The essentials of cytology : an introduction to the study of living matter, with a chapter on cytological methods

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

CH AP.

I. INTRODUCTORY

II. THE STRUCTURE AND PARTS OF THE CELL .

Chapter I. Introductory

Our knowledge of the properties of living matter generally 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 connection, 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

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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 undifferentiated 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.

Living organisms, whether animal or vegetable, may exist in the form of single independent cells — unicellular organisms, or may he composed of man}" 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 excepting 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 necessary 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

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THE ESSENTIALS OF 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 multicellular 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.

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 the result is also comparable — the fertilized individuals are 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 a.

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Fig. 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 reproduction 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 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 multicellular 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 conditions, 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 w^as 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.

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 hut 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, dependent 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,

THE ESSENTIALS OF CYTOLOGY.

and so on.1 If, however, the four cells be artificially separated without injury and are kept under suitable conditions, 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 the}r are dissociated. This reciprocal influence is called Somatic Co-ordination.

1 These derivatives of the four blastomeres are given merely as a hypothetical examj)le, to facilitate the explanation.

Chapter II. The Structure And Parts Of The Cell

We have seen that the whole soma or body of the multicellular 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, hut 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

THE ESSENTIALS OF CYTOLOGY.

under a microscope the nucleus appears denser in structure than the rest of the cell, and if the cell he 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 appearing practically homogeneous. Some kind of structure is the more usual form, but the nature of this structure varies considerabl}T. Perhaps it most frequently presents a finely granular appearance, or that of a fine reticulum or mesliwork. 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 membrane, the nucleus proper, may vary considerably. The most usual appearance however, 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.

STEUCTUEE AND PAETS OF THE CELL. 11 threadwork is called “ linin,” and the darkly staining substance is called “ chromatin.”

It may here he 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, hut the chromatin

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Fig. 4. — A semi-diagrammatic representation of a cell. nuclear membrane ; linin reticulum ; c, chromatin masses contained in envelopes of linin (chromatin nucleoli) ; d, true nucleolus ; e , vacuole ; f, plastids ; <7, 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 dye 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,

THE ESSENTIALS OF CYTOLOGY.

was the chromatin. Very strong evidence has however come to light which seems to show that the chromatin is prohahly no more than a secretion of the linin, and that it is the linin, if it he 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 sometimes 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 distinguish 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 contained 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 they 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 which 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 “ macronucleus ” seems to control various functions of the cell,

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 he absolutely constant. Apart from the fact that it disappears, as we shall see later, during the process of mitosis in cells where it is usualty present, some forms have been described in which there appears to he 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 protoplasm 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 tlie most striking modifications, as, for instance, in tlie 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 “chromato-

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.

Lying 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 be 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

THE ESSENTIALS OF CYTOLOGY.

the higher plants, though this is not quite conclusive evidence that the}7" 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 generation 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 be 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 condition 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 ma}T even go further, and say that only the limn with its chromatin and the general protoplasmic ground work are necessary, for the nuclear membrane disajjpears 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 connected 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 performed 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

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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 op the Process of Mitosi^ in a Cell Exhibiting 8 Chromosomes.

the form of a coiled-up thread (spireme). The

is in a more advanced stage of formation, ironed.

Metaphase. [Lateral view.]

i;d

Fig. 6. — A cell with the nucleus in the vegetative condition.

Fig. 6. — Early propbaseof division. The linin nnd chromatin have adopt( asters and spindle are forming in connection with the centrosomes.

Fig. 7. — The spireme has broken up into the chromosomes, and the spindle 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 opposite poles of the spindle. [Early anaphase. J Fig. 13.— Later stage of anaphase.

Fig. 14. — The daughter-cells are separated from each other. [Telophase.]

Fig. 15. — The reconstruction of the daughter nuclei.

[ To face p. 19

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 be 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