International Review of Cytology, Volume 10

INTERNATIONAL

REVIEW OF CYTOLOGY VOLUME10

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INTERNATIONAL

Review of Cytology EDITED BY

G. H. BOURNE

J. F. DANIELLI

Department of.Anatomy Emory University Atlanta, Georgia

Department of Zoology King's College London, England

VOLUME 10

Prepared Under the Auspices of The International Society for Cell Biology

ACADEMIC PRESS, New York and London 1960

Copyright 0, 1960,

by

ACADEMIC PRESS INC. All Rights Reserved NO PART OF THIS BOOK MAY BE REPRODUCED I N ANY FORM, BY PHOTOSTAT, MICROFILM, OR ANY OTHER MEANS, WITHOUT WRITTEN PERMISSION FROM THE PUBLISHERS.

ACADEMIC PRESS INC. 111 FIFTHAVENUE NEW YORK3, N. Y . United Kingdom Edition Published by ACADEMIC PRESS INC. (LONDON) LTD. 17 OLDQUEEN STREET, LONDON S.W. 1

Library of Cortgress Catalog Card Nurnbcr 52-5203

PRINTED I N THE UNITED STATES OF AMERICA

Contributors to Volume 10 F. DOLJANSKI, Departwent of Experimental Medicine and Cancer Research, Hebrew University, Jerusalem, Israel FREDERICK H. KASTEN,Department of Biology, Agricultural and Mechanical College of Texas, College Station, Texas

A. GEDEON MATOLTSY, Department of Dermatology, University of Miami, School of Medicine, Miami, Florida YOSHIMINAGATANI, Biological Institute, Faculty of Literature and Science, Yamaguchi University, Yamaguchi, Japan ARCHANASHARMA,Cytogenetics Laboratory, Department of Botany, Calcutta University, Calcutta, Indict ARUNKUMARSHARMA, Cytogenetics Laboratory, Department of Botany, Calcutta University, Calcutta, India SAULWISCHNITZER, Department of Anatomy, N e w York Medical College, Flower and Fifth Avenue Hospitals, N e w York, N e w York LEWISWOLPERT, Zoology Department, Kings College, London, England

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CONTENTS

...............................................

v

CONTENTSOF PREVIOUS VOLUMES..........................................

xi

TO VOLUME 10 CONTRIBUTORS

The Chemistry of Schiff’s Reagent

FREDERICK H . KASTEX

. . . .

I I1 I11 IV V. VI VII VIII IX . X XI XI1

.

. .

. . .

Introduction ...................................................... Historical Background ............................................ Methods of Preparing Reagent .................................... Factors Affecting Sensitivity of Reagent .......................... Specificity of Reagent ............................................ Chemical Nature of Schiff’s Reagent .............................. Reaction with Aldehydes .......................................... Development of Schiff -Type Reagents ............................. Kinetics of the Schiff-Aldehyde Reaction .......................... Applications of the Reagent to Cytochemistry ...................... Absorption Curve Analyses of Schiff-Polyaldehyde Binding in Situ Conclusions ...................................................... Addendum ....................................................... References .......................................................

1 2

4 11 22

34 37

46

..

56 65 81 89 91 93

Spontaneous and Chemically Induced Chromosome Breaks ARUNKUMARSHARMA A N D ARCHANA

.

I I1. I11 IV V VI VII . VIII IX X.

. . . .

. .

SHARMA

Introduction ...................................................... Spontaneous Breakage ............................................ Technical Limitations in the Study of Chromosome Breaks ........ Action of Alkaloids. Pigments. and Coumarin Derivatives Action of Vegetable Oils. Fats. and Essences ...................... Action of Drugs and Bacterial Products .......................... Action of Hormones and Other Growth-Promoting Substances Action of Mustards. Related Compounds. and Phenols .............. Other Compounds ................................................ Conclusion ....................................................... References .......................................................

.......... ....

101 102 107 110 112 112 113 116 119 122 123

T h e Ultrastructure of the Nucleus and Nucleocytoplaemic Relations

SAUL WISCHNITZER I. I1 I11 IV . V. VI VII VIII.

. .

. .

Introduction ...................................................... Nomenclature .................................................... Nuclear Envelope ................................................ Nucleolus ........................................................ Chromosomes .................................................... Nucleoplasm ...................................................... Discussion ....................................................... Conclusion ........................................................ References ........................................................ Addendum .......................................................

137 138 139 142 143 148 148 158 158 162

The Mechanics and Mechanism of Cleavage

LEWISWOLPERT

. . . . . . .

Introduction ...................................................... Geometry of Cleavage ............................................ Theories of Cleavage ............................................ Mechanical Properties of the Cell Surface ........................ Discussion of Surface Force Theories ............................ Astral Relaxation Theory ........................................ Application of Astral Relaxation Theory to Other Cells and Forms of Cleavage .................................................... VIII. Biochemistry of Cleavage .......................................... I X. Summary and Conclusions ........................................ References ....................................................... I I1 I11 IV V VI VII

164 165 169 175 183 186 195 205 211 213

The Growth of the Liver with Special Reference to Mammals

F. DOLJANSKI I. Introduction ...................................................... I1. Development of the Liver during Embryonic and Postnatal Growth of the Organ ...................................................... I11. Liver Growth Response to Various Conditions in the Body .......... I V Liver Regeneration after Partial Hepatectomy ...................... V Concluding Remarks and Summary ................................ References ........................................................

. .

217 218 234 236 236 239

Cytological Studies on the Affinity of the Carcinogenic Azo Dyes for Cytoplasmic Componentn YOSHIMINAGATANI

.

Introduction ...................................................... Methods for Demonstrating the Cellular Affinity of Carcinogenic Azo Dyes for Cytoplasmic Components .......................... I11 Affinity of the Carcinogens for Cytoplasmic Components of Amphibian Erythrocytes .................................................... IV Mitochondria of Mammalian Differentiated Erythrocytes .......... V Affinity of Carcinogens for Amphibian Somatic Cells ............... V I Conclusion ........................................................ References ........................................................

I I1

243

. .

. . .

244 246 255 257 305 306

Epidermal Cells in Culture A . GEDEON MATOLTSY

I. I1. I11. I V. V.

Introduction ...................................................... Properties of Epidermal Cells hi V i m ............................ Properties of Embryonic Epidermal Cells in Vitro .................. Properties of Postnatal Epidermal Cells in Vitro .................. Remarks on Keratinization ........................................ References ........................................................

315 317 323 331

344 348

AUTHORINDEX...........................................................

353

SUBJECT INDEX..........................................................

368

..................................

377

CUMULATIVE SUBJECT INDEX. VOLUMES 1-9

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Contents of Previous Volumes Aspects of Bacteria as Cells and as Volume 1 Organisms - STUART MUDDAND Some Historical Features in Cell EDWARD D. DELAMATER Biology -ARTHURHUGHES Nuclear Reproduction- C. LEONARD Ion Secretion in Plants- J. F. SUTCLIFFE Hus~ms Enzymic Capacities and Their Rela- Multienzyme Sequences in Soluble Extracts -HENRYR. MAHLER tion to Cell Nutrition in Animals The Nature and Specificity of the GEORGEW. KIDDER Feulgen Nucleal Reaction--. A. The Application of Freezing and DryLESSLER ing Techniques in Cytology-L. G. Quantitative Histochemistry of PhosE. BELL phatases -WILLIAML. DOYLE Enzymatic Processes in Cell Membrane Penetration -TH. ROSENBERCAlkaline Phosphataee of the Nucleus M. CH~VREMONT A N D H. FIRKET A K D W. WILBRANDT Gustatory and Olfactory Epithelia Bacterial Cytology - K. A. BISSET A. F. BARADI AND G. H. BOURNE Protoplast Surface Enzymes and Absorption of Sugar-R. BROWN Growth and Differentiation of ExReproduction of Bacteriophage -A. planted T i m e s - P. J. GAILLARD D. HERSHEY Electron Microscopy of Tissue SecThe Folding and Unfolding of Protions-A. J. DALTON tein Molecules as a Basis of Os- A Redox Pump for the Biological motic Work-R. J. GOLDACRE Performance of Osmotic Work, and Nucleo-Cytoplasmic Relations in AmIts Relation to the Kinetics of Free phibian Development -G. FAKK- Ion Diffusion Across Membranes HAUSER E. J. CONWAY Structural Agents in Mitosis--. M. A Critical Survey of Current ApSWANK proaches in Quantitative Histo- and Factors Which Control the Staining Cytochemistry DAVIDGLICK of Tissue Sections with Acid and Nucleo-Cytoplasmic Relationships in Basic Dyes -MARCUSSINGER the Development of Acetabularia. The Behavior of Spermatozoa in the Report of the Conference of Tiwue Neighborhood of Eggs -LORD Culture Workers Held at CoopersROTHSCHILD town, New York- J. HXMMERLING The Cytology of Mammalian Epider- AUTHOR INDEX-SUBJECT INDEX. mis and Sebaceous Glands -WILLIAM MONTAGNA Volume 3 The Electron-Microscopic InvestigaThe Nutrition of Animal Celh tion of Tissue Sections-L. H. CHARITYWAYMOUTH BRETSCHNEIDER Caryometric Studies of Tissue CulThe Histochemistry of Esterases - G. t u r e ~ OTTU BUCHER GOMORI The Properties of Urethan ConaidAUTHOR INDEX-SUBJECT INDEX. ered in Relation to Its Action on Volume 2 Mitosis -IVOR CORNMAN Quantitative Aspects of Nuclear Nu- Composition and Structure of Giant Chromosomes -MAX ALFERT cleoproteins - HEWSONSWIFT Ascorbic Acid and Its Intracellular How Many Chromosomes in MamLocalization, with Special Refermalian Somatic Cells?-R. A. ence to Plants- J. CHAYEN BEATTY

-

-

xi

xii

CONTENTS O F PREVIOUS V O L U M E S

The Significance of Enzyme Studies Volume 5 on Isolated Cell Nuclei - ALEXAN- Histochemistry with Labeled AntiDER L. DOUNCE ALBERT H. COONS The Use of Differential Centrifuga- The Chemical Composition of the tion in the Study of Tissue EnBacterial Cell Wall-C. S. CUMzymes-CHR. DE DUVEAND J. MINS BERTHET Theories of Enzyme Adaptation in Enzymatic Aspects of Embryonic Microorganisms -J. MANDELSTAM Differentiation -TRYGGVE The Cytochondria of Cardiac and GUSTAFSON Skeletal Muscle- JOHN W. HARAzo Dye Methods in Enzyme HistoMAN chemistry -A. G. EVERSON PEARSEThe Mitochondria of the Neuron Microscopic Studies in Living MamWARREN ANDREW mals with Transparent Chamber The Results of Cytophotometry in Methods -ROY G. WILLIAMS the Study of the Deoxyribonucleic The Mast Cell - G. ASBOE-HANSEN Acid (DNA) Content of the NuElastic Tissue -EDWARD W. DEMPSEY cleus--. VENDRELY AND C. VENAND ALBERTI. LANSING DRELY The Composition of the Nerve Cell Protoplaamic Contractility in Relation Studied with New Methods- SVEN- to Gel Structure: TemperatureOLOFBRATKARDAND HOLCER HYDEN Pressure Experiments on CytokineAUTHOR INDEX-SUBJECT INDEX. ds and Amoeboid Movement DOUGLAS MARSLAND Volume 4 Intracellular pH - PETER C. CALDWELL Cytochemical Micrurgy - M. J. KOPAC The Activity of Enzymes in MetaboAmoebocytea -L. E. WAGGE lism and Transport in the Red Cell Problems of Fixation in Cytology, T. A. J. PRANKERD Histology, and Histochemistry Uptake and Transfer of MacromoleM. WOLMAN cules by Cells with Special ReferBacterial Cytology -ALFRED MARence to Growth and Development SHAK A. M. SCHECATMAN Histochemistry of Bacteria -R. Cell Secretion; A Study of Pancreas VENDRELY and Salivary Glands - L. C. U. Recent Studies on Plant MitochonJUNQUEIRA AND G. C. HIRSCA dtia-DAVID P. HACKETT The Acrosome Reaction- JEAN C. The Structure of Chloroplasts -K. DAN MUHLETH ALER Cytology of Spermatogenesis Histochemistry of Nucleic Acids -N. VISHWA NATH B. KURNICK The Ultrastructure of Cells as ReStructure and Chemistry of Nucleoli vealed by the Electron Microscope W. S. VINCENT FRITIOFS. SJOSTRAND O n Goblet Cells, Especially of the Intestine of Some Mammalian Species AUTHOR INDEX-SUBJECT INDEX. HARALD MOE Volume 6 Localization of Cholinesterases at The Antigen System of Paramecium Neuromuscular Junctions -R. aurelia-G. H. BEALE COUTEAUX Evidence f o r a Redox Pump in the The Chromosome Cytology of the Ascites Tumors of Rats, with SpeJ. Active Transport of Cations--. cial Reference to the Concept of the CONWAY Stemline Cell - SAJIRO MAKINO AUTHOR INDEX-SUBJECT INDEX.

CONTENTS OF PREVIOUS VOLUMES

...

s111

The Structure of the Golgi Apparatus Hypothalamo-neurohypophysial NeuARTHURW. POLLISTER AND PRISCILLA rosecretion- J. C. SLOPER F. POLLISTER Cell Contact - PAULWEISS An Analysis of the Process of Fertili- The Ergastoplasm: Its History, U1zation and Activation of the Egg trastructure, and Biochemistry A. MONROY FRAN~OISE HACUENAU The Role of the Electron Microscope Anatomy of Kidney Tubules in Virus Research-ROBLEY C. JOHANNES RHODIN WILLIAMS Structure and Innervation of the InThe Histochemistry of Polysacchaner Ear Sensory Epithelia - HANS rides - ARTHURJ. HALE ENCSTROM AND JAN WERSALL The Dynamic Cytology of the Thy- The Isolation of Living Cells from roid Gland- J. GROSS Animal Tissues--. M. J. RINALRecent Histochemical Results of DIN1 Studies on Embryos of Some Birds AUTHOR INDEX-SUBJECT INDEX. and Mammals - ELIOBORCHESE Carbohydrate Metabolism and Em- Volume 8 bryonic Determination -R. J. The Structure of Cytoplasm O’CONNOR CHARLESOBERLINC Enzymatic and Metabolic Studies on Wall Organization in Plant Cells Isolated Nuclei - G. SIEBERT AND R. D. PRESTON R. M. S. SMELLIE Recent Approaches to the Cytochemi- Submicroscopic Morphology of the Synapse - EDUARDO DE ROBERTIS cal Study of Mammalian Tissues GEORGEH. HOGEBOOM, EDWARD L. The Cell Surface of Paramecium AND E. L. POWERS KUFF,AND WALTER C. SCHNEIDER C. F. EHRET The Kinetics of the Penetration of The Mammalian Reticulocyte- LEAH MIRIAM LOWENSTEIN Nonelectrolytes into the Mammalian The Physiology of Chromatophores BOWYER Erythrocyte - FREDA MILTON FINCERMAN AUTHOR INDEX-SUBJECT INDEX. CUMULATIVE SUBJECT INDEX The Fibrous Components of Connec(VOLUMES 1-5). tive Tissue with Special Reference to the Elastic F i b e r - D A ~ D A. Volume 7 HALL Some Biological Aspects of Experi- Experimental Heterotopic Osdfication- J. B. BRIDGES mental Radiology: A Historical ReA Survey of Metabolic Studies on Isoview--. G. SPEAR lated Mammalian Nuclei-D. B. The Effect of Carcinogens, HorRWDY N mones, and Vitamins on Organ Cultures -ILSE LASNITZKI Trace Elements in Cellular Function BERTL. VALLEEAND FREDERIC L. Recent Advances in the Study of HWH Kinetochore -A. LIMA-DE-FARIA Autoradiographic Studies with S35- Osmotic Properties of Living Cells D. A. T. DICK Sulfate -D. D. DZIEWIATKOWSKI The Structure of Mammalian Sperma- Sodium and Potassium Movements in Nerve, Muscle, and Red Cells-I. tozoon -DON W. FAWCETT M. GLYNN The Lymphocyte-0. A. TROWELL The Structure and Innervation of Pmocytosis - H. HOLTER Lamellibranch Muscle -J. BOWDEN AUTHOR INDEX-SUBJECT INDEX.

xiv

CONTENTS OF PREVIOUS VOLUMES

Volume 9 The Influence of Cultural Conditions on Bacterial Cytology- J. F. WILKINSON AND J. P. DUGUID Organizational Patterns Within Chromosomes- BERWINDP. KAUFMANN, HELENGAY, AND MARGARETR. McDONALD Enzymic Processes in Cells- JAY BOYDBEST The Adhesion of Cells-Lm~Am WEISS

Physiological and Pathological Changes in Mitochondrial Morphology - CH. ROUILLER The Study of Drug Effects at the Cytological Level -G. B. WILSON Histochemistry of Lipids in Oogenesis -VISHWANATH Cyto-Embryology of Echinoderm and Amphibia -KATSUMADAN The Cytochemistry of Non-Enzyme R. COWDEN Proteins-RONALD AUTHOR INDEX-SUBJECT

INDEX.

The Chemistry of Schiff's Reagent FREDERICK H. KASTEN Department of Biology. dgricirlhral arid Mechanical College of Texas. College Station. Texas Page I . Introduction ....................................................... 1 I1. Historical Background ........................................... 2 I11. Methods of Preparing Reagent ..................................... 4 A . Sources of Sulfur Dioxide ..................................... 4 B. Use of Strong Acids in Reagent ............................... 5 C. Stability of Reagent .......................................... 10 I V. Factors Affecting Sensitivity of Reagent ........................... 11 A,. Basic Fuchsin and Impurities ................................. 11 B. Treatment of Reagent with Charcoal ......................... 15 C. Influence of Acids and Salts. ................................. 16 D. S&r Dioxide Concentration ................................. 19 E. Influence of Heat ............................................ 21 V . Specificity of Reagent ............................................ 22 V I . Chemical Nature of Schiffs Reagent ............................. 34 VII . Reaction with Aldehydes ......................................... 37 A . Condensation Theory ......................................... 37 B . Abnormal Schiff Reactions with Certain Aromatic A.ldehydes ... 38 C. Alkyl-Sulfonic Acid Theory .................................. 39 D. Wieland and Scheuing Theory ................................ 42 VIII . Development of Schiff-Type Reagents ............................. 46 IX: Kinetics of the Schiff-Aldehyde Reaction ........................... 56. 56 A . Studies in Vitro ............................................. B. Studies of Stained Tissues ................................... 62 X . Applications of the Reagent to Cytochemistry ....................... 65 A . Use in Feulgen Reaction ..................................... 65 B. Use in Plasma1 Reaction ..................................... 71 C. Use in the Bauer and Casella Oxidizing Techniques ............ 73 D . Use in the Periodic Acid-Schiff Reaction ...................... 74 E. Use in Performic Acid- and Peracetic Acid-Schiff Techniques . . 76 F. Use in Detection of Proteins ................................. 77 G. Use in Direct Staining of Lipids ............................... 78 H. Recent Use in Multiple-Staining Reactions .................... 78 X I . Absorption Curve Analyses of Schiff -Polyaldehyde Binding in Sittr 81 XI1. Conclusions ...................................................... 89 Addendum ........................................................ 91 Acknowledgments ................................................. 93 93 References ........................................................

I . Introduction Schiff's reagent has played a major role in the development of cytochemical techniques used in the study of deoxyribonucleic acid (DNA) and polysaccharides. Other methods of detecting lipid aldehydes and pro1

2

FREDERICK €I. KASTEN

teins also utilize this reagent. The widespread use of Schiff’s reagent in cytochemistry has heightened the need for further information concerning reaction mechanisms. A more complete understanding of the basic principles underlying any cytochemical reaction is of paramount importance, especially when quantitative applications are made. The complexities of the Feulgen reaction are not adequately understood even though vigorous studies were carried out about 35 years ago. Past review articles and recent histochemistry texts have summarized the Feulgen reaction and the application of the technique to biological problems (Swift, 1953 ; Lessler, 1953; Swift 1955 ; Kurnick 1955 ; Leuchtenberger, 1958 ; Casselman, 1959 ; Walker and Richards, 1959 ; Pearse, 1960). The histochemistry of polysaccharides was recently reviewed (Hale, 1957). This article will attempt to provide a fuller account of Schiff’s reagent from the standpoint of historical backgrdbnd and theories of reactions with aldehydes. Other major topics will include applications in cytochemistry, development of Schiff-type reagents, and absorption curve analyses of stained cells. Cytochemical techniques which make use of Schiff’s reagent will not be considered in detail here since the emphasis will be on those phases of the reaction that directly involve the reagent and polyaldehyde moieties. A complete review of the Feulgen reaction is in preparation (Kasten, 1960~).

11. Historical Background Before his death in 1915 at the age of 81, Hugo Schiff had written about 250 scientific papers which had appeared in French, German, and Italian chemical journals. Born in Frankfurt am Main, he obtained a Ph.D. in chemistry at the age of 24 from the University of Turin. H e was associated during his scientific career with the Institute of Higher Studies at Florence, Italy. Here he had the position of Professor of Chemistry and became Director of the Institute. In addition to articles published in scientific journals, he wrote three books and contributed to the Italian Encyclopedia of Chemistry. Schiff’s contributions to organic chemistry embraced several areas. Any detailed discussion of his work would go beyond the boundaries of this article. However, it is significant that he carried out extensive research involving reactions of aromatic amines with aldehydes; known today as Schiff-base reactions (Schiff, 1865a, b). One of the amines he studied was the triphenylmethane basic dye, rosaniline, also known as fuchsin. The name fuchsin was given to the dye because of its fuchsia or rose color, but magenta was a name also used, in honor of the victory of Napoleon I11 in the Battle of Magenta in 1859. In 1 8 6 1 8 6 7 Schi< reported that a

THE CHEMISTRY OF SCHIFF'S REAGENT

3

red-violet color returned to the dye solution, decolored by SOZ, upon addition of a few drops of aldehyde. His interpretation of the mechanism of formation of the re-formed dye was that there was a direct combination of dye and aldehyde, regardless of the presence or absence of HzS03 in the reacting solution. This interpretation is now known to be incorrect, although there is still considerable debate about the true mechanism of reaction between the SO2-treated dye solution and aldehydes. In subsequent discussion, the term Schiff's reagent or fuchsin sulfurous acid (FSA) refers to the colorless derivative produced by the action of sulfur dioxide in water on basic fuchsin or any of its component dye -.Yl'E&ies including pararosaniline, rosaniline, and new fuchsin. The terms leucofuchsin or leuco dye have also been used, but can be misleading, since other leuco derivatives may be obtained from basic fuchsin. Treatment with a r-ng agent forms a leuco dye which is easily oxidized to basic fuchsin. The ca%il base of fuchsin is obtained by treating the dye with ammonium hydroxide or other alkali. The leuco compound formed from ammonium hydroxide reacts with aldehydes to form a colored product (Wang, 1932) but this reaction is unrelated to the Schiff-aldehyde reaction. The attachment of sulfite ester, -O-SOzH, to the methane carbon of fuchsin produces a colorless compound that is nonreactive with aldehydes (Wieland and Scheuing, 1921). The early popularity of Schiff's reagent was due to a large extent to some publications by Schmidt (1880, 1881). Schiff had little to say about the reagent. Many other studies involving Schiff's reagent were reported, especially in the chemical literature. Of special importance to biologists and biochemists was the use of this reagent in cytology to detect deoxyribonucleic acid in nuclei (Feulgen and Rossenbeck, 1924). Feulgen and Voit (1924) also found a substance in the cytoplasm of cells which differed from nucleic acid but gave a positive Schiff reaction. This reaction became known as the plasma1 reaction. I n 1933, Bauer employed Schiff's reagent in conjunction with chromic acid to detect glycogen in tissue sections. Some years later the periodic acid-Schiff ( P A S ) technique was developed (McManus, 1946; Lillie, 1947a, b ; Hotchkiss, 1948) for the histochemical visualization of 1,2 glycols or aminohydroxyl compounds in tissues. For all practical purposes, polysaccharides are the usual compounds stained. Unsaturated fatty acids and keratin have been studied using the performic acid-Schiff ( P F A S ) and peracetic acid-Schiff (PAAS) reactions (Pearse, 1951). An ultraviolet Schiff method, claimed to specifically demonstrate double bonds, was published (Belt and Hayes, 1956). In recent years Schiff's reagent has been applied to the cytochemical detection of proteins after treating sections with oxidative deaminating agents such as ninhydrin, alloxan, or chloramine T (Yasuma and Ichikawa, 1953 ; Burstone, 1955).

4

FREDERICK H. KASTEN

111. Methods of Preparing Reagent

SOURCES OF SULFUR DIOXIDE The original reagent reported by Schiff was prepared by saturating an aqueous solution of fuchsin with sulfur dioxide. The literature is now replete with dozens of modifications. It is surprising that users of Schiff’s reagent have been so willing to work with solutions that contain added ingredients, since the only essential components are fuchsin and sulfurous acid. Many of these modifications are shown in Table I, listed according to sulfur dioxide source and date of. publication. Unfortunately, many of thein involve minor variations of previously published formulas. Some of the “recipes” were designed for special characteristics such as extra sensitivity, reduced sensitivity, easy preparation, etc. For strictly chemical studies, SO2 gas has -used by many workers. Although the method has not been easy to standardize, some have bubbled sufficient gas into the dye solution to add the desired number of grams of SOz,or have titrated it with iodine to obtain the desired concentration. The usual procedure has been to add SOz sufficient to saturate the solution and remove excess gas by a vacuum (Josephson, 1923). This was one of the methods used by Feulgen and Rossenbeck (1924) in their pioneering work on cytochemical detection of DNA. It has been claimed (Tobie, 1942) that Schiff’s reagent, prepared with a definite amount of SOz, 0.2 gm./100 ml. (0.03 M),is supersensitive. In Tobie’s study, the reagent gave pink colors with aldose sugars, whereas a conventional reagent gave a negative reaction. Rafalko also reported (1946) that small and diffuse chromatin elements could be detected in the Feulgen reaction by using a fuchsin solution saturated with SOz. He could not stain these same elements using the DeTomasi ( 1936) reagent, which contains sodium metabisulfite. Ris and hlirsky (1949) later showed that Rafalko’s reagent did not give as intense a Feulgen reaction in mouse liver as did the metabisulfite reagent of Coleman ( 1938). However, the comparison may not be justified, since the Coleman reagent employs carbon to remove interfering impurities. Of particular interest is some nice work reported recently by Elftman ( 1959a), who adjusted the sensitivity of his Schiff’s reagent by titrating with Lugol’s iodine solution. In this way, he obtained maximal histochemical staining of PAS-reactive material in rat pituitary. H e employed a 0.2 M solution of HzS03 in the Schiffs reagent; this was sensitive enough for mod histochemical purposes. H e found that the most sensitive histochemical reagent staining rat pituitary had a 0.02 M SO2 concentration. This agreed very well with the concentration used by Tobie (1!342), A.

THE CHEMISTRY OF SCHIFF’S REAGENT

5

which was 0.03 M. It would be interesting to use Schiff reagents of different sensitivities to determine whether maximal staining of different aldehyde elements can be achieved at the same SO2 concentration. Sodium bisulfite, NaHS03, was used as long ago as 1890 (Mohler) in combination with sulfuric acid and fuchsin solution to provide H2S03. A reagent containing these components, with HC1 in place of H2SOa, was used in cytochemistry by Feulgen and Rossenbeck (1924) and by other workers in the field. Various other sulfur compounds have been used in place of sodium bisulfite. A sensitive reagent containing sodium hydro-, NazS204, was introduced by Prud’homme (1904). A reagent with this compound and a very low amount of fuchsin was reported by Wertheim (1922) to have very desirable properties. The reagent does not require the use of carbon, boiled water, or hydrochloric acid in preparation, is cololless, will not stain the container or the hands, may be kept in clear glass bottles in the laboratory without refrigeration, and can be prepared very quickly. Except for a recent report confirming these advantages and the application to histo- and cytochemistry (Kasten and Burton, 1959), the reagent has not been used in this field. Sodium sulfite, Na2S03, was used as a source of SO2 in the reagent by a few chemists, but has been little used in histochemical applications (note Mowry, 1958). The sulfur compound that seems to be commonly used is potassium metabisulfite, K2S206. It was first used by DeTomasi ‘ (1936), who pointed out that sodium bisulfite is hard to keep anhydrous. Because sodium metabisulfite, Na2Sz06, is unstable, DeTomasi prepared a reagent with potassium metabisulfite. The reagent was popularized by Stowell ( 1945). Thionyl chloride, SOC12, was employed in another modification of Schiff’s reagent by Barger and DeLamater (1948). This compound reacts with water to produce HCl and S 0 2 . It is a convenient way to make a Schiffs reagent since one only adds a few drops of thionyl chloride to the dye solution. It is evident from Table I that there is wide variation in the amounts of the various sulfur compounds used in different Schiff reagents. The optimal concentrations for producing a sensitive solution have been studied (Atkinson, 1952 ; Longley, 1952) and will be discussed in another section. B. USEOF STRONG ACIDSIN REAGENT Almost all sulfur compounds used in Schiff reagents require some acid to generate the important component SO2. The exception is sodium hydrosulfite, which gives good results without acid (Wertheim, 1922 ; Alexander et al., 1950; Kasten and Burton, 1959). Sulfuric acid has been used in a few cases, but most techniques call for 10 to 20 meq. hydrochloric acid in

TABLE I COMPOSITION OF SCHIFF'S REAGENTS IN 100 ML. Source of SO, and references

Sulfur compound (gm.)

Basic fuchsin km.1

Schiff, 1866 Chautard, 1886

-

?

-

0.05

Francois, 1897

-

0.012

OF

SOLUTION

Acid

-

-

1.2 ml. H,SO,

Bitto, 1897 Tolman and Trescott, 1906 Chace, 1906

-

0.25

-

0.5

0.05 0.05

-

1.6

Woodman and Lyford, 1908

2.0

0.05

__

Mulliken, 1914 Josephson, 1923

-

Feulgen and Rossenbeck, 1924 Cracker, 1925 Porter, et al., 1927 Widstrom, 1928

-

0.5

-

0.06 0.1 0.35

-

Schibsted, 1932

0.085

1.o

-

Tobie, 1938

0.5

0.05

-

-

o. 1 0.025

-

t-

-

Comments

To detect acetone in urine T o detect aldehyde in ether (H,SO, reduces sensitivity)

-

Aldehyde in whiskey Citral in lemon extracts Benzaldehyde in almond extracts Excess SO, removed by vacuum Excess SO, removed by vacuum

-

-

Excess SO, removed by vacuum 50% ethanol soh. used for fatty aldehydes 0.1 gm. carbon

*I

i

m

E

G w

c

2

TABLE I (continued) Source of SO., and references-

Sulfur compound km.>

Basic fuchsin km.)

Schreiner and Fuson, 1940 Tobie, 1942

Acid

Comments

1

SO, (continued) half-saturated at room temp. 0.2

0.05 0.1

Rafalko, 1946

0.5

Moree, 1917

0.1

Adams and Johnson, 1949 Cason and Rapoport, 1950 Wild, 1953 Itikawa and Ogura, 1954

0.05 0.05 0.05 0.5

11.3

-

1.5 ml. of 5.6% H2S0,

Rosin, 1955 Feigl, 1956 Vogel, 1956 Linstead and Weedon. 1956 Staple, 1957

-

0.5 0.025 0.1 0.1 0.1 0.5

Na2S20.4 Prud’hornme, 1904 Wertheim, 1922

0.87 2.0

0.013 0.002

Alexander et al., 1950

0.5

0.5

-

0.2 gm. carbon; supersensitive For small and diffuse chromatin elements For sensitive staining of chromatin

-

E i! 0

e

z

-

0

For acetaldehyde Stop at red-violet with SO,; decolors later

v)

-

-

70% ethanol is solvent

1 ml. H2S0, -

Sensitive reagent To make more sensitive, boil 1 min.

-

e

w

c, q W”

m

-

w

Source of SO, and references

Sulfur compound (gm.)

Basic fuchsin

(gm.1

Acid

Comments

1.5 ml. concentr. HCl 1 ml. concentr. HCl 2 ml. concentra. HCl 4 ml. concentr. HCl

-

Na,SO, Fincke, 1914 Elvove, 1917 Scott, 1945 Mowry, 1958 SOCl, Barger and DeLamater, 1948

2.5 1.0 1.o 2.5

0.1 0.1 0.1 1.o

Modified cold Schiff; store at room temp.

H

m tl

m

0.25 ml.

0.25

-

0.45 0.95 0.91 0.98 0.98 1.9 0.5 0.98 0.55 0.3

0.45 0.48 0.45 0.49 0.49 1.0 0.5 0.49 0.29 0.3

10 ml. 1 N HCl 10 ml. 1 N HCI 10 ml. 1 N HCl 10 ml. N HCl 10 ml. 2 N HCI 100 ml. 0.15 N HCl 100 ml. 0.15 N HCl 10 ml. 2 N HCl 1.25 ml. concentr. HCI 10 ml. N H,SO,

-

K2S206

(Na,S,O, sometimes used) DeTomasi, 1936 Coleman, 1938 Stowell, 1945 Hotchkiss, 1948 Ritter and Oleson, 1950 Lillie, 1951a Longley, 1952 Glegg et al., 1952 Mowry et al., 1952 Hormann ct 01.. 1958

~

b

Adds carbon

-

Cold Schiff

-

70% ethanol is solvent Na,SO, 10 H,O as buffer with H,SO,, pH 1.7

-

E

w3: P e E

rn

TABLE I (coittinzced) Source of SO, and references NaHS03 Mohler, 1890

Sulfur compound (gm.)

Basic fuchsin km.)

8.6

0.013

1

Acid

Comments +I

2.0 0.45

0.1

1.5 ml. H,SO,

Bitto, 1897 Feulgen and Rossenbeck. 1924 Margolena, 1932 Bauer, 1933 Carey et al., 1933 Snell and Snell, 1937

0.5 1.7 2 ml. satur. 1.o

0.45 0.83 0.1 0.1

20 ml. 5 N HCI 1.4 ml. concentr. HCl 1 ml. concentr. HCl 1 ml. concentr. HCI

Benseley, 1939 Blaedel and Blacet, 1941

0.83 1.0

0.83

0.1

20 ml. 1 N HCl 17 ml. 6 N HCl

Lillie, 1947a McManus, 1948 Atkinson, 1952 Wild, 1953

1.7 0.45 1.04 0.5

0.83 0.45 0.39 0.125

Kramm ct al., 1955 Vogel, 1956

0.62 1.o

0.29 0.1

0.45

2 ml. concentr. HCl 20 ml. 1 N HCI

k kz

To detect aldehydes in ethyl alcohol

c)

-

Y

v)

+I

20 20 10 1 ml.

ml. 1 N HCl ml. 1 N HCl ml. 2 N HCl concentr. H,SO,

27 ml. 6 N H,SO, 1 ml. HCl

-

2 0

w

-

v)

For methanol by oxidiz. to formaldehyde

4 q-

Add little H2S0, to reduce sensitiv. -

-

For methanol by oxidiz. to formaldehyde

-

-

v)

56

PI

>

c)

M

z

+I

10

FREDERICK H. KASTEN

100 ml. of reagent. Unreacted acid in the reagent undoubtedly reduces the sensitivity (Biddle, 1913). This accounts in part for the fact that the extra-sensitive Schiff reagents are those produced by bubbling in SO2 (Tobie, 1942 ; Rafalko, 1946 ; Elftman, 1959a) without introducing mineral acid. Although Wertheim’s reagent contains only 2 mg. of fuchsin in 100 ml. of 2% hydrosulfite, it seems to be an adequate histochemical reagent (Kasten and Burton, 1959). One probable reason is that it does not contain any strong acid. Blaedel and Blacet (1941) took advantage of the effect of strong acids to reduce the sensitivity of their reagent. They added a little sulfuric acid to a solution already containing 100 meq. of hydrochloric acid. Since many Schiff reagents are prepared with pararosaniline hydrochloride, it is possible that a small amount of hydrochloric acid is released. OF REC. STABILITY The longevity of Schiff’s reagents varies from several hours to 6 months or longer, depending on the ingredients and the method of storing. The useful life of Schiff’s reagents has been reported as 6 hours (Alyea and Backstrom, 1929), 2 days (Chace, 1906), 10 days (Woodman and Lyford, 1908), 6 weeks (Elvove, 1917), several months (Mulliken, 1914), 5 months (Tobie, 1938), 6 months (Lhotka and Davenport, 1949), and 13 months (Tobie, 1942). In the writer’s laboratory, large quantities of the Barger and DeLamater variety of Schiff’s reagent are stored in stoppered dark bottles in the refrigerator. The reagent gives good staining results for at least six months. Some of the factors involved in preservation were considered by Elftman (1959a). They include the evaporation of SO2 and the chemical change of sulfite to sulfate. Loss of SO2 raises the pH, while decrease in sulfite due to oxidation lowers it. The presence of a pink or red color is usually a sign of dissociation of sulfurous acid from the dye; the reagent deteriorates and loses its specificity. Various methods have been employed to prevent loss of S02. Two of the obvious methods are storing in a stoppered bottle (Chautard, 1886; Mulliken, 1914 ; Feulgen and Rossenbeck, 1924 ; Finholt and Thorvick, 1943) and maintaining the solution at a low temperature (Ely and Ross, 1949; Elftman, 1959a). The solubility of SO2 increases with a decrease in temperature as one might expect. Evaporation is also minimized by covering the surface with xylene (Alexander et al., 1950) or mineral oil (Elftman, 1959a). Oxidation of sulfite to sulfate is lessened by using inhibitors such as isopropanol and secondary butanol ( Alyea and Backstrom, 1929), using boiled water in the preparation of the reagent, storing the reagent in completely filled dark bottles (Finholt and Thorvick, 1943; Ely and Ross, 1949), adding antioxidizing agents, such as 0.1%

THE CHEMISTRY OF SCHIFF’S REAGENT

11

pyrogallol (Middleton and Hymas, 1931) and 0.5% hydroquinone (Elftman, 1959a), or using very acid solutions (Reinders and Vles, 1925). Storing in the refrigerator (Ely and Ross, 1949 ; Elftman, 1959a) is simple and probably is the most effective method. Some dye occasionally precipitates from Schiff’s reagent during storage. According to Longley (1952), this results in part from the use of hot solutions during preparation and in part from the use of 1.0% dye solutions. He did not get any precipitation from a 0.5% “cold Schiff” stored in the refrigerator for 3 months. Unfortunately, it does not seem possible to prepare a solid form of Schiff’s reagent that is stable. The solid may be precipitated (Wieland and Scheuing, 1921), but it quickly turns pink. Apparently, SO2 groups are attached loosely to the amine groups and are lost easily. In solution the reagent will retain its staining efficiency for at leasXmo?ths, if kept in well-filled and tightly corked bottles at 0-5OC (Lhotka and Davenport, 1949). It was proposed recently (Beutner, 1954) that a standardized form of Schiff’s reagent be made available commercially. This would involve mixing basic fuchsin, charcoal, and sulfite and adding it to a dilute acid solution. It was suggested by Beutner that formaldehyde could be used on samples of the solution to standardize it before sealing and storing it. The method suffers from the disadvantage that charcoal is used during the sulfite treatment rather than afterwards (Longley, 1952). A simpler approach here might be to use a variation ’of the reagent described by Wertheim (1922). This reagent has sodium hydrosulfite and fuchsin but no charcoal or acid. A stable reagent of controlled sensitivity was reported, which gave reproducible analytical results for 23 days (Kramm and Kolb, 1955).

IV.

Factors Affecting Sensitivity of Reagent

A. BASICFUCHSIN A N D IMPURITIES Rosaniline was used by Schiff (1866) in his original formulation of the aldehyde reagent. It is a triphenylmethane dye containing three amine groups (Conn, 1953). It is commonly called fuchsin or magenta. It is often found associated with the closely related dyes pararosaniline or parafuchsin and, possibly, with magenta 11. The mixture is known as basic fuchsin (C. I. 42510) ; it contains pararosaniline in the greatest concentration, some rosaniline, and small amounts of magenta I1 (Conn, 1953). The chemical structures of these dyes are shown on page 12. In addition to these dyes, colorless Schiffs reagents may be obtained with new fuchsin, C. I. 42520, and Doebner’s violet, both of which are triphenylmethane derivatives. According to Prud’homme (1904),

12

FREDERICK H. KASTEN

7-

H2N=

c)@

NH*

Pararoeaniline

o=c