> • • U -7- -ll mmm R.C.P. EDINBURGH LIBRARY R28183J0236 THE ESSENTIALS OF CYTOLOGY THE ESSENTIALS OF CYTOLOGY AN INTRODUCTION TO THE STUDY OF LIVING MATTER WITH A CHAPTER ON CYTOLOGICAL METHODS BY CHARLES EDWARD WALKER ASSISTANT DIRECTOR OF THE CANCER RESEARCH, LIVERPOOL, AND HONORARY LECTURER IN CYTOLOGY TO THE SCHOOL OF TROPICAL MEDICINE IN THE UNIVERSITY OF LIVERPOOL, FORMERLY DEMONSTRATOR OF ZOOLOGY IN THE ROYAL COLLEGE OF SCIENCE, LONDON LONDON ARCHIBALD CONSTABLE & CO. LTD. IO ORANGE STREET LEICESTER SQUARE W.C. BRADBURY, AGNEW, & CO. LD., PRINTERS, LONDON AND TONBRIDGE PREFACE Cytology is a distinctly specialised study both in the scope of its observations and in the means of making them. For this reason a book specially describing its achievements and technique will be welcome to many. To the student of biology the interest of cytological facts is at the present time especially great, since their bearings upon problems of heredity and disease were never more evidently significant than now. A clear outline therefore of this relatively young branch of the tree of modern knowledge will, given as it is by one well acquainted with it at first hand, be a boon not only to the student of science but to general readers. The book will fulfil yet another admirable purpose if it attract to the field of cytological research, rich as that is in promise of harvest of discovery, additional workers in this country. C. S. SHERRINGTON. AUTHOR’S NOTE The author wishes to express his gratitude to Dr. Murray Cairns, of Liverpool, and to Mr. George Arnold, for the very material assistance they have given him with regard to the proofs of this book. Liverpool, 1907. CONTENTS CHAP. PAGE I. INTRODUCTORY 1 II. THE STRUCTURE AND PARTS OF THE CELL .... 9 III. CELL DIVISION 19 IV. THE MEIOTIC PHASE 31 V. POST- MEIOTIC DIVISIONS ....... 46 VI. THE MALE SEXUAL ELEMENTS ...... 51 VII. THE MATURATION OF THE OVUM ...... 61 VIII. FERTILIZATION IN MULTICELLULAR FORMS .... 66 IX. FERTILIZATION IN UNICELLULAR FORMS .... 74 X. THE PROBABLE INDIVIDUALITY OF THE CHROMOSOMES . 85 XI. THE MORPHOLOGICAL ASPECT OF THE TRANSMISSION OF HEREDITARY CHARACTERS 98 XII. CYTOLOGICAL METHODS . . . . . . .109 INDEX 131 THE ESSENTIALS OF CYTOLOGY. CHAPTER I. INTRODUCTORY. Our knowledge of the properties of living matter gener- ally as distinct from any other form of matter is very limited, but there is one fundamental character which, though it is perhaps the most important of any in this con- nection, does not always appear to be properly appreciated. This fundamental fact is that living matter, as far as our knowledge goes, exists only in one form. Whether it he in the shape of animals or vegetables, living matter is always in the form of definite and concrete masses of a complex material, each mass being composed of certain definite and distinct parts. The material of which these masses are composed is known as protoplasm. The masses themselves have, somewhat inaptly, been called cells. Living matter then can apparently only exist in the form of cells. Until quite recently protoplasm was regarded as being the simplest form of living matter, “ the physical basis of life ” of Huxley. Recent research, however, has shown that no homogeneous mass of protoplasm is really alive. Unless e.c. B '2 THE ESSENTIALS OF CYTOLOGY. the constant constituents of a cell are present in a mass of protoplasm it will disintegrate with greater or less rapidity,1 and the phenomena exhibited by living matter will never be exhibited by protoplasm unless the protoplasm be in the form of cells or a cell. All the supposed cases of undifferen- tiated protoplasm being alive, such as Huxley’s Bathybius, have broken down under examination. Bathybius was first abandoned by Huxley himself. Haeckel’s Monera have never been rediscovered. Having realised that the cell is the unit of living matter, the simplest form in which it can exist, the conclusion naturally follows that most problems relating to living matter must ultimately resolve themselves into cell problems. Physiology is the study, the recording, and the measuring of the phenomena and processes taking place normally in groups of cells. Pathology is the study and recording of departures from the normal in similar groups of cells. That such problems are ultimately cell problems becomes evident when we realise that every part of the animal and vegetable body which is alive is formed of cells, and of cells only. Those parts which are not composed of cells, such as the hard parts of bones and the wood of trees, are not alive, but are merely the dead secretions or excretions of cells. The similarity in structure, however, does not by any means end the likeness between the different forms of living matter. As we shall see later, some of the most complicated cellular phenomena, including many which are of the most fundamental importance, are similar, even to minute details, in both animal and vegetable organisms. 1 See p. 17 INTRODUCTORY. 3 Living organisms, whether animal or vegetable, may exist in the form of single independent cells — unicellular organ- isms, or may he composed of many cells associated together and forming an individual — multicellular organisms. In either case, however, these cells — units of living matter — have always been derived from pre-existing cells. We have no knowledge of any origin of cells at the present time except- ing from other cells, and no alleged instance of any other origin of living matter has hitherto been able to bear investigation. The whole of the cells forming the various parts of the multicellular organism are derived from a single cell, the ovum. We have seen that so far as our knowledge goes, no cells can arise from any other source than from pre-existing cells. New individuals of various kinds of multicellular organisms are produced from cells derived from similar multicellular organisms. Under normal conditions the new individual is produced from a single cell derived from an individual of the same species. This single cell divides into two cells, each of these two again divides into two more, and so on, until the whole soma or body of the individual is built up. At some stage in the life of the organism, cells are thrown off — separated from — the body of the parent to form new individuals in their turn. In the case of most multicellular organisms, however, two individuals are neces- sary to produce a new organism. This production of the new individual is brought about by two cells, one derived from each parent, fusing to form a single cell. This process is called fertilization, and from this single cell, the fertilized ovum, the body of the new individual is formed. The new individual thus formed will in its turn produce cells which b 2 4 THE ESSENTIALS OE CYTOLOGY. will be cast off from its body, and which will produce new individuals, and so on through each fresh generation. If this series of events be represented diagrammatically (Fig. 1) it will be seen that there is a direct line of cell generations running through successive generations of multi- cellular individuals. Some of the cells, in fact, the repro- Fig. 1. — A, represents a fertilized ovum which has given rise to successive generations of cells, and thus has built up a multicellular organism. The cells forming the body or soma are represented by white circles, the line of cell generations destined to produce sexual elements by black. A sexual cell, B, is represented as having fused with a sexual cell C, derived from another individual. As the result of this fusion a new individual is being built up. (Modified from J. E. S. Moore.) ductive cells and the intermediate generations of cells between them and the ovum, are potentially immortal. In most unicellular animals of whose life-cycles we have any accurate knowledge a very similar series of events takes place. A number of generations of individuals is produced by simple division of pre-existing individuals. Then a stage intervenes where the individuals conjugate,, INTRODUCTORY. 5 exchanging portions of their nuclei with each other, or actually fusing into one cell (Fig. 2). This corresponds to the process of fertilization in multicellular organisms, and able to go on dividing for a large number of generations, until conjugation again takes place. In organisms which consist of one cell only all the Pick 2. — A series of divisions beginning in the unicellular organism A, has produced a large number of individuals. After many generations have been produced in this manner, the individuals conjugate. The black individuals are represented as having conjugated, and a fresh series of generations is being produced in each case by simple divisions. (After J. E. S. Moore.) functions necessary to the well-being of the individual and to the reproduction of the species are performed by the one cell. Digestion, secretion, excretion, movement, and repro- duction are all carried on by it. In multicellular organisms, on the other hand, the different functions necessary to the individual are carried out by different cells or groups of the result is also comparable — the fertilized individuals are 6 THE ESSENTIALS OF CYTOLOGY. cells. The members of these different groups differ in appearance and structure within certain limits, always retaining, however, the essential parts found in all cells. The differentiation in fact lies in the addition of various structures to these essential parts. Thus in the multi- cellular animal we find various groups of cells which normally carry out the different processes involved in the digestion of food, other groups which are so modified as to enable the animal to move, others which enable it to appreciate its surroundings, and so on. In every case, under normal con- ditions, the activities of the cells thus differentiated are confined to certain particular functions. Every multicellular organism, however, as we have already seen, begins its existence as a single cell. Thus it is evident that the ovum possesses all the potentialities subsequently exhibited by the cells derived from it. It may, in fact often does, possess all the properties of a unicellular organism. Now the differentiated cells of the multicellular organism appear to retain, to a greater or less extent, this general potentiality of reproducing the various other differentiated cells found in the particular organism. This is very obvious in the case of some plants. It has been proved experimentally that it is retained by the cells of some vertebrate animals to a certain extent. If, for instance, the lens be removed from the eye of a salamander a fresh lens will grow. The original lens wras developed from the outer layer of cells, which were differentiated from the rest of the cells of the embryo at a very early period. The new lens, however, is developed from another group of cells which were differentiated also at an early period along quite different lines, and which normally would never have produced anything at all like the INTRODUCTORY. 7 lens. Again, the phenomena observed in cancer, as we shall see later on, show that even in mammalia differentiation does not entirely destroy the general potentiality of the cells forming the various tissues of the body. In spite of this greater or less potentiality to form different kinds of tissues retained by the differentiated cells of the multicellular organism, the fact remains that, under normal conditions, cells belonging to differentiated groups retain their special characteristics and properties, and, when they divide, produce cells similar to themselves. It is therefore evident that in the multicellular organism there is some kind of interaction or mutual regulation which limits the characters and properties of each individual cell and each group of cells in some special manner in relation to the rest of the cells forming the organism. For want of a better term we will call this influence “ Somatic Co- ordination.” We know but little more with regard to it than that it is an interaction or common influence dominating the individual cells of a group or groups which causes them to be, to a considerable extent, depen- dent upon each other — in fact, to form an individual multicellular body or soma. For instance, if a fertilized ovum has divided into two daughter-cells and each of these into two more, on the way to forming the body of a multicellular animal, each of the four cells, if the embryo is allowed to develop in the normal manner, will produce different tissues possessing different functions. Thus the derivatives of A (Fig. 3) may produce nervous tissue, and will eventually convey nervous impulses ; B may, among other things, produce the liver and eventually secrete bile ; C may produce the kidneys and secrete urine, 8 THE ESSENTIALS OF CYTOLOGY. and so on.1 If, however, the four cells be artificially separated without injury and are kept under suitable con- ditions, it has been proved experimentally that each of the four cells may produce a complete embryo possessing a full complement of tissues and functions.2 A, therefore, in association with B, C, and D, .will produce only one group of tissues. Out of association with them it will produce not only its own proper tissues, but also all those which would have been produced by B, C, and D. In the one case the tissues produced by A will be confined to one set of functions ; in the other they will perform all the functions of the body. It is obvious then that there is some influence which dominates A, B, C, and D which interacts between them, and which limits the characters and functions of the tissues produced by each when they are associated, but which ceases to influence them or allow them to mutually influence each other when they are dissociated. This reciprocal influence is called Somatic Co-ordination. 1 These derivatives of the four blastomeres are given merely as a hypothetical example, to facilitate the explanation. 2 Roux, Wilson, Driesch, Morgan, Zoja, and others. CHAPTER II. THE STRUCTURE AND PARTS OF THE CELL. We have seen that the whole soma or body of the multi- cellular organism is built up by generations of cells produced from the ovum. The process of cell division was regarded by the older observers as a very simple matter, no more complicated than the division of a drop of viscous fluid into two drops. Occasionally cells do divide in a manner that is not apparently very much more complicated than this, but it is now known that the phenomenon of division in the great majority of cases involves a very complicated process. The apparently simple form of cell division just referred to is known as “amitotic” division or “ amitosis.” It will be dealt with in more detail later on. The usual and more complicated mode of division is known as “mitotic” division, “ mitosis,” or “ karyokinesis.” Before considering the details of this process it is necessary to have some knowledge of the structure and constituent parts of a cell. Cells are, unless they become specially differentiated or until they are pressed upon by contiguous cells or other things, more or less spherical in shape. Within the cell is a mass of protoplasm which is different in structure from the rest of the protoplasm which forms the cell. This contained mass is called the nucleus, and, like the cell itself, is usually spheroidal or spherical in shape. When the cell is examined 10 THE ESSENTIALS OF CYTOLOGY. under a microscope tlie nucleus appears denser in structure than the rest of the cell, and if the cell be stained, this difference becomes more marked. The protoplasm which surrounds the nucleus, the rest of the cell in fact, is called the “ cytoplasm.” Both nucleus and cytoplasm are composed of protoplasm. The protoplasm itself is a viscous translucent substance, sometimes showing a definite structure, sometimes appear- ing practically homogeneous. Some kind of structure is the more usual form, but the nature of this structure varies considerably. Perhaps it most frequently presents a finely granular appearance, or that of a fine reticulum or mesh- work. A foam structure, like a number of minute bubbles crowded together, is not uncommon. The structural basis of the protoplasm forming both nucleus and cytoplasm is almost certainly the same, for, as we shall see later, at a certain period in the process of cell division, those portions of the protoplasmic ground substance which are separated into nucleus and cytoplasm become inextricably mingled together. When, however, the cell is not in actual process of division but is in a vegetative condition1 the nucleus is surrounded by a membrane which separates it from the cytoplasm. The appearance of the contents of this mem- brane, the nucleus proper, may vary considerably. The most usual appearance howTever, is a reticulum ormeshwork. In the substance of the threads of this reticulum, or attached to them, are masses of a darkly staining substance. This 1 A cell which is not in any of the several phases which immediately precede, accompany, and follow the process of division, is sometimes described as being in a state of rest. This is an obvious inaccuracy, and is likely to be misleading, as a living cell can hardly, if ever, be in a resting condition : it will always be doing something. STRUCTURE AND PARTS OF THE CELL. 11 threadwork is called “ linin,” and the darkly staining substance is called “ chromatin.” It may here be pointed out that the different parts of the cell react differently to different stains. The cytoplasm and most of the structures contained in it and derived from it take an acid in preference to a basic stain. The linin of the nucleus also takes an acid stain, but the chromatin Fig. 4. — A semi-diagrammatic representation of a cell, a, nuclear membrane ; linin reticulum ; c, chromatin masses contained in envelopes of linin (chromatin nucleoli) ; d, true nucleolus ; e, vacuole ; f, plastids ; g , centrosomes ; h , archoplasm ; i, food particles. stains with a basic rather than with an acid dye. All the structures contained in a cell will stain with either an acid or basic d}^e to a greater or less extent, but if a basic and an acid stain be both used the different structures contained in the cell react to them as is here indicated.1 For a long time it was believed, indeed it is still very commonly held, that the one constant part of the nucleus, the substance through which all the hereditary characters were transmitted from one generation of cells to another, 1 See p. 122. 12 THE ESSENTIALS OF CYTOLOGY. was the chromatin. Very strong evidence has however come to light which seems to show that the chromatin is probably no more than a secretion of the linin, and that it is the linin, if it be any definite substance contained in the cell, which performs the functions generally attributed to the chromatin.1 The linin, as has been already said, generally forms a reticulum in the ground substance of the nucleus. Often the linin is apparently in the form of threads or ribbons, hut it may also, particularly during certain stages in the process of division, be in the form of tubes. The chromatin is, as a rule, distributed in granules of varying sizes along the threads or tubes of the linin. In most nuclei chromatin is also found in a varying number of more or less irregular masses of a larger size, these masses some- times occupying an appreciable portion of the space contained in the nucleus. These masses were called “nucleoli” by the earlier observers, hut it was found that they were only some among several other kinds of structures to which this term was applied. They were then called “ chromatin nucleoli,” but as they are simply masses of chromatin of a large size it seems unnecessary to add a fresh word to an already large and rapidly increasing vocabulary to distin- guish chromatin masses which are indistinguishable from their fellows except by their size. It would indeed be impracticable to specify what size gave a right to the special name. The chromatin masses are almost certainly generally con- tained in an envelope of linin which is continuous with the threadwork or tubes. 1 See p. 37, “ Meiotic Phase.” STRUCTURE AND PARTS OF THE CELL. 13 There are other bodies in the nucleus which are commonly called “true nucleoli.” They generally stain with an acid stain, in contrast to the chromatin masses which take a basic stain. Though there are many speculative theories as to the nature and function of these bodies, nothing certain is known with regard to them. It is even doubtful whether they are all similar. They are almost always spherical in shape, and generally appear practically homogeneous in texture. As the}r are very often absent altogether, and as during the process of cell division they generally seem to be absorbed or disintegrated completely, they are evidently not necessary constituent parts of all cells. The spaces between the linin meshwork are occupied by a clear viscous fluid or jelly in w7hich there is no apparent structure. This is known as the “ nuclear sap.” The size of the nucleus in proportional relation to the size of the cell varies considerably in different classes of cells. It is usually large in those cells in which any active metabolic process is taking place, such as gland cells, and also in those cells which are destined to give rise to the reproductive elements. Its shape, though on the whole very constant, may also vary. Usually spherical, it may be elongated in highly differentiated cells such as those forming muscle fibres. It may also be definitely lobular, as is the case in some cells found in cancer, in bone-marrow, and in some leucocytes. The nucleus may also change in shape by movements which have been described as amoeboid. In some unicellular organisms ( e.g ., Infusoria) two nuclei are present. These two nuclei are of different sizes and apparently perform different functions. The larger “ macro- nucleus ” seems to control various functions of the cell, 14 THE ESSENTIALS OF CYTOLOGY. sucli as digestion and excretion. The smaller “ micro- nucleus” seems to be concerned in the reproduction of the species. The nuclear membrane does not appear to be absolutely constant. Apart from the fact that it disappears, as we shall see later, during the process of mitosis in cells where it is usually present, some forms have been described in which there appears to be no nuclear membrane. In these organisms the nucleus is described as being distributed in the form of chromatin granules, often collected in masses. If this description is accurate the chromatin masses are probably enveloped and possibly connected by linin. Although this class of nucleus requires further investigation, there is nothing with regard to it which is at all improbable. Indeed, it may be that this is the most primitive form of nucleus. The cytoplasm seems to vary considerably in structure in different classes of cells. In some cells it is apparently almost homogeneous, in others it is granular, reticular, or of a formation which appears like a delicate foam. Though some cells, such as leucocytes, have been held to be naked and to possess no membrane enclosing the cytoplasm, it is almost certain that in all cells there exists a layer of differentiated protoplasm upon the surface. There certainly is such a layer in leucocytes. Between this differentiated layer of cytoplasm and a very definite cell wall, all stages exist. In animal organisms a differentiated layer of proto- plasm is the more usual, while in plants a cell wall is the commoner. Where a cell wall exists it is regarded by most observers as having been produced by a secretion of the cell. The differentiated cell organs, such as cilia and flagellse, arise from the cytoplasm, and the form of the STRUCTURE AND PARTS OF THE CELL. 15 cytoplasm itself frequently undergoes the most striking modifications, as, for instance, in the case of the striped muscle fibres. Various cell secretions also appear to arise in the cytoplasm either in the form of granules or as liquids filling hollow spaces known as vacuoles. The digestion of food particles takes place in the cytoplasm, and masses of food material of varying sizes are frequently seen lying in its substance. Other bodies, known as “ plastids,” are sometimes found in the cytoplasm. Several different kinds of plastids exist. Among those about which we know most are the “cliromato- phores.” These are usual in plant cells, though they occur in but few animals. They arise, in some cases at any rate, if not in all, as colourless plastids in the embryonic cells, and, dividing with the other parts of the cell, go on from one cell generation to the next, eventually forming the pigment bodies in the adult cell. L}ring in the cytoplasm, as a rule near the nucleus, is a pair of minute bodies which are of great importance. These are the “ centrosomes.” They are very small as a rule, often being on the extreme limit of microscopic vision. So far as can be seen they are usually homogeneous in structure, though occasionally they appear to he granular. In the latter case they are generally comparatively large. They are usually kidney or bean shaped. Sometimes only one is present, but in other cells there may be several, as in certain leucocytes and the red blood corpuscles of Axolotl. In a few cases the centrosomes are stated to be inside the nuclear membrane, but this statement requires further confirmation. Centrosomes have not been demonstrated in the cells of 16 THE ESSENTIALS OF CYTOLOGY. the higher plants, though this is not quite conclusive evidence that they are not present. They may be multiple and below the range of microscopic vision. They are present in the lower plants and in animal cells, and it is possible that they may have been lost in the higher plants in the process of differentiation. When they are present, centrosomes go on from genera- tion to generation dividing with the cell, one going to each daughter-cell produced by the division of the parent. They play a very important part in the phenomenon of mitosis, as will he seen later. Often the centrosomes are surrounded by an area of protoplasm which is denser than the rest of the cytoplasm. This condensed mass is called the “ archoplasm.” The archoplasm is often particularly well marked in the case of cells which are about to produce sexual elements, in some leucocytes, and in some of the cells found in cancer. When no archoplasm is to be seen, the centrosomes are sometimes surrounded by radiations. This formation is known as the ‘‘aster.” Frequently in cells that are in a vegetative con- dition the centrosomes lie in the cytoplasm with practically no differentiated area around them, and they may then be almost impossible to demonstrate. Of all these constituent parts the two which are of vital necessity to the cell are the nucleus and cytoplasm. As we have seen, all the other cell structures may be absent. We may even go further, and say that only the linin with its chromatin and the general protoplasmic ground work are necessary, for the nuclear membrane disappears during the process of division, and seems to be absent altogether in some unicellular forms. STRUCTURE AND PARTS OF THE CELL. 17 Besides this morphological evidence as to the necessity for both nucleus and cytoplasm there is a considerable amount of experimental evidence which appears to prove this necessity. At any rate the evidence that a portion of cytoplasm that does not contain a portion of nucleus upon separation from a living cell will certainly die, is conclusive. The majority of these experiments have been made with unicellular animals, and in them it has been found that while a portion of cytoplasm containing even a minute portion of nucleus will, when separated from the animal, develop into a complete animal, portions of cytoplasm devoid of any portion of nucleus, though they are able to move about and engulf food particles, are unable to digest, unable to regenerate the cell membrane or organs, and die in a comparatively short time.1 An example of a similar piece of evidence with regard to multicellular forms is the well-known fact that, if a nerve fibre in one of the higher animals be cut, the portion which is separated from the nucleus of the nerve cell proceeds to degenerate, while a new fibre grows out from the part of the cell which is con- nected with the nucleus to take the place of the degenerated portion. These facts indicate that the active metabolism of the cell, that is such processes as digestion and secretion, is per- formed or controlled by the nucleus. It also appears that growth and the regeneration of organs, or indeed of any part of the cell which may be destroyed, is dependent upon the presence of the nucleus. Chemical evidence, with 1 Brandt, 1877 ; Nussbaum, 1884 ; Gruber, 1885 ; Verworn, 1888 ; Lillie, 1896. E.C. C 18 THE ESSENTIALS OF CYTOLOGY. which it is not proposed to deal, also seems to corroborate this view. We shall see also that there is very strong evidence that certain hereditary characters, or rather the potentiality of producing them, is transmitted by the linin and possibly to a lesser extent by the chromatin of the nucleus. Semi-Diagrammatic Representation of the Process of Mitosis Fig. 5. — A cell with the nucleus in the vegetative condition. Fig. 6. — Early prophase of division. The linin nnd chromatin have adopt :d asters and spindle are forming in connection with the centrosomes. Fig. 7. — The spireme has broken up into the chromosomes, and the spindli Fig. 8. — The nuclear membrane has disappeared. The spindle is fully for Fig. 9. — Each of the chromosomes has become attached to a spindle fibre. Fig. 10.— Metaphase. [Polar view.] Fig. 11. — The chromosomes are splitting lengthwise. Figs. 11a and 11b. — The different ways in which the chromosomes split. Fig. 12. — The halves of the chromosomes are travelling towards the oppos t Fig. 13. — Later stage of anaphase. Fig.' 14.— The daughter-cells are separated from each other. [Telophase,] FlQ. 15.— The reconstruction of the daughter nuclei. Cell Exhibiting 8 Chromosomes. the form of a coiled-up thread (spireme). ' is in a more advanced stage of formation, led. Metaphase. [Lateral view.] e poles of the spindle. [Early anaphase.] [To face p. 19 T CHAPTER III. CELL DIVISION. It is of the greatest importance to the right understanding of the phenomenon of mitosis that all mental concepts of the various phases of the process should be definitely three dimensional. It is extremely difficult to convey the idea of three dimensions in a diagram or even in the portrait of a cell. Under the high powers of the microscope, which are necessary for the observation of cell phenomena, but a very thin optical section of a cell can be seen at a time, so that actual observation of the mitotic figures themselves is frequently of but little help unless a mental picture he built up from the various optical sections as they successively come into focus. The cell is generally roughly spherical, and always extends in three dimensions. Therefore all the figures which are produced in the various cell processes occupy three dimensions, and this fact must be kept in mind in considering them.1 When a cell is in a vegetative condition a reticulum of linin containing granules and masses of chromatin is usually found in the nucleus. When, however, the cell is about to divide into two daughter- cells, the linin and chromatin 1 The stereoscopic illustrations on cards in the cover of the book, will, when looked at through a stereoscope, give the reader a proper conception of the phenomenon of mitosis. A more complete set of sterescopic illustrations are published separately. (See advertisement at end of book.) 20 THE ESSENTIALS OF CYTOLOGY. undergo some very striking changes in form and arrange- ment. Following the nomenclature introduced by Strasburger, Flemming, and others, the phenomenon of mitosis may be divided into (1) “ Prophase,” (2) “ Meta- phase,” (3) “ Anaphase,” and (4) “ Telophase.” The most usual sequence of events is as follows — The chromatin and linin gradually lose their irregular arrangement, and adopt the form of a coiled-up thread. This thread is much thicker and more definite than any of the threads of chromatin and linin that are seen in the vegetative condition (Fig. 6). It is usually formed of a ribbon or tube of linin, in which are distributed chromatin granules. These granules are much more regularly arranged along this coiled thread than is the case with the threads of the resting nucleus. Sometimes the arrangement of the chromatin is so close that the thread appears almost solid. Again, in other cases the granules coalesce in small masses, so that the coil has the appearance of a thread of beads. In many cases the coils of the thread can apparently be followed indefinitely, and many observers hold that it may be con- tinuous— endless, in fact. This coiled thread is called the “ spireme.” In other cases, however, the spireme is divided into definite segments from the time of its first appearance. Frequently the spireme is at first thin and very closely coiled, but later it spreads out and grows thicker and shorter. From the time of the appearance of the spireme the chroma- tin seems to increase in quantity, to become denser in texture, and to stain more darkty. Eventually the spireme thread breaks transverse^ in several places, thus forming a number of rod-like bodies which often retain the curve of the spireme, being in the shape of U’s (Fig. 7). Frequently> CELL DIVISION. 21 however, they are V-shaped, and sometimes they are nearly straight. These rod-like bodies are called “ chromosomes,” and in them the chromatin appears to reach its maximum of density and staining reaction. The formation of a spireme is by no means a constant prelude to cell division. Sometimes the chromosomes appear directly, without any coiled thread being observable. Here the usual sequence of events is that certain areas in the nucleus appear to grow denser through the accumula- tion of linin and chromatin. At first more or less nebulous, these masses gradually acquire a definite shape, and become dense in structure. Though generally in the form of rods bent into the form of U’s or V’s these chromosomes sometimes appear as oval or round masses. One of the most striking facts connected with the chromo- somes is that every species of animal or plant appears to possess a characteristic number. Thus in man we find 32, in a mouse 24, in a donkey 86, in a cockroach 32, in Ascaris megalocephala, var. univalens 2, var. bivalens 4, and so on in different organisms. This characteristic number appears in all the cells forming the soma or body of the organism. Though dividing cells may exhibit a number of chromo- somes which differs from the characteristic number of the organism to which it belongs, the number is extraordinarily constant, much too constant to be a matter of coincidence. The number of the chromosomes is known to be modified in the most marked manner by pathological conditions, so that a few cases of departure from the usual number among the cells of an apparently healthy multicellular organism is not in the least a surprising matter. While the chromosomes have been in process of formation 22 THE ESSENTIALS OF CYTOLOGY. very striking changes have been taking place with regard to the centrosomes. It has been stated that the centrosomes are sometimes surrounded by radiations, forming what is known as the aster. In the prophase of mitosis an aster generally appears, round the centrosomes if they are present, round a clear area of cytoplasm close to the nucleus in the case of the higher plants where no centrosomes are dis- cernible. As the changes proceed in the nucleus the centrosomes begin to separate and travel away from each other rapidly. Besides the radiations extending into the cytoplasm around the centrosomes, fibres may be seen extending from one centrosome to the other, lengthening as the centrosomes get further and further apart (Figs. 6 and 7). From the form assumed by these fibres they have been called collectively the “ spindle,” and are known as “ spindle fibres.” In plants which exhibit no centro- somes the spindle is formed in an exactly similar manner, the areas of clear cytoplasm at the centres of each set of radiations acting apparently after the manner of centrosomes. By the time that the chromosomes have been fully differentiated the centrosomes have become so far separated that they are often as distant from each other as the length of the diameter of the nucleus. Often the spindle partially envelops the nucleus, pressing in the membrane where it touches it. At this stage the chromosomes are generally found at the periphery of the nucleus, lying just under the nuclear membrane, and then the nuclear membrane disintegrates, setting free the chromosomes (Fig. 8). The nuclear sap and the substance of the cytoplasm are thus obviously mingled CELL DIVISION. 23 together by the disappearance of the nuclear membrane. This is the end of the prophase of mitosis. As soon as the chromosomes are liberated by the dis- appearance of the nuclear membrane each of them becomes attached to one of the spindle fibres. The centrosomes are usually at opposite poles of the cell, and the chromosomes are always attached to the fibres at the equatorial plane of the spindle. This stage, when the chromosomes are attached to the spindle fibres at the equatorial plane, is the metaphase (Fig. 9). It can easily be demonstrated by comparing the appear* ance of a cell in this stage of mitosis as regarded from the point of view of one of the centrosomes (polar view) with the appearance it presents when looked at from a point of view level with the equatorial plane (lateral view), that the U or V shaped chromosomes all tend to lie flat upon the equatorial plane (Figs. 9 and 10). Very soon after the chromosomes have got into position upon the equatorial plane of the spindle, they begin to split lengthwise (Fig 11). A very common mode of splitting is that illustrated in Fig. 11 A, but often they split as shown in Fig. 11 b. This splitting is the commencement of the anaphase, during which the chromosomes are individually split into halves which are destined to be distributed, a half of each chromosome to each of the daughter-cells produced by the division. Each of the halves produced by this longitudinal splitting travels along the fibre to which it is attached towards the nearest centrosome at the pole of the spindle until a half of each chromosome is quite close to each of the centrosomes (Figs. 12 and 13). There is thus a group of the longitudinal 24 THE ESSENTIALS OF CYTOLOGY. halves of each chromosome collected round each of the centrosomes. The cell at the same time becomes elongated and eventually contracts in the middle in the situation of the equatorial plane, assuming, more or less, an hour-glass shape. This contraction continues, pressing in the spindle fibres which have become parallel, until the two parts of the cell are completely separated from each other (Fig. 14). Thus the two daughter-cells arise, each containing an exact half of each of the chromosomes contained in the parent cell. The stage of actual separation is the telophase. The spindle fibres are still apparent, even after the two daughter-cells are completely cut off from each other. As the two halves of each chromosome separate and travel towards the respective centrosome they are still connected by a spindle fibre, and these fibres are at first parallel with each other, being subsequently pressed together at what was the equatorial plane of the spindle. They often show very marked thickenings at the point where the plane crossed them, and so, when they are all pressed together at one point when the two daughter-cells are separated from each other, a body is formed by these thickenings (Figs. 13 and 14). This is the “ mid-body ” or cell plate.1 Though com- mon and very well marked in the case of many plant cells this phenomenon is often not observable in any marked degree in the case of animal cells, and is frequently absent. The asters round the centrosomes, both before and after the disappearance of the nuclear membrane, are generally 1 In animals the bodies thus formed are known as mid-bodies, in plants as cell plates. Though far more definite, the cell plate in the case of the plant cell is probably analogous to the mid-body occurring in the animal cell. CELL DIVISION. 25 extremely well marked in all divisions occurring among embryonic cells. As tlie organism approaches the adult condition the asters are often less and less marked and in the division figures seen in the cells forming adult tissues they are frequently absent altogether. The chromosomes in the two daughter-cells are congre- gated together round the centrosomes. Here, as soon or even before the cytoplasm has contracted so far as to be definitely separated into two masses, they begin to form new nuclei. There are two usual modes by which this is accom- plished, though there are many variations from them. The congregated chromosomes may form a figure which closely resembles the spireme of the prophase of division, except that the coil never seems to be continuous, and is comparatively very thick. The new nuclear membrane is then formed. In Ascaris this is apparently brought about by processes of linin growing out from the chromosomes to form the new nuclear membrane. Other processes grow between the different segments of the coil, the chromatin is distributed in them, and the nucleus gradually resumes the normal vegetative appearance (Fig. 15). The chromosomes may never assume such a position as to simulate a spireme, but processes may simply grow out from them in the way just described. In many cells on the other hand, each chromosome seems to become more or less vesicular, appearing to some extent like a small nucleus. These small structures then fuse to form the nucleus of the new cell. It is most important to realise thoroughly that each daughter-cell receives an exact half of each of the chromo- somes that is contained in the parent-cell. It is upon this 26 THE ESSENTIALS OF CYTOLOGY. ground alone that any probable morphological theory as to the mode in which certain hereditary characters are trans- mitted can at present he based, as we shall see later. The single cell from which the multicellular organism is built up divides, and half of each chromosome contained in it goes to each of the daughter-cells produced. This process is repeated in each succeeding cell division, the substance of which the chromosomes are formed apparently growing in bulk at each succeeding cell generation. The obvious result is that eveiy cell forming every tissue of the multi- cellular organism possesses individual chromosomes, each of which is probably derived by a direct succession of divisions with intervening periods of growth from the actual substance of the corresponding individual chromosome present in the single cell from which the organism was built up. It must be remembered too, that the division of the chromosomes being longitudinal there is apparently a derivative of every transverse section of every chromosome present in the parent-cell in all the cells produced in subsequent generations. The general constancy in the number of the chromosomes exhibited b}^ the cells of any given organism is so great as to completely outweigh the occasional departures from this rule. The fact that pathological conditions will produce irregular mitoses makes it evident that many exceptions to the normal number of chromosomes ought to be expected when many cells are examined. The fact that a num- ber of exceptions are found cannot therefore be used as a valid argument that the number of the chromosomes varies normally to any extent in the cells of similar organisms, at least not on the evidence that is available at the present CELL DIVISION. 27 time. On the other hand, as we shall see later, the varia- tions that do occur, under exceptional circumstances, from the normal number of chromosomes in the cells of any given organism may have a special significance of their own. The process of mitosis is subject to several modifications, some of which are normal, others being caused by abnormal conditions, some of which are pathological. Sometimes the chromosomes divide and the daughter nuclei are formed without the cytoplasm of the cell dividing. A condition is thus produced in which two or more nuclei are included in a common cytoplasm. When these daughter nuclei, in their turn divide, the several spindles become intermingled, though there are three, four, or more poles. Such forms are known as “ pluripolar mitoses ” (Fig. 16). In organisms where multinucleate cells are common, pluri- polar mitoses are also common. Though they have long been recognised as normal in the cells of certain plants, they were regarded as abnormal in the cells of the higher animals until quite recently. They have been shown, Fig. 16. 28 THE ESSENTIALS OF CYTOLOGY. however, to occur normally in certain cells even among mammals ( e.g ., Myeloplaxes — certain large cells found in the bone marrow). Certain cells in many organisms divide amitotically, that is, without the appearance of chromosomes or the formation of a spindle. In such cells the nuclei often divide without the cytoplasm dividing, and this is another cause of pluripolar mitoses. The result of a pluripolar mitosis is usually that the cell divides into the same number of daughter-cells as there are poles in the figure. Sometimes however it may be that a similar number of nuclei are produced without a division of the cytoplasm occurring. Among the pathological causes of irregular mitoses are certain poisons, which if allowed to act upon the cells without killing them, cause an unusual number of mitoses, many of which will be abnormal. Nicotine, cocaine, iodide of potassium, antipyrine, quinine, and other substances have been used experimentally for this purpose. Some- times the irregularity of the mitoses is due to one centro- some dividing and not the other, which causes tripolar, quadripolar and other forms of spindles. In other cases the mitosis is asymmetrical, more chromosomes going to one pole than to the other, and so an unequal number of chromosomes in the daughter-cells results. Again, some of the chromosomes may fail to become attached to spindle fibres, and degenerate without being involved in the process of mitosis. It would seem probable that the irregular mitoses observed in many diseases are due to the presence of a poison produced by parasitic organisms or by some other cause. At any rate it is obvious from the results of CELL DIVISION 29 experiments that conditions that are only slightly abnormal may produce abnormal mitoses. As has already been pointed out, though cells generally divide by the process of mitosis, some cells divide by the c Fig-. 17. — Amitotic division, a, a large multinucleate cell from the bone-marrow of a guinea-pig (myeloplax). The nuclei are dividing amitotically ; b , a similar cell where a portion of a cytoplasm is dividing off amitotically ; c, the nucleus of a cell in the testis of Triton dividing amitotically. apparently simpler process of amitosis. Here both nucleus and cytoplasm divide in a similar manner to a drop of viscous fluid. The contents of the nucleus remain in the vegetative condition, and the nuclear membrane does not disappear. Both nucleus and cytoplasm are constricted at 30 THE ESSENTIALS OF CYTOLOGY. one part until they separate. Generally the process occurs first in the nucleus independently of the cytoplasm, and thus we find inultinucleate cells produced (Fig. 17). In most cases there is no evidence as to whether the centro- somes play any part in amitosis, though it would appear that they frequently divide at or about the same time as the nucleus. It was generally believed, until quite recently, and many observers still hold, that amitosis never occurs except as a prelude to degeneration. There is, however, no doubt whatever that, in very many cases, amitosis cannot in any way be regarded as a sign of approaching degeneration. In certain animals (Amphibia), among the cells that are destined to produce the male sexual elements, amitotic generations are followed by several generations of mitotic divisions, and the mature sexual elements thence derived fuse with others to produce new individuals. Again, among the cells in the bone marrow, amitotic are followed by mitotic divisions. As far as we can see at present, amitosis involves the division of the nucleus in bulk, just as though a portion were cut off hap-hazard. If this is really the case, amitosis is one of the most inexplicable phenomena exhibited by cells, when it is remembered how accurately individual parts of the cells involved are sorted out and preserved at each succeeding division both before and after this apparently undiscriminating mode of division occurs. It may be found eventually, however, that amitosis is anything but the simple process it appears to us at present. CHAPTER IV. THE MEIOTIC PHASE. It lias already been explained how, during the pheno- menon of mitosis, all cells exhibit a definite number of chromosomes, and that though in different species of both animals and plants this number varies, in the cells of the same species the number of the chromosomes remains the same. This constancy in the number of the chromosomes holds good for all the cells of the co-ordinate soma or body of the multicellular organism. A period is reached, however, in the life of the organism when certain cells depart from this uniformity of behaviour and in these the phenomenon of division differs widely from what is seen to occur in the cells forming the somatic or body tissues. The period at which this divergence on the part of certain cells takes place is when the organism reaches sexual maturity, and the result is the production of cells which are in a suitable condition to fuse with cells that have passed through the same series of changes in a similar individual of the opposite sex, and so to create a fresh individual. The main feature of the form of mitosis by which these sexual cells are produced is, that the number of chromosomes is reduced to one-half of that found in dividing cells forming the somatic tissues of the same organism. This makes the utility of the phenomenon obvious. If the number of the chromosomes exhibited by the sexual elements remained 82 THE ESSENTIALS OF CYTOLOGY. the same as that of the cells forming the somatic tissues, the fusion of two cells would double their number, and all the cells forming the soma of the new individual would exhibit this double number. Not only would this happen but the number of the chromosomes would be doubled at each suc- ceeding generation. We know that in the same species the chromosomes are not increased by the fusion of the sexual elements, and this fact is explained by the reduction to one- half in the number of the chromosomes that occurs in the production of the mature sexual cells. The terminology with respect to this phenomenon of reduction is in a state of considerable confusion. In 1887 Flemming first described a form of mitosis which differed materially from the usual somatic form. This he called the “heterofcype ” mitosis. It has since been shown that it is by this peculiar form of mitosis that the chromosomes in the daughter-cells produced by it are reduced to one-half of the somatic number, and that this form of division always precedes the production of sexual elements. In the following pages it is proposed to use the term “Meiotic Phase ” for the whole period during which reduc- tion is taking place, and “ Meiotic Division ” for that particular division which is the culminating point in the Meiotic Phase. Though the series of phenomena observed during the Meiotic Phase is extremely complex, a great similarity exists between that occurring in animals and in plants, a similarity so striking that it may justly be said to differ only in minor details and not in essentials or in general outline. It is necessary, in order to obtain a clear conception, not Fig. 27. The Meiotic Phase Fig. 18. — The early fine spireme'. Fig. 19. — ' Thickening and double beading of the loops. Fig. 21.— The coarse spireme. Fig. 22.— Beg Fig. 23. — The second synaptic contraction fi Fig. 25. — The nuclear membrane has disappearec fibres. Fig. 27.— Separation of the meiotic genii In vertebrates and, in ” ‘ represented in Figs. 20 to 25 somes leave the archoplasm. The archoplasm c double beading. Minute flagellse grow from thee (Semi-diagrammatic). FIG. 26.— The e F io. 30. [To face p. 33. THE MEIOTIC PHASE. 33 only of the Meiotic Phase itself, but of the multicellular organism as a whole, to divide the several series of celk generations into Pre-Meiotic, Meiotic, and Post-Meiotic. The Pre-Meiotic generations are all those extending from the first segmentation of the fertilized ovum to the prophase of the Meiotic division. Thus all the cells forming the various tissues of the organism that are not passing or have not passed through the Meiotic Phase must be classed as Pre-Meiotic. The Meiotic Phase includes the series of changes follow- ing the last Pre-Meiotic division and the Meiotic division itself. The generations following the Meiotic Phase are Post- Meiotic, and the cells of these generations retain the reduced number of chromosomes. Post-Meiotic cells may form actual sexual elements, or may form tissues possess- ing the characters and functions of tissues formed of Pre- Meiotic cells, that is, tissues similar to those of the co-ordinate soma. The essential characters of the Meiotic Phase, common to both animals and plants, though very complex, are also very definite. It was seen that in the pre-meiotic cells the prophase of division is comparatively simple and brief. The linin and chromatin generally, though not always, appear in the form of a more or less definite thread or spireme, which immediately breaks up into a definite num- ber of segments (chromosomes). The prophase of the meiotic division, on the other hand, exhibits a number of extremely characteristic and very striking stages. The cells immediately destined to go through the meiotic phase grow considerably, thus being larger than most of the E.C. D 34 THE ESSENTIALS OF CYTOLOGY. somatic cells. The nuclear reticulum becomes closer, more chromatic and often polarised. The polarisation increases, and eventually it is seen that the linin and its contained chromatin is arranged in the form of a definite number of loops or rods. Where these loops can be counted, it is seen that there are half as many loops as there are chro- mosomes in the pre-meiotic cell when it divides. In those cases where special observations have been made, it is found that these loops have a particular origin and arrangement.1 2 In such cells, in the species which have been specially observed, it is found that a more or less constant number of chromatin masses are found at this stage and just before. The number of these masses, which in some organisms are in pairs, is generally one half of that of the somatic number of chromosomes.3 Where the number varies it is due either to the coalescence or to the unusually early division of some of these masses. From each mass!- a thread of linin, containing a varying amount of chromatin, passes into the substance of the nucleus, and if the course of these threads is traced it is seen that their other ends return into some part of the same chromatin mass from which they started (Fig. 18). This gives the appearance of two threads leading from each chromatin mass. In reality these threads are the ends of the loops whose number is half that of the somatic chromosomes. Shortly after this stage has been reached the loops begin to contract away from the nuclear membrane. As the con- traction takes place in these threads, the chromatin masses 1 Moore and Walker. 2 Where the masses are in pairs, the appearance of one quarter of the somatic number of chromosomes is given. THE MEIOTIC PHASE. 85 migrate to one part of the nuclear membrane, and that curious and striking arrangement of the nuclear elements arises to which the term “Synaptic ” was originally applied.1 This is the first “ synaptic contraction ” (Fig. 19). Some of the chromatin masses frequently coalesce at this period, and the loops become thicker and better defined owing to the presence of an increased amount of chromatin in the linin forming them. After a short time the chro- matin granules in the threads divide and give an appear- ance of splitting or double-beading to each individual loop2 (Fig. 20). Shortly after this has happened the chromatin masses divide, and it is eventually seen that each consists of the peripheral end of a loop. This implies that each original mass has divided into its two constituent parts, and that subsequently two ends of each loop are separated, each end carrying with it that part of the chromatin mass to which it was attached. There results, therefore, the same number of chromatic ends to the loops as there are somatic chro- mosomes in the pre-meiotic cell. Further, it is often easy to make out that each loop is more or less divided in the middle, so that there are the same number of bodies com- posed of linin containing chromatin in the cell at this stage of the meiotic phase as there are chromosomes in the pre- meiotic cell, though in the meiotic cell they adhere to each other in pairs (Fig. 21). As we shall see later, if it has not already become evident, each of these two components of the synaptic loops probably represents a pre-meiotic chro- mosome. 1 Moore, 1895. 2 Farmer and Moore D 2 36 THE ESSENTIALS OE CYTOLOGY. The moving apart of the ends of the loops is accompanied by a considerable increase in their thickness, and an accentuation of the splitting or double-beading, so that the threads are apparently longitudinally divided from end to end.1 The ends of the loops are apparently attached to the nuclear membrane, and at these ends the splitting of the chromatin is often as distinct as it is at any other part of the thread. The divergence of the ends of the loops also causes the loss of that appearance of polarity that was so striking during the first synaptic contraction. The arrange- ment of the nuclear contents appears to be quite irregular, the threads seeming, upon anything but a most detailed examination, to be continuous, which has led to this stage being called the “coarse spireme.” The details with regard to the loops and segments of the coarse spireme being separate from the commencement of the meiotic prophase have not as yet been followed in many forms. Except for this, however, the process appears to be uniform. This so-called coarse spireme stage is in many cases succeeded by another contraction in the loops in which the double beading is lost, and the two constituent parts of each are joined more or less completely end to end (Figs. 22 and 23). In this, the “second synaptic contraction,” the loops become much thickened and their ends often travel to one area of the nuclear membrane, so that an apparent polarity in the nuclear contents is again observed. The second con- traction figure lasts but a very short time in every case. While it is usual in plants, it is very often absent in animals. It appears to be absent in all vertebrates, but is very well 1 Farmer and Moore. THE MEIOTIC PHASE. B7 marked in the cockroach. Gradually each loop assumes a definite shape, and in assuming these shapes the changed loops, which are pairs of pre-meiotic chromosomes, hereafter called “ c « ro- #0 N f N *tr «* 'v’ N «5 CV)» 8 * X=* M t3 N jrj «. St « o ^ O * o * 0 7. Man 2. Rat 3. Triton, sp. 4. Cockroach 5. Gryllus assimilis 'SS'S'S O *3 d „ 0 g bc^j ^ ’d d 13 . «n « d -S-S*^ ^i— < q} d co _a M n -P W H 3 *43 g ~&§J 5 ®S • f-H 'U1 2 c3 ° ° 1 G .3 S « ^ d & g £ bcW d +3 0 fl g 43^ a go^ g* CD ’“l -u aj? § ^ d - •9 g * > •h^* d .S d M-rt c &C