control the Staining of Tissue Sections with Acid and Basic Dyes*

Factors Which Control the Staining of Tissue Sections with Acid and Basic Dyes* MARCUS SINGER? Department of Anatomy, Harvard Mcdical School, Bostotc, Massachusetts CONTENTS I. Introduction ....................................................... 11. The Influence of pH of the Staining Solution on the Interaction of Dye and Protein ................................................. 111. The Nature of the Influence of pH on Staining ....................... IV. The Site of Dye Binding and the Nature of the Bond between Dye and Protein ................................. V. The Relation between the Isoelectric Point and Staining . . . . VI. The Ionic Strength of the Dye Solution ............................. VII. The Influence of Dye Concentration ................ VIII. The Afinity of Dyes ............................................. IX. The Influence of Fixation and Other Modifications of Tissues on Subsequent Staining ............................................ X. The Influence of Temperature of the Staining Solution . . . . . . . . . . . . . . . XI. Some Observations on the Kinetics of Staining ....................... XII. The Reversibility of Staining Reactions ; Equilibrium of Staining and Other Factors which Influence Staining .................. ... .............................. ... XIII. References .............

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I. INTRODUCTION Acid and basic dyes are employed in histology mainly to reveal the morphology of cells and tissues. Yet, these stains may also be used in the histochemical study of cellular and tissue proteins, an application which was appreciated in a number of early works on the staining reaction (for example, Miescher, 1874 ; Ehrlich, 1879a, b ; Lilienfeld, 1893 ; Michaelis, 1900, 1901, 1911 ; Pappenheim, 1901, 1917; Heidenhain, 1902, 1903; Mann, 1902; Magnus, 1903; Bethe, 1905). Mann (1902) in his remarkable book on histology noted: “It is not enough to regard dyes as simply acids or bases, as oxidizers or reducers, but we must aim at microchemical methods, and endeavour to know the composition of the dye and the tissue, to apply tests in a purposive way. Not till then will progress be made in the most difficult of all branches of Physiology, namely inicrochemistry.’’ But relative to the total literature on staining, interest in the

* This work was supported in part by funds received from the Eugene Higgins Trust. t Present address: Cornell University, Ithaca, N. Y . 21 1

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use of acid and basic dyes as histochemical tools has always been a limited one. A major reason for this has been the purely morphological demands which such growing fields as genetics, embryology, experimental inorphology, and endocrinology have hitherto made on the use of dyes and staining techniques. Another and perhaps more important reason for the limited histochemical application of staining procedures has been the absence of adequate information on the nature of the staining reaction. A prerequisite of a histochemical analysis of tissue sections with dye is, of course, a working knowledge of the mechanism of the staining reaction. The present review hopes to define the more important factors which govern interactions of acid and basic dyes with proteins and is preliminary to a second article, now in preparation, which applies this information to histochemical study of proteins of cells and tissues. Many facts of interest have appeared in the literature on staining of proteins, and it is one of the purposes of the present work to review the most salient of these and, whenever possible and necessary, to reinterpret them in the light of modern concepts. The review of literature is not confined to histological sources but also considers pertinent works in the field of protein, dye and textile chemistry. Much work has been done in the latter field on the protein-dye interaction, but these works have been largely ignored by histologists except for the detailed references and the comparison of textile and tissue staining of Pappenheim (1901; see also Baker, 1945) and for casual notations by other workers. The textile literature is particularly instructive since, as in the case of tissues, the substrate is also in a solid, insoluble state. Indeed, even studies of dyeing of cellulose derivatives and polyamide fibers are instructive for tissue staining. In contrast to that of textile materials, however, the stainable substance of cells and tissues is much more complex and heterogeneous and shows considerable variation from one region to the next in the kind, the interrelations, and the arrangement of substances. Much of the early literature on staining was devoted to the controversy, then extant, on whether the staining process is physical or chemical in nature. These speculations are adequately covered in one sense or another in many works such as those of Hofmeister (1891), Fischer (1899), Pappenheim (1901), Heidenhain (1902, 1903), Mann (1902), Dreaper (1906), Schwalbe (19O7), Zacharias ( 1908), Michaelis ( 1911), Gee and Harrison (1910), Pelet-Jolivet (1910), Harrison (1911), Bancroft (1914a, b, c, 1915 a, b), Briggs and Bull (1922), von Mollendorff and Krebs (1923), Unna (1928), Conn and Holmes (1928), Holmes

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(1929), Stearn and Stearn (1929, 1930), Zeiger (1938), Conn (1940), Baker (1945), Bourne (1951), and therefore no attempt will be made to review and evaluate these works systematically. However, a few comments taken from the literature may be pertinent in placing this controversy in proper perspective for the ensuing discussion of the forces which influence staining. In the light of modern information on intermolecular forces “the different theories are merely different ways of looking at the same phenomenon” (Vickerstaff, 1950). Mann (1902) wrote in similar vein fifty years ago when, in describing his views on the nature of staining, he noted : “Before discussing whether chemical or physical factors are at work in staining, the question must be asked, Is there any difference between chemistry and physics? There is not. Frankland used to teach that chemistry is but a branch of physics, and the whole recent development of chemistry points to the importance of studying chemical interaction apart from those conditions where substances join in definite molecular proportions. Even adsorption, which is perhaps the best example of physical action, has certain resemblances to chemical action. This matter has been gone into to make the reader realize that hard and fast lines cannot be drawn between chemical and physical action.” These sentiments have since been reemphasized in views of others. For example, the working assumption of Rawlins and Schmidt (1930) in analyzing the mode of combination between certain dyes and gelatin granules (see also Goldstein, 1949) was the fundamental statement of Langmuir ( 1916, 1917) that “there is no present justification for dividing interatomic (or intermolecular) forces into physical and chemical forces. It is much more profitable to consider all such forces as strictly chemical in nature. Evaporation, condensation, solution, crystallization, adsorption, surface tension, etc., should all be regarded as typical chemical phenomena.” Michaelis (1920) in a theoretical work on staining pointed out that practically all phenomena of staining with the exception, perhaps, of dye uptake of charcoal, could be described in chemical terms. He later noted ( 1926) that processes of adsorption, generally considered by physical theorists to be the mechanism of interaction between dye and substrate, “do not represent a fundamental contrast to chemical union.” This view was stressed by Stearn and Stearn (1930). Similar thoughts and sentiments concerning the significance of this controversy have been expressed by Dubos (1945), Baker (1945), Dempsey and Wislocki (1946), Rideal (1950), Vickerstaff (1950), Bourne (1951), and others. Studies of the interaction of dyes and proteins, although originally directed toward elucidating the physical or chemical nature of staining, un-

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covered many facts which are of greater interest here than the controversy which originally stimulated them. These facts will be focused upon in some detail. The experiments were varied and considered one or another aspect of the staining process. Some were concerned especially with the structure of dyes ; others with the nature of the dye reaction ; and still others dealt primarily with the physical-chemical nature of the protein interactant. In analyzing the staining of protein, many substances were employed as “models,” and along with tissue sections, subjected to a variety of dyes and conditions of staining. The model substances included, among others, gelatin, glue, wool, charcoal, celloidin, fibrin film, filter paper, and soluble proteins (e.g., casein, albumin). From these many and varied investigations the obvious fact emerges that there are numerous and diverse factors which profoundly influence the dye uptake of proteins of cells and tissues. The more salient of these factors are considered in detail in this review since they throw light upon the nature of the staining mechanism. The variable which affects dyeing most profoundly is pH, and consequently most extensive treatment is given it. Other factors of importance are ionic strength of the dye medium, concentration of dye, nature and affinity of the dye, fixation and other chemical modification of the protein, temperature, diffusion, rate of staining, and the chemical and physical characteristics of the protein. With few exceptions, the discussion is confined to aqueous solutions of molecularly dispersed acid and basic dyes. Staining under conditions other than these is frequently done in the histological laboratory, and it is appreciated here that the forces which operate under such conditions cannot be equated with those described here and are worthy of separate consideration at another time. Finally, as for the chemistry and constitution of acid and basic dyes, no attempt will be made here to discuss this subject in detail. Instead, the reader is referred to appropriate works. A brief summary of features of these dyes important for the following discussion is, however, pertinent here. Works which treat this subject in proper detail are the concise and illuminating descriptions by Conn ( 1940), Baker ( 1945), and Bourne (1951), and the more detailed ones by Rowe (Colour Index, 1924), Mayer ( 1934), Pratt ( 1947), Fierz-David and Blangey ( 1949). Acid (anionic) and basic (cationic) dyes are aromatic compounds of various complexities containing a water-solubilizing group, which in the case of acid dyes is frequently a sulfonic radical (--SOsH) but which also may be a carboxyl or hydroxyl group. In the latter event a nitroso, nitro, or another hydroxyl group is present. The solubilizing group of the basic

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dye is an amino group or one of its derivatives. These acid and basic groups are also in large part the groups responsible for the reaction of the dye with the protein, for which activity they have been termed auxochronzes. The acid dyes are generally used as the sodium salt of the dye acid ; basic dyes are prepared as chlorides, sometimes as acetates, sulfates, or as still other salts. There may be both acid and basic groups in a dye molecule, in which instance the overall acid or basic tendency is determined by the relative strength of these groups. Certain unsaturated groups of the dyes are chiefly responsible for their color, although the auxochrome may influence the quality and intensity of the color. Groups which impart the color are called chronzophorcs and include among others the carboxyl group >C = 0 ; the azo group -N = N- ; the nitroso group -N = 0 ;

the nitro group -N Go ; the quinoid group 0 = <=> 0 = <=> = N ; and the ethylene group -CH = CH -. 1 0

=

OF p H OF THE STAINING SOLUTION ON 11. THE INFLUENCE INTERACTION OF DYE A N D PROTEIN

0 or

THE

Control of pH, although reached on a highly empirical basis, is implicit in many staining techniques where acid or alkali is used to enhance or depress staining. Instances of such control are abundant among the methods listed in books of histological technique (e.g., see Baker, 1945, Bourne, 1951). For example, among the various methylene blue solutions recommended by Mallory (1944) for staining of basophilic substances are Loeffler’s solution which includes sodium hydroxide, Kiinne’s with phenol, Gabbett’s with sulfuric acid, Unna’s with potassium carbonate, Goodpasture’s with potassium carbonate and acetic acid, and Sahli’s with borax. Examples of substances which are used to control staining with acid dye may be found in the work of Maneval (1941) on bacteria. References in early histological literature to acidic or basic substances which enhance dye uptake (“accentuators”) or which otherwise control the intensity of staining were frequent. The mechanism of action of these substances was not agreed upon, but it was evident to many (see Mann, 1902) that alkaline substances accentuate basic dye uptake and acid substances favor the acid one. Examples of basic accentuators listed by Mann (1902) are bicarbonate, soap, pyridine, sodium borate, aniline, and potassium or sodium hydroxide. Some acid ones are phenol and sulfuric, acetic, and oxalic acids. There are many other examples in the literattire where the effective mechanism of enhancing or otherwise controlling staining is the use of acid or alkali. The influence of acid

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and base on staining has also been appreciated by wool and silk dyers who have employed various acids as well as salts to enhance and control dyeing of textiles (see e.g., Brown, 1901b), an effect which today is still the subject of considerable interest and research. A number of early studies were devoted to the experimental analysis of the effect of acid and base on staining. Mathews (1898) observed that proteins could be precipitated from solution as the salt of acid dyes such as acid fuchsin, erythrosin, orange red, and methyl blue if a few drops of dilute acetic acid were added to the dye-albumin mixture. Basic stains did not give this reaction under such conditions, but when brought into protein solutions made alkaline with sodium carbonate a precipitate was formed of the protein in combination with the dye. H e stated, in summary, “The acid stains will combine with albumoses only in acid solutions and the basic stains will combine with the albumoses only in alkaline solution, when they form insoluble colored compounds.” That these reactions held true for solid, coagulated protein as well as protein in solution was also demonstrated by Mathews. “If two pieces of coagulated egg albumin be brought the one into slightly acid and the other into alkaline solutions of thionin, the stain poured off after a few seconds, and the albumin washed in water, the piece that has been in the alkaline solution will be an intense purple, the other barely tinged with color.” H e observed similar results when he extended his observations to tissue sections of liver, kidney, and muscle of the frog. “I find that sections of the above-mentioned tissue, if immersed for an instant in one-tenth per cent sodium carbonate solution before staining or if stained in solutions of the basic stains made slightly alkaline with sodium carhomte show the cytoplasm deeply stained, as well as the chromatin. These reactions, which are identical with those of the albumoses, show that in alkaline solution many of the basic dyes will combine with the albumin molecule whether in cytoplasm or nucleus.” This work of Mathews which was written at a time when little was known about the dissociation and reaction of proteins was one of the earliest important contributions to our understanding of acid-base relations in staining ; yet, except for the detailed treatment which Mann (1902) gave it, little reference has been made to it. Mann (1902) was also impressed by the effect of acidity or alkalinity of the staining solution on the degree of dye-binding. H e believed that in cases where staining was favored the acid or alkali prepared the protein to receive the dye radical. At that time proteins were described as pseudoacids and pseudobases whose acidic or basic nature could be brought out by various substances or conditions. An alternative explanation ad-

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vanced by some textile chemists at this time was that the accentuators acted upon the dye ; thus, in the case of the accentuating action of acid in dyeing of wool with acid dyes, the acid accentuator caused the liberation of the dye acid which then reacted with the basic groups of the protein to form a stable salt. Bethe (1905) observed that the degree of staining of tissue sections varied with the acidity of the staining solution. He stained sections of spinal cord, sublingual gland, mammary gland, and kidney in toluidine blue solutions of constant dye concentration but containing different amounts of acid or alkali. Alkali favored and acid inhibited the uptake of the basic dye. This was true for all structures of cells and tissues, but the degree of staining at any given level of acid or base varied from one structure to another. A e described the staining of given structures by curves which depicted the intensity of staining as a function of the amount of acid or alkali. The curves showed that in highly alkaline solution all structures stained with the basic dye ; with decreasing alkalinity the tissue substances gradually lost their affinity for toluidine blue. The order of loss varied, however, with the tissue structure. For example, in weakly alkaline solution tracts and glia of the central nervous system no longer stained, whereas solutions of approximate neutrality were required before fibrous connective tissue and colostrum of mammary gland failed to stain. Cartilage and mucus continued to stain even in very acid solutions. Other early workers who stressed the importance of acid and alkali in dyeing were Spiro (1897), Brown (1901a), Heidenhain (1902, 1903), Halphen and Riche ( 1904, 1905), Pelet- Jolivet ( 1910), and Harrison (1911). As information was accumulated on the dissociation of protein in aqueous solution interest in the effect of acid and alkali increased greatly. Michaelis (1920) in a theoretical work on the nature of the staining reaction was among the first to emphasize the relation between the binding of acid and basic dye at different concentrations of hydrogen ion and the amphoteric nature of the protein. Loeb (1922, 1924) showed that the binding of acid or basic dye by gelatin followed the pH of the dye bath, high p H favored basic dye uptake, and low p H enhanced staining with acid dye. Staining with either dye appeared to be minimal at the isoelectric point, but some interaction with acid dye occurred above and with basic dye below the isoelectric point. Staining of collagen powder also varied with p H (Thomas and Kelly, 1922). Briggs and Bull (1922) concluded from their studies of dyeing of wool that the “hydrogen ion concentration of the dye-bath is the most important single factor affecting the process of dyeing” and that “many of the assistants or re-

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strainers, used in dyeing, produce an appreciable and often a great change in the hydrogen ion concentration of the dye-bath and their action in many cases is due more to this change than to any other specific action.” They observed that high pH favored binding of the basic dye and low pH that of the acid one. Hobbins (1923, 1924, 1926) studied the influence of acidity on the staining of plant tissues and reported that potato tuber tissue, Elodea leaves, and the mycelium of fungi responded as amphoteric colloids being stained with either acid or basic dye according to the p H of the solution. A number of important works bearing upon the influence of p H in staining bacteria were published by Stearn and Stearn (1924, 1925, 1928a, b). In solutions of varying p H the bacteria stained in a fashion similar to the protein structures discussed above; at low pH, acid fuchsin (acid dye) was taken up, but at higher ,pH, gentian violet (basic dye) was bound. When the intensity of staining was plotted as a function of pH, curves decreased with increasing p H for the acid dye, but were the reverse for basic dyes. Furthermore, such staining curves were displaced along the pH axis for different bacterial species (see also McCalla and Clark, 1941). Stearn and Stearn (1928a, b, c) also investigated the action of various chemical decolorizers on the dye fastness of stained bacteria and observed that decolorizers which were especially effective were acidic and basic ones, The acidic ones, for example phenol aldehyde, selectively removed basic dye, and basic decolorizers removed acid dyes. Pischinger (1926) , a student of Bethe, reexamined the influence of pH on staining and applied the method of p H control for the histochemical analysis of tissue structure. He studied quantitatively the binding of acid and basic dye by gelatin, egg white, thymus, and cartilage over a range of pH. The amount of bound dye was determined colorimetrically after extraction from the stained product. In each instance dye uptake followed the p H relations described above, namely that basic dye binding was inhibited as the pH of the staining solution was lowered until finally, at a given pH and below, no dye was bound; and staining with acid dye occurred most readily at low p H but dropped progressively and eventually ceased with gradual elevation of pH. Differences were observed among these protein substances in the degree of staining at any given p H or in the critical pH which marked the boundary between loss of staining capacity and increasing dye uptake. The proteins of tissue sections when subjected to p H variations responded in a manner similar to that of the model substances. As in Bethe’s earlier work, Pischinger plotted somewhat quantitatively the intensity of staining of given tissue structures

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against pH. The curves obtained thereby were different for each tissue structure, a fact which led Pischinger and others to introduce pH staining control as a method of histochemical characterization and study of tissue proteins. The importance of p H in staining of tissue sections and in histochemical study of tissue proteins was independently recognized and reported by other workers at about the time of Pischinger’s publications. A series of publications, in which dyes were used in the study of tissue proteins emanated from the laboratories of the University of Missouri (Robbins, 1923, 1924, 1926; Naylor, 1926; Stearn, 1931, 1933; Stearn and Stearn, 1924, 1925, 1928a, b, c, 1%9, 1930; and Levine, 1940). These studies sought to extend to the proteins of cells and tissues the information on protein reactions published by Loeb (1922). Robbins (1923, 1924) applied the staining reaction at different p H to study of the isoelectric point of the proteins of fungi. Naylor (1926) showed that the staining of plant tissues followed the p H relations reported by Loeb for gelatin staining. In addition to studying dye binding from solutions of an individual dye, he analyzed staining from solutions containing both an acid (eosin) and basic (methylene blue) dye. At high p H only methylene blue was bound, but as the acidity of the staining solution was increased, the sections at first became purplish because of the partial binding of eosin as well and, then, at a low p H the sections were stained exclusively with eosin. The use of an eosin-methylene blue combination over a p H range was also applied with similar results to staining of tissue, particularly blood cells, and of bacteria by Tolstoouhov (1927, 1928, 1929), who also evaluated independently the use of dyes in the study of proteins of cells and tissues. Other contributions at this time to the study of the relations between p H and dye uptake of tissue and cellular proteins were made by Pulcher (1927) and Haynes (1928). The control of acidity has a longer history in textile dyeing, and it is evident in reviewing the literature that some of the experiences of textile colorists have been drawn upon by histologists. The history of pH control in dyeing of textile fibers has been summarized recently by Seymour, Agnew, Crumley, and Kelly (1948). The influence of pH and other factors on the dyeing of wool and various textile fibers is reviewed in a number of articles, important among which are works by Rose (1942), Abbot, Crook, and Townend (1947), and a journal-sponsored review article in the American Dyestuff Reporter (1948). Wool is dyed with acid dyes which are generally applied in acid solution between p H 2 and 5,

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depending on the type of dye employed. Sulfuric acid is used to establish the lower p H and acetic acid the higher. Finally, studies have been reported recently on the influence of pH and other factors on the binding of dye in a relatively pure protein system (Singer and Morrison, 1948). Films of fibrin of known protein content, purity, and thickness prepared from products of fractionation of human blood (Cohn, 1945; Ferry and Morrison, 1946, 1947) were used as a model substance. This model protein was of particular value because as initially prepared it was native and undenatured. Moreover, it was in a solid and insoluble form and thus was similar in condition to cellular and tissue proteins of histological sections. Some films were modified by chemical or physical denaturants such as are used in fixing and then subjected to progressive staining under a variety of conditions until equilibrium was reached. The dye uptake was measured quantitatively by photometric means and expressed in terms of the amount of fibrin. Many factors influenced staining of fibrin, but primary among these was pH. The relations observed with pH variation are exemplified in the curves of Fig. 1. The curves resemble those for the titration of protein with CAST GREEN

MmFNNE U E

vn

FIG.1. The influence of pH on the staining of fibrin film with acid (fast green) and basic dye (methylene blue). Film fixed in formaldehyde (10 per cent Tor 10 hours) dye concentration, 5 X 10-6 M.;ionic strength, 0.02. (Singer and Weiss, unpublished.)

acid and base. The importance of pH for staining is emphasized particularly by the fact that there are p H levels where the protein shows no affinity for a dye even though immersed for very long periods in a solution of high concentration. The results of these studies were applied to the histochemical characterization and identification of proteins of cells and tissues (Dempsey and Singer, 1946; Dempsey, Wislocki, aiid Singer, 1946; Dempsey, Bunting, Singer, and Wislocki, 1947 ; Wislocki, Weather-

22 1

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ford, and Singer, 1947; Singer and Wislocki, 1948; Wislocki, Singer, and Waldo, 1948 ; Singer, 1949 ; Dempsey, Singer, and Wislocki, 1950).

111. THENATUREOF

THE

INFLUENCE OF pH

ON

STAINING

The profound influence which p H exerts on staining reflects in large part the sensitivity of the dissociation characteristics of proteins to alterations in the solution environment. Before proceeding to a description of the dissociation of proteins under various conditions of p H it is well to summarize at first some chemical information about proteins pertinent to the discussion. A characteristic feature of proteins in solution is that they are amphoteric, that is, they contain at the same time both basic and acidic groups which by their dissociation give rise respectively to positive and negative charges on the protein molecule. These acidic and basic groups comprise the free side groups of certain amino acids, the terminal amino and carboxyl groups of the protein molecule, and, finally, the charged substances which niay be conjugated to the protein. Free basic side groups (substituted ammonium : NH3+) are found in the amino acids-lysine, histidine, and arginine ; and acidic ones (carboxyl : COO-) in glutamic, hydroxyglutamic, and aspartic amino acids. Another acid group in addition to free carboxylic acid is the hydroxyl one of certain amino acids (e.g., of tyrosine, serine) . Many amino acids have no dissociating group other than their single amino and carboxyl ones which are used in the peptide linkage and, therefore, do not impart a charge to the protein unless they are located terminally in the polypeptide chain. Of special interest among the conjugated proteins are those which contain acid groups such as nucleoprotein (phosphoric acid) and mucoprotein (uronic and sulfuric acids). Proteins differ according to the nature and number of their constituent amino acids and their conjugated substances. For example, free basic groups may be relatively more abundant in one protein and acidic ones in another. -kcording to the relative number of acidic and basic groups and their degree of dissociation, the net or overall charge on the protein mole-’ cule at a particular time will be positive (excess basic dissociation), negative (excess acidic dissociation), or zero (isoelectric point). Positive and negative charges exist in the protein molecule even at the isoelectric point (defined as the p H of a solution in which the protein does not migrate in an electrical field and therefore in which it is electrically neutral), although the net charge is zero. Thus, the electrically neutral form of the protein molecule is a dipolar ion (Zwittem’on or amphion) and its where R structure may be represented in general as H,+N-R-COO-,

z 2

MARCUS SINGER

represents the polypeptide chain and NHs', COO- the ionized basic and acidic side and free terminal groups of the constituent amino acids. The degree of ionization of the free acidic or basic groups of proteins depends on the pH of the solution. When acid is added to the solution, the dissociation of the free carboxylic acid group (as well as any conjugated acid group, e.g., phosphoric acid) is decreased and that of the free amino is increased, and the protein becomes less negatively charged. The reverse tendency in ionization and charge follows upon addition of alkali to the solution. The responses of protein to changes in p H may be indicated according to the following formulations : Isoelectric condition

A

B

NHs+-R-COONH3+-R-COO-

acid alkali

NHs+-R-COOH NHs -R-COO-

If the pH of the solution is below the isoelectric point the protein tends to respond increasingly in the direction described by formula A and above the isoelectric point according to B. It is presumed that at extremes of p H (approximately 2 and 11) complete ionization of the substituted ammonium or carboxylic groups is attained, and in these regions the respective net positive or net negative charge on the protein is maximal. At p H levels intermediate between these extremes and the isoelectric point, the net charge on the protein falls somewhere between the zero of the isoelectric point and the maximum, being positive below the isoelectric point and negative above. The ability of proteins to take up acid or basic dye according to the acidity of the environment is an expression of these amphoteric properties and of the charge on the dye ion. Basic dyes are generally chloride salts of a dye base, whereas acid dyes are in general the sodium salts of a dye acid. In the following discussion these salts are assumed to be completely ionized. Actually a small amount of the dye exists in solutions of ordinary pH as the undissociated dye acid or base whose concentration is probably influenced by alterations in p H of the solution. In those cases where dye ion association is prevalent at the p H of staining it is important to stress that the degree of dissociation of the dye would play an important part in the extent of staining. But, with few exceptions, the dissociation constants of these dye acids or bases fall in the extremes of p H and consequently ordinary levels of pH, such as those considered here, will hardly influence the degree of dye dissociation. Indeed, the variations in dissociation of the dye with alteration of p H are evidently insignificant when compared with that of the protein. Consequently, variations in ioniza-

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tion of the dye may be ignored in most cases in the influence of p H on staining. The uptake of dye over a range of p H (Fig. 1) follows fairly closely the alterations in electrostatic charge on the protein. Basic dye, being cationic, may be expected to be attracted to protein with an excess negative charge. And, the staining conditions which favor increase in the net negative charge, such as low acidity (high p H ) of the staining solution, should also favor basic dye uptake-a relation borne out by previously described studies of staining as a function of pH. On the other hand, increase in the net positive charge, as the p H is lowered, favors the increased attraction of acid (anionic) dye. As dissociation proceeds and the groups are uncovered, the amount of dye bound increases until at the extremes of p H complete dissociation occurs and the maximum dye binding is attained. The above description of the effect of electrostatic alterations of the protein molecule on staining is based on the experiences and ideas of many workers. The early works of Brown (1901a), Mann (1902), Gee and Harrison (1910), and Harrison ( 1911) explained staining as an electrostatic attraction between dye and protein. These early interpretations were further elaborated as more detailed information on the nature of the bond between organic substances was obtained (Michaelis, 1920 ; Loeb, 1922; Stearn and Stearn, 1928a; Tolstoouhov, 1928; Craig and Wilson, 1937; Kelley, 1939b; Zeiger, 1938; Conn, 1940; Levine, 1940; McCalla, 1941 ; Rose, 1942 ; Neale, 1947 ; Gerstner, 1949 ; Vickerstaff, 1950). It will be shown later that there are other forces besides electrostatic ones which operate in the interaction of dye and protein. Yet, the electrostatic ones explain most effectively the observed variation of staining with pH and cannot be ignored even if it is finally shown that other forces provide the bond which combines the dye and protein. In the latter event, the electrostatic forces would play the important role of attracting the dye and protein within the range of the binding forces (see below). Finally, it should also be noted here that alteration in p H of the dye liquor also affects staining in another, albeit less important way. Swelling of the protein framework may occur as the p H is shifted beyond the isoelectric point. The increased separation of the micellae attendant upon swelling facilitates penetration of dye ions, particularly aggregates, which otherwise might be excluded from more central binding sites (Gerstner, 1949). Further thoughts on swelling phenomena as they relate to dyeing are noted below.

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IV. THESITEOF DYEBINDING AND BETWEEN

THE

NATURE OF

DYEAND PROTEIN

THE

BOND

Electrostatic forces operate over relatively long distances and, consequently, are particularly effective in attracting the dye ion to the protein. The actual site on the protein to which the dye is bound and the nature of the bond have been frequently speculated upon. The most prevalent and firmly established view holds that the site of binding is the charged group of opposite sign which has attracted the dye ion and that the bond is an electrostatic one (also called salt linkage or primary zdence, coulombic, or ionic bond). Accordingly, the actual combination may be described by ordinary laws of chemical combination as a salt formation between dye and protein (Loeb, 1922). The idea that the dye-protein reaction is a salt formation was advanced by textile chemists more than a hundred years ago and is also prevalent in early histological literature (see reviews of Pappenheim, 1901; Mann, 1902 ; Heidenhain, 1902; Pelet-Jolivet, 1910). Mann ( 1902) repeatedly described staining phenomena as salt linkages ; for example, he stated that “Proteid, changed in this manner, can readily interact with the ions derived from the salt which we employ as a dye, and in consequence chemical union between the kat-ions of the tissue and the an-ions of the dye (or the an-ions of the tissue with the kations of the dye) can readily take place, provided the tissue and the dye are brought into contact with one another in a common solvent, i e . , in a fluid which allows of electrical dissociation of both the proteid and the dye.” And elsewhere he said, “The conception I have formed of precipitated proteids is that each component molecule, in addition to adhering probably only physically to its neighbour, is still endowed with a number of side chains, which are unsatisfied after the removal of the fixing reagent, and which, under suitable conditions, may attract toward themselves color radicals of the opposite electrical sign. Thus, for example, a tissue side-chain may be a sufficiently strong kat-ion to combine with the an-ion radical of-picric acid, and the unsatisfied nucleic acid radical in nucleins may withdraw the kat-ion on methylene-blue from its an-ion chlorine. Thus dyes and tissue-molecules can adhere chemically to one another by their side-chains.” Salt-like combinations between dye and protein were also emphasized by Mathews (1898), and, Nietzke ( 1901) observed in wool dyeing that “Certain facts speak for the view that the unions of dyes with fibres are nothing but salt-like unions, in which the fibre, analogously to an amido acid, plays in the one case the part of an acid, in the other case that of a base.” Weber (1894) concluded that “the amido group of the wool combines with the sulphonic group of the dye, while the

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carboxyl group of the wool remains unaffected.” H e believed, moreover, that basic dye reacted with the carboxyl group, leaving the amino group unaffected. In the early literature on the affinity of chromatin for basic dye a number of works appeared which described the interaction between nucleic acid and basic dye as salt formation (see for example Miescher, 1874; Lilienfeld, 1893 ; Mathews, 1898). Description of dye binding as a salt linkage involving primary valence forces has been repeatedly advanced for reaction between various dyes and soluble proteins such as casein and albumin, or solid ones like gelatin and wool (some references are : Michaelis, 1920, 1947; Loeb, 1922, 1924 ; Chapman, Greenburg, and Schmidt, 1927 ; Hewitt, 1927 ; Rakusin, 1928; Rawlins and Schmidt, 1929, 1930; Stearn and Stearn, 1929, 1930; Stearn, 1931 ; Craig and Wilson, 1937; Ender and Miiller, 1937; Fraenkcl-Conrat and Cooper, 1944; Peters, 1945; Schmidt, 1945; Abbot, Crook, and Townend, 1947 ; Sokolova, 1948; Veller, 1948). The charged groups which woul_d form salts with acid dye are the free basic groups of the amino acids lysine, histidine, and arginine. Those to which basic dye would be bound are the free carboxyl groups of aspartic, glutamic, and hydroxyglumatic acids, the hydroxy groups of certain other amino acids, and the free acidic groups of phosphoproteins and mucoproteins. If a salt linkage is formed between dye and protein, one would expect that the amount of dye bound corresponds to the number of free acid or basic groups on the protein molecule. Such stoichiometric proportions between the quantity of dye and the number of binding .sites in the protein molecule have been looked for by many workers to support the view of salt linkages by primary valence bonds. The total number of acidic or basic groups available for interaction with dye may be calculated from the amino acid content of the protein or may be determined by titration of the protein with acid or alkali. In determining the number of dyeing sites for comparison with the calculated number of available sites, staining is done at extremes of pH where there is presumably maximum dissociation of acidic and basic groups of the protein and, consequently, where maximum uncovering of the binding sites occurs. The extremes of p H at which these groups are maximally dissociated are respectively 2 and 11 for the free amino and carboxyl groups. If the reaction is a stoichiometric one, there should presumably be a one-to-one combination between dissociating groups of the protein and dye ion at these extremes of pH. Such a result obtained in many of the works cited above where it was demonstrated that the amount of dye bound was equivalent to the calculated number of basic or acid groups.

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Other methods have been used for determining the stoichionietry of dye binding by protein. A unique one was employed by Stearn (1931) in his conductometric titrations of sodium gelatinate and sodium nucleinate with basic dye. H e observed alterations in electrical conductivity of the solution as the basic dye displaced hydrogen ion and calculated therefrom the amount of dye bound. His results supported those obtained by direct measurement of the dye taken up by protein. McCalla (1941) described the stoichiometry of the reaction of dye and protein in bacteria treated with MgS04 by observing the amount of Mg++displaced by the basic dye, methylene blue. The stoichiometry of staining was further studied in bacteria by observing the amount of hydrogen ion ( p H ) released in the course of staining with methylene blue (McCalla, 1941; McCalla and Clark, 1941). If combination of acid dye is determined by basic groups of the protein, then destruction of these groups should be followed by loss in capacity to bind acid dye. Experiments directed toward this end have been reported for wool in which the fiber was deaminated with nitrous acid (Speakman and Stott, 1934; Speakman and Elliott, 1943). But, deamination though extensive was never complete, and a small amount of acid dye was still taken up by the fiber. Further loss in acid dye binding was obtained by acetylation of the wool after deamination. Early experiments along these lines were done by Gelmo and Suida (1905), who looked for various groups in the fiber by first neutralizing them in various ways and then observing the alteration in dyeing. While a large body of information has been accumulated to support the view of dye binding in stoichiometric proportions by primary valence forces, there are many instances, however, where the amount of dye bound does not reflect the number of dissociating groups on the protein. Because of some of these results, as well as still other information to be presented below, the possibility has been advanced that forces other than coulombic ones may also operate in staining, though to various degrees depending on the substrate and the dye. In studies of dye binding of the solid protein, fibrin, much less dye appeared to be bound at extremes of pH than the number of groups available in the fibrin molecule (Ferry, Singer, et al., 1947; Singer and Morrison, 1948). Indeed, the quantity of dye bound at these levels of p H varied according to conditions of staining other than pH, such as ionic strength and dye concentration (see also studies with safranine 0 of Fraenkel-Conrat and Cooper, 1944). Another situation in which the number of dye equivalents probably differs from the number of binding sites obtains whenever an aggregate of dye ions rather than a single one is bound. Aggregation is quite common in solutions of dye,

STAINING O F TISSUE SECTIOXS

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and dye is frequently taken up by the substrate in the form of aggregates ranging from dimers to colloidal particles. In the staining of agar and other sulfated substances the basic dye is probably bound as an aggregaterather than a molecular unit-to the charged side chains of the substrate, a mode of binding which would explain the metachromasia of the stained product (Michaelis and Granick, 1945 ; Michaelis, 1947). Another example, where stoichiometric proportions with the primary charged groups do not obtain, appeared in studies of the interaction of various inorganic and organic acids and wool at low p H where more acid was bound than expected from the basic amino acid content; and the conclusion was drawn that either feeble basic groups, such as the amide groups of glutamine and asparagine and of peptide nitrogen or an entirely different mechanism of binding was responsible (Steinhardt and Harris, 1940; Peters, 1945 ; Carlene, Fern, and Vickerstaff, 1947 ; Abbot, Crook, and Townend, 1947; Vickerstaff, 1950). The amide groups may become charged at very low pH and thus become sites for ionic interaction with acid dye, as is shown by a rise in binding of certain acid dyes between p H 1 and 2 (Abbot, Crook, and Townend, 1947). Strong support for binding by amide nitrogen has been obtained in the study of nylon dyeing. Vickerstaff (1950) described interaction of dye and amide groups by way of hydrogen bonding (see below) and believed that amide combination is the source of main affinity of acid dyes and protein. According to him, combination with the amide group would occur particularly in neutral solution. He described two sites of amide binding; one which is adjacent to positively charged basic sites and the other not. In solutions of low p H the former sites are more effective and have the higher affinity for acid dye by virtue of the electrostatic attraction of dye ions to these regions. In this way he explained the stoichiotnetric correspondence often reported between amount of dye bound and the number of basic sites. Interest in amide groups as sites of dye attachment has been emphasized in recent years since the advent of polyamide fibers. Nylon contains some free amino and carboxyl groups, but various lines of evidence suggest that the amide groups with which this fiber abounds are particularly active as dye-binding sites. Another possible exception to stoichiornetric proportions and salt linkages is the observation, frequently made, that protein can bind acid dye, albeit minimally, above the isoelectric region where dye and protein have a similar charge, and basic dye below the isoelectric region where the interactants are both positive (Atkin and Douglas, 1924; Loeb, 1924; Grollman, 1925 ; Robbins, 1926; Chapman, Greenherg, and Schmidt, 1927 ;

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Kelley, 1939a; Schmidt, 1945 ; Skinner and Vickerstaff, 1945 ; Klotz, 1946; Neale, 1946, 1947; Singer and Morrison, 1948; and others). These relations are evident in Fig. 1 where both cationic and anionic dye are bound in the pH range above and below the point at which the curves cross. It can be argued that the limited binding of dye at a p H where staining is opposed by electrostatic repulsion may be due to interaction of dye ions with occasional and isolated protein groups of opposite charge. Yet other possibilities have been advanced to explain dye uptake under circumstances in which dye and substrate have the same electrical sign and, indeed, under ordinary conditions of staining, too. Dye ions which reach the protein surface may be anchored there by specific and powerful short range forces such as van der Waals (Neale, 1947; see discussion below of cellulose dyeing in section on dye affinity). If such bonds are important in binding of dye, then the electrostatic forces would operate in assisting or opposing diffusion of dye to the binding sites. In their analysis of the interaction of wool and dye, Gilbert and Rideal, (1944) also suggested that other forces besides primary valence bonds may be effective, such as resonance bonds, van der Waals forces, and coordinate links (covalent bonds), perhaps by way of the chromophore and auxochrome groups of the dye. The fact that large molecules have a greater affinity for wool fibers than small ones, would be expected if forces in addition to ionic ones, as for example van der Waals forces, obtain in the binding of the dye (Steinhardt, Fugitt, and-Harris, 1941a, 1942). Moreover, it is possible that forces operating between protein and dye vary according to the dyestuff, being a simple salt linkage in the case of some, such as small ions, but including additional forces in the case of others such as those mentioned above (Skinner and Vickerstaff, 1945). The important hydrogen bond, already mentioned above, may be particularly effective in the binding of certain dyes (Vickerstaff, 1950). In hydrogen bonding, hydrogen acts as a bond between two atoms, especially highly electronegative ones such as oxygen and nitrogen. Hydrogen bond formation has been postulated between the amide group of the protein and the hydroxyl, amine, or azo group of the dye (Vickerstaff, 1950). Additional discussion of this method of dye binding is given below (see section on affinity of dyes). Another possible method of linking dye and protein emerged from study of shifts in the spectral absorption curves of azo dyes during certain reactions (Gerstner, 1949) ; in addition to ordinary ionic reactions the carboxyl groups of protein may bind dye by linkage with the azo group (also refer to Gillet, 1889, 1890). There are, conceivably, still other ways by which dye and protein may be

STAINING OF TISSUE SECTIONS

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combined than those listed above. And, indeed, the forces involved in dye binding may vary as staining progresses, since there is some evidence that dye ions which are first bound influence the binding of subsequent ones in various ways, for example by repelling oncoming ones electrostatically (Klotz, Walker, and Pivan, 1946). The nature of the binding site and the forces involved may vary according to the characteristics and state of the protein. In the study of the stoichiometry of staining, soluble proteins were mainly used. Yet, there is good evidence to show that binding in a solid system, such as is encountered in histological sections, is different, and consequently the mechanism of staining of soluble proteins cannot be equated precisely with that for solid ones. Major differences are evident between the titration curves of dissolved and solid proteins (Speakman and Hirst, 1933; Lloyd and Bidder 1934 ; Speakman and Stott, 1934, 1935 ; Steinhardt and Harris, 1940; Steinhardt, Fugitt, and Harris, 1941b). The p H levels over which the titration curve shows little change with added acid is much greater than for soluble proteins. Moreover, whereas the pK values of soluble proteins are very similar to the corresponding amino acids, they are shifted to higher and lower p H levels for the basic and acidic groups respectively of the solid protein (Vickerstaff, 1950). These differences between insoluble and soluble proteins have been attributed to structural differences, to the effect of a two-phase system on the reaction as explained by the Donnan equilibrium, or to other means. It is quite evident that the conditions under which dye is bound in a three-dimensional solid protein are quite different from those for protein free in solution. I n the former instance Donnan effects operate just as effectively as though the protein were in solution and separated from the staining solution by a semipermeable membrane (Eliid, 1933), and, consequently, the solution within the protein meshwork differs from that without in terms of p H as well as ionic distribution. These differences have been emphasized and quantitated by Speakman and Peters (1949), who applied the Donnan theory of membrane equilibrium to titration of wool with acids and observed that the internal p H of wool differed from that of the external solution. They were able to calculate the internal p H but could not measure it directly (see also Vickerstaff, 1950; Gilbert and Rideal, 1944; Kitchener and Alexander, 1949 ; Peters and Speakman, 1949). There is further evidence to complicate and question the view that salt linkages and stoichionietric proportions are alone characteristic of staining. This other evidence is best considered in relation to the nature of the affinity of dyes, which is discussed below. Particularly instructive is the

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MARCUS SINGER

information on cellulose derivatives where forces other than primary valence bonds explain dyeing most logically.

BETWEEN T H E ISOELECTRIC POINTAND STAININC; V. THERELATION The dyeing of protein in the isoelectric condition was studied by Loeb (1922) who emphasized that in this condition gelatin was relatively inert ; but, he later reported (1924) that gelatin could bind acid dye, albeit minimally, above or basic dye below, the isoelectric point. Nevertheless, the close relation between the isoelectric point and the relative absence of protein reactivity appeared to offer a means of defining the isoelectric point by staining procedures. Many attempts were subsequently made to apply such determinations of isoelectric points to the protein complexes of cells and tissues. Robbins (1923, 1924, 1926) stained the mycelium of fungi and tissue of the potato tuber with acid or basic dye and then washed them in buffer solutions of various p H and observed the degree of dye loss in each solution. The p H region of minimum retention of dye was considered the apparent isoelectric point of the protein complex. Because of the relation between dissociation of the protein at dieerent pH and combination with dye Stearn and Stearn (1924) concluded that the isoelectric point of bacteria could be determined from curves of staining with acid and basic dye. They reported that the crossing point of staining curves of acid and basic dye reflects more accurately the isoelectric condition than the region of minimal anion or cation retention. Therefore, they defined the isoelectric point ( 1928c) as “the hydrogen-ion concentration at which there is equal retention of cation and of anion.” Tolstoouhov ( 1929) , using a mixture of eosin and methylene blue buffered to various pH, defined the isoelectric point of blood cells and other tissue proteins as that p H where staining was an approximately equal combination (purple color) of the red and blue dye components. The isoelectric point of structures of plant cells was described by Naylor (1926) and Robbins (1926) , following observations on the pH of minimal acid and basic dye uptake, as a p H range rather than a specific point. The isoelectric point of hemoglobin in hemoglobin-containing cells was observed in fixed and unfixed specimens with acid and basic dye (Kindred, 1932, 1935). The crossing point of p H curves of acid and basic dye binding, or the region of minimal basic or acidic dye uptake was used as the criterion of the isoelectric condition by Pischinger (1926, 1927a) in histochemical studies of the proteins of cells and tissues. A whole series of studies of ‘%oelectric points” on a variety of tissue and cellular components was initiated by Pischinger’s work (Mommsen, 1927; Pulcher, 1927a ;

STAINING OF TISSUE SECTIONS

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Schwarz-Karsten, 1927 ; Ochs, 1928 ; Pfeiffer, 1929 ; Seki, 1933c, 1934 ; Yasuzumi, 1933a, 1933b, 1934 ; Nishimura, 1934 ; Achard, 1935 ; Ikeda, 1935, 1936a, b ; Sturm, 1935; Fautrez, 1936; Yasuzumi and Matsumoto, 1936; for review of these and other works see Zeiger, 1938 and Levine, 1940). More recently the isoelectric point of structures of skin were defined using fluorescent dyes over a pH staining range (Bejdl, 1950; Stockinger, 1950). The accuracy of isoelectric determinations of proteins by staining procedures has been questioned. The close relation between the isoelectric point and the minimum of acid and basic dye uptake observed by Loeb for gelatin did not obtain for powdered hide collagen (Thomas and Kelly, 1922). When isoelectric point determinations were attempted from titration data or from study of the interaction of the protein with various substances, a number of difficulties appeared (Speakman and Stott, 1934). Between p H 5 and 7 there was no significant binding of alkali or acid by wool, and consequently the isoelectric point of wool could not be exactly defined but rather the values 5 to 7 were considered an isoelectric range. Comparison of the staining of extracted nucleoprotein with that of tissue nuclei showed that the isoelectric point of the nucleoprotein of the cell could not be determined by staining with the basic dye, toluidine blue (Kelley, 1939a, b). According to Kelley, staining depended on the amount of nucleic acid in the nucleoprotein and not on the isoelectric point. H e observed that toluidine blue was bound to nucleoprotein below its cataphoretic isoelectric point. Levine ( 1940) reexamined the concept of isoelectric point‘ determination of tissue proteins with dyes and concluded that the relatively qualitative curves of staining with p H are inadequate sources of such information. H e found that the crossing points and other characteristics of acid and basic dye curves upon which “isoelectric point” determinations were based varied with such factors as the nature and concentration of dye and buffer salts of the staining solution. Isoelectric point determinations varied by as much as 2 p H units when different dye pairs were used. Pfeiffer (1929, 1931) also questioned the belief that the point of‘ intersection of the dye curves represents the isoelectric point. Some pertinent observations concerning isoelectric determinations by staining procedures may be drawn from studies of fibrin film, an isolated and relatively pure protein system. The isoelectric point of powdered samples of fibrin film was determined electrocataphoretically and the molar binding of orange G and methylene blue analysed quantitatively over a range of p H (Singer and Morrison, 1948). The cataphoretic iso-

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MARCUS SINGER

electric point of freshly prepared fibrin was 6.0,but the p H of crossing of orange G and methylene blue curves was 6.5 (Fig. 5, see Fig. 3A of Singer and Morrison, 1948). When denatured by heating (20 minutes at lZO”C), the apparent isoelectric point was 5.5 but the crossing point of acid and basic dyes was 6.3;for films heated for lesser times (1 minute at 100°C)the values were 5.7and 6.4respectively (refer to same figures). The disparity between the two values, although thoroughly apparent, is not great so that the p H of crossing diverges between one-half and one p H unit from the cataphoretic isoelectric point. If such quantitative procedures could be applied as readily to the microscopic and, yet, highly heterogeneous protein systems of tissue sections, it would be possible to approximate the isoelectric conditions of tissue elements by determining the pH at which there is equivalent binding of acid and basic dye. It must be stressed that such determinations would be only approximations. The divergence between the isoelectric point and the region of crossing of the curves may be due to binding of dye by forces other than coulombic ones. These additional forces do not determine the cataphoretic isoelectric point and, consequently, binding by them should only increase the disparity between the values. The deviation between the cataphoretic isoelectric point of fibrin and the pH a t which the acid and basic dye curves intersect one another varies with the dye pairs used (unpublished results). The pH of equimolar dyeing of formalin-fixed fibrin (cataphoretic isoelectric point of 5.2) with methylene blue and the following acid dyes were: orange 11, 5.0;picric acid, 5.1; orange G, 5.2; ethyl orange, 5.3; fast green, 5.5 ; sodium 2, 4-dihydroxyazobenzene-4-sulfonate, 5.6; light green, 5.7. These results are in agreement with the more qualitative observations of Levine ( 1940), who demonstrated variation of the intersection point for dye pairs in tissue staining. There are other factors as well which influence the crossing point. It is evident, therefore, from these observations that caution must be exercised in defining the isoelectric condition of proteins by their staining characteristics, particularly in tissue sections where the conditions of binding are not easily controlled and where adequate procedures for determining the ’quantity of bound dye are not yet developed. At best, perhaps, a broad isoelectric range may be described in which the actual isoelectric point would probably fall (“isoelectric range” of Naylor, 1926;“isoelectric zone” of Stearn, 1933 ; Speakman and Stott, 1934;see also discussion by Dubos, 1945,p. 68). Perhaps too much emphasis has been placed upon the relation between the isoelectric point and dye uptake. There are characteristics about the pH curves of staining which can be drawn upon in analyzing the protein

STAINING OF TISSUE SECTIONS

233

without any particular reference to the isoelectric point. For example, the region of the curve showing greatest rate of change with alteration in pH reflects a most sensitive and characteristic response of the protein to alterations in its electrostatic environment. This region can be defined by that p H at which a tangent along the steep part of the curve intercepts the pH axis; or it can be described as the p H which bisects the steepest part of the curve. If dye pairs are used then the pH at which curves cross may be used as a characterization point of the protein structure. There are still other ways of characterizing proteins according to the dye curves which they yield. Although the isoelectric point of the protein cannot be precisely defined by p H staining characteristics, yet it is possible to compare curves of different proteins and to draw conclusions therefrom on the relative position of the isoelectric point of these substances. From previous considerations of dissociation of proteins in relation to staining, it would follow that staining curves displaced to regions of higher p H reflect higher isoelectric proteins and those which lie lower on the p H axis represent lower isoelectric proteins. It is also possible to determine the direction of shift of the isoelectric point of a protein or protein complex following a physiological modification by comparing staining curves of the substance before and after modification (Levine, 1940; Singer and Morrison, 1948 ; Singer and Wislocki, 1948; Singer, 1949). A point which requires some emphasis here is that studies with dyes of the “isoelectric point” or other characteristics of proteins invariably and by necessity have been made on fixed proteins. Such chemical modification undoubtedly affects the protein, and the characteristics of dye binding does not reflect those of the native undenaturecl system, as will be evident in the discussion of the effect of fixation on staining (see below). The isoelectric point may be influenced profoundly by such chemica1 modification. Pischinger ( 1926, 1927a) and many others (see e.g., Zeiger, 1930b ; Seki, 1933b) have assumed in their studies that the isoelectric point of tissue proteins is not altered by alcohol fixation, a conclusion which has been criticized by Yasuzumi (1933a). VI. THE IONICSTRENGTH OF

THE

DYE SOLUTION

The foregoing discussion of the influence of p H on staining has emphasized that the protein amphion is sensitive to alterations in the pH of its environment and reflects these alterations by a change in charge. This being the case, it may be expected that factors other than acid or alkali which alter the electrolytic environment should also affect the electro-

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MARCUS SINGER

static condition of the ampholyte and, thereby, its interaction with dye. The amount of dissolved salt represented either as neutral or buffer salts is such a factor. The activity of salt ions in solution is best expressed by the ionic strength (see Cohn and Edsall, 1943) rather than by other concentration expressions. Salt solutions of equal concentration measured by the latter means may have different ionic activities by virtue of a difference in number and valence of dissociable ionic groups. The ionic strength ( u ) of a solution of electrolyte is defined according to Lewis and Randall (1923) as half the sum of the concentration (molality) of each ion multiplied by the square of its valence: u = 8 mi Zf Increasing ionic strength of the dye solution decreases staining with both acid and basic dye, a result which is exemplified in Fig. 2 for the stainORANGE G

0.4

k

g a3 W

n J

a

-0

oa 0.1

s a 10-5 W.

-.\.

+:, ,.'

o -\ . 0 . 0

0

Ionic strength

' 0

\e

0

g

METHYLENE BLUE

I I 10-sY.

3

9@Y

4

%,bc@ /O s

6

I

7

0 8 0

0.01 0.04 0.15

I.o

3.8 36

.d

0.4

/ E

1.2

01

9

P"

FIG.2. The influence of ionic strength on staining of fibrin film with acid (orange G ) and basic dye (methylene blue). Taken from Singer and Morrison, J. B i d . Chem., 176, 1948. ing of fibrin with methylene blue and orange G. Steinhardt, Fugitt, and Harris (1941a) described such a depressing effect on the titration of soluble and fibrous proteins with various anions. Levine (1940) observed that the staining of tissue sections decreased with increasing concentration of buffer salt. A number of reasons have been advanced for this effect of the electrolyte on staining, among which are the remarks of Singer and Morrison (1948) on the dye binding of fibrin and the more detailed discussion of Elod (1933) in his work on the effect of neutral salt on the binding of dye anions by wool at low pH. According to Donnan's theory of membrane equilibrium, salt ions may influence the staining of solid proteins by altering the distribution of dye ions between the external solution and the solution within the protein itself. For this reason, the effective

STAINING OF TISSUE SECTIONS

235

staining concentration, namely the concentration of dye in the solution between and within the fibrillae of the protein probably differs from that of the dye bath. In solutions of high ionic strength the internal dye concentration in equilibrium with the stained micellae is evidently much less than in solutions where little or no salt is present. A quantitative interpretation of the effect of different ionic srengths on the interaction of solid protein and various acids according to Donnan’s theory has been elaborated by Speakman and Peters (1949) in studies of the binding by wool of sulfuric acid and hydrochloric acid singly or in the presence of different concentrations of KCl. The analysis of dyeing equilibria according to the Donnan theory has been summarized and evaluated by Vickerstaff (1950). In addition to the Donnan effect it is also possible that the salt ion competes with the color ion for the binding site on the protein molecule and thereby limits the quantity of dye bound (Pelet-Jolivet, 1910; Briggs and Bull, 1922; Elod, 1933; Speakman and Clegg, 1934; Skinner and Vickerstaff, 1945). I n describing such competition Elod (1933) showed that in dyeing of wool the small salt ion penetrates the fiber most rapidly by virtue of its greater diffusibility and is bound, but then secondarily is replaced by the larger dye ion which shows less tendency to dissociate after binding (see discussion below on dye affinity). When the concentration of salt ion is increased, less of the dye ion in competition with it is bound. Neale (1946, 1947) concluded that salt ions serve to suppress the electrostatic forces on the protein, thus decreasing the attraction to acid dye below the isoelectric point and basic above. It is interesting, as Neale and others have emphasized, that higher ionic strengths have the reverse effect on dye binding when the sign of the charge on the dye and the protein is the same (above the isoelectric point for acid dyes and below for basic ones). In these instances the positive effect of the salt on staining results from suppression of forces which would normally repel the dye ion. Since he attributed dye binding, in part, to the more powerful but short-range forces, such as hydrogen bonds and covalent links, the effect of the decreased potential of the protein surface is to allow more dye ions to come within the range of action of these forces (see discussion on dye affinity). I n this way the activation energy of the dyeing process is reduced and dyeing increased with added salt (Vickerstaff, 1950). Electrolyte also plays an important role in the dyeing of cellulose fibers, but in a manner quite different from that for ordinary dyeing of wool. Cellulose derivatives are negatively charged and yet are generally stained

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MARCUS SINGER

with acid dyes. The affinity of the color anion for cellulose is quite low in the absence of electrolytes. As electrolytes are added to the color bath, dyeing increases markedly despite the similarity in charge of the color anion and the plant fiber. The effect of the salt ions is to dampen the mutual repulsion and allow dye to approach the cellobiose chains closely enough to be bound by hydrogen linkages. The effect of electrolyte on the staining of cellulose resembles its effect on dyeing of proteins above the isoelectric point with acid dye and below this point with basic dye. In both instances salt serves to facilitate dyeing. Finally, it is also possible that salt influences staining by acting on the dye itself. Increased salt causes the dye to form colloidal aggregates and even eventually to precipitate from solution (Michaelis, 1947). Large aggregates of the dye by virtue of their size find less ready access to intermicellar regions of the protein than single ions or dimers. Consequently, the effective concentration of dye in solution is lowered thereby, and the staining decreased.

VII. THEINFLUENCE OF DYE CONCENTRATION Variation in the amount of dye taken up by tissue sections according to the concentration of the dye bath is a matter of common knowledge to histologists. Greater amounts of dye are bound with increasing concentration as shown in Fig. 3 for both acid and basic dye. The amount of dye which is bound with increasing dye concentration is limited by the number of available binding sites (Knecht, 1889, 1904; Hofmeister, 1891, p. 224; ORANGE G

44.

METHYLENE BLUE Dye concentration

o

2.

- 1.2 -

I r ~ ~ M.- 5 0 SXIO'~ M. 0 2.5 ~ 1 0 M. . ~

-

x

0.8

a6

-.0.4

- 02 2

3

4

5

6

P"

7

8

9

FIG.3. The influence of dye concentration on staining of fibrin film with acid (orange G ) and basic dye (methylene blue). Taken from Singer and Morrison, J. Biol. Chem., 176, 1948.

STAINING OF TISSUE SECTIONS

237

Steinhardt, Fugitt, Harris, 1941a; Skinner and Vickerstaff, 1945). Except for the activity of other forces which may bind dye such as hydrogen bonds and van der Waals forces, once coulombic forces are satisfied little or no additional dye is bound. Limitation in the amount of dye taken up as staining proceeds is due, however, not merely to occupation of the dyeing sites by dye molecules but also to the effect of bound dye on the oncoming dye molecules. Klotz, Walker, and Pivan (1946) have pointed out in their studies of the adsorption of dye by serum albumin that steric hindrance or electrical effects on approaching dye molecules may result from already bound ones. The change in the rate of increase of bound dye with increase in dye concentration has been explained by Elod (1933) in terms of the Donnan equilibrium. Other experiments on the influence of dye concentration on staining are those of Craig and Wilson (1937).

VIII. THEAFFINITYO F DYES By ufinity is generally meant the tendency of a dye to combine with a given tissue structure. However, the term is userl quite loosely by histologists and may have a variety of implications. Often it implies a specificity between the substrate and a particular dye not shared by other dyes. Yet, a given protein may be stained by any one of a number of acid or basic dyes provided conditions of staining, particularly such as pH, are adequate. And, consequently, from this viewpoint, specific affinities are not the rule and molecularly dispersed dyes of quite different character are taken up by the same protein. For example, the basic dye methyl green which is ordinarily considered very specific for desoxyribonucleoprotein will interact with proteins of cells and tissues in general in aqueous solution, provided the appropriate p H conditions are used (compare Michaelis, 1947). And other basic dyes will stain the same nucleoprotein quite well. The acid stain, aniline blue, will also stain cells and tissues widely when applied under conditions of low pH, although as employed in triple staining methods it is considered fairly specific for collagen. Mathews (1898) appreciated this similarity in staining capacity of dyes at a time when dfferences or “specific affinities” were especially highlighted. Although protein shows little tendency to bind a particular acid or basic dye exclusively and reject others, nevertheless the degree of binding varies from one dye to the next. Some dyes are bound in greater amount than others. If the equivalent of acid dye or of other acid substance taken up by the protein is plotted against pH, notable differences in the number of equivalents of each substance are apparent, even though the conditions of staining are the same. Steinhardt, Fugitt, and Harris (1941a) recorded

238

MARCUS SINGER

differences between a variety of substances which at the extremes reached 2 units on the p H coordinate between the curves for HC1 and flavianic acid when the p H values were compared a t which half the maximum amount of acid was bound. Such differences in the curves of staining of various dyes were also recorded by Levine (1940) and Elod (1933) and are exemplified in Fig. 4 for staining of fibrin film.

PH

FIG. 4. Differences in the affinity of two acid dyes (light green and picric acid) revealed by their pH staining curves. Note the differences in the crossing points of these curves with the curve of methylene blue (see text on discussion of isoelectric points). Formaldehyde-fixed film (10 per cent for 10 hours) ; dye concentration, 5 X 10-6 M . ; ionic strength, 0.02. (Singer and Weiss, unpublished.)

The differences in affinity of dyes for solid proteins evidently depends on a number of factors (see also review of the factors which govern the affinity of soluble proteins for dyes and other interactants by Goldstein, 1949). As mentioned previously, it is conceivable that other groups on the protein molecule than amino or carboxyl ones may be involved in the reaction with different dyes. Some forces may be more available for combination with one dye than with another. For example, combination of protein with simple acids may involve primary valence bonds with substituted ammonium groups, whereas with other substances forces such as covalent links or hydrogen bonds may be involved simultaneously or alternatively (Klotz, Walker, and Pivan, 1946; Neale, 1946, 1947; Klotz and Walker, 1947). Neale (1947) in his study of affinity and its meaning in terms of electrochemistry of staining (see also Harrison, 1948) concluded that short-range forces such as are present in covalent links or hydrogen bonds are responsible for the specific affinity of a dye for a particular fiber. The long-range electrostatic forces oppose or assist these more powerful short-range forces. But, even with electrostatic repulsion of the dye (e.g., below the isoelectric point with basic dye or above with

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acid dye) some dye is bound, nevertheless, by the short-range forces, although work evidently must be done (thermal agitation) to bring the dye ion to the surface of the fiber. Another explanation of differences in affinity is based on studies of the degree of dissociation of various protein-anion combinations ( Steinhardt, 1940; Steinhardt and Harris, 1940; Steinhardt, Fugitt, and Harris, 194la, b, 1942). When the dissociation was great, as occurred after reaction with chloride ion, the affinity as recorded in the titration curve was much less than when the dissociation was slight as occurred with protein-dye combinations (compare Chapman, Greenberg, and Schmidt, 1927). The dissociation constants, calculated for various combinations of protein and anion, showed wide variations (also Steinhardt, 1942). Therefore, affinity of the dye was directly related to the degree of association of the color ion with the binding site of the protein. The differences in affinity were correlated with differences in size of the anion, and it was noted that with few exceptions increasing order of affinity followed increase in molecular weight and was higher in aromatic than in aliphatic ions of the same size (compare Klotz and Walker, 1947; Klotz, Triwush, and Walker, 1948). In the exceptions, considerations of shape of the molecule and its relation to steric hindrance were offered as an alternative possible explanation of differences in affinity (cf. Goldstein, 1949, p. 146). The difference in combining capacity of various acids with protein was great. When chloride ion was taken as unity, then the combining capacity of picric acid was 758 and that of Orange I1 over 23400 (Rose, 1942). It has also been stated by textile chemists as a general rule that affinity increases with molecular weight and, moreover, with complexity of the dye ion and with the introduction of additional polar groups (Abbot, Crook, and Townend, 1947; Lemin and Vickerstaff, 1947). In connection with size differences of dye molecules, Speakman and Clegg (1934) and Speakman and Smith (1936) believed that in the case of wool the cystine (-S-S-) and salt linkages (-COO-, -NH3+) offer resistance to the penetration of large dye molecules, but at low pH, salt linkages are broken since the carboxyl groups are displaced from combination and the freed amino groups combine with the added acid. As a result of lowered cohesion of the micelles the structure swells with water and is now accessible to large dye molecules which are then trapped in the protein fiber. The tendency of dye ions to aggregate in solution and to stain as aggregates will also affect the penetration and therewith the affinity of the dye for the protein. There are evidently other possibilities involving the number and kind

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of reacting groups on the dye molecule (Speakman and Clegg, 1934; Townend and Simpson, 1946 ; Gerstner, 1949) and the physical structure of the protein as well as that of the dye (see also Seki, 1933a; Zeiger, 1938). For each dye with more than one binding site in its molecule it would be important to inquire how many of these sites are involved in the actual attachment of the dye molecule to the protein. For example, a dye molecule with two charges may be bound in equivalent or molecular fashion (Loeb, 1922; Elod, 1933; Speakman and Stott, 1935), and its mode of attachment may vary according to the protein. Abbot, Crook, and Townend (1947) have described this effect for the dyeing of nylon. When the number of acid groups of the dye molecule is increased from one to two, the affinity of the dye decreases since it is now spread over two sites rather than one. Or, dye with three or even four charged groups has been reported to occupy an equivalent number of oppositely charged sites. Because of the probable disparity in spatial arrangement of the charges on the dye and those on the protein, it is possible that an equivalent number of charged sites are neutralized rather than occupied by the dye ion (Vickerstaff, 1950). However, if only one binding group of the dye molecule is involved, the remaining groups may influence adversely the binding of oncoming ions by electrical effects or steric hindrance (Klotz and Walker, 1947; Vickerstaff, 1950). I n these instances the affinity of dye for protein must be weighed not merely in terms of its combining capacity but also, once bound, in relation to its effect on the approach of another dye ion to an adjacent site. Therefore, as staining proceeds, the available sites are no longer equivalent to the initial ones and the dyeing mechanism may be profoundly influenced and altered. In connection with these thoughts it may be well to note that there is no need to assume that all dyeing sites are equivalent, even at the onset of dyeing (Vickerstaff, 1950). Some may not be accessible to certain dye molecules, but readily available to others. Some correlation has been drawn between the shape of the dye molecule and the affinity for protein in that planar molecules are believed to have more affinity for wool than three-dimensional or linear ones (Steinhardt and Harris, 1940) ; but in the case of protein staining in general little information is available relating shape and affinity (Vickerstaff, 1950). The relation between the structure of the substrate and the shape and constitution of the dye has been studied in most detail in direct dyeing of cellulose fibers. Cellulose is dyed by color ions (anions) having the same charge (negative) as the fiber, and consequently, other forces than

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electrostatic ones must be invoked to explain the uptake and fixing of the dye. Indeed, the electrostatic forces serve in this case to repel the dye ion rather than attract it, as in the case of ordinary protein staining. Dyes which combine directly with cellulose are generally sodium salts of sulfonated aromatic azo dyes. Once the dye anion is brought to the surface of the fiber (as described in preceding pages, factors of thermal agitation and addition of large quantities of electrolytes to the dye bath are important in this movement) and penetrates the water swollen regions between the cellobiose chains, the dye molecule is bound by short-range forces to the cellulose molecules (Neale, 1947 ; Vickerstaff, 1950). The nature of these forces and the problems involved in binding dyes of various structure and configuration have been speculated upon. Among the theories is that of hydrogen bonding (Rose, 1935) in which hydrogen of certain groupings of the fiber or of the dye acts as the electron acceptor. According to Rose at least two hydrogen links are required to bind a colored ion. Such linkages between the color ion and the linearly arranged cellobiose chains should be more readily formed with dye ions of certain shape and structure. Indeed, dye molecules used in direct dyeing of cellulose are in general long and chain-like and thus can contact the cellulose micellae more closely than non-linear ones (Meyer, 1928). Coplanarity of the various ring nuclei (benzene and naphthalene) also determines affinity of the dye (Hodgson, 1933) ; dyeing is favored when the rings lie in one plane. Finally, a high number of double bonds in the dye molecule seems also to be important (Schirm, 1935). This description of some of the theories of cellulose dyeing emphasizes that dye interaction depends to some degree upon the shape, size, and constitution of the dye molecule. The problem of the affinity of dye is undoubtedly complicated by still other factors than those described above. In non-aqueous solutions or under conditions of staining quite different from those considered in the present review, the extent of interaction may be still further influenced; and the reaction between a particular substrate and a dye may be specifically favored. Relatively little attention has been paid to the influence of special media, such as an alcoholic or phenolic one, as is employed, for example in methyl green-pyronin staining of nucleoprotein. Perhaps the function of the special media or other peculiar conditions of staining is specifically to enhance or to facilitate forces which may favor a particular dye and protein combination.

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IX. THEINFLUENCE OF FIXATION AND OTHERMODIFICATIONS OF TISSUES ON SUBSEQUENT STAINING The obvious and important effect of fixation in histology is to denature and thereby render insoluble both the solid and dissolved proteins. In addition, fixation and other chemical or physical modification influence profoundly the subsequent binding of dye. In general, the living cell shows a selective affinity for some dyes which it concentrates and stores. This selective affinity is an expression of vital activities of living cells and bears little relation to forces which control the binding of dye in fixed and histologically prepared cells and tissues. After death, dyes of various character readily penetrate the cell, but the capacity to bind them is slight. However, when the cell is subjected to physical or chemical denaturing agents there is an immediate and pronounced increase in staining. The extracellular protein matrix of tissues also shows a limited affinity for dye until it is fixed. The precise nature of the effect of fixatives on proteins is not known. Elucidation of the alterations suffered by the protein during fixation is a problem of major importance in the study of the physical chemistry of tissue proteins since it is the modified and not the native protein which is studied under the microscope. Yet, relatively little work is being or has been done in recent years on fixation. Early workers devoted a considerable time to the elaboration of different fixatives designed to preserve the morphology of the cell and tissue with little change and yet to favor the staining of one or another morphological component. A considerable number of procedures was elaborated empirically or on the basis of certain chemical information. The procedures were reviewed admirably by Mann (1902), who also discussed, according to the information then available, the chemical and physical significance of the techniques. A more recent evaluation of fixation is given by Zeiger (193Oa, b, 1938). References to various methods of fixation and their general application may be obtained in technical works on histological procedures (for example Baker, 1945 ; McClung’s Handbook, Jones, 1950; Lee’s Vade-Mecum, Gatenby and Beams, 1950 ; Bourne, 1951). No attempt will be made to cover these works here. Instead, some general statements will be made about fixation based especially upon chemical information on denaturation of proteins and related information that is available in the histological literature, The manner in which fixation influences the subsequent staining is exemplified in experiments recently reported on films of fibrin (Singer and Morrison, 1948). These films are particularly suited for studies of

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fixation, since the constituent fibrin of freshly prepared films may be considered native (Ferry, Singer, et al., 1947). Moreover, the protein is solid and insoluble having the three-dimensional relations typical of the structural proteins of cells and tissues. Consequently, the factors which obtain in fixation of fibrin film resemble somewhat those for cells and tissues. The interaction with dye of native fibrin and fibrin modified by various procedures is compared in Fig. 5. Freshly prepared fibrin has

i

I 2

10

08

06

0.4

0. I 09.

t

3

6

4

9

PH

FIG.5. The influence of different fixatives on subsequent staining of fibrin film. Note how the acid (orange G ) and basic (methylene blue) dye-binding curves shift according to the fixative (see text on discussion of isoelectric points and of fixation). Taken from Singer and Morrison, J. Biol. Chem., 176, 1948. little capacity to bind dye and resembles unfixed tissue protein in this way. In general, a notable increase in stainability with both acid and basic dye occurs after fixation, whether such fixation be p%sical (heat) or chemical in nature. However, the increase in acid and basic dye-binding capacity is not an equal one and in most cases varies according to the treatment. For example, there is a relatively greater increase in basic than in acid dye uptake following formaldehyde fixation. O n the other hand, the reverse is true after fixation with HgClz or some other salt of a heavy metal (cf. Kelley, 1939a). Fixatives appear to have two major effects on protein as reflected in the dye reaction. There is an initial effect of increase in affinity for both classes of dye attributable presumably to a physical reorganization of the protein, whereby charged and other groups to which the dye ion may attach are rendered more available to the dye (Singer and Morrison, 1918). This effect is in general shared by all fixatives. An increase in

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availability of reacting groups resulting from denaturation of proteins has been frequently described in the chemical literature (see reviews of Neurath, Greenstein, Putnam, and Erickson, 1944 ; and Anson, 1946). Alteration in physical structure of the protein implies also an alteration in the permeability of the protein meshwork for dye molecules. The second effect is peculiar to the particular fixative and results in an alteration in the relative uptake of acid and basic dye. This effect may be attributed to a specific influence of the fixative on groups which bind the dye, or on other groups whose proximity or mere presence influences the binding of dye to adjacent sites. The fixative, depending on its nature, may introduce other ionizing and, therefore, dye-binding sites in the protein. It may cover specifically certain groups or in some other manner prevent dye interaction with these sites. The two effects of the fixative are well exemplified in heat denaturation of fibrin film (Ferry, Singer, et al., 1947). As a result of brief heating the acid and basic dye-binding capacity increased remarkably (see also Herrmann, Nicholas, and Boricious, 1950), an effect which corresponds to the first postulate. Upon prolonged heating a second effect appeared and gradually became pronounced ; the affinity for basic dye increased whereas that for acid dye declined. Associated with the second change there was a gradual drop in the isoelectric point of the protein (Singer and Morrison, 1948). Prolonged heating probably causes a gradual and progressive deamination of the protein and thereby a decrease in the number of positive groups available for acid dye. A similar drop in the isoelectric point and a similar alteration in staining is observed following denaturation with formaldehyde. But, in this instance, the secondary effect is accomplished by combination and therefore covering of the amino groups. A secondary effect in which metal ions combine with carboxyl groups presumably occurs with HgCl? and other heavy metal fixatives. In this way the basic dye affinity is relatively diminished and the acid one is increased. Other alterations, such as mordanting, which are secondarily imposed upon the primary fixation will further influence the staining of proteins by modifying the dyeing sites or so changing the physical characteristics of the protein structure (such as degree of swelling) as to alter the extent of penetration by the dye ion. Staining intensity varies with changes in the protein and, consequently, is a sensitive criterion of the modifications to which the protein was previously subjected. Indeed, slight differences in heat treatment of fibrin film were detected through alterations in dye uptake (Ferry, Singer, et al., 1947). Another study of staining of modifietl proteins was that of Fraenkel-Conrat (1944) ; and the effect of various chemical modifications

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on the dye binding of wool was reviewed by Kienle, Royer and McCleary (1945), Lemin, Vickers and Vickerstaff (1946) ; Vickerstaff ( 1950). A number of interesting studies of the variation of staining with fixation have been reported in histological literature (see reviews by Zeiger, 193Oa, 1938). Tolstoouhov (1928) recorded the staining of blood cells in mixtures of eosin and methylene blue of various pH and following various fixations. The p H of approximately equal binding of these two dyes depended upon previous fixation. After fixation in solutions of salts of heavy metals, the cells had much less affinity for basic dye and the p H of equal binding rose. The reverse occurred after formalin fixation, whereas fixation with ethyl alcohol yielded stain affinities of an intermediate character. Zeiger (1930) studied alcohol and formalin fixation and believed that there was less shift in the isoelectric point of tissue proteins after alcohol fixation than with other common fixatives. Yasuzumi ( 1933) studied the effect of alcohol fixation on the isoelectric point of red blood cells. An extensive series of experiments has been done with various fixatives on egg albumen and tissue sections (Seki, 1933b) and on extracted nucleoprotein (Kelley and Miller, 1935 ; Kelley, 193913).

X. THEINFLUENCE OF TEMPERATURE OF THE STAINING SOLUTIOK The temperature of the staining solution may influence staining in a number of ways. Probably the most pronounced effect is on the rate of diffusion or movement of the dye within the protein and, therefore, the rate of staining. The rate of dyeing is increased progressively with increased temperature so that equilibrium staining is reached much faster at higher temperatures than at lower ones. The notable effect of temperature on equilibrium staining is illustrated well in the example which Vickerstaff (1950) gives for the dyeing of wool fiber. Five months at 20°C would be required for equilibrium dyeing of wool which can be dyed in 1 hour at 100°C. This effect of temperature on the diffusivity of the dye has been treated quantitatively by determining the activation energy of dyeing (Vickerstaff, 1950). Dyes of poor diffusivity are affected most by temperature changes (Abbot, Crook, and Townend, 1947). Temperature of the dye bath is believed to have other influences besides alteration in the rate of diffusion of the dye. It may influence the affinity of the dye-protein interactants since the amount of dye bound at equilibrium decreases with increasing temperature (Boulton, Delph, Fothergill, Morton, 1933 ; Neale, 1933 ; Vickerstaff, 1950). At low temperatures equilibrium may be so slow in attainment that the erroneous impression is thereby given that less dye is bound at low temperatures (Vickerstaff,

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1950). Steinhardt ( 1940) and Steinhardt, Fugitt, and Harris ( 1940b, c, 1912) studied the effect of three temperatures (0,25, and 50°C) on the affinity of various anions and wool. They observed that the p H association curves of these anions showed different degrees of sensitivity to temperature change. Larger, more tightly bound anions have greater heats of dissociation, and thus their affinity was altered less by increase in temperature than smaller more readily dissociated anions. Altered temperature may be expected to produce essential responses and chemical changes in the protein itself (Elod, 1933). Heating may cause a swelling of the fiber due to loosening of the micellar structure as has been reported by Speakman and Smith (1936) for wool dyeing. Penetration of the protein by the color ion is thereby facilitated. The effect would be particularly pronounced for large dye ions or for colloidal aggregates (Gerstner, 1949). The separation of the micellar sheets can occur by loosening of the covalent bonds between them but also by destruction of the disulfide linkages. At elevated temperature there may also be a decomposition of protein (see previous discussion of heat modification of fibrin). In the case of colloidally dispersed dyes, the effect of temperature is very marked since swelling of the protein which results from increased temperature allows greater penetration of dye into the protein. Moreover, the dye is more finely dispersed at higher temperature and aggregation tendencies are diminished (Speakman and Smith, 1936; Goodall, 1938, 1947). For most successful dyeing, dyes of low dispersivity require higher temperatures. This information explains the importance in tissue staining of elevated temperatures with colloidal . solutions of dye (for example, azocarmine in triple acid staining techniques). Other works of interest for further references and description of temperature effects in dyeing are those of Brown (1901a, b), Sheppard, Houck, and Ditmar (1942) ; and Royer, Zimmerman, Walter and Robinson (1948). The latter workers described the effect of extremely elevated temperatures (200"and 300°F) on dye uptake of textiles. In the histological literature there is relatively little other than empirical studies on temperature effects on staining. Some references may be had in the work of Ochs (1928), who also described temperature alterations of the staining of blood cells and gelatin.

XI. SOMEOBSERVATIONS ON THE KINETICS OF STAINING In preceding pages attention was focused primarily on the forces which bind molecularly dispersed dye to solid protein and on the various factors

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which influence these forces or otherwise alter the dyeing process. Another important aspect of the staining process concerns forces involved in the movement of dye ions to the surface of the protein and their distribution to the binding sites. Histological staining is invariably done in concentrated solutions with a great excess of dye so that there is essentially no decrease in concentration as staining proceeds. Consequently, diffusion of dye ions to the protein surfaces must be extremely rapid and the movement must continue at a high level particularly when the solution or tissue is agitated during staining. Moreover, coulombic forces acting between dye and protein would serve to hasten the movement. Under conditions where the dye bath is gradually exhausted during the course of dyeing as is done in textile coloring, the problem of diffusion of dye in solution to the surface of the fiber is of greater import. Diffusion of the dye ion from the surface of the protein into the internal meshwork and thence into the intermolecular spaces of tissue structures to more deeply placed dyeing sites, constitutes, however, an important factor which evidently exerts a profound control on the rate of staining. An early and particularly lucid discussion of diffusion factors is given by Pappenheim (1901 ) . The importance of factors of diffusion in staining was stressed particularly in early works which supported the physical mechanism of staining (Gierke, 1885 ; Fischer, 1899; Knoevenagel, 1911 ; Pappenheim, 1917; von Mollendorff, 1923; von Mollendorff and Krebs, 1923; von Mollendorff and von Mollendorff, 1924; excellently reviewed by Zeiger, 1938). The importance of dye particle size and pore size in the protein was speculated upon as a mechanism of staining (e.g., the “Durchtrankungsfarbung” of von Mollendorff, 1923). Problems of diffusion of dye to the binding sites are also of importance in textile dyeing since the fiber and fibril sizes to be penetrated by the dye ion are large and the micellar network dense (Vickerstaff, 1949, 1950). Once the dye reaches the surface of the tissue section it is free to react with sites available at that position. Penetration of other dye ions to deeper staining sites must occur through this layer in which staining forces are already at least partly satisfied. Since the interaction of the dye with the surface sites is probably immediate, the rate of staining is determined in large part by the time for the dye ions to reach more central regions of the protein (Vickerstaff, 1950). The movement of dye ion to deeply placed sites is much slower than to the surface because of mechanical obstruction of the protein micellae and because of other forces acting between dye and protein and, indeed, between bound and free dye. The protein structure may provide an effective barrier to some dye ions and not others. Large dye ions may

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penetrate with difficulty or not at all and dye aggregates may penetrate the interstices of certain meshworks but be excluded from others. The speed of penetration may vary from one region of the tissue to another according to the density of the charge in these regions. It may change as dyeing proceeds because of the gradual alteration of charge on the protein with staining. The ease of diffusion may also be affected by dye ions which are already bound according to conditions described in a preceding section on the affinity of dye. For example, dye ions with residual unsatisfied charges would repel oncoming ions. Such a repulsion would be particularly effective in influencing the rate of staining if the first dye reaction occurs at or near the surface of the tissue section. The rate of dyeing may follow closely the rate of diffusion of the dye ion into the protein but also may depend upon the affinity of the dye for the protein and upon the various conditions of staining such as pH, temperature, ionic strength, and dye concentration of the dye bath. The physical state of the protein is important not only as it may interfere with movement of dye but also in other ways. Degree of orientation of the fiber has been shown to affect greatly the degree of dye binding by cotton (Preston and Pal, 1947). This effect has also been described for nylon since dyeing may be enhanced by “relaxing” the fiber with heat treatment (Fidell, Royer, and Millson, 1948). Thus, highly oriented fibers show less affinity for dye than less oriented ones. XII. THE REVERSIBILITY OF STAINING REACTIONS ; EQUILIBRIUM OF STAINING A N D OTHER FACTORS WHICH INFLUENCE STAINING It is important to stress that staining is a reversible reaction and that when the solution environment of tissue sections is changed, there is a corresponding alteration in the equilibrium concentration of dye within the tissue. Dye may then be lost to the solution or removed from it. It is possible to wash out the stain in a solution free of dye particularly if the p H is adjusted upward in the case of acid dyes or downward in the case of basic ones-pH regions which would favor dissociation of the dye-protein combination. Although the reaction may be reversed and dye washed from the stained protein structure, the extent and rate of “desorption” varies with the histological structure, with the dye and with the washing conditions. Washing from tissue in which the protein is densely packed is conceivably more difficult than where the protein is more dispersed particularly when the dye has aggregated upon binding. And, dye of great affinity for a particular protein will show greater fastness than another dye and is removed only

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with difficulty (Gerstner, 1949). Conditions of p H which do not favor binding of acid or basic dye are most favorable for stripping the dye from the protein. As already mentioned, elevated p H is most conducive for decolorizing acid dyes and low pH basic ones. The effect of acidity or alkalinity of the washing medium on destaining is considered in the early review of Pappenheim (1901) and the more recent work of Stearn and Stearn (1928a, b). Reversibility of the staining reaction is important for another reason, namely the redistribution of dye so that final staining is relatively even in a given protein structure despite rapid staining or destaining procedures which might be expected to favor uneven localization of the dye. In the redistribution, dye ions are “desorbed” from one site and transferred to another more deeply placed one. The ease of desorption and transference determines the final uniformity of the distribution. I t is known in textile staining that the migrating or leveling power of dyes differs greatly. The leveling capacity of dyes influences the course of the reaction. Dye which satisfies surface sites without tending to shift to deeper regions delays or prevents the expression of full staining capacity of the protein. Such staining stands in contrast to that with dye which rapidly migrates and distributes itself uniformly as staining proceeds. I n the former case dyeing is uneven and slow in reaching equilibrium. Various procedures of gradual alteration in dyeing conditions are used in the textile industry to improve and hasten the leveling of dye. Of interest among these is that leveling is favored by p H regions at which the charge of the protein is not extreme. There are still other factors which operate in the staining reaction. Most of these are poorly understood and, therefore, are only briefly considered here. If the dyeing time is lengthy and the temperature is elevated, decomposition of the protein may set in. In some instances the dye has been said to have a catalytic effect on degradation of the protein (Lemin and Vickerstaff, 1947). Ionic exchange has been described for the staining of ligno-cellulose with methylene blue (Sarkar and Chatterjee, 1948) and may operate more widely. The problems of staining with dye aggregates or suspensions which are widely used in histology (for example, Congo red, azocarmine, and trypan blue) have barely been touched upon in this review. The profound influence of mordants, media other than water, the effect of specific ions and the competition between dye ions of mixtures for similar sites deserve special study. Finally, the influence of brief staining times on dyeing must be touched upon. In progressive staining low dye concentrations are employed and

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staining is allowed to proceed to equilibrium. Staining is more easily controlled under such circumstances where the time of staining is not a variable. If staining is not carried to equilibrium, then the time of immersion in the dye solution is an important factor in dye uptake. Short staining times at elevated dye concentrations are quite popular in many histological techniques. Since the rate of staining is greatest during the first few minutes of reaction, and levels off slowly thereafter, tissues may be effectively stained during brief immersion. The tissue is removed when the desired intensity of staining is reached or the tissue is overstained and then secondarily destained. The amount of dye taken up in a given time will depend on a number of factors, including the mobility of the dye ion in the tissue substrate, the speed of interaction, the leveling capacity, the affinity of the dye, and the conditions of staining. Consequently, the time of dyeing must vary with each dye. In order to compensate for individual differences in dyes the concentration of the dye may be changed or other conditions of staining varied. XIII. REFERENCES Abbot, E. B., Crook, H., and Townend, F. (1947) J . SOC.D y . Col., Bradford, 63. 462. Achard, J. (1935) 2. Zellforsch., 23, 573. Arner. Dyestuff Reporter. Proc. of Airtrr. Asso. Textile Chem. Col. (1918) drncr. Dyestuff Rep., 37, 149. Anson, M. L. (1946) Protein denaturation and the properties of protein groups. Advances in Protein Chem., 2. Atkin, R. W., and Douglas, F. W. (1924) J. Airzer. Leather Chem. Asso., 19, 528. Baker, J. R. (1945) Cytological Technique. Methuen Monog., London. Bancroft, W. D. (1914a) J. phys. Chem., l8, 1. Bancroft, W.D. (1914b) J . phys. Chem., l8, 118. Bancroft, W.D. (1914~) J. phys. Chem., 18, 385. Bancroft, W.D. (1915a) 1. phys. Chem., 19, 50. Bancroft, W.D. (1915b) J . phys. Chem., 19, 145. Bejdl, W. (1950) Mikroskopie, 6, 83. Bethe, A. (1905) Beitr. Chem. Physiol. Pathol., 6, 399. Bonin, W.,Frappier, J., and LararnCe, A. (1944) Rev. Canad. BioJ., C. R., 3. 481. Boulton, J., Delph, A. E., Fothergill, F., and Morton, T. H. (1933) J. Textile Inst., 24, 113. Bourne, G. (1951) Cytology and Cell Physiology, 2nd ed. Clarendon Press, Oxford. Briggs, T. R., and Bull, A. W. (1922) J. phys. Chem., 26, 844. Brown, R. B. (1901a) J. SOC.Dy. Col., Bradford, 17, 92. Brown, R. B. (1901b) J . SOC.Dy. Col., Bradford, 17, 125. Carlene, P. W., Fern, A. S., and Vickerstaff, T. (1947) J . SOC.D y . Col., Bradford, 0S, 388. Chapman, L. M., Greenberg, D. M., and Schmidt, C. L. A. (1927) J . biol. Chem., 72, 707.

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Cohn, E. J. (1945) Blood and Blood Derivatives. Smithsonian Rep., Smithsonian Inst., Wash., p. 413. Cohn, E. J., and Edsall, J. T. (1943) Proteins, Amino Acids and Peptides. Reinhold Co., New York. Conn, H. J. (1940) Biological Stains. Humphrey Press Inc., Geneva, New York. Conn, H. J., and Holmes, W. C. (1928) Stain Tech., S, 94. Craig, R., and Wilson, C. (1937) Stain Tech., Za, 99. Dempsey, E. W., Bunting, H., Singer, M., and Wislocki, G. B. (1947) Anat. Rec., 98, 417. Dempsey, E. W., and Singer, M. (1946) Endocrin., 38, 270. Dempsey, E. W., Singer, M., and Wislocki, G. B. (1950) Stain Tech., 26, 73. Dempsey, E. W., and Wislocki, G. B. (1946) Physiol. Rev., 26, 1. Dempsey, E. W., Wislocki, G. B., and Singer, M. (1946) Anat. Rec. 96, 221. Dreaper, W. P. (1906) The Chemistry and Physics of Dyeing. London. Dubos, R. J. (1945) The Bacterial Cell. Harvard University Press, Cambridge, Massachusetts. Ehrlich, P. (1879a) Arch. Physiot., 166. Ehrlich, P. (1879b) Arch. Physiol., 571. Elod, E. (1933) Trans. Faraday SOC.,29, 327. Ender, W., and Miller, A. (1937) Melliand TextilDer., 18, 633. Fautrez, J. (1936) Bull. Histol. Appl., l8, 202. Ferry, J. D., and Morrison, P. R. (1946) Znd. Eng. Chem., 38, 1217. Ferry, J. D., and Morrison, P. R. (1947) J. Amer. Chem. SOC.,69, 400. Ferry, J. D., Singer, M., Morrison, P. R., Porsche, J. D., and Kutz, R. L. (1947) 1. Amer. Chem. Soc., 69, 409 Fidell, L. I., Royer, G. L., and Millson, H. E. (1948) Amer. Dyestuf Rep., 37, 166. Fierz-David, H. E., and Blangey, L. (1949) Fundamental Processes of Dye Chemistry. Interscience, New York. Fischer, A. (1899) Fixierung, Farbung, Bau des Protoplasmas. Jena. Fraenkel-Conrat, H. (1944) J. biol. Chem., 164, 227. Fraenkel-Conrat, H., and Cooper, M. (1944) J. biol. Chenz., 164, 237. French, R. W. (1930) Stain Tech., 6, 87. Gatenby, J. B., and Beams, H. W. (1950) The Microtomist's Vade-Mecum (Bolles Lee), Blakiston, Philadelphia. Gee, W. W. H., and Harrison, W. (1910) Trans. Faraday SOC.,6, 42. Gelmo, P., and Suida, W. (1905) Sitz. Akad. Wiss. Wien. Jan. Math.-nat. Kt. 114, Quoted from Pelet-Jolivet, 1910. Gerstner, H. (1949) Melliand Textilber., 30, 253; 302. Gierke, H. (1885) 2. m'ss-Mikr., 2, 13, 164. Gilbert, G. A., and Rideal, E. K. (1944) Proc. roy. SOC.,8182, 335. Gillet, C. (1889) Rev. gen. mat. cot., pp. 15, 189. Gillet, C. (1890) Rev. gen. mat. col., p. 339. Goldstein, A. (1949) 1. Pherm. exp. Therap., Pt. 11, 96, 102. Goodall, F. L. (1938) J. SOC.Dy. Col., Bradford, 64, 45 Goodall, F. L. (1947) Am. Dyestuf Rep., 36, 380. Grollman, A. (1925) J. biol. Chem., 64, 141. Halphen, G., and Riche, A. (1904) C. R. SOC.Biol., 140, 1408. Halphen, G., and Riche, A. (1905) Rev. gen. mat. cot., p. 200.

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