Talk:Book - A textbook of histology, including microscopic technic (1910) General Histology 1

From Embryology



DURING the latter part of the seventeenth century, Hooke, Malpighi, and Grew, making observations with the simple and imperfect microscopes of their day, saw in plants small compartment-like spaces, surrounded by a distinct wall and filled with air or a liquid ; to these the name cell was applied. These earlier observations were extended in various directions during the latter part of the seventeenth and the eighteenth century. Little advance was made, however, until Robert Brown (1831) directed attention to a small body found in the cell, previously mentioned by Fontana, and known as the nucleus. In the nucleus Valentin observed (1836) a small body known as the nucleolus. In 1838 Schleiden brought forward proof to show that plants were made up wholly of cells, and especially emphasized the importance of the nuclei of cells. In 1839 Schwann originated the theory that the animal body was built up of cells resembling those described for plants. Both Schleiden and Schwann defined a cell as a small vesicle, surrounded by a firm membrane inclosing a fluid in which floats a nucleus. This conception of the structure of the cell was destined, however, to undergo important modification. In 1846 v. Mohl recognized in the cell a semifluid, granular substance which he named protoplasm. Other investigators (Kolliker and Bischoff ) observed animal cells devoid of a distinct cell membrane. Max Schultze (1861) attacked vigorously the older conception of the structure of cells, proclaiming the identity of the protoplasm in all forms of life, both plant and animal, and the cell was defined as a nucleated mass of protoplasm endowed zvith the attributes of life. In this sense the term cell is now used.

The simplest forms of animal life are organisms consisting of only one cell (protozoa). Even in the development of the higher animals, the first stage of development, the fertilized egg, is a single cell. This by repeated division gives rise to a mass of similar cells, which, owing to their likeness in shape and structure, are said to be undifferentiated. As development proceeds, the cells of this mass arrange themselves into three layers, the germ layers, the outer one of which is the ectoderm, the middle one the mesoderm, and the inner one the entoderm. In the further development, the cells of the germ layers change their form, assume new qualities, adapting themselves to perform certain definite functions ; a division of labor ensues, the cells become differentiated. Cells having similar shape




and similar function are grouped to form tissues, and tissues are grouped to form organs.

We shall now consider the structure of the cell. Every cell consists of a cell-body and a nucleus.


The body of the cell consists of a substance known as protoplasm or cytoplasm. This is not a substance having uniform


Chromatin network.

Linin network. Nuclear fluid.

Nuclear membrane. Cell-membrane.


Spongioplasm. Hyaloplasm.

Nucleolus. Chromatin net-knot.

Centrosome. Centrosphere.

Foreign inclosures. Metaplasm. Fig. 10. Diagram of a cell.

physical and chemical qualities, but a mixture of various organic compounds concerning which knowledge is not as yet conclusive, but which in general are proteid bodies or albumins in the widest sense.

In spite of the manifold differences in its composition, protoplasm exhibits certain general fundamental properties which are always present wherever it is found. Ordinarily, protoplasm exhibits certain structural characteristics. In it are observed two constituents, threads or plates, which are straight or winding, which branch, anastomose, or interlace, and which are generally arranged in a regular framework, network, or reticulum. These threads probably consist of or contain small particles arranged in rows, called cellmicrosomes (vid. van Beneden, 83 ; M. Heidenhain, 94; and others). Benda, who has devoted much time to the study of certain protoplasmic structures, has found in these threads small granules or



rod-shaped structures to which he has given the name "threadgranules" or mitochondria. The mitochondria can be differentially stained and are not distributed irregularly through the cell protoplasm, but in certain definite regions. They are regarded as in part identical with the microsomes. This thread-like substance is known as protoplasm in the stricter sense (Kupffer, 75); also as spongioplasui, or the fibrillar mass of Flemming (82). The other constituent of the cytoplasm is a more fluid substance lying between the threads in the meshes of the spongioplastic network, and is known as paraplasm (Kupffer), hyaloplasm, cytolymph, or the interfibrillar substance of Flemming. According to most investigators, the more important vital processes of the cell are to be identified with the spongioplasm, and are controlled by the nucleus, while the paraplasm assumes an inferior or passive role. With special methods Altman (94) was able to demonstrate granules in the protoplasm, associated with, but not in the spongioplastic threads. To these he gave the name bioblasts, and referred the vital qualities of the protoplasm to them. Butschli believes the protoplasm to consist of


HdH-.. Cell-body.

" ~ Nucleus.

Fig. 1 1. Cylindric ciliated cells from the primitive kidney of Petromyzon planeri ;

X 1200.

separate, honeycomb-like spaces, which give it a foam-like structure foam-structure of protoplasm.

Protoplasm displays phenomena of motion, shown on the one hand by contraction, and on the other by the formation of processes that take the form either of blunt projections or lobes, or of long, pointed, and even branched threads or processes known as pseudopodia. The extension and withdrawal of the pseudopodia enable the cell to change its position. The point of such a process fastens to some object and the rest of the cell is drawn forward, thus giving the cell a creeping motion ivandering cells. Certain cells take up and surround foreign bodies by means of their pseudopodia. If these bodies are suitable for nutrition, they are assimilated ; if not, they can, under certain circumstances, be deposited by the cell in certain localities (Metschnikoff 's phagocytes). Similar thread-like processes which, however, can not be drawn into the cell, occur in some cells in the shape of cilia, which are in constant and energetic motion ciliated cells. Certain cells possess only a single long process, by means of which unattached cells are capable of direct or rotating motion -flagellate cells, spermatozoa.


Inside of the cell-body the protoplasm also shows phenomena of motion, the streaming of the protoplasm. In plant cells there is often a noticeable regularity in the direction of the current. Mention should not be omitted of the so-called molecular or Broivnian movement in the cells, which consists in a rapid whirling motion of particles or granules suspended in the protoplasm (Brown).

Living protoplasm is irritable in the highest degree, and reacts very strongly to chemic and physical agents. It is very sensitive to changes in temperature. All the phenomena of life occur in greater intensity and more rapidly in a warm than in a cold temperature, this fact being very strikingly shown by the phenomena of motion in the cell, as also in its propagation. By subjecting protoplasm to different temperatures, its various movements can be slowed or quickened. It dies in too high or too low a temperature.

Certain substances coming in contact with the cell from a given direction have on it an attracting or repelling action. These phenomena are known as positive and negative chemotropism (chetnotaxis}. The action of chemic agents on the different wandering cells of the body and on certain free-swimming unicellular organisms naturally varies to a great degree. Among these phenomena must be included those produced by water (hydrotropism) and light (heliotropism). It is very probable that all these phenomena are of importance to the proper appreciation of some of the processes going on in the vertebrate body (as, for instance, in the origin of diseases caused by micro-organisms).

Protoplasm may contain various structures. Of these, the vacuolcs deserve special mention. They are more or less sharply defined cavities filled with fluid, and vary considerably in number and size. The fluids that they contain differ somewhat, but are always secreted by the protoplasm, and are, as a rule, finally emptied out of the cell. As a consequence, vacuoles are best studied where the function of the cell is a secretory one. Here they are often large, and sometimes fill up the whole cell, the contents of which are then emptied out (glandular cells).

Contents of a solid nature, such as fat, pigment, glycogen, and crystals, are peculiar to certain cells. By these deposits the cell is more or less changed, the greatest variation in form taking place in the production of fat. The latter, as a rule, takes the shape of a globule, and greatly modifies the position of the normal constituents of the cell. Deposits of pigment alter the cells to a less degree. This substance occurs in the protoplasm either in solution or in the form of fine crystalline bodies. Glycogen is more generally diffused, occurring very generally in embryonal cells and in the liver- and cartilage-cells of the adult. Occasionally we find larger crystals in animal cells, as, for instance, in the red blood-corpuscles of the teleosts. So-called margarin crystals sometimes occur in large numbers as stellate figures in dead fatty tissues kept at low temperatures.


Many cells are without a distinct cell membrane, another constituent of the protoplasm. In such cells the outer layer of the protoplasm is often more homogeneous and less dense than that lying more centrally, which has often a more granular appearance; the outer layer of the protoplasm is in such cells known as the exoplasm, in contradistinction to the more granular endoplasm.

In other cells, however, the outer layer of the cell-protoplasm shows differentiation, leading to the formation of a distinct cell-membrane (as in fat-cells, cartilage-cells, goblet-cells, etc.). F. E. Schulze has given it the name pcllicula in cases where the entire cell is surrounded by a homogeneous layer, and ciiticula or cuticle where only one side of the cell is supplied with the membrane (as in the intestinal epithelium). It is assumed that both spongioplasm and paraplasm are concerned in the formation of this membrane.

In the protoplasm of many cells there is found a small body known as the centrosome. This is usually situated near the nucleus of the cell, occasionally in the nucleus. Generally, it has the appearance of a minute granule, sometimes scarcely larger than a microsome. It is often surrounded by a small area of a granular or finely reticular or radially striated cytoplasm, ( known as the attractionsphere or centrosphere.


The second constant element of the cell is the nucleus. As a rule, it is sharply defined, and in its simplest form consists of a round vesicle of a complicated structure composed of several substances. The form of the nucleus corresponds in general to the shape of the cell ; in an elongated cell, it is correspondingly long, and flattened where the cell is plate-like in shape. The nucleus of a wandering cell that is in the act of passing through a narrow intercellular cleft adapts itself to the changes of form in the cell without being permanently altered in shape. In other words, the nucleus is soft, and can be easily distorted by any solid substances within or without the protoplasm, only to resume its original form when the pressure is removed. It possesses, then, a certain amount of elasticity. Movements of certain nuclei, entirely independent of the surrounding protoplasm, have often been observed. It is only rarely that the general form of the nucleus differs materially from the general form of the cell. This, however, occurs in the nuclei of leucocytes and many of the giant cells of bone-marrow, which are often irregular, and may even be ring-shaped. In certain arthrozoa, branching forms of nuclei occur, as also in the skin glands of turtles. The proportionate size of nucleus to cell-body varies greatly in different cells. Especially large nuclei are found in immature ova, in certain epithelial cells, etc.

The contents of the nucleus consist of a framework or reticulum, in the meshes of which there is found a semifluid substance.


In treating the nuclei with certain stains, the nuclear reticulum will be seen to consist of two constituents, a substance appearing in the form of variously shaped, minute granules, which stains deeply, and is, therefore, known as chromatin. This is imbedded in and deposited on a less stainable network, the linin. The meshes of this network are occupied by a transparent, semifluid substance, which does not stain easily, and is known as the achromatic portion of the nucleus. It is also known as paralinin, nuclear sap, karyolymph, or nucleoplasm. Chemically, cliromatin belongs to those albuminous substances known as nucleins.

In well-stained nuclei of considerable size the chromatin granules are seen closely placed in a continuous row throughout the network of linin, which penetrates the nuclei in all directions. In every resting nucleus one or more small round bodies are found imbedded in the nucleoplasm. These are known as true nuclcoli, and do not stain quite so deeply as the chromatin. The fact that certain reagents dissolve the chromatin, but not the true nucleoli, proves that the substance of which the latter are composed is not identical with chromatin, and is, therefore, known as paramtclcin (F. Schwartz).

In many cases we find in the linin, granules of a substance known as lanthanin, which displays a marked affinity for the socalled acid anilin stains, in contradistinction to chromatin, which stains principally with the basic anilin colors. These are known as oxychromatin granules in contradistinction to the basichromatin granules of the chromatin (M. Heidenhain, 94).

The true nucleoli should not be confused with the slight swellings of the chromatin network found at the junction of the threads, and known as net-knots, or karyosomes.

Surrounding the resting nucleus is usually a nuclear membrane (amphipyrenin) resembling in many respects chromatin. As a rule, it does not form a continuous layer, but is perforated, having openings that contain nuclear fluid. We have, then, both substances, chromatin and nucleoplasm, as elements of the nuclear membrane. Besides this, the nuclear membrane receives an outer layer, differentiated from the protoplasm. Later Investigations have shown that even during a period of rest the relationship of the nucleus to the protoplasm of the cell is much more intimate than was heretofore believed (yid. Reinke, 94).

A resting nucleus i.e., one not in process of division usually consists, therefore, of a sharply defined membrane (amphipyrenin), which has in its interior a chromatic (nuclein) and an achromatic (linin) network, a nuclear fluid (paralinin), and nucleoli (paranuclein).

The chromatin of the nucleus is not always in the form of a network. In some cases as, for instance, in the premature ova of certain animals (O. Hertwig, 93. II) and in spermatozoa it is collected in compact bodies. In the ova it may often be mistaken for a true nucleolus (germinal spot). In this case, however, it consists of nuclein, and not of paranuclein.



The founders of the cell theory believed in what may be known as a modification of the theory of spontaneous generation, stating that cells might originate from a structureless substance known as kytoblastema or blastema, in which a nucleus was formed by precipitation. Henle (1841) drew attention to the fact that cells might multiply by the separation of small portions of the cell-body, a process known as budding; and Barry (1841) stated that during the multiplication of cells the nuclei divided. The same year Remak observed division of cells in the blood of embryos. Goodsir (1845) originated the theory that all cells were developed from preexisting cells. This was first clearly stated as a general law by Virchow (1855), and his saying, " Omnis cellula e cellula," is constantly being verified. Our more accurate knowledge of cell-division dates, however, from more recent times (187380), when Schneider, Fol, Strasburger, Flemming, and many others demonstrated that during the division of the cell the nucleus passed through a series of complicated changes which resulted in an exact division of the chromatin.

The phenomena which usher in cell-division are especially noticeable in the nucleus, the elements of which are arranged and transformed in a typic manner. During the division of the nucleus the nuclear membrane is lost, and the relationship of the substances of the nucleus to the protoplasm of the cell is a very intimate one. As a consequence, during the middle phases of division there is no well-defined demarcation between the nucleus and the cell-body. As a rule, the mother cell and nucleus divide into two daughter cells, each having a nucleus, alike in every particular. It was early observed, however, that occasionally cells divided by a much simpler process, in which case the nucleus did not pass through such complicated changes. Accordingly, two distinct types of celldivision are recognized, which are distinguished as mitosis, karyokinesis, or indirect cell-division, and amitosis, or direct cell-division. Both lead to the formation of two nuclei, which are known as daughter nuclei as distinguished from the original mother nucleus.


The description of the process of mitotic cell-division is complicated by the fact that structural changes are observed which occur simultaneously in the nucleus, centrosome, and cytoplasm. This fact should be borne in mind, as, for the sake of clearness, a separate description of the changes involving each of these structures seems demanded. The process of mitotic cell-division may be divided into four periods or phases, which follow one another without clearly defined limits :

The prophascs, in which the nuclear membrane disappears, the chromatin is transformed into definite threads, and the centrosome



Figs. 12-21. Ten stages of mitotic nuclear division from the oral epithelium of the larva of a salamander. (Plate I, Sobotta and Huber's " Atlas and Epitome of Human Histology," 1903) : Fig. 12, Cell with resting nucleus ; Fig. 13, cell with nucleus at the beginning of mitosis; Fig. 14, nuclear membrane has disappeared, chromosomes in a loose skein, pole field at the left; Fig. 15, monaster viewed from above ; Fig. 16, monaster viewed from the side, achromatic spindle is also shown ; Fig. 17, monaster viewed from the side, with chromosomes crowded closely about the equator of the spindle ; Fig. 18, stage of metakinesis ; Fig. 19, diaster with beginning constriction of the cell-body: Fig. 20, dispirem with completion of the cell division ; Fig. 21, telophase.

and centrosphere undergo important changes. This is the preparatory stage.

The metaphases, in which the division and the separation of the chromatin take place.

The anaphascs, in which the daughter nuclei are formed and the' cell-protoplasm begins to divide.

The telopliases, in which the division of the cell is completed. 5



Fig. 22

Fig. 23.

Fig. 24.

Fig. 25. Fig. 26.

Figs. 22-26. Mitotic cell-division of fertilized whitefish eggs Coregonus albus.

Fig. 22, Cell with resting nucleus, centrosome, and centrosphere to the right of the nucleus ; Fig. 23, cell with two centrospheres, with polar rays at opposite poles of nucleus; Fig. 24, spirem; Fig. 25, monaster; Fig. 26, metakinesis stage.

To give a better understanding of the process we have inserted a series of figures in which several phases of mitotic division are portrayed. In figures 12-21 are shown ten stages of mitotic nuclear division from the oral epithelium of the larva of a salamander, in which changes undergone by the nucleus and centrosome are clearly brought out. And, further, a series of figures (2229) showing the different phases of mitotic cell-division of the fertilized eggs of the whitefish {Coregonus albas} ; the changes involving the centrosome, centrosphere, and cytoplasm are illustrated. Figure 30, showing a small portion of a section through the testis of the salamander, the object in which Flemming first observed this complicated series of changes, presents the appearance more generally seen during mitotic cell-division of the tissue cells of the higher vertebrates.

(a) Prophases. -The changes occurring in the nucleus will be considered first. At the beginning of the process of mitosis, the chromatin network, consisting of chromatin granules, is transformed into a twisted skein of threads, beginning at the periphery of the


Fig. 27.

Fig. 28.

Fig. 29.

Figs 27-29. Mitotic cell-division of fertilized whitefish eggs Coregonus albus. Fig. 27, Metakinesis stage; Fig. 28, diaster; Fig. 29, late stage of dispirem, the cell-protoplasm almost divided.

nucleus. This skein of threads is known as the spirem or mother skein, and may appear as a single thread, which breaks up into a definite number of segments, or the segments may appear as such when the skein is forming. . At first the threads are coarse and often somewhat irregular, staining much more deeply than the linin network. The separate segments of chromatin are known as chromosomes (Waldeyer, 88). They appear, as a rule, in the form of rods varying in length and thickness, and staining very deeply, and often bent into characteristic U-shaped loops. The bent portion of each loop is called its crown. " Every species of plant or animal has a fixed and characteristic number of chromosomes, which regularly recurs in the division of all its cells ; and in all forms arising by sexual reproduction the number is even" (Wilson, 96). In man the number of chromosomes is given as sixteen by Bardeleben (92) and Wilson (96), and as twenty-four by Flemming (98). During the formation of the spirem the nuclear membrane, as a rule, disappears. The nucleolus is also lost sight of, although the manner of its disappearance can not be definitely stated. The netknots are no doubt taken up by the chromosomes. The chromo


somes are now free in the protoplasm ; gradually the crown of each chromosome approaches the center of the space occupied by the nucleus, and the chromosomes form a characteristic, radially arranged stellate figure, known as the monaster, in the equatorial plane of the cell. During the progress of the changes affecting the chromatin of the nucleus and resulting in the formation of the chromosomes, important phenomena are observed, connected partly with the achromatic substance of the nucleus, more especially with the centrosome, centrosphere, and cytoplasm of the cell. These phenomena result in the formation of a complicated structure known as the acliromatic spindle or amphiaster. Its development is as follows : The centrosome and centrosphere, as has been stated, usually lie in the protoplasm to one side of the nucleus. If, at the beginning of the division, the centrosome be single, it divides, and the two centrosomes begin to separate, causing a division of the centrosphere. Between the centrosomes are usually seen finely drawn-out connecting threads. The centrosomes, each of which is surrounded by a centrosphere, now move apart, and a structure known as the central spindle, and consisting of fine threads arranged in the form of a spindle, develops between them. At each end of the central spindle is found a centrosome surrounded by a centrosphere from which radiate into the cytoplasm fine fibers known as polar rays. During the formation of the achromatic spindle the nuclear membrane disappears and the chromosomes develop, as above described. Some fibers, which seem to have their origin from the centrosphere, grow into the spirem formed of chromosomes, which they appear to pull into the equatorial plane of the cell, which is also the equator of the central spindle. Thus, the nuclear figure above described as the monaster is formed. In other cases the centrosomes and centrospheres continue moving apart until opposite each other and separated by the nucleus (Figs. 23, 24). As the nuclear membrane disappears and the spirems and chromosomes are forming, the central spindle develops, its fibers running from centrosphere to centrosphere. The polar rays also develop in the cytoplasm at the same time. As the central spindle develops, the chromosomes arrange themselves or are arranged about its equator monaster.

(//) Metaphases. Usually, during the formation of the monaster, or immediately after its formation (sometimes in the spirem stage or even earlier), the most important process of cell -division takes place. Each chromosome divides longitudinally into two daughter chromosomes. The loops first divide at the crown, the cleft extending up either limb until the free ends are reached. The smallest particle of chromatin divides, retaining the exact relative' position in the twin chromosomes that it possessed in the mother chromosome. The daughter chromosomes now wander over the central spindle, their crowns presenting, in opposite directions toward the poles of the cell. This process is known as metakinesis. Two stel


6 9

late figures are developed about the respective poles of the central spindle. The appearance presented is known as a diaster. Our knowledge of the part taken by the amphiaster or achromatic spindle in metakinesis is not above controversy. It would appear, however, that certain cytoplastic fibers, which arise from the centrosphere and hang over the central spindle and chromosomes, designated as mantle fibers, assist in drawing the daughter chromosomes toward the poles of the central spindle.

(c] Anaphases. After the formation of the diaster, the loops belonging to each stellate figure are joined together to form a skein, thus forming the dispirem. The chromatin threads of the two skeins gradually assume the disposition found in the resting nucleus. This process takes place in such a way that the threads of the





Resting nucleus. Metakinesis.


- Daughter cells.

Spirem. Fig. 30. Mitotic division of cells in testis of salamander (Benda and Guenther).

skeins (or the single thread) send out lateral processes. These interlace, and little by little reproduce the network of the resting nucleus ; at the same time the nuclear membrane and the nucleolus reappear. In this stage the changes that lead to the division of the cell-body are observed. In some cases the division of the cell-body is ushered in by an equatorial differentiation of the connecting threads of the central spindle. Chains of granules, arranged in double rows, are seen to appear in this region. The cell now begins to contract at its equator, the contraction extending between the two chains of granules until the cell is completely divided. At this time, also, the threads of the amphiaster disappear or are drawn into the nucleus. The centrosomes, with centrospheres, again lie by the side of the daughter nuclei.


According to the opinion of C. Rabl (85), there remains in the nucleus, even after it has fully returned to a state of rest, a polar arrangement of the chromatin loops that is, an arrangement of the axis of the loops in the direction of the centrosphere. The area toward which the crowns of the loops point is known as the polar field.

The equatorial differentiation of the connecting threads of the central spindle, above mentioned, was first observed in vegetable tissue, and is known as the cell-plate. (Fig. 29.) In animal cells such a plate is relatively rare, and, when seen, is found developed in a rudimentary form (v. Kostanecki 92, I).

(d} Telophases (M. Heidenhain 94). In these phases of mitosis the cell divides completely. The daughter nuclei and centrospheres, which do not yet occupy their normal position in the daughter cells, show movements that result in their assuming their normal positions.

From our description it is seen that the anaphases represent the same stages as the prophases, only in an inverted sequence. In the latter case, the result is the resting nucleus, while the prophases lead to the metaphases.

The fertilized ovum also divides by indirect nuclear division. (Figs. 22-29.) From it are derived, by this process, the segmentation cells, or blastomeres, from which the whole embryo is developed.

(<?) The Heterotypic Form of Mitosis. The above-described type of indirect or mitotic nuclear division (Jwvieotypic mitosis) is the usual one. Variations, however, occur, as, for instance, in the so-called heterotypic form of division (Flemming 87), which occurs in certain cells of the testes (spermatocytes). In this form the first stages are lacking, the nucleus possessing from the beginning a skein-like structure. The longitudinal splitting and division of the chromatin threads take place during the first spirem stage, after which there is a phase in which the figure may be compared with an aster of ordinary mitosis, although the free ends of the threads in this case are seldom observed. The latter is due to the fact that after the longitudinal splitting, the ends of the chromosomes remain united, or, if entire separation occurs, they are again joined. In this way closed loops are formed extending from pole to pole. Later the threads break at the equator and move toward the poles, again dividing to form the daughter stars.


Very different from the indirect form of nuclear division is the direct or amitotic. It appears to occur seldom as a normal process, and is only exceptionally followed by a subsequent cell-division (yid. Flemming, 91, III). As a consequence, this process, in most cases, results in the formation of polynuclear cells (polynu clear leucocytes, giant-cells, etc.). The complicated nuclear figures of


indirect division are here entirely absent. The nucleus merely contracts at a certain point and separates into two or more fragments (direct fragmentation, Arnold) ; often the nucleus first assumes an annular form and then breaks up into several fragments, which remain loosely connected (polynuclear cells). Centrospheres are also present, and appear to take a prominent part in the whole process, although the exact relationship between the achromatin and chromatin has not as yet been determined.

Xemiloff has recently called attention to two locations where amitotic divisions may readily be observed namely, in the large surface cells of transitional epithelium of the bladder of mammals and in the lymphoid tissue layer of the liver of amphibia. In the cells of the former type the nuclear division is initiated by a division of the nucleolus which is followed by a division of nucleus and later the protoplasm. Centrosomes and attraction spheres were not noticed in these cells. The division of the lymphoid cells of the amphibian liver is initiated by a depression found in one side of their spherical nuclei. This depression deepens until the nuclei become perforated and assume an annular shape. These ring-shaped nuclei then break through in two or more places and two or more daughter nuclei are formed. During the process of division a centrosome with attraction sphere may often be observed, generally situated in the depression which initiates the division and later in the center of the perforated nucleus. Its role in the division of the nucleus and the .cell-body is, however, not fully understood.


The sexual cells form a special group among cells in general. Before the division of the egg-cell leading to the development of the embryo can take place, the ovum must be impregnated (the socalled parthenogenetic ova are an exception to this rule). Fertilization is produced by the male sexual cell, the spermatozoon.

The process of fertilization consists in a conjugation of two sexual cells, and in this process certain peculiarities in the behavior of both cells must be mentioned.

The cell forming the ovum and the one forming the spermatozoon must pass through certain stages before fertilization can be accomplished. These consist in the loss of half their chromosomes by the nuclei of both sexual cells. In this way are produced the matured sexual cells (ova and spermatozoa), which retain only half of the number of chromosomes of a somatic (body-) cell. In the conjugation of the male and female sexual cells their nuclei unite to form a single nucleus, known as the segmentation nucleus. Consequently, this nucleus contains the same number of chromosomes as does that of a somatic cell.

In its earlier developmental stages the ovum is an indifferent cell, the nucleus of which is known as the germinal vesicle. As the


Membrane of ovum.

Nucleus of

ovum. Spermatozoon


Protoplasm of ovum with deutoplastic granules.

Fig. 31.

Female pronucleus.

Head of spermatozoon with centrosome.

Female pronucleus.



Fig- 32. Fig. 33:

Figs. 31-33. Diagrams of the process of fertilization, after Boveri. Figure 31, the ovum is surrounded by spermatozoa, one of which is in the act of penetration. Toward it the yolk is pushed forward in a short, rounded process. Figure 32, the tail of the spermatozoon has disappeared. Beside the head is a centrosome with polar radiation. Figure 33, the pronuclei approach each other.

ovum matures the germinal vesicle approaches the periphery, and a peculiar metamorphosis, which may be regarded as a double, unequal division of the egg-cell, takes place. One portion, in the case of both divisions, is much smaller than the other, and is known as a polar body. At the close of these divisions, during which the chromosomes have been reduced to half the original number, there are, therefore, two polar bodies and the matured ovum, which is now ready for impregnation.

The development of the male sexual cell in its earlier stages is similar to that of the ovum. They are derived from cells known as spermatogones. These divide into equal parts, forming the cells of a second generation, the spermatocytes. From a further division of the spermatocytes, during which division the chromosomes are reduced to half the number, the spermatids are produced. These latter are then changed directly into spermatozoa. The reduction division of the egg-cell and that of the spermatocytes is in principle the same, except that in spermatogenesis all cells become matured sexual cells




Female pronucleus.

Chromosomes of egg-nucleus.

Chromosomes of male pronucleus.


Fig. 34

Fig. 35

Chromosomes from egg-nucleus.

'."}-- Chromosomes from spermnucleus (male pronucleus).

-- Centrosome.

Fig. 36.

Figs. 34-36. Diagrams of the process of fertilization, after Boveri. Figure 34, from the spirems in the pronuclei, chromosomes have been formed. The centrosphere has divided. Figure 35, the double chromosomes of the two pronuclei lie in the equatorial plane of the ovum. Figure 36, the ovum has divided. Chromosomes from the male and female elements are seen in equal numbers in both daughter nuclei.

(spermatozoa). In short, there is here an absence of structures analogous to the polar bodies, which degenerate after maturation of the ovum.

The spermatozoa are flagellate cells. The head consists principally of nuclear substance, to which is added a smaller middlepiece containing, according to the investigations of Pick, the centrosome. These two portions of the male sexual cell, the head- and middle-piece, are the most important, and are exclusively concerned in fertilization, the flagellum or tail playing no part in this process.

The spermatozoon usually penetrates the ovum after the first polar body has been extruded. The tail disappears during this process, being either left at the periphery of the egg or dissolved in the protoplasm. From this time the head represents the so-called male pronucleus t and the middle-piece the centrosome. From this stage the male pronucleus undergoes changes, the first of which consists of a loosening of the chromatin. Chromatin granules are


formed, which later arrange themselves in the form of chromosomes.

After the second polar body has been extruded, the chromatin remaining in the ovum is transformed into the female pronucleus. The latter then approaches the male pronucleus, the membranes of both nuclei disappearing. The chromosomes of the two nuclei thus formed are of equal number, and now come to lie together. After a longitudinal division of the chromosomes, the daughter chromosomes glide along the filaments of the achromatic spindle, developed from the centrosome of the male pronucleus, toward its two poles, as in ordinary mitosis. This they do in such a manner that an equal distribution of the male and female daughter chromosomes results. Then follow the stages of the anaphase.

From the above description of the process of fertilization it is seen that it consists, in the end, of a union of the nuclei of both sexual cells.

If paternal qualities are inherited by the offspring, this can only take place through the nucleus, or through the centrosome of the male sexual cell. In other words, it can be safely said that these structures, or the nucleus alone, are the principal means of transmitting inherited qualities. The same may also be said of the female pronucleus. There is no doubt that the first two segmentation cells of the ovum are equally provided with male and female nuclear elements. Since all future cells are derivatives of these two, it is possible that the nucleus of every somatic cell (body-cell) is hermaphroditic.


In the living organism many cells are destroyed during the various physiologic processes and replaced by new ones. On the death of a cell, changes take place in its nucleus which result in its gradual disappearance. These processes, which seem to follow certain definite but as yet unfamiliar laws, have been known since their study by Flemming (85, I) by the name of cliromatolysis (karyolysis). The nuclei during the course of these changes show many varied pictures.


In a fresh condition, cells do not show much of their internal structure. Epithelial cells of the oral cavity, which can easily be obtained and examined in the saliva, show really nothing except the cell outlines and the nuclei. More, however, can be seen in young ova isolated from the Graafian follicles of mammalia ; or the examination may be facilitated by using the ovary of a young frog. Tissues that are especially adapted for the observation of cells in a fresh condition are small ova, blood-corpuscles, and epithelia of certain invertebrate animals (shellfish,


etc.). Unicellular organisms such as amebae, infusoria, and many low forms of vegetable life make also good material for this purpose.

Protoplasmic currents are best seen in the tactile hairs of the nettle. Should fresh animal cells be desired, amebae can occasionally be found in muddy or marshy water. The same phenomena may be observed in the leucocytes of the frog or, better still, in the blood of the crab.

In order to make a detailed study of the minute relationship of the different cellular structures, it is necessary to fix the cells ; the same is true of nuclear division and cell proliferation. Although this process has been observed in living cells, it was not until it had been thoroughly worked out in preserved preparations. The best results in the study of the cell are obtained by methods that will be subsequently described. Fresh tissues are absolutely essential.

According to Hammer, mitosis in man does not cease immediately after death. The nuclei suffer chromatolytic destruction, and the achromatic spindle is the last element to disappear.

Flemming's solution here deserves first mention as a fixative. The tissues are imbedded, sectioned, and stained with safranin. An equally good fixative is Hermann's solution, which may be combined with a subsequent treatment with pyroligneous acid. Rabl fixes with a o. i-o. 12 '-/< solution of chlorid of platinum, washes with water, passes into gradually stronger alcohols, then stains with Delafield's hematoxylin, and finally examines the preparation in methyl alcohol.

Mitoses can also be seen by fixing in corrosive sublimate, picric acid, chromic acid, etc., and staining in bulk with hematoxylin or carmin, although perhaps not so well as by the preceding method. The objects to be examined are best when obtained from young and growing animals, especially those possessing large cells. Above all are to be recommended the larvae of amphibia, like the frog, triton, and salamander. If examination by means of sections be undesirable, thin structures should be procured, such as the mesentery, alveoli of the lungs, epithelium of the pharynx, urinary bladder, etc. These have the advantage of enabling one to observe the whole cell instead of parts or fragments of cellular structures. In sections of a larva that has been fixed in toto, mitotic figures can be seen in almost all the organs, and are particularly numerous in the epithelium of the epidermis, gills, central canal of the brain and spinal cord, etc. Other organs, such as the blood, liver, and muscle, also show mitoses.

Certain vegetable cells are peculiarly adapted to the study of mitosis, as, for instance, those in the ends of young roots of the onion. The onion should be placed in a hyacinth glass filled with water and kept in a warm place. After two or three days numbers of small roots will be found to have developed. Beginning at the points, pieces 5 millimeters in length are cut, which are treated in the same manner as animal tissues. These are then cut, either transversely or longitudinally, into very thin sections (not over 5 // in thickness). In one plane, polar views of the mitoses are obtained ; in the other, lateral views.

The methods used for demonstrating the remaining parts of the cell and its nucleus (except the chromatin) are, as a rule, more complicated, and. consequently less reliable. In order to see the centrosome, the spindle fibrils, the linin threads, and the polar rays, one of the


methods already described may be used; viz., the treatment with pyroligneous acid of objects previously fixed in osmic acid mixtures.

According to Hermann (93, II), sections from such preparations can be double-stained as well as those that have not been treated with pyroligneous acid. They are accordingly stained with safranin in the usual manner, and afterward treated from three to five minutes with the following solution of gentian violet : 5 c.c. of a saturated alcoholic solution of the stain is dissolved in 100 c.c. of anilin water. The 'latter is composed of 4- c.c. of anilin oil in 100 c.c. of distilled water. This is shaken in a test-tube and then filtered through a wet filter. The sections are then placed in a solution of iodin and iodid of potassium (iodin i gm., iodid of potassium 2 gm., water 300 c.c.) until they have become entirely black, after which they are immersed in alcohol until they receive a violet tinge with a slight dash of brown. By this means the chromatin network, the resting nuclei, and the chromosomes in both of the spirem stages appear bluish-violet, while the true nucleoli are pink. The chromosomes of the aster and diaster are colored red.

Flemming (91, III) recommends the following method: Fixation by his mixture ; the specimens or thin sections are then placed in safranin from two to six days, washed for a short time in distilled water, and then immersed in absolute alcohol weakly acidulated with hydrochloric acid (i : 1000), until no more color is given off. They are then washed again with distilled water and placed in a concentrated solution of anilin-watergentian-violet from one to three hours. After a third rinsing in distilled water, they come into a concentrated aqueous solution of orange G, until they begin to assume a violet color. Then wash with absolute alcohol, clear in clove or bergamot oil, and mount in Canada balsam.

A comparatively simple method showing the different structures of the cell and its nucleus with great clearness consists in staining with Heidenhain's hematoxylin.

Solger (89, I and 91) has discovered that both chromosomes and polar rays are shown in an exquisite manner in the pigment cells of the skin (corium) of the frontal and ethmoidal regions of the common pike (vid. Fig. 37). The preliminary treatment is optional, Flemming's solution or corrosive sublimate being the best. These cells illustrate the stability of the radiate structures of protoplasm, the polar rays showing as parallel rows of pigment granules.

The various structures of resting and dividing nuclei and cells are of such a complicated nature that they can be observed only with great difficulty in ordinary objects, because of the crowding of so many elements into a comparatively small space. For example, salamandra maculosa, which has become a classic histologic object through the researches of Flemming, possesses somatic cells whose nuclei have no less than twenty-four chromosomes. (It may here be remarked that, curiously enough, salamandra atra has only half this number. ) Consequently, van Beneden's discovery (83), that the somatic cells of ascaris megalocephala have only four primary chromosomes, is a fact of considerable importance. Boveri (87, II and 88) has even found an ascaris showing only two chromosomes. As these animals also show distinct achromatic figures in the protoplasm of their ova and sperm cells, they are certainly worthy of being regarded as typic specimens for laboratory __ purposes. The processes of cell-proliferation are almost diagrammatic in their distinctness.



After opening the abdominal wall of the animal, the ovisacs are removed, their numerous convolutions separated as much as possible, and then fixed for twenty-four hours in a picric-acetic acid solution (a concentrated aqueous solution of picric acid diluted with 2 vols. of water to which i per cent, glacial acetic acid is added). Then follows washing for twenty-four hours with water, after which the specimen is transferred to increasing strengths of alcohol (Boveri, ibid.^). Different regions of the ovisacs contain ova in various stages of development, those nearest the head containing cells ripe and ready for fecundation, while in the more posterior regions are ova in varying stages of segmentation showing mitoses. Specimens fixed in the manner above described can be stained with a borax-carmin solution. After staining, the ova are gently pressed out with needles upon a slide, separated, covered with a cover-glass, and cleared by gradual irrigation with glycerin. The ova, especially the segmentation spheres, are very small, and can be examined only under high magnification. In spite of the minuteness of the object and the fact that the yolk does not take the stain, and, on account of



':; :;; (U__Centrosphere.

- 37- Pigment cell from the skin of the head of a pike ; X 650.

its high refractive index, distorts the picture to a considerable extent, the mitotic figures are beautifully distinct.

Certain methods of treatment bring out in both cells and nuclei the presence of peculiar granules. The latter have been especially studied and described by v. Altmann (94, 2d ed. ). The methods that he applies are as follows : The specimens of organs of recently killed animals are fixed in a mixture consisting of equal volumes of a 5% aqueous solution of potassium bichromate and a 2% solution of osmic acid, remaining in the mixture for twenty-four hours. They are then washed for several hours in water and treated with ascending strengths of alcohol ; viz., 70, 90, and 100%. The specimens are now placed in a solution of 3 parts of xylol and i part of absolute alcohol, then in


pure xylol, and finally in paraffin. The tissues imbedded in paraffin must not be cut thicker than i to 2 //.

Altmann mounts according to the following method : A rather thick solution of caoutchouc in chloroform (the so-called traumaticin of the Pharmacopeia i vol. guttapercha dissolved in 6 vols. chloroform) is diluted before use with 25 vols. of chloroform and the resulting mixture poured upon a slide. The latter is tilted, and after evaporation of the chloroform, heated over a gas flame. The paraffin sections are mounted upon the slides so prepared and then painted with a solution of guncotton in aceton and alcohol (2 gm. guncotton dissolved in 50 c.c. of aceton, 5 c.c. of which is diluted with 20 c.c. of absolute alcohol). After painting with this solution, the sections are firmly pressed upon the slide with tissue paper, and after drying are made to adhere more closely by slight warming. Fixation to the slide with water is equally good. The sections can now be treated with various staining solutions without becoming detached from the slides. The paraffin is gotten rid of by immersing in xylol, after which the specimens are placed in absolute alcohol. Fuchsin S. can be used as a stain (20 gm. fuchsin S. dissolved in 100 c.c. anilin water). A small quantity of this solution is placed upon the section, and the slide warmed over a flame until its lower surface becomes quite perceptibly warm and the staining solution begins to evaporate. The slide is then allowed to cool, washed with picric acid (concentrated alcoholic solution of picric acid diluted with 2 vols. of water), after which it is covered with a fresh quantity of picric acid, and again, but this time vigorously, heated (one-half to one minute). Occasionally the same results can be obtained by covering the section for five minutes with a cold solution of picric acid of the above strength. This last procedure has a decided influence upon the granula, and gives rise to a distinct differentiation between them and the remaining portions of the cell, the latter appearing grayish-yellow, while the granula themselves appear bright red. In some cases where the granula can not be sharply differentiated from the remaining structures, it may be necessary to repeat the staining process. Xylol-Canada balsam should not be used for mounting, as it has a bleaching effect upon the osmic acid in the specimen. Mount either in liquid paraffin (Altmann) or in undiluted Canada balsam, which is easily reduced to a fluid state, whenever needed, by heating.

There is another method used by Altmann which deserves mention, but practical application of which must be improved upon in the future ; this consists in freezing the specimens and drying them for a few days in the frozen condition in a vacuum over sulphuric acid at a temperature of about 30 C.

According to Fischer, dilute solutions of pepton when treated with various reagents (especially with a potassium bichromate -osmium mixture) form precipitates and granules which are remarkable in that they react to stains exactly as do Altmann' s granula. It is, therefore, doubtful whether Altmann 's granules should be regarded as vital structures.

Altmann (92) has also devised a simpler negative method for demonstrating the granula. Fresh specimens are placed for twenty -four hours in a solution consisting of molybdate of ammonium 2.5 gm., chromic acid 0.35 gm., and water 100 c.c. ; then treated for several days with absolute alcohol, sectioned in paraffin, and colored with -a nuclear stain such as hematoxylin or gentian. The intergranular network


is colored, while the granula remain colorless. The amount of chromic acid used (0.25 to i%) varies according to the object treated ; if molybdate of ammonium alone be used, the nuclei will appear homogeneous, while if an excess of chromic acid be employed, the nuclei will appear coarsely reticulated. This method leads to the formation of granula in the cells as well as in the nucleus.

Biitschli's Foam-structure. Fixing is done either in picric acid solution or in weakly iodized alcohol. The specimens are then stained with iron-hematoxylin /'. c., first treated with acetate of iron, rinsed in water, and transferred to a 0.5^ aqueous solution of hematoxylin (similar to the method of R. Heidenhain). Very thin sections are required (^ to i //). Mounting is done, when the lighting is good, in media having low refractive indices, which emphasize the alveolar or foam-like structure of the protoplasm. Of various animal objects, Biitschli especially recommends young ovarian eggs of teleosts, and blood -cells and intestinal epithelium of the frog, etc. It is still a matter of uncertainty whether or not the structures are actually present in living protoplasm.