HISTOLOGY THE ESSENTIALS OP HISTOLOGY DESCRIPTIVE AND PRACTICAL FOR THE USE OF STUDENTS BY E. A. F.R.S., JODRELL PROFESSOR OF PHYSIOLOGY IN UNIVERSITY COLLEGE, LONDON ; EDITOR OF THE HISTOLOGICAL PORTION OF QUAIN’S “ANATOMY” ILLUSTRATED BY MORE THAN 300 FIGURES. FOURTH EDITION LEA BROTHERS & CO., PHILADELPHIA AND NEW YORK. 1894. o, ’ 1 PREFACE. This Book is written with the object of supplying the student with directions for the microscopical examination of the tissues. At the same time it is intended to serve as an Elementary Textbook of Histology, comprising all the essential facts of the science, but omit- ting unimportant details, the discussion of which is only calculated to confuse the learner. For a similar reason references to authorities have also generally been omitted. Most of the illustrations are taken from Quain’s Anatomy. Of the remainder, those which have been selected from other authors are duly indicated; the rest have either been drawn expressly for this work, or have been transferred to it from the author’s Course of Practical Histology.’ I am indebted to Dr. Ferrier for permission to use the illustrations of the structure of the spinal cord and cerebral cortex which have been contributed to the second edition of his book, The Functions of the Brain, by Mr. Bevan-Lewis. For conveniently accompanying the work of a class of medical students, the hook is divided into forty-five lessons. Each of these may be supposed to occupy a class from one to three hours, according to the extent to which the preparations are made beforehand by the VI PREFACE. teacher or are prepared during the lesson by the students. A few of the preparations—e.g. some of those of the sense-organs—cannot well be made in a class, but it has been thought advisable not to injure the completeness of the work by omitting mention of them. Only those methods are recommended upon which experience has proved that full dependence can be placed, but the directions given are for the most part capable of easy verbal modification in accordance with the ideas or experience of different teachers. CONTENTS. PAGE Enumeration of the Tissues—General Structure of Animal Cells, 1 LESSON I. Use of the Microscope—Examination of Common Objects, . . 6 LESSON II. Study of the Human Blood-Corpuscles, 10 LESSON III. Action of Reagents upon the Human Blood-Corpuscles, . . 15 LESSON IY. Study of the Blood-Corpuscles of Amphibia, . . . . 18 LESSON V. The Amieboid Phenomena of the Colourless Blood-Corpuscles, . 20 LESSON VI. Epithelium—Cell-Division, 24 INTRODUCTORY. CONTENTS. LESSON VII. PAGE Columnar and Ciliated Epithelium and Transitional Epithelium, 30 LESSON VIII. Study of Cilia in Action, 34 LESSON IX. The Connective Tissues : Areolar and Adipose Tissue, . . 37 LESSON X. The Connective Tissues (continued) : Elastic Tissue, Fibrous Tissue, Development of Connective Tissue, .... 45 LESSON XI. The Connective Tissues (continued) : Articular Cartilage, . . 50 LESSON XII. The Connective Tissues (continued): Costal Cartilage, Fibro- Cartilage, 54 LESSON XIII. The Connective Tissues (continued): Bone and Marrow, . . 57 LESSON XIV. The Connective Tissues (continued) : Development of Bone, . 64 LESSON XV. Structure of Striated Muscle, 72 LESSON XVI. Structure of Striated Muscle (continued), 75 CONTENTS. IX LESSON XVII. PAGE Connection of Muscle witii Tendon—Blood-Vessels of Muscle— Cardiac Muscle—Development of Muscle—Plain Muscle, . 79 LESSON XVIIT. Structure of Nerve-Eibres, 83 LESSONS XIX. and XX. Structure of Ganglia—Structure of Nerve-Cells—Structure of Neuroglia - Cells — Development of Nerve - Fibres — Wallerian Degeneration, 90 LESSON XXI. Modes of Termination of Nerve-Fibres, 98 LESSON XXII. Structure of the Larger Blood-Vessels, 107 LESSON XXIII. Smaller Blood-Vessels, Lymphatic Vessels, Serous Membranes, Synovial Membranes, 112 LESSON XXIV. Lymphatic Glands, Tonsil, Thymus, 121 LESSON XXV. The Skin, 127 LESSON XXVI. Structure of the Heart, . . . 139 LESSON XXVII. The Trachea and Lungs, 143 CONTENTS. LESSON XXVIII. PAGE Structure of the Teeth, the Tongue, and Mucous Membrane of the Mouth, 149 LESSON XXIX. The Salivary Glands, 161 LESSON XXX. The Structure of the Stomach, 167 LESSONS XXXI. and XXXII. Structure of Small and Large Intestine, 173 LESSON XXXIII. Structure of the Liver and Pancreas, 181 LESSON XXXIV. Structure of the Spleen, Suprarenal Capsule, and Thyroid Body, 187 LESSON XXXV. Structure of the Kidney, 192 LESSON XXXVI. Structure of the Ureter, Bladder, and Male Generative Organs, 200 LESSON XXXVI1. Generative Organs of the Female, and Mammary Glands, . 209 LESSON XXXVIII. Structure of the Spinal Cord, 216 LESSONS XXXIX. and XL. The Medulla Oblongata, Pons Varolii, and Mesencephalon, . 227 CONTENTS. XI LESSON XLI. PAGE Structure of the Cerebellum and Cerebrum, .... 241 LESSONS XLII. and XLIII. Structure of the Eyelids and of the Parts of the Eyeball, . 257 LESSON XLIY. Structure of the Olfactory Mucous Membrane and of the External and Middle Ear, 270 LESSON XI, V. Structure of the Labyrinth, 280 APPENDIX. Methods used in Preparing Sections, 290 Index, • 299 THE ESSENTIALS OF HISTOLOGY. INTRODUCTORY. ENUMERATION OF THE TISSUES AND THE GENERAL STRUCTURE OF ANIMAL CELLS. Animal Histology 1 is the science which treats of the minute struc- ture of the tissues and organs of the animal body ; it is studied with the aid of the microscope, and is therefore also termed Microscopical Anatomy. Every part or organ of the body, when separated into minute fragments, or when examined in thin slices (sections), is found to consist of certain textures or tissues, which differ in their arrange- ment in different organs, but each of which exhibits characteristic structural features. The foflowing is a fist of the pt'incipai tissues which compose the body :— 1. Epithelial. 2. Connective : Areolar, Fibrous, Elastic, Adipose, Lymphoid, Cartilage, Bone. 3. Muscular : Voluntary, Involuntary or plain, Cardiac. 4. Nervous. Some organs are formed of several of the above tissues, others contain only one or two. It is convenient to include such fluids as the blood and lymph amongst the tissues, because they are studied in the same manner and contain cellular elements similar to those met with in some of the other tissues. The elements which compose the tissues are of the nature either of fibres or cells. Some tissues are composed almost entirely of fibres with relatively few cells interspersed amongst the fibres ; this is the case with most of the connective tissues. Others, such as the epithelial tissues, are composed entirely of cells, whilst nervous and muscular tissues are formed of cells which are partly or wholly extended to form fibres. Cells.—A cell is a minute portion of living substance or protoplasm, which is sometimes inclosed by a cell-membrane and always contains a vesicle which is known as the nucleus. The protoplasm of a cell (fig. 1, p) is composed of albuminous 1 From taros, a web or texture. THE ESSENTIALS OF HISTOLOCxY. substance, which is characterised in typical cells by possessing the property of spontaneous movement. When the cell is uninclosed by a membrane a change in the shape, or even in the position of the cell, may be thereby produced (amoeboid movement, see Lesson V.). The protoplasm often exhibits a granular appearance, which, under high magnifying powers, is seen to be due to the fact that it is composed of two distinct substances (fig. 1), one a reticulum or spongework, which appears under the microscope in the form of a network, and the other a clear soft substance which occupies the interstices of the reticulum, and may also cover the surface or project beyond the rest of the cell. The granular appearance above mentioned is caused by the knots in the network appearing when imperfectly observed as separate granules. The material which forms the reticulum is termed spongio- plasm; the clear material Avhich occupies its meshes is hyaloplasm. The proto- plasm often includes actual granules of albuminous or fatty nature, or globules of watery fluid (vacuoles) containing glycogen or other substances in solu- tion. Materials which are thus included in the protoplasm of a cell are either stored up for the nutrition of the cell itself, or are converted into substances which are eventually extruded from the cell in order to serve some purpose useful to the whole organism, such as the secretion which is furnished by the cells of a gland. The term paraplasm has been given by Kupffer to any such material within a cell other than the actual protoplasm. Paraplasm is often present in sufficient amount to reduce the protoplasm to a relatively small amount, the bulk of the cell being occupied by other material, as when starch becomes collected within vegetable cells or fat Avithin the cells of adipose tissue. In some cells there are fine but distinct strife or fibrils running in definite directions. These are very commonly met with in fixed cells, such as various kinds of epithelium-cells, nerve- and muscle-cells. But besides this special differentiation, which appears to be related to the special function of the cell, and is not universal, there is another definite structure in the cell-protoplasm, which is known as the attraction-sphere (fig. 2). This consists of a wheel like arrangement of fine fibrils or rows of granules, which radiate from a clear area, in the middle of which lies a central particle—the attraction-particle. The attraction-spheres were dis- ' Fig. 1.—Diagram of a cell. p, protoplasm composed of spongio- plasm and hyaloplasm; n, nucleus with intranuclear network; n', nucleolus. ANIMAL CELLS. 3 covered by v. Beneden in the ovum or egg-cell, and were at first supposed to be peculiar to the ovum, but they have now been re- cognised (by Flemming and others) in many cells, and are probably of universal occurrence. They are very often double, the twin Fig. 2.—A cell (white blood cor- puscle) SHOWING ITS ATTRACTION- SPHERE. In this, as in most cases, the attraction- sphere lies near the nucleus. Fig. 3.—Ovum of ascaris, showing A TWIN ATTRACTION-SPHERE. (v. Beneden.) The nucleus with its contorted filament of chromoplasm is represented, but the protoplasm of the cell is not filled in. spheres being connected by a spindle-shaped system of delicate fibrils (achromatic spindle): this duplication invariably precedes the division of a cell into two (fig. 3). A cell-membrane is rarely distinct in animal cells, nor has its chemical nature been sufficiently investigated. It is formed by the external layer of the protoplasm. The nucleus of the cell (fig. 1, n) is a minute vesicle, spherical, ovoidal or elongated in shape, embedded in the protoplasm. It is bounded by a membrane which incloses a clear substance (nuclear matrix), and the whole of this substance is generally pervaded by an irregular network of fibres, some coarser, others finer (intranuclear network). This intranuclear network often exhibits one or more en- largements, which are known as the nucleoli. The nuclear membrane, intranuclear fibres and nucleoli all stain deeply with haematoxylin and with most other dyes; this property distinguishes them from the nuclear matrix, and they are accordingly spoken of as chromatic, composed of chromoplasm, the matrix as achromatic. Sometimes in- stead of uniting into a network the intranuclear fibres take the form of convoluted filaments, having a skein-like appearance. This is always the case when a nucleus is about to divide, but it may also occur in the resting condition. These filaments may sometimes be seen with very high magnifying powers to be made up of fine juxta- posed particles arranged either in single or multiple rows; thus imparting a cross-striated appearance to the filament (see fig. 4, B, c). The fibres within the nucleus have been observed to undergo spon- taneous changes of form and arrangement, but these become much 4 THE ESSENTIALS OF HISTOLOGY. more evident during its division. The division of the protoplasm is always preceded by that of the nucleus, and the intranuclear fibres undergo during its division a series of remarkable changes which are known collectively by the term karyokinesis (Schleicher). These changes may most easily be studied in the division of epithelium- cells (see Lesson VI.), but exactly similar phenomena have been shown to occur in cells belonging to the other tissues. a, cell from the marrow ; p, protoplasm with fine reticulum ; n, nucleus, long and folded, with intranuclear network, b, gland-cell from a larva of Nemocera; in, cell-membrane ; p, protoplasm; n, nucleus with convoluted filament, c, part of the nuclear filament in b, greatly magnified, d, an amoeboid-cell (white blood-corpuscle) of the newt, very highly magnified, showing a double nucleus with reticulum of chromoplasm, and the protoplasm composed of two substances (spongioplasm and hyaloplasm), d is from a drawing by Mr. D. Gunn; a, b, and c are from Camoy. Fig. 4.—To illustrate the structure of cells and nuclei Iii the early embryo the whole body is an agglomeration of cells. These have all been formed from the ovum or e. Mount two or three hairs from the head in water and look at them, first with the low, then with the high power. Examine also some fibres from any woollen material and compare them with the hairs. They have the same structure, although the wool is finer and is curled ; its structure may be partly obscured by the dye. Draw one or two woollen fibres. 6. Examine some dust of the room in water with a high power. In addition to numerous groups of black particles of carbon (soot) there will probably be seen fibres of linen, cotton, or wool, and shed epithelium-cells derived from the epidermis. 10 THE ESSENTIALS OF HISTOLOGY. LESSON II. STUDY OF THE HUMAN BLOOD-CORPUSCLES. 1. Having cleaned a slide and cover-glass, prick tlie finger and mount a small drop of blood quickly, so tliat it lias time neither to dry nor to coagulate. Examine it at once with the high power. Note (a) the coloured corpuscles, mostly in rouleaux and clumps, but some lying apart seen flat or in profile ; (b) the colourless corpuscles, easily made out if the cover-glass is touched by a needle, on account of their tendency to stick to the glass, whilst the coloured corpuscles are driven past by the cur- rents set up ; (c) in the clear spaces, fibrin-filaments and elementary particles or blood-tablets. Sketch a roll of coloured corpuscles and one or two colourless corpuscles. Count the number of colourless corpuscles in a field of the microscope. 2. To be made like 1, but the drop of blood is to be mixed upon the slide with an equal amount of normal saline solution,1 so that the red corpusles tend to be less massed together, and their peculiar shape is better displayed. Sketch a red corpuscle seen on the flat and another in profile (or optical section). Also a crenated corpuscle. Measure ten red corpuscles, and from the results ascertain the average diameter of a corpuscle. Measure also the largest and the smallest you can find. 3. Make a preparation of blood as in § 1 and put it aside to coagulate. After fifteen minutes allow a drop of a strong solution of neutral carminate of ammonia to run under the cover-glass. This decolorises the red corpuscles, but stains the nuclei of the white corpuscles and brings the network of fibrin- filaments and the elementary particles clearly into view (fig. 10, a). When the fibrin is fully stained, a drop of glycerine is allowed to diffuse into the fluid. The cover-glass may then be cemented with gold-size and the pre- paration labelled and kept. 4. Enumeration of the blood-corpuscles. This is done by some form of blood-counter such as the hsemacytometer of Gowers. This instrument con- sists of a glass slide (fig. 7. c), the centre of which is ruled into millimeter squares and surrounded by a glass ring l mm. thick. It is provided with measuring pipettes (a and b), a vessel (d) for mixing the blood with a saline solution (sulphate of soda of sp. gr. 1015), glass stirrer (e) and guai’ded needle (f). 995 cubic millimeters of the saline solution are placed in the mixing jar ; 5 cubic millimeters of blood are then drawn from a puncture in the finger and blown into the solution. The two fluids are well mixed by the stirrer and a small drop of this dilution (1 to 200) is placed in the centre of the cell, the cover-glass gently laid on (so as to touch the drop, which thus forms a layer I mm. thick between the slide and cover-glass) and pressed down by two brass springs. In a few minutes the corpuscles have sunk to the bottom of the layer of fluid and rest on the squares. The number in ten squares is then counted, and this, multiplied by 50 gives the number in a cubic milli- 1 Made by dissolving 6 grammes of common salt in 1 litre of ordinary water. STUDY OF THE HUMAN BLOOD-CORPUSCLES. 11 meter of the mixture, or if multiplied by 50x200 ( = 10,000) the number in a cubic millimeter of blood is obtained. Fig. 7.—H.emacytometeb of gowers. Fig. 8.—Human blood as seen on the warm STAGE. (Magnified about 1200 diameters.) Fig. 9.—Human bed corpus- cles LYING SINGLY AND COL- LECTED into ROLLS. (As seen under an ordinary high power of the microscope.) r, r, single red corpuscles seen lying flat; r', r1, red cor- puscles on their edge and viewed in profile ; r", red corpuscles arranged in rouleaux : c, c, crenate red cor- puscles ; p, a finely granular pale corpuscle; g, a coarsely granular pale corpuscle. Both have two or three distinct vacuoles, and were undergoing changes of shape at the moment of observation ; in g, a nucleus also is visible. 1, On the flat; 8, in profile. 12 THE ESSENTIALS OF HISTOLOGY. The coloured blood-corpuscles.—Under the microscope the blood is seen to consist of a clear fluid (plasma), in which are suspended the blood-corpuscles. The latter are of two kinds : the red or coloured (fig. 8, r, r'), which are by far the most numerous, and the white, pcdc, or colourless (p, g), which from their occurrence in the lymph are also known as lymph-corpuscles. When seen singly the coloured cor- puscles are not distinctly red, but appear of a reddish-yellow tinge. In the blood of man and of all other mammals, except the Camelidse, they are biconcave circular disks. Their central part usually has a lightly shaded aspect, under the ordinary high power (fig. 9, 1), but this is due to their biconcave shape, not to the presence of a nucleus. They have a strong tendency to become aggregated into rouleaux and clumps when the blood is at rest, but if it is disturbed they readily become separated. If the density of the plasma is increased in any way, as by evapora- tion, many of the red corpuscles become shrunken or crenated (c). The average diameter of the human red corpuscles is 0-007o milli- meter (about inch).1 There are from four to five millions of coloured corpuscles in a cubic millimeter of blood. The colourless corpuscles of human blood are protoplasmic cells, averaging OOl mm. inch) in diameter when spheroidal, but they vary much in size. They are far fewer than the coloured corpuscles, usually numbering not more than ten thousand in a cubic millimeter. Moreover, they are specifically lighter, and tend to come to the surface of the preparation. If ex- amined immediately the blood is drawn, they are spheroidal in shape, but they soon become irregulai (fig. 8, p, g), and their outline con- tinually alters, owing to the amoeba- like changes of form to which they are subject. Some of the colourless corpuscles are very pale and finely granular, others contain coarser and more distinct granules in their protoplasm. The protoplasm may also Fig. 10.—Fibrin-fit,amemts and blood- tablets. A, network of fibrin, shown after washing away the corpuscles from a preparation of blood that has been allowed to clot; many of the filaments radiate from small clumps of blood- tablets. B (from Osier), blood-corpuscles and elementary particles of blood-tablets, within a small vein. 1 The following list gives the diameter in parts of a millimeter of the red blood- corpuscles of some of the common domestic animals :—l)og, 0-0073; rabbit, 0 '0069; cat, 00065 ; sheep, 0*0050 ; goat, 0*0041. COLOURLESS CORPUSCLES. 13 contain clear spaces or vacuoles, and it has a reticular structure. Each pale corpuscle has one or more nuclei, which are difficult to see without the aid of reagents. In the clear fluid in which the corpuscles are suspended, a network of fine straight intercrossing filaments (fibrin) soon makes its appearance (fig. 10, A). There are also to be seen a certain number of minute round colourless discoid particles, either separate or collected into groups or masses, which may be of considerable size. These are the elementary particles or blood-tablets. Their meaning is not known. Fatty particles, derived from the chyle, may also occur in the plasma. Development of blood-corpuscles.—-In the embryo, the first-formed coloured blood-corpuscle* are amoeboid nucleated cells, the protoplasm Fig. 11.—Development of blood-vessels and blood-corpuscles in the vascular AREA OF THE GUINEA-PIG. bl, blood-corpuscles becoming free in the interior of a nucleated protoplasmic mass. of which contains haemoglobin. These embryonic blood-corpuscles are developed within cells of the mesoblast, which are united with one another to form a protoplasmic network (fig. 11). The nuclei of the cells multiply, and around some of them there occurs an aggregation of coloured protoplasm. Finally the network becomes hollowed out by an accumulation of fluid in the protoplasm, and thus are produced a number of capillary blood-vessels, and the coloured nucleated portions of protoplasm are set free within them as the embryonic blood-corpuscles (fig. 11, hi). In later embryonic life, nucleated coloured corpuscles disappear from mammalian blood, and are replaced by the usual discoid corpuscles. These are formed within certain cells of the connective tissue, a portion of the substance of the cell becoming coloured by haemoglobin, and separated into globular particles (fig. 12, a, b, c), which are gradually moulded into disk-shaped red corpuscles. In the meantime the cells 14 THE ESSENTIALS OF HISTOLOGY. become hollowed out, and join with similar neighbouring cells to form blood-vessels (fig. 13, a, b, c). The process is therefore the same as before, except that the cell-nuclei do not participate in it. Although no nucleated coloured corpuscles are to be seen in the blood in post-embryonic life, they continue to be formed in the marrow of the bones (see Lesson XIII.), and in some animals they have also been found in the spleen. It is thought probable that the red disks may be formed from these by the nucleus disappearing and the coloured Fig. 12.—Blood-corpuscles developing within connective-tissue cells. a, a cell containing diffused haemoglobin; b, a cell filled with coloured globules ; c, a cell con- taining coloured globules in the protoplasm, within which also are numerous vacuoles. protoplasm becoming moulded into a discoid shape. Others have sup- posed that the red disks are derived from the white corpuscles of the blood and lymph, and others again that they are developed from the blood-tablets ; but the evidence in favour of these views is insufficient. The white blood-corpuscles and lymph-corpuscles occur originally as Fig. 13.—Further development of BLOOD-CORPUSCLES IN CONNECTIVE- TISSUE CELLS, AND TRANSFORMA- TION OF THE LATTER INTO CAPIL- LARY BLOOD-VESSELS. a, an elongated cell with a cavity in its protoplasm occupied by fluid and by blood-corpuscles mostly globular; 6, a hollow cell the nucleus of which has multiplied. The new nuclei are arranged around the wall of the cavity, the corpuscles in which have now become discoid ; c shows the mode of union of a ‘hasmapoietic’ cell, which in this instance contains only one corpuscle, with the prolongation (bl) of a previously existing vessel, a, and c, from the new-born rat; b, from a foetal sheen. free unaltered embryonic cells, which have found their way into the vessels from the circumjacent mesoblast. Later they become formed in lymphatic glands and other organs composed of lymphoid tissue, and pass from these directly into the lymphatics and so into the blood. HUMAN BLOOD-CORPUSCLES. 15 LESSON III. ACTION OF REAGENTS UPON THE HUMAN BLOOD- CORPUSCLES. 1. Make a preparation of blood as in Lesson II. 1, and apply a drop of water, at one edge of the cover-glass. Examine at a place where the two fluids are becoming mixed. Notice particularly the first effect of water upon both red and white corpuscles, as well as the ultimate action. Sketch both kinds of corpuscles under the action of water. 2. Repeat on another preparation, using very dilute alkali (02 per cent, potash in salt solution) instead of water. Notice the complete solution first of the white and then of the coloured corpuscles as the alkali reaches them. 3. Repeat on another preparation, using dilute acetic acid (1 per cent.). Observe that the effect of the acid upon the coloured corpuscles is similar to that of water, but that it has a different action upon the colourless corpuscles. Sketch two or three of the latter after the action is completed. 4. Make a preparation of blood mixed with salt solution as in Lesson II. 2, and investigate the action of tannic acid (1 part tannic acid to 100 of dis- tilled water) in the same way. Sketch two or three coloured corpuscles after the reaction is complete. The action of reagents upon the human red blood-corpuscles shows that, although to all appearance homogeneous, they in reality consist of an external envelope of colourless material which forms a thin film enclosing the dis- solved colouring matter or haemoglobin. Thus, when water reaches the corpuscle, it passes through the film by osmosis and swells the corpuscle, causing it to become globular; eventually the film is burst through, and the colouring matter escapes into the serum. Salt, on the other hand, by increasing the density of the fluid in which the corpuscles float, causes a diffusion of water out of the corpuscle, and a consequent shrinking and cor- rugation of the surface, the crenated form (fig. 8, c; fig. 14,/) being thereby produced. The separation of the haemoglobin from the cor- puscle can be effected not only by water (fig. 14, a-e), but also by dilute acids, by the action of heat (60° C.), the freezing and thawing Fig. 14. a-e, successive effects of water upon a red corpuscle; /, effect of solution of salt; g, effect of tannic acid. 16 THE ESSENTIALS 01" HISTOLOGY. of blood, the vapour of chloroform, and the passage of electric shocks through blood.1 The mixing of human blood with the blood or serum of various animals also has a similar action, probably owing to differences of density or alkalinity. Tannic acid produces a peculiar effect (fig. 14, g); the haemoglobin is discharged from the corpuscle, but is immediately altered and precipitated, remaining adherent to the envelope in the form of a round or irregular globule of a brownish tinge (hematin ?). Some of these reactions occur by process of osmosis as in the case of water, but in others a physical or chemical solution of the envelope of the corpuscle is produced, and the haemoglobin is thus allowed to escape. The film or envelope is probably in large measure composed of lecithin and cbolesterin (along with a little cell-globulin—Halliburton), and these are substances which possess many of the physical properties of fats, although of a different chemical composition. If we assume this to be its composition the running of the red disks into rouleaux can readily be explained, since it has been shown by Norris that disks of any material, e.g. cork, floating in a fluid, tend in the same way to adhere in rouleaux, provided their surfaces are covered with a layer which is not wetted by the fluid. The envelope of the red corpuscle is often termed the stroma (Rollett), but this name rests upon an entirely false conception of the structure of the cor- puscle, and although of late years almost universally used, it ought to be entirely abandoned. In adopting the name, it was supposed that the corpuscle is formed of a homogeneous porous material (stroma), in the pores of which the haemoglobin is contained, but there is no reasonable foundation for this belief, whereas the supposition that there exists a delicate external film or envelope enclosing a coloured fluid is in accordance with all the known facts regarding the action of reagents upon these bodies. The structure of the colourless corpuscles is also brought out by the action of some of these reagents. As the water reaches them their amoeboid movements cease ; they become swollen out into a globular 1, first effect of the action of water upon a white blood corpuscle ; 2, 3, white corpuscles treated with dilute acetic acid ; n, nucleus. Fig. 15. form by imbibition of fluid (fig. 15, 1), and the granules within the protoplasm can be seen to be in active Brownian motion. Their nuclei 1 Iii the blood of some animals crystals of haemoglobin readily form after its separation by any of these means from the i'ed corpuscles. These crystals are rhombic prisms in most animals, but tetrahedra in the guinea-pig, and hexagonal plates in the squirrel. They are most appropriately studied along with the chemical and physical properties of blood, and are therefore omitted here. The same remark applies to the minute dark-brown rhombic crystals (hamin), which are found when dried blood is heated with glacial acetic acid, and to the reddish- yellow crystals of hivmatoiilin, which are found in old blood extravasations. COLOURLESS CORPUSCLES. 17 also become clear and globular, and are more conspicuous than before. With the further action of the water, the corpuscle bursts and the granules are set free. Acids have an entirely different action upon the white corpuscles. Their nuclei become somewhat shrunken and very distinct (fig. 15, 2 and 3), and a granular precipitate is formed in the protoplasm around the nucleus. At the same time, a part of the protoplasm generally swells out so as to form a clear bleb-like expansion (an appearance which often accompanies the death of the corpuscle from other causes). 18 THE ESSENTIALS OF HISTOLOGY. LESSON IV. STUDY OF THE BLOOD-CORPUSCLES OF AMPHIBIA. 1. Mount a drop of newt’s blood obtained from the cut end of the tail. It may be allowed to mix with a very small quantity of salt solution. Examine with the high power. Notice the shape of the coloured corpuscles both when seen flat and edgeways, and the nucleus within each. Measure ten corpuscles (long and short diameters), and from the results obtain the average dimensions of the newt’s blood-corpuscle. Notice also the colourless corpuscles, smaller than the red, but considerably larger than the pale corpuscles of human blood, although otherwise resembling these. Sketch two or three red corpuscles and as many white. Be careful not to mistake the liberated nuclei of crushed red corpuscles for pale corpuscles. Enormous cells and nuclei belonging to the cutaneous glands as well as the granular secretion of those glands may be present in this preparation. 2. Apply a drop of water to the edge of the cover-glass of the same pre- paration and notice its action upon the corpuscles. Sketch two or three corpuscles altered by the action of the water. 3. Mount another drop of blood, and apply dilute acetic acid (1 per cent.) instead of water at the edge of the cover-glass. Make sketches showing the effect of the acid upon both red and white corpuscles. 4. Examine the corpuscles of newt’s blood which has been allowed to flow into boracic acid solution (2 per cent.). Notice the effect produced upon the coloured corpuscles. Sketch one or two. The coloured blood-corpuscles of amphibia (fig. 16), as well as of most vertebrates below mammals, are biconvex elliptical disks, con- siderably larger than the biconcave circular disks of mammals.1 In addition to the coloured body of the corpuscle, which consists, as in mammals, of haemoglobin inclosed within an envelope, there is a colourless nucleus, also of an elliptical shape, but easily becoming 1 The following are the dimensions in parts of a millimeter of some of the cor- puscles of oviparous vertebrates :— Long diameter Short diameter Pigeon, . 0-0147 0-0065 Frog, . . 0-0223 0-0157 Newt, . 0-0293 0-0195 Proteus, . 0-0580 0-0350 Amphiuma, . 0-0770 0-0460 COLOURED BLOOD-CORPUSCLES OF AMPHIBIA. globular, especially if liberated by any means from the corpuscle. The nucleus resembles that of other cells in structure, being bounded by a membrane, and having a network of filaments traversing its interior (fig. 17). It is not very distinct in the unaltered corpuscle, but is brought clearly into view by the action of reagents, especially acetic acid. The action of reagents upon the red corpuscle of am- phibia is otherwise similar to that produced upon the mammalian 19 Fig. 17.—Coloured CORPUSCLE OF SA- LAMANDER, SHOW- ING INTRANUCLEAR NETWORK. (Flem- ming. ) Fig. 16.—Frog’s blood. (Ranvier.) a, red corpuscle seen on the flat; v, vacuoles in a cor- puscle ; b, c, red corpuscles in profile ; k, n, pale cor- puscles at rest; m, pale corpuscle exhibiting amoe- boid movements ; p, colourless fusiform corpuscle. •corpuscle, water and acetic acid causing it to swell into a globular form and then to become decolorised; solution of salt causing wrink- ling of the envelope, and so on. Boracic acid acts like tannic acid in causing the haemoglobin to be withdrawn from the envelope; but it becomes partially or wholly collected around the nucleus, which may then be extruded from the corpuscle. The colourless corpuscles (fig. 16, k, m, n), although larger, are very similar to those of mammals. Like them, they are either wholly pale or inclose a number of dark granules. They vary much in size and in the activity of their amoeboid movements. They may have one or several nuclei. Reagents have the same effect upon them as on those of mammals. The presence of glycogen may be demonstrated in them by its reaction with iodine (port-wine colour). 20 THE ESSENTIALS OF HISTOLOGY. LESSON V. THE A MCE BO ID PHENOMENA OF THE COLOURLESS BLOOD-CORPUSCLES. 1. Make a preparation of blood from the finger in the usual way. Draw a brush just moistened with oil around the edge of the cover-glass to check evaporation. Place the preparation upon a ‘ warm stage,’ and heat this to about the temperature of the body (38° C.). Bring a white corpuscle under observation with the high power, and watch the changes of shape which it undergoes. To become convinced of these alterations in form, make a series of outline sketches of the same corpuscle at intervals of a minute. The simplest form of ‘ warm stage ’ is a copper plate of about the size of an ordinary slide, perforated in the centre and with a long tongue of the same Fig. 18.—Simple warming apparatus, complete, shown in operation. metal projecting from the middle of one edge (fig. 18). The copper plate rests upon the stage of the microscope with a piece of cloth or other non- conducting material between. The preparation is made upon an ordinary slide, which is placed upon the warm stage and pressed into contact with AMOEBOID PHENOMENA. 21 it by the brass clips. Heat is applied to the copper tongue by a small spirit- lamp flame, and a greater or less amount is conducted to the warm stage and the superjacent preparation according to the point to which the flame is applied. To ascertain that the right temperature is got and maintained, put two pieces of paraffin, one melting at 35° C. (95° F.) and another at 38° C. (100° F.), on the slide, one on either side of the preparation. The tempera- ture must be such that the first piece is melted and remains so whilst the second remains solid.1 2. Mount a drop of newt’s blood diluted with an equal amount of salt solution, and examine it in the same manner upon the copper stage, at first cold, afterwards warm ; the temperature must, however, be kept below 30° C. Observe the effect of heat in accelerating the amoeboid movements of the pale corpuscles. Sketch one at intervals of a minute (a) in the cold, (b) whilst warmed. Fig. 19.—White corpuscles op frog’s blood migrated from shrunken clot within a capillary tube. (From Sanderson’s Handbook for the Physiological Laboratory.) 3. Take some yeast which lias been mixed with salt solution, and mix a little of the yeast and salt solution with a fresh drop of newt’s blood, slightly oiling the edge of the cover-glass as before. Endeavour to observe the incep- tion of torulse by the white corpuscles. Sketch one or two corpuscles con- taining torulse. Milk-globules or particles of carbon or of vermilion may also be used for this experiment, but the process of inception is most readily observed with the yeast particles. 1 For exact work, an apparatus somewhat more complex than the above is required. For description of such see A Course of Practical Histology. 22 THE ESSENTIALS OF HISTOLOGY. 4. At the beginning of the lesson collect a drop of newt’s blood into a fine capillary tube, seal the ends of the tube, and mount it in a drop of oil of cedar or Canada Balsam. Towards the end of the lesson examine it again to see white corpuscles emigrating from the shrunken clot (see fig. 19). 5. To obtain a specimen showing white corpuscles in amoeboid condition, make a preparation of newt’s blood, mixed with salt solution, and set it aside for ten minutes. By this time the corpuscles will be freely amoeboid, and will probably show well-marked pseudopodia. To fix them in this condition let a jet of steam from the spout of a kettle play for two or three seconds upon the cover-glass. The heat instantaneously kills the corpuscles, and they are fixed in the form they presented at the moment the steam was applied. They may now be stained by passing dilute hsematoxylin, eosin, methyl violet or fuchsin under the cover-glass, and the stain may be replaced by dilute glycerine, after which the cover' may be cemented and the prepara- tion kept. The amoeboid phenomena which are exhibited by the protoplasm of the colourless blood-corpuscles consist, in the first place, of spon- taneous changes of form, produced by the throwing out of processes or pseudopodia in various directions. When first thrown out the pseudopodia are composed of hyaloplasm alone, and they are prob- ably produced by a flowing of the hyaloplasm from out the meshes of the protoplasm (see p. 2). If the corpuscle is stimulated, either mechanically, as by tapping the coArer-glass, or electrically, the hyaloplasm is withdrawn again into the spongioplasm, and the pseudopodia are thereby retracted, the corpuscle becoming spherical. A change of form, caused by the protrusion of the pseudopodia, may, when active, be followed by changes in place or actual locomotion (migration) of the corpuscle. When a pseudopodium, or the external surface of the corpuscle, comes in contact with any foreign pai'ticle, the hyaloplasm tends to flow round and enwrap the particle, and particles thus incepted may then be conveyed by the corpuscle in its locomotory changes from one place to another. This property appears to play an important part in many physiological and patho- logical processes. It is probable that particles of organic matter which are taken up by the pale corpuscles may undergo some slow process of intra- cellular digestion within their protoplasm. The processes of the granular corpuscles are generally quite clear at first, and the granules afterwards flow into them. The migration of the colourless corpuscles from the blood-vessels into the surrounding tissue, or from a blood-clot into the surrounding serum (fig. 19), is owing to these amoeboid properties. The conditions which are most favourable to this amoeboid activity of the white corpuscles are (1) the natural slightly alkaline medium, AMCEBOID PHENOMENA. 23 such as plasma, serum, or lymph, or faintly alkaline normal saline solution. Any increase of density of the medium produces a diminu- tion of amoeboid activity, whilst, on the other hand, a slight decrease in its density has the opposite effect; (2) a certain temperature. In warm-blooded animals the phenomena cease below about 10° C. When gradually warmed they become more and more active up to a certain point, the maximum being a few degrees above the natural tempera- ture of the blood. Above this point they become spheroidal and at Fig. 20.—Changes op porm op a white corpuscle op newt’s blood, sketched at INTERVALS OP A PEW MINUTES, SHOWING THE INCEPTION OP TWO SMALL GRANULES AND THE CHANGES OF POSITION THESE UNDERWENT WITHIN THE CORPUSCLE. Fig. 21.—Three amceboid white corpuscles of the newt, killed by INSTANTANEOUS APPLICATION OF STEAM. a, a coarsely granular cell; b, c, ordinary cells. The nuclei appear multiple, but are seen to be connected by fine filaments of chromoplasm traversing the protoplasm. a somewhat higher temperature their protoplasm is coagulated and killed. Acids at once kill the corpuscles and stop the movements. Narcotic gases and vapours, such as carbonic acid gas or chloroform vapour, also arrest the movement, but it recommences after a time if their action is discontinued. THE ESSENTIALS OF HISTOLOGY. 24 LESSON VI. EPITHELIUM. 1. Mount a drop of saliva and examine first with a low, afterwards with a high power. Observe the nucleated epithelium-cells, some single, and others still adhering together by overlapping edges. Measure three or four, and also their nuclei. Sketch one or two on the flat and one edgeways. Notice the salivary corpuscles, which are like white blood-corpuscles swollen out by imbibition of water. 2. Put a small shred of human epidermis into a drop of strong caustic potash solution for five minutes. Then break it up in water with needles, cover and examine. Observe the now isolated swollen cells. Measure some. 3. Study the arrangement of the cells in a section through some stratified epithelium, such as that of the mouth, skin, or cornea.1 Notice the changes in shape of the cells as they are traced towards the free surface. Measure the thickness of the epithelium. Count the number of layers of cells. 4. Study the minute structure of epithelium-cells and their nuclei, both at rest and dividing, in sections of the skin of the newt’s tail or in shreds of epidermis of the salamander-tadpole. The preparation may, for this purpose, be stained either with haematoxylin or with some aniline dye such as saffranin.2 Sketch an epithelium-cell with resting nucleus, and others with nuclei in different phases of karyokinesis. An epithelium is a tissue composed entirely of cells separated by a very small amount of intercellular substance (cement-substance), and generally arranged so as to form a membrane covering either an external or an internal free surface. The structure of epithelium-cells, and the changes which they undergo in cell-division, are best seen in the epidermis of the newt or of the salamander-tadpole; in the latter especially the cells and nuclei are much larger than in mammals. Structure of the cells.—Each epithelium-cell consists of protoplasm containing a nucleus. The protoplasm may either look granular, or it may have a reticulated appearance. In some kinds of epithelium it is striated. The nucleus is a round or oval vesicle lying in the pro- toplasm. Usually there is only one, but there may be two or more. 1 The methods of preparing sections are given in the Appendix. 2 The methods which serve the purpose of exhibiting the division of nuclei are given in the Appendix. EPITHELIUM. 25 The cell-substance is often modified in its chemical nature; its external layer may become hardened to form a sort of membrane, or the whole cell may become horny (keratinised); or the cell may develop fibrils within it, and passing from it into adjacent cells, or lastly, there may be an accumulation of materials within the cell which are ultimately either used by the organism, as in the ordinary secreting glands, or eliminated as waste products as in the kidney. Fig. 22.—Epithelium-cells of salamander larva in different phases of division by karyokinesis. (Flemming.) Division of the cells.—The division of a cell is preceded by the division of its attraction-sphere, and this again appears to determine the division of the nucleus. The latter, in dividing, passes through a series of remarkable changes (fig. 22), which may thus be briefly summarised:— 1. The network of chromoplasm-filaments of the resting nucleus 26 THE ESSENTIALS OF HISTOLOGY. becomes transformed into a sort of skein, formed apparently of one long convoluted filament; the nuclear membrane and the nucleoli disappear or are merged into the skein (fig. 22, b, c, d). Sometimes the skein becomes looped in and out of a central space ; this form is termed the rosette (e). Fig. 23.—A dividing cell, showing attraction-particle at either pole of nucleus FROM WHICH THE GRANULES OF THE PROTOPLASM ARE SEEN RADIATING, AND WITH WHICH ALSO THE SPINDLE-SHAPED SYSTEM OF ACHROMATIC FIBRES TRAVERSING THE NUCLEUS IS CONNECTED. THE CHROMOSOMES, SIX IN NUMBER, ARE ARRANGED ASTRALLY AT THE EQUATOR OF THE SPINDLE. (Rabl.) Fig. 24.—A nucleus at a stage similar to that shown in the last figure, but SEEN FROM ONE OF THE POLES INSTEAD OF IN PROFILE. THE SPINDLE IS REPRESENTED FORESHORTENED. EIGHT CHROMOSOMES ARE REPRESENTED. (Rabl.) 2. The filament breaks into a number of separate portions, often V-shaped, and termed chromosomes. The number of chromosomes varies with the species of animal; in some animals the dividing nuclei may contain at this stage only four chromosomes, in others 24 or more. As soon as they become distinct they are usually arranged radially like a star [aster, f g). 3. Each of the chromosomes splits longitudinally into two, so that they are now twice as numerous as before (stage of cleavage, g, h). 4. The fibres separate into two groups, the ends being for a time interlocked (stage of metakinesis, i, j, k). 5. The two groups pass to the opposite poles of the now elongated nucleus and form a star-shaped figure (l) at each pole (dyaster). Each of the stars represents a daughter-nucleus. 6. 7, 8. Each star of the dyaster goes through the same changes as EPITHELIUM. 27 the original nucleus, but in the reverse order—viz., skein at first more open and rosette-like (m), then closer (n), then a network {o, p, q); passing finally into the typical reticular condition of a resting nucleus. The protoplasm of the cell divides soon after the formation of the dyaster (m). During division fine lines are seen in the protoplasm, radiating from the ends of the nucleus. Other lines produced by a spindle-shaped system of achromatic fibres lie within the nucleus, diverg- ing from the poles towards the equator (figs. 23, 24); they are far less easily seen than the other or chromatic fibres, but are not less important, for they are derived from the attraction-spheres, which, as we have seen, always initiate the division of a cell. Moreover, the achromatic fibres within the nucleus appear to form guides along which the chromo- somes or chromoplasmic filaments are conducted towards its poles. Classification of epithelia.—Epithelia are classified according to the shape and arrangement of the component cells. Thus we speak of scaly or pavement, cubical, columnar, polyhedral, and spheroidal epithelium. All these are simple epithelia, with the cells only one layer deep. If forming several superposed layers, the epithelium is said to be stratified, and then the shape of the cells differs in the different layers. Where there are only three or four layers in a stratified epithelium, it is termed transitioned. Stratified epithelium covers the anterior surface of the cornea, lines the mouth, pharynx (lower part), and gullet, and forms the epidermis Fig. 25.—Section of the stratified epithelium covering the front of THE CORNEA OF THE EYE. c, lowermost columnar cells ; p, polygonal cells above these ; Jl, flattened cells near the surface. Between the cells are seen intercellular channels bridged over by processes which pass from cell to cell. which covers the skin. In the female it lines the vagina and part of the uterus. The cells nearest the surface are always flattened and scale-like (fig. 25, Jl; fig. 26), whereas the deeper cells are more rounded or polyhedral, and those of the deepest layer generally somewhat columnar in shape (fig. 25, c). Moreover, the deeper cells are soft and protoplasmic, and are separated from one another by a 28 THE ESSENTIALS OF HISTOLOGY. system of intercellular channels, which are bridged across by numerous fibres passing from cell to cell, and giving the cells, when separated, the appearance of being beset with short spines (prickle-cells of Max Schultze). The deeper cells multiply by division, the nuclei first dividing in the manner just described. The newly formed cells tend as they enlarge to push those external to them nearer to the surface, from which they are eventually thrown off. As they approach the surface they become hard and horny, and in the case of the epidermis lose entirely their cellular appearance, which can, however, be in a measure restored by the action of potash (§2). The cast-off superficial cells of the stratified epithelium of the mouth, which are seen in abundance in the saliva (§ 1), are less altered, and the remains of a nucleus is still visible in them (fig. 26). Simple scaly or pavement epithelium is found in the saccules of the lungs, in those of the mammary gland when in- active, in the kidney (in the tubes of Henle), and also lining the cavities of serous membranes (fig. 27), and the heart, blood-vessels, and lymphatics. When occurring on internal surfaces, such as those of the serous membranes, blood-vessels, and lymphatics, it is often spoken of as endothelium. Fig. 26.—Epithelium-scales from the inside of the mouth. (Mag- nified 260 diameters.) Fig. 27.—Pavement epithelium or endothelium of a serous MEMBRANE. NITRATE OF SILVER PREPARATION. POLYHEDRAL OR SPHEROIDAL EPITHELIUM. Polyhedral or spheroidal epithelium is characteristic of many secret- ing glands. Columnar and ciliated epithelium are for the most part found covering the inner surface of mucous membranes ; which are membranes moistened by mucus and lining passages in communication with the exterior, such as the alimentary canal and the respiratory and generative passages. The detailed study of most of these may be reserved until the organs in which they occur are respectively dealt with. The hairs and nails and the enamel of the teeth are modified epithelial tissues. 30 THE ESSENTIALS OF HISTOLOGY. LESSON VII. COLUMNAR AND CILIATED EPITHELIUM, AND TRA NSITIONA L EPITHELIUM. 1. Take a piece of rabbit’s intestine which has been two days in chromic acid solution (1 part chromic acid to 2,000 normal saline solution). Scrape the inner surface with a scalpel, break up the scrapings in a drop of water on a slide. Add a small piece of hair to avoid crushing, and cover the prepara- tion. The tissue may then be still further broken up by tapping the cover- glass. Sketch one or two columnar cells and also a row of cells. Measure two or three cells and their nuclei. To keep this preparation, place a drop of very dilute haematoxylin solution at one edge of the cover-glass. When the limmatoxylin has passed in and has stained the cell-nuclei, place a drop of glycerine at the same edge and allow it slowly to diffuse under the cover-glass. Cement this another day. Osmic acid (1 per cent.) may be used in place of hhematoxylin. 2. Break up in glycerine a shred of epithelium from a piece of frog’s intestine that has been treated with osmic acid, and has subsequently macerated in water for a few days. The cells easily separate on tapping the cover-glass. They are larger than those of the rabbit and exhibit certain points of structure better. Measure and sketch one or two cells. The cover-glass may be at once fixed by gold size. 3. Prepare the ciliated epithelium from a trachea that has been in chromic acid solution (1 to 2,000 normal saline) for two days, in the same way as in § 1. Measure in one or two of the cells (a) the length of the cells, (6) the length of the cilia, (c) the size of the nucleus. Sketch two or three cells. This preparation is to be stained and preserved as in § 1. 4. Make a similar teased preparation of the epithelium of the urinary bladder, which is to be distended with bichromate of potash solution (1 part to 800 of water), and after an hour or two cut open and placed in more of the same solution. Observe the large flat superficial cells, and the pear- shaped cells of the second layer. Measure and sketch one or two of each kind. The cells will vary greatly in appearance according to the amount of distension of the organ. Stain and preserve as in §§ 1 and 3. All the above varieties of epithelium will afterwards be studied in situ when the organs where they occur come under consideration. Columnar epithelium.—The cells of a columnar epithelium (fig. 28) are prismatic columns, which are set closely side by side, so that when seen from the surface a mosaic appearance is produced. They often taper somewhat towards their attached end, which is generally trun- cated, and set upon a basement membrane. Their free surface is COLUMNAR EPITHELIUM. 31 covered by a thick striated border (fig. 29, str), which may sometimes become detached in teased preparations. The protoplasm of the cell is highly vacuolated and reticular, and fine longitudinal striae may be seen in it, which appear continuous with the striae of the free border. The nucleus (n) is oval and reticular. The lateral borders of the cells are often somewhat irregular or jagged, the result of the pressure of amoeboid lymph-cells, which are generally found between the columnar cells, at least in the intestine. After a meal containing fat the cells may contain fat globules, which become stained black in the osmic preparation. Fig. 28.—A row of columnar cells prom the intestine of the rabbit. Smaller cells are seen between the epithelium-cells ; these are lymph-corpuscles. Columnar epithelium-cells are found lining the whole of the interior of the stomach and intestines : they are also present in the ducts of most glands, and sometimes also in their secreting tubes and saccules. The epithelium which covers the ovary also has a modified columnar shape, but cells having all the structural peculiarities indicated above are found only in the alimentary canal and in its diverticula. Fig. 29.—Columnar epithelium-cells op the rabbit’s intestine. The cells have been isolated after maceration in very weak chromic acid. The cells are much vacuolated, and one of them has a fat-globule near its attached end ; the striated border (str) is well seen, and the bright disk separating it from the cell-protoplasm ; n, nucleus with intranuclear network ; a, a thinned-out wing-like projection of the cell which prob- ably fitted between two adjacent cells. G-oblet-cells.—Some columnar cells, and also cells of glandular, ciliated and transitional epithelia, contain mucigen, which is laid down within the cell in the form of granules (fig. 33, m1, m2) and may greatly distend the part of the cell nearest the free border. When the mucigen is extruded as mucus, this border is thrown off, and the cell takes the form of an open cup or chalice (fig. 30 and fig. 33, m3). 32 THE ESSENTIALS OF HISTOLOGY. Ciliated epithelium.—The cells of a ciliated epithelium are also usually columnar in shape (fig. 31), but in place of the striated border the cell is surmounted by a bunch of fine tapering filaments which, during life, move spontaneously to and fro, and serve to produce a current of fluid over the surface which they cover. The cilia are to be regarded as active prolongations of the cell- protoplasm. The border upon which they are set is bright, and appears formed of little juxtaposed knobs, to each of which a cilium is attached. In the large ciliated cells which line the alimentary canal of some molluscs (fig. 32), the knob may be observed to be prolonged into the protoplasm of the cell as a fine varicose filament, termed the rootlet of the cilium. These filaments perhaps represent the longi- Fig. 30.—Goblet-cell FROM THE TRACHEA. (Klein.) Fig. 31.—Columnar cili- ated EPITHELIUM-CELLS FROM THE LOWER PART OF THE NASAL PASSAGES. Examined fresh in serum. (Sliarpey.) Fig. 32.—Ciliated cell, FROM THE INTESTINE OF A MOLLUSC. (Engel- mann.) tudinal striae often seen in the protoplasm of the columnar cell, the bunch of cilia being homologous with the striated border. The pro- toplasm and nucleus have a similar vacuolated and reticular structure in both kinds of cell. CILIATED EPITHELIUM. 33 Ciliated epithelium is found throughout the whole extent of the air-passages and their prolongations (but not in the part of the nostrils supplied by the olfactory nerves, nor in the lower part of the pharynx); in the Fallopian tubes and the greater part of the uterus; in some of the efferent ducts of the testicle (where the cilia are longer than elsewhere in the body); in the ven- tricles of the brain, and the central canal of the spinal cord; and, according to some authorities, in the convoluted tubules of the kidney. Transitional epithelium is a stratified epithelium consisting of only two or three layers of cells. It occurs in the urinary bladder, the ureter, and the pelvis of the kidney. The superficial cells (fig. 34, a) are large and flattened; they often have two nuclei. On their under surface they exhibit depressions, into which fit the larger ends of pyriform cells, which form the next layer (fig. 34, b). Between the tapered ends of the pyriform cells one or two layers of smaller polyhedral Fig. 33.—Ciliated columnar EPITHELIUM, FROM THE TRACHEA OF A RABBIT. mi1, mi2, m3, mucus-secreting cells in various stages of mucigen formation. The preparation was treated with dilute chromic acid in the manner recommended in the instructions for practical work. Fig. 34.—Epithelial-cells from the bladder of the rabbit. (Klein.) (Magnified 500 diameters.) «, large flattened cell from the superficial layer, with two nuclei and with strongly marked ridges and intervening depressions on its under surface ; b, pear-shaped cell of the second layer adapted to a depression on one of the superficial cells. cells are found. The epithelium is renewed by division of these deeper cells. 34 THE ESSENTIALS OF HISTOLOGY. LESSON VIII. STUDY OF CILIA IN ACTION. 1. Mount in sea-water one or two bars of the gill of the marine mussel (fig. 35). Study the action of the large cilia. Now place the preparation upon the copper warm stage (see Lesson V.) and observe the effect of raising the temperature. Fig. 35.—Valve of mussel (mytilus edulis) showing hr, hr, the expanded GILLS OR BRANCHIAE, WHICH, OWING TO THE LITTLE BARS OF WHICH THEY ARE COMPOSED, PRESENT A STRIATED ASPECT. ml mantle ; m, cut adductor muscle ; i, mass of viscera; the dark projection just above is the foot. Keep this preparation until the end of the lesson, by which time many of the cilia will have become languid. When this is the case pass a drop of dilute potash solution (1 part KHO to 1,000 of sea-water) under the cover- glass and observe the effect. Fig. 36.—Moist chamber adapted for passing a gas or vapour to PREPARATION UNDER THE MICROSCOPE. 2. Cement with sealing-wax a piece of small glass tubing to a slide so that one end of the tube comes nearly to the centre of the slide. To do this STUDY OF CILIA IN ACTION. 35 effectually the slide must be heated and some sealing-wax melted on to it and allowed to cool. The glass tube is then made hot and applied to the slide, embedding itself as it does so in the sealing-wax. On this put a ring of putty or modelling wax (half an inch in diameter and rising above the glass tube) so as to include the end of the tube. Make a deep notch in the ring opposite the tube. Place a small drop of water within the ring (fig. 36). Put a bar from the gill upon a cover-glass in the least possible quantity of sea-water ; invert the cover-glass over the putty ring, and press it gently down. The preparation hangs in a moist chamber within which it can be studied through the cover-glass, and into which gases or vapours can be passed and their effects observed. Pass C02 through the chamber, and after observing the effect replace it by air (see fig. 37). Repeat with chloroform vapour instead of C02. The movement of cilia.—When in motion a cilium is bent quickly over in one direction with a lashing whip-like movement, immediately recovering itself. When vigorous the action is so rapid, and the rhythm so frequent (ten or more times in a second) that it is im- possible to follow the motion with the eye. All the cilia upon a ciliated surface are not in action at the same instant, but the move- Fig. 37.—Method of subjecting a pbepabation to a stream of carbonic ANHYDRIDE. b, bottle containing marble and hydrochloric acid: wash-bottle, connected by indiarubber tube, t, with the moist chamber, s. ment travels in waves over the surface. If a cell is detached from the general surface, its cilia continue to act for a while, but at once cease if they are detached from the cell. The rhythm is slowed by cold, quickened by warmth, but heat beyond a certain point kills the cells. The movement will con- tinue for some time in water deprived of oxygen. Both C02 gas and chloroform vapour arrest the action, but it recommences on re- 36 THE ESSENTIALS OF HISTOLOGY. storing air. Dilute alkaline solutions quicken the activity of cilia, or may even restore it shortly after it has ceased. Various attempts have been made to explain the manner in which cilia act, some supposing that they are themselves contractile, others that their movement is a passive one, and that the real movement is at their rootlets in the protoplasm of the cell. The bending-over action can also be supposed to be due to the alternate flowing and ebbing of hyaloplasm from the body of the cell into hollow permanent cell- processes, i.e. the cilia ; if we assume that one side of each cilium is less extensible than the other, it must necessarily be bent over in the manner usually observed. Some cilia, however, have a spiral action rather than the simple to and fro movement; in this case we may assume that the line of less extensibility passes not straight along one side of the cilium, but spirally round it. This hypothesis has the advantage that it permits ciliary motion to he brought into the same category as amoeboid movements, in so far that both are explicable by the flowing of hyaloplasm out of and into the reticulum of spongioplasm. THE CONNECTIVE TISSUES. 37 LESSON IX. THE CONNECTIVE TISSUES. AREOLAR AND ADIPOSE TISSUE, RETIFORM TISSUE. 1. Take a little of the subcutaneous tissue or of the intermuscular connective tissue of a rabbit or guinea-pig and spread it out with needles on a dry slide into a large thin film. Keep the centre moist by occasionally breathing on it, but allow the edges to dry to the slide. Before commencing put a drop of salt solution on a cover-glass, and now invert this over the film. Examine with a high power. Sketch one or two bundles of white fibres and also one or two elastic fibres, distinguishable from the former by their sharp outline, isolated course, and by their branching. Sketch also one or more connective- tissue corpuscles, if any such are visible in the clear interspaces. Look also for migratory cells (lymph-corpuscles). Next carefully remove the cover- glass and replace the salt solution by dilute acetic acid (1 per cent.). Watch its effect in swelling the white fibres and bringing more clearly into view the elastic fibres and corpuscles. Look for constricted bundles of white fibres. 2. Make another film in the same way, but mount in dilute magenta solution1 instead of saline solution. The elastic fibres are deeply stained by the dye ; the cells are also well shown. Cement the cover-glass at once with gold size. 3. Prepare another film of the subcutaneous tissue, including a little adipose tissue. Mount, as before, in dilute magenta solution, with a piece of hair under the cover-glass to keep this from pressing unduly upon the fat-cells. Cement at once with gold size. Examine first with a low and afterwards with a high power. The nucleus and envelope of the fat-cell are well brought out by the magenta, and if from a young animal, fat-cells will be found in process of formation. Measure and sketch two or three of the cells. 4. Spread out another large film of connective tissue, letting its edges dry to the slide, but keeping the centre moist by the breath. Place on its centre a large drop of nitrate of silver solution (1 per cent.). After ten minutes wash this away with distilled water, and expose to direct sunlight until stained brown. Then dehydrate with alcohol, replace the alcohol by clove- oil, and this by Canada balsam dissolved in xylol. Cover2 and examine. Sketch the outlines of two or three of the cell-spaces. 5. Mount in dilute glycerine and water, coloured by magenta, a section of lymphatic gland which has been immersed for a few minutes in 0-5 per cent, solution of caustic potash. The alkali destroys the cells, and thus allows the network of fibres which compose the retiform tissue to be seen. They are in all respects like the fibrils of areolar tissue. 1 See Appendix. - Preparations which are mounted in Canada balsam solution will soon become fixed by the hardening of the Canada balsam at the edges of the cover-glass. They must on no account be cemented with gold size. 38 THE ESSENTIALS OF HISTOLOGY. The connective tissues include areolar tissue, adipose tissue, elastic tissue, fibrous tissue, retiform and lymphoid tissue, cartilage and bone. All these tissues agree in certain microscopical and chemical charac- ters. They, for the most part, have a large amount of intercellular substance in which fibres are developed, and these fibres are of two kinds—white and yellow or elastic. Moreover, there are many points of similarity between the cells which occur in these several tissues ; they are all developed from the same embryonic formation, and they tend to pass imperceptibly the one into the other. Besides this, their use is everywhere similar; they serve to connect and support the other tissues, performing thus a passive mechanical function. They may therefore be grouped together, although differing considerably in ex- ternal characters. Of these connective tissues, however, there are three which are so intimately allied as to be naturally considered together, being composed of exactly the same elements, although differing in the relative development of those elements; these are the areolar, elastic, and fibrous tissues. Adipose tissue and retiform tissue may both be looked upon as special modifications of areolar tissue. Areolar tissue being the commonest and, in a sense, the most typical, its structure may be considered first. Fig. 38.—Bundles of the white fibres of areo LAR TISSUE PARTLY UNRAVELLED. (Sliarpey.) Fig. 39.—Ground substance of CONNECTIVE TISSUE STAINED BY silver. (The cell-spaces are left white.) Areolar tissue.—The areolar tissue presents to the naked eye an appearance of fine transparent threads and laminae which intercross in every direction with one another, leaving intercommunicating meshes, or areolae, between them. When examined with the microscope, these AREOLAE TISSUE. 39 threads and fibres are seen to be principally made up of wavy bundles of exquisitely fine transparent fibres (white fibres, fig. 38). The bundles run in different directions, and may branch and intercommunicate with one another; but the individual fibres, although they pass from one bundle to another, never branch or join other fibres. The fibres are cemented together into the bundles by a clear substance containing mucin, and the same clear material forms also the basis or ground- substance of the tissue, in which the bundles themselves course, and in which also the corpuscles of the tissue lie embedded. This ground- substance between the bundles can with difficulty be seen in the fresh tissue on account of its extreme transparency ; but it can be brought to view by staining with nitrate of silver, as in § 4. The whole of the tissue is thereby stained of a brown colour, with the exception of the spaces which are occupied by the corpuscles (cell-spaces, fig. 39). Fig. 40.—Elastic fibres of areolar tissue. From the subcutaneous TISSUE OF THE RABBIT. Besides the white fibres of con- nective tissue here described, fibres of a different kind (fig. 40) may be made out in the preparations; these are the elastic fibres. They are especially well seen after treatment with acetic acid, and after staining with magenta; but they can be detected also in the fresh preparation. They are characterised by Fig. 41.—A white bundle swollen by acetic acid. From the subarach- noid TISSUE AT THE BASE OF THE BRAIN. (From Toldt.) 40 THE ESSENTIALS OF HISTOLOGY. their distinct outline, their straight course, the fact that they never run in bundles, but singly, and that they branch and join neighbour- ing fibres. If broken by the needles in making the preparation, the elastic recoil causes them to curl up, especially near the broken ends. Besides the microscopical differences, the two kinds of fibres differ also in their chemical characters. Thus the white fibres are dissolved by boiling in water, and yield gelatin; whereas the substance of which the elastic fibres are composed (elastin) resists for a long time the action of boiling water. Moreover, the white fibres swell and become indistinct under the action of acetic acid; the elastic fibres are un- altered by this reagent. The bundles of white fibres which have been swollen out by acid sometimes exhibit curious constructions (fig. 41). These are due either to elastic fibres coiling round the white bundles, or to cell-processes encircling them, or to an investment or sheath which remains un- broken at certain parts, and thus prevents the swelling up of the bundle at these places. Fig. 42.—Subcutaneous tissue from a young rabbit, prepared as directed in § 1. (Highly magnified.) The white fibres are. in wavy bundles; the elastic fibres form an open network, p, p, plasma- cells ; g, granule-cell; c, c', lamellar-cells ; /, fibrillated-cell. The cells of areolar tissue.—Several varieties of connective-tissue cells are distinguished, viz. : (1) Flattened lamellar-cells, which are often branched (fig. 42, c, c') and may be united one to the other by their AREOLAR TISSUE. 41 branches, as in the cornea, or are unbranched and joined edge to edge like the cells of an epithelium ; the cell-spaces have in all cases a similar arrangement. (2) Plasma-cells of Waldeyer (fig. 42, p), which are com- posed of a soft much-vacuolated protoplasm, rarely flattened, but other- wise varying greatly in shape and size. (3) Granule-cells (a), usually spheroidal or ovoidal in shape, and formed, like the plasma cells, of soft protoplasm, but thickly occupied with albuminous granules, which are deeply stained by eosin and by most aniline dyes. Migratory lymph- corpuscles may also be seen here and there in the areolar tissues {wander-cells). In the middle coat of the eye the connective-tissue cells are filled with granules of pigment {pigment-cells). The cells lie in spaces in the ground-substance between the bundles of white fibres. In some parts of the connective tissue the white bundles are developed to such an extent as to pervade almost the whole of the ground-substance, and then the connective-tissue corpuscles become squeezed into the interstices, flattened lamellar expansions of the cells extending between the bundles, as in tendon (see next Lesson). The cells and cell-spaces of areolar tissue come into intimate relation with the cells lining the lymphatic vessels and small blood-vessels. This connection can best be seen in silvered preparations; it will be again referred to in speaking of the origin of the lymphatics. Fig. 43.—A small fat-lobule fbom the subcutaneous tissue of the GUINEA-PIG. ( "T0-.) ft, small artery distributed to the lobule ; 6, small vein ; the capillaries within the lobule ;ire not visible. Adipose tissue consists of vesicles filled with fat (figs. 43, 44), and collected into lobules, or into tracts which accompany the small blood- vessels. The vesicles are round or oval in shape, except where closely packed, when they become polyhedral from mutual compression. The fat-drop is contained within a delicate protoplasmic envelope (fig. 44, m) which is thickened at one part, and here includes an oval flattened 42 THE ESSENTIALS OE HISTOLOGY. nucleus. The vesicles are supported partly by filaments of areolar tissue, but chief!y by a fine network of capillary blood-vessels. The fat when first formed is deposited within granular cells of areolar tissue (fig. 45). It appears to be produced by a transformation of Fig. 44.—A few cells from the margin of a fat-lobule. f.g, fat-globule distending a fat-cell; n, nucleus ; m, membranous envelope of the fat-cell; c r, bunch of crystals within a fat-cell; c, capillary vessel; v, venule ; c t, connective- tissue cell; the fibres of the connective tissue are not represented. albuminous granules into droplets of fat. As these droplets increase in size they run together into a larger drop, which gradually fills the cell /, a cell with a few isolated fat-droplets in its protoplasm ; /', a cell with a single large and several minute drops; fusion of two large drops ; g, granular cell, not yet exhibiting any fat-deposition; c t, flat connective-tissue corpuscle ; c, c, network of capillaries. Fig. 45.—Deposition op fat in connective-tissue cells, more and more, swelling it out so that the cell-protoplasm eventually appears merely as the envelope of the fat-vesicle. ADIPOSE TISSUE. 43 Fat is found most abundantly in subcutaneous areolar tissue, and under the serous membranes; especially in some parts, as at the back of the peritoneum around the kidneys, under the epicardium, and in the mesentery and omentum. The yellow marrow of the bones is also principally composed of fat. There is no adipose tissue within the cavity of the cranium. Retiform or reticular tissue (figs. 46, 47) is a variety of connective tissue in which the intercellular or ground substance has mostly dis- appeared or is replaced by fluid. There are very fevr or no elastic Fig. 46.—Retiform tissue from a lymphatic gland, from a section which has BEEN treated in the manner described in § 5. (Moderately magnified.) Ir, a trabeculum of connective tissue ; r, r', retiform tissue, with more open meshes at r and denser at r'. fibres in it, and the white fibres and bundles of fibres form a dense net- work, the meshes of which vary in size, being very small and close in Fig. 47.—Portion of the above, more highly magnified. some parts; more open and like areolar tissue in other parts. In some places where the tissue occurs the fibres are almost everywhere en- 44 THE ESSENTIALS OF HISTOLOGY. wrapped by battened branched connective-tissue cells, and until these are removed it is not easy to see the fibres. Lymphoid or adenoid tissue is retiform tissue in which the meshes of the network are largely occupied by lymph-corpuscles. This is by far the most common condition of a retiform tissue, and is met with in the lymphatic glands and allied structures (see Lesson XXII.), and also in the tissue of the alimentary mucous membrane, and in some other situations. Basement membranes (membranae propriae) are homogeneous-looking membranes, which are found forming the surface-layers of connective- tissue expansions in many parts, especially where there is a covering of epithelium, as on mucous membranes, in secreting glands, and else- where. They are generally formed of flattened connective-tissue cells joined together to form a membrane ; but, in some cases, they are evidently formed not of cells, but of condensed ground-substance, and in others they are of an elastic nature. Jelly-like connective tissue, although occurring largely in the embryo, is found only in one situation in the adult—viz., forming the vitreous humour of the eye. It seems to be composed mainly of soft ground-substance, with cells scattered here and there through it, and with very few fibres, or none at all. These several varieties of connective tissue will be more fully described in connection with the organs where they occur. THE CONNECTIVE TISSUES. 45 LESSON X. THE CONNECTIVE TISSUES (continued). ELASTIC TISSUE, FIBROUS TISSUE, DEVELOPMENT OF CONNECTIVE TISSUE. 1. Tease out as finely as possible a small shred of elastic tissue (ligamentum nuchse of the ox or ligamenta subflava of man) in glycerine and water, slightly coloured by magenta. Cover and cement the preparation. Note the large well-defined fibres constantly branching and uniting with one another. Look for transverse markings on the fibres. Measure three or four. Sketch a small part of the network. Note the existence of bundles of white fibres amongst the elastic fibres. 2. Examine a thin transverse section of ligamentum nuchse which has been hardened in 2 per cent, solution of bichromate of potash. The section is to be stained with hsematoxylin and mounted in Canada balsam by the usual process,1 or simply in glycerine and water. Observe the grouping of the fibres and their angular shape. Notice also the nuclei of connective- tissue cells amongst the fibres. Sketch one or two groups. 3. Pinch off the end of the tail of a dead mouse or rat, draw out the long silk-like tendons and put them into saline solution. Take two of the longest threads and stretch them along a slide, letting the ends dry firmly to the slide but keeping the middle part moist. Put a piece of hair between them and cover in saline solution. Observe with a high power the fine wavy fibrillation of the tendon. Draw. Now run dilute acetic acid (0’75 per cent.) under the cover-glass, watch the tendons where they are becoming swollen by the acetic acid. Notice the oblong nucleated cells coming into view between the tendon bundles. Sketch three or four cells in a row. Lastly, lift the cover-glass, wash away the acid with distilled water, place a drop of Delafield’s hsematoxylin solution on the tendons, and leave the pre- paration until it is deeply stained ; then wash away the logwood and mount the preparation in faintly acidulated glycerine. Cement the cover-glass with gold size. 4. Take one or two other pieces of tendon, and, after washing them in distilled water, stretch them upon a slide as before, fixing the ends by allowing them to dry on to the slide. Put a drop of nitrate of silver solution (1 per cent.) on the middle of the tendons, and leave it on for five to ten minutes, keeping the preparation in the dark. Then wash off the silver nitrate with distilled water, and expose the slide to direct sunlight. In a very few minutes the silvered part of the tendons will be brown. As soon as this is the case, dehydrate the tendons with alcohol in situ upon the slide, run off' the alcohol, and at once put a drop of clove-oil on the preparation. In a minute or two the clove-oil can be replaced by Canada balsam in xylol, and covered. 5. Stain with magenta solution1 a thin section of a tendon which has been hardened in 70 per cent, alcohol. Mount in dilute glycerine and cement the cover-glass at once. Sketch a portion of the section under a low power. 1 See Appendix. 46 THE ESSENTIALS OF HISTOLOGY. Elastic tissue is a variety of connective tissue in which the elastic fibres preponderate. It is found most characteristically in the liga- mentum nuchae of quadrupeds and the ligamenta subflava of the vertebrae, but the connective tissue of other parts may also have a considerable development of elastic fibres. It occurs also in an almost pure form in the walls of the air-tubes, and uniting the cartilages of the larynx. It also enters largely into the formation of the walls of the blood-vessels, especially the arteries. In the ligamentum nuchae the fibres are very large and angular (fig. 48); they often exhibit cross-markings or even transverse clefts. When dragged asunder, they break sharply across ; they constantly branch and unite, so as to form a close network. In transverse section they are seen to be separated into small groups (fig. 49) by intervening white bundles of connective tissue. Fig. 49.—Cross-section of elastic FIBRES FROM THE LIGAMENTUM NUCH.E OF THE OX. Fig. 48.—Elastic fibres from the liga- MENTUM NUCH.E OF THE OX, SHOWING TRANSVERSE MARKINGS ON THE FIBRES. Elastic tissue does not always take the form of fibres, but may occur as membranes (as in the blood-vessels). Sometimes the fibres are very small, but their microscopical and chemical characters are always very well marked (see p. 40). Fibrous tissue is almost wholly made up of bundles of white fibres running in a determinate direction. These again are collected into FIBROUS TISSUE. 47 larger bundles, which give the fibrous appearance to the tissue. The bundles are constantly uniting with one another in their course, although their component fibres remain perfectly distinct. The interspaces between the larger bundles are occupied by areolar tissue (fig. 50) in which the blood-vessels and lymphatics of the fibrous Fig. 50.—Part of a large tendon in transverse section. (Moderately magnified.) a, areolar sheath of the tendon, with the fibres for the most part running transversely, but with two or three longitudinal bundles, b ; l, lymphatic cleft in the sheath ; immediately over it a blood-vessel is seen cut across, and on the other side of the figure a small artery is shown cut longitudinally ; c, large septum of areolar tissue ; d, smaller septum ; e, still smaller septum. The irregularly stellate bodies are the tendon-cells in section. tissue are conveyed. The interstices between the smallest bundles are occupied by rows of lamellar connective-tissue corpuscles (tendon-cells), Fig. 51.—Tendon of mouse’s tail ; showing chains of cells between the tendon-bundles. (175 diameters.) which from being squeezed up between three or more bundles become flattened out in two or three directions. In transverse section the cells appear somewhat stellate (figs. 50, 52), but when seen on the flat they appear lamellar (fig. 51), and from this aspect their general shape is 48 THE ESSENTIALS OF HISTOLOGY. square or oblong. They lie, as before said, in rows between the tendon- bundles, and the nuclei of adjacent cells are placed opposite one another Fig. 52.—Transverse section of tendon of mouse’s tail, stained. (175 diameters.) The flattened processes of the tendon-cells appear in section as lines, frequently coming off at right angles from the body of the cell. in pairs (fig. 53). The cell-spaces correspond in general figure and arrangement to the cells which occupy them (fig. 54). Fig. 53.—Eight cells from the same tendon as represented in fig. 51. (425 diameters.) The dark lines on the surface of the cells are the optical sections of lamellar extensions directed towards or away from the observer. Fibrous tissue forms the tendons and ligaments, and also certain membranes, such as the dura mater, the fibrous pericardium, the fascke of the limbs, the fibrous covering of certain organs, etc. It is found wherever great strength combined with flexibility is concerned. It Fig. 54.—Cell-spaces of tendon of mouse’s tail, brought into view by TREATMENT WITH NITRATE OF SILVER. (175 diameters.) receives a few blood-vessels, disposed longitudinally for the most part, and contains many lymphatics. Tendons and ligaments also receive nerve-fibres, which, in some cases, end in small localised ramifications like the end-plates of muscle, while others terminate in end-bulbs or in simple Pacinian corpuscles. These will be described along with the modes of ending of nerve-fibres. Development of connective tissue.—Connective tissue is always FIBROUS TISSUE. 49 developed in the mesoblast or mesoderm of the embryo. In those parts of this layer which are to form connective tissue, the embryonic Fig. 55.—Jelly of Wharton. (Ranvier.) r, ramified cells intercommunicating by their branches ; l, a row of lymph cells; /, fibres developing in the ground-substance. cells become separated from one another by a muco-albuminous semi- fluid intercellular substance (ground-substance), but the cells generally remain connected by their processes. The connective- tissue fibres, both white and elastic, are deposited in this ground-substance, the elastic substance usually in the form of granules (fig. 56, g), which subsequently become connected together into elastic fibres or laminae, as the case may be, the white fibres appearing at first in the form of very fine bundles, which after- wards become gradually larger; so that in fibrous tissue the whole ground substance is eventually pervaded by them, and the cells of the tissue become squeezed up into the intervals between them. Before any considerable development of fibres has taken place, the embryonic connective tissue has a jelly-like appearance; in this form it occurs in the umbilical cord, where it is known as the ielln of Wharton (fig. 55). Fig. 56.—Development of elastic tissue by DEPOSITION OF FINE GRANULES. (Ranvier.) g, fibres being formed of rows of ‘ elastin ’ granules; p, flat plate-like expansion of elastic substance formed by the fusion of ‘ elastin ’ granules. 50 THE ESSENTIALS OF HISTOLOGY. LESSON XI. THE CONNECTIVE TISSUES {continued). ARTICULAR CARTILAGE. 1. Cut two or three very thin tangential slices of the fresh cartilage of a joint, mount them in saline solution or serum, and examine with a high power. Observe carefully the form and grouping of the cells. Look at the thin edge of the section for spaces from which the cells have dropped out. Measure two or three cells and their nuclei, and sketch one or two groups. Now replace the saline solution by water and set the preparation aside for a little while. On again examining it, many of the cartilage-cells will be found to have shrunk away from their containing capsules. 2. Make other sections of the cartilage (1) from near the middle, (2) from near the edge. Place the sections for two or three minutes in acetic acid (1 per cent.), wash them with water, and stain with dilute hsematoxylin solution. When stained mount in dilute glycerine and cement the cover- glass. In (2) look for branched cartilage-cells. Draw one or two. 3. Make vertical sections of articular cartilage from a bone which has been for several days in per cent, chromic acid solution, and mount the sections in glycerine and water, or, after staining, in Canada balsam.1 Sketch the arrangement of the cells in the different layers. 4. Wash a fresh joint with distilled water ; drop 1 per cent, nitrate of silver solution over it; after five to ten minutes wash away the nitrate of silver and expose in water to direct sunlight. When browned, place in spirit for half an hour or more, and then with a razor wetted with spirit cut thin sections from the surface and mount in Canada balsam after passing through clove-oil. The cells and cell-spaces show white in the brown ground-substance. Draw. Cartilage or gristle is a translucent bluish-white tissue, firm, and at the same time elastic, and for the most part found in connection with bones of the skeleton, most of which are in the embryo at first repre- sented entirely by cartilage. Two chief varieties of cartilage are distinguished. In the one, which is termed hyaline, the matrix or ground-substance is clear, and free from obvious fibres; in the other, which is termed fibro-cartilage, the matrix is everywhere pervaded by connective-tissue fibres. When these are of the white variety, the tissue is white fibro-cartilage; when they are elastic fibres, it is yellow or elastic fibro-cartilage. 1 See Appendix. HYALINE CARTILAGE. 51 Hyaline cartilage occurs principally in two situations—namely (1) covering the ends of the bones in the joints, where it is known as articular cartilage ; and (2) forming the rib-cartilages, where it is known as costal cartilage. It also forms the cartilages of the nose, the external auditory meatus, the larynx, and the windpipe ; in these places it serves to maintain the shape and patency of the orifices and tubes. Articular cartilage.—The cells of articular cartilage are mostly scattered in groups of two or four throughout the matrix (fig. 57). The latter is free from fibres, except at the extreme edge of the Fig. 57.—Articular cartilage from head of metatarsal bone of man (OSMIC ACID PREPARATION). THE CELL-BODIES ENTIRELY FILL THE SPACES in the matrix. (340 diameters.) a, group of two cells; b, group of four cells ; h, protoplasm of cell, with g, fatty granules ; n, nucleus. cartilage, where the connective-tissue fibres from the synovial membrane extend into it; and here also the cartilage cells are often branched, and offer transitions to the branched connective-tissue corpuscles of that membrane (transitional cartilage, fig. 58). By long maceration, however, evidence of a fibrous structure may be obtained, even in the matrix of true hyaline cartilage. Some histologists also describe fine communica- tions in the matrix uniting the cartilage-cells with one another, but these are of doubtful occurrence. 52 THE ESSENTIALS OF HISTOLOGY. The matrix immediately around the cartilage-cells is often marked off from the rest by a concentric line or lines, this part of the matrix, Fig. 58.—Border of articular cartilage showing TRANSITION OF CARTILAGE CELLS INTO CONNECTIVE- TISSUE CORPUSCLES OF SYNOVIAL MEMBRANE. FROM HEAD OF METATARSAL BONE, HUMAN. (About 340 diameters.) a, ordinary cartilage-cells ; 6, b, with branching processes. Fig. 59.—A cartilage-cell IN THE LIVING STATE, FROM THE SALAMANDER. (Flemming.) (Very highly magnified.) which is the latest formed, being known as the capsule of the cell. The cells are bluntly angular in form, the sides opposite to one another in Fig. 60.—Vertical section of articular cartilage covering the lower end OF THE TIBIA, HUMAN. (Magnified about 30 diameters.) a, cells and cell-groups flattened conformably with the surface; 6, cell-groups irregularly arranged; c, cell-groups disposed perpendicularly to the surface; d, layer of calcified cartilage; e, bone. the groups being generally flattened. The protoplasm is very clear, but it may contain droplets of fat; and with a high power fine inter- AETICULAE CAETILAGE. 53 lacing filaments and granules have been observed in it (fig. 59). During life the protoplasm entirely fills the cavity or cell-space which it occupies in the matrix; but after death, and in conse- quence of the action of water and other agents, it tends to shrink away from the capsule. The nucleus is round, and shows the usual intranuclear network. In vertical section (fig. 60) the deeper cell-groups (c) are seen to be arranged vertically to the surface, the more superficial ones (a) parallel to the surface; whilst in an intermediate zone the groups are irregu- larly disposed (b). In the deepest part of the cartilage, next the bone, there is often a deposition of calcareous salts in the matrix (calcified cartilage, d). The disposition of the cells of cartilage in groups of two, four, and so on, is apparently due to the fact that these groups have originated from the division of a single cell first into two, and these again into two, and so on (fig. 61). It would seem that the matrix is formed of Fig. 61.—Plan of the multiplication of cells of cartilage. (Sharpey.) a, cell in its capsule; b, divided into two, each with a capsule ; c, primary capsule dis- appeared, secondary capsules coherent with matrix ; d, tertiary division ; e, secondary capsules disappeared, tertiary coherent with matrix. successive portions, which are deposited around each cartilage-cell as the so-called ‘capsules,’ each newly formed portion soon blending in its turn with the previously formed matrix, whilst a new capsule is formed within it. The division of the cartilage-cell, like that of other cells, is accompanied by a process of karyokinesis. Embryonic cartilage is characterised by the cells being usually more sharply angular and irregular, being even in some cases, markedly branched, like those which occur at the junction of cartilage and synovial membrane in the adult. The cells are also more closely packed, the matrix being in relatively less amount. 54 THE ESSENTIALS OF HISTOLOGY. LESSON XII. TIIE CONNECTIVE TISSUES {continued). COSTAL CARTILAGE. FIBRO-CARTILAGE. 1. Make transverse and tangential sections of a rib-cartilage, which may either be fresh, or may have been preserved in spirit. Stain them with hmmatoxylin (if fresh, after treatment with acetic acid as in Lesson XI., § 2), and mount in glycerine. Sketch a part of a transverse section under a low power and a cell-group from one of the tangential sections under a high power. Notice especially the arrangement of the cells, somewhat con- centric near the surface but radial near the centre. The costal cartilages are often ossified near the middle in animals, but in man when ossification occurs it is the superficial layer which is invaded. 2. Make sections of the cartilage of the external ear, either fresh or after hardening in alcohol. Mount in dilute glycerine faintly coloured with magenta. If from the ox, notice the very large reticulating elastic fibres in the matrix. Notice also the isolated granules of elastin, and around the cartilage-cells the area of clear ground-substance. Draw a small portion of the section. 3. Mount a section of the epiglottis in the same way. Notice the closer network of much finer fibres in its cartilage. 4. Cut sections of white fibro-cartilage (intervertebral disk), which has been hardened in saturated solution of picric acid, followed by spirit, or in spirit only. Stain the sections with dilute hsematoxylin. Mount in dilute glycerine. Observe the wavy fibres in the matrix and the cartilage-cells lying in clear areas often concentrically striated. Look for branched carti- lage-cells. Sketch three or four cells and the adjoining fibrous matrix. Costal cartilage.—In the costal cartilages the matrix is not always so clear as in the cartilage of the joints, for it often happens that fibres become developed in it. The cells are generally larger and more angular than those of articular cartilage, and collected into larger groups (fig. 62). Near the circumference, and under the perichon- drium or fibrous covering of the cartilage, they are flattened and parallel to the surface, but in the deeper parts they have a more irregular or a radiated arrangement. They frequently contain fat. The cartilages of the larynx and windpipe and of the nose resemble on the whole the costal cartilages, but the study of them may be deferred until the organs where they occur are dealt with. FIBRO-CARTILAGE. 55 Elastic or yellow fibro-cartilage occurs in only a few situations. These are, the cartilage of the external ear and that of the Eustachian tube, and the epiglottis and cartilages of Santorini of the larynx. The Fig. 62.—Section op kib-cartilage, showing cells and cell-groups in a SOMEWHAT FIBROUS-LOOKING MATRIX. Two or three empty cell-spaces are seen from which the cells have dropped out in preparing the section. matrix is everywhere pervaded with well-defined branching fibres, which unite with one another to form a close network (fig. 63). These fibres resist the action of acetic acid, and are stained deeply by magenta; they are evidently elastic fibres. In the ox they are very large, but smaller in man, especially in the cartilage of the epiglottis (fig. 64). They appear to be developed, as with elastic tissue elsewhere (see p. 49), by the deposition of granules of elastin in the matrix, which at first lie singly, but afterwards become joined to form the fibres. White fibro-cartilage is found wherever great strength combined with a certain amount of rigidity is required : thus we frequently find fibro-cartilage joining bones together, as in the case of the intervertebral disks and other symphyses. Fibro-cartilage is frequently employed to line grooves in which tendons run, and it may also be found in the tendons themselves. It is also employed to deepen cup-shaped articular surfaces ; and in the case of the interarticular cartilages, such as those of the knee and lower jaw, to allow greater freedom of movement whilst diminishing the liability to dislocation. Under the microscope white fibro-cartilage looks very like fibrous tissue, but its cells are 56 THE ESSENTIALS OF HISTOLOGY. cartilage-, not tendon-, cells (fig. 65). They are rounded or bluntly angular and surrounded by a concentrically striated area of clear Fig. 64.—Section of part of the carti- lage of the epiglottis. (Kanvier.) Fig. 63.—Section of the elastic carti- lage of the EAR. (Hertwig.) (Highly magnified.) a, cartilage cell in clear area; b, granular-looking matrix near the middle of the cartilage, the granular appearance being due partly to the fine reticulum of elastic fibres, partly to the presence of granules of elastic substance in the matrix; c, clearer matrix with longer fibres. cartilage-matrix. In some parts of the intervertebral disk many of the Fig. 65.—White fibko-cartilage from an intervertebral disk, human. (Highly magnified.) The concentric lines around the cells indicate the limits of deposit of successive capsules. One of the cells has a forked process which extends beyond the hyaline area surrounding the cell, amongst the fibres of the general matrix. cells are branched, and may be looked upon as transitional forms to connective-tissue corpuscles. BONE AND MARROW. 57 LESSON XIII. BONE AND MARRO W. 1. Ik thin sections of hard bone made by grinding, observe the Haversian canals, lamellae, lacunae, canaliculi, etc. Make a sketch first under a low and afterwards under a high power. 2. With fine forceps strip off a thin shred from the superficial layers of a bone which has been decalcified in dilute nitric acid and afterwards kept for some time in dilute alcohol. Mount the shred in water. Observe the fibrous structure of the lamellae. Look for perforating fibres or the holes from which they have been dragged out. Sketch a small piece of the thin edge of a lamella. 3. Stain with dilute magenta very thin sections of compact bone which has been decalcified in chromic or picric acid, and mount in dilute glycerine, cementing at once. Look for fibres of Sharpey piercing the circumferential lamellae. The elastic perforating fibres are more darkly stained than the others. Notice the stained nuclei of the bone-corpuscles in the lacunae. In the thinnest parts of the sections try to make out the blood-vessels and other structures in the Haversian canals. 4. Mount in Canada balsam sections of marrow (from a long bone of a rabbit) stained with haematoxylin.1 Observe the fat-cells, the reticular tissue supporting them, the proper marrow-cells in this tissue, etc. 5. Tease in salt solution or serum some of the red marrow from the rib of a recently killed animal. Observe and sketch the proper marrow-cells and look for myeloplaxes and nucleated coloured blood-corpuscles. If examined carefully, amoeboid movements may be detected in the latter and in the marrow-cells. Bone is a connective tissue in which the ground-substance is im- pregnated with salts of lime, chiefly phosphate, these salts consti- tuting about two-thirds of the weight of the bone. When bones are macerated this earthy matter prevents the putrefaction of the animal matter. When bones are calcined they lose one-third of their weight, owing to the destruction of the animal matter; when steeped in acid the earthy salts are dissolved and only the animal matter is left. This, like areolar and fibrous tissue, is converted into gelatine by boiling. Bony tissue is either compact or cancellated. Compact bone is dense like ivory ; cancellated is spongy with obvious interstices. The outer layers of all bones are compact, and the inner part is generally can- xSee Appendix. 58 THE ESSENTIALS OF HISTOLOGY. cellated, but the shaft of a long bone is almost entirely made up of compact substance except along the centre, which is hollow and filled with marrow. The interstices of cancellated bone are also occupied by marrow. Externally bones are covered except at the joints by a vascular fibrous membrane, the periosteum. True bone is always made up of lamellce, and these again are com- posed of fine fibres lying in a calcified ground-substance. Between the lamellae are branched cells, the bone-corpuscles, which lie in cell-spaces or lacunae. The ramified passages which contain the cell-processes are termed canaliculi. In cancellated bone the blood-vessels run in the interstices supported by the marrow. In compact bone they are contained in little canals— the Haversian canals—which everywhere pervade the bone. These canals are about 0-05 mm. inch) in diameter, but some are Fig. 66.—Transverse section of a bone (ulna). (Skarpey.) (Magnified 20 diameters.) The openings of the Haversian canals are seen encircled by concentric lamellae. Other lamellae run parallel with the surface (a). smaller, others larger than this. Their general direction is longitudinal, i.e. parallel to the long axis of the hone, but they are constantly united by transversely and obliquely running passages. In a section across BONE. 59 the shaft of a long bone they are seen as small rounded or irregular holes (fig. 66). When the section has been made by grinding, the holes get filled up with air and debris, and they then look black by transmitted light, as do also the lacunae and canaliculi (fig. 67). Most of the lamellae in compact bone are disposed concentrically around the Haversian canals; they are known as the Haversian lamellae, and with Fig. 67.—Transverse section of compact tissue (of humerus.) (Sharpey.) (Magnified about 150 diameters.) Three of the Haversian canals are seen, with their concentric rings; also the lacunae, with the canaliculi extending from them across the direction of the lamellae. The Haversian apertures had become filled with air and debris in grinding down the section, and therefore appear black in the figure, which represents the object as viewed by transmitted light. the included canal form what is known as a Haversian system. The lacunae of a Haversian system communicate with one another and with the Haversian canal, but not as a rule with the lacunae of other Haversian systems. The angular interstices between the Haversian systems are generally occupied by bony substance, which is fibrous but not lamellar. Besides the lamellae of the Haversian systems there is a certain thickness of bone at the surface, immediately underneath the periosteum, which is composed of lamellae arranged parallel with the surface; these are the circumferential or periosteal lamellce (fig. 66, a). They are pierced here and there by canals for blood-vessels, which are proceeding from the periosteum to join the system of Haversian canals, and also by calcified bundles of white fibres and by elastic fibres-which may also be prolonged from the periosteum. These are the perforating fibres of Sharpey (fig. 68). The lamellae of bone are fibrous in structure. This may be seen in 60 THE ESSENTIALS OF HISTOLOGY. Fig. 68.—Transverse section of decalcified human tibia, from near the SURFACE OF THE SHAFT. h, Haversian canals, with their systems of concentric lamellse; in all the rest of the figure the lamellse are circumferential; s, ordinary perforating fibres of Sharpey; e, e, elastic perforating fibres. Drawn under a power of about 150 diameters. Fig. 69.—Lamella: torn off from a decalcified human parietal bone at SOME DEPTH FROM THE SURFACE. (Sliarpey.) a lamellae, showing decussating fibres; b, b, thicker part, where several lamellae are super- ’ posed; c, c, perforating fibres; the fibrils which compose them are not shown in the figure. Apertures through which perforating fibres had passed are seen, especially in the lower part, a, a, of the figure. Magnitude as seen under a power of 200, but not drawn to a scale. (From a sketch by Allen Thomson.) BONE. 61 shreds torn off from the superficial layers of a decalcified bone (fig. 69). The fibres often cross one another in adjacent lamellae, and in the Haversian systems they run in some lamellae concentrically, in others parallel with the Haversian canal. In shreds of lamellae which have been peeled off from the surface the perforating fibres may some- times be seen projecting from the surface of the shred, having been torn out of the deeper lamellae (fig. 69, c, c). Where tendons or ligaments are inserted into bone, their bundles of white fibres are prolonged into the bone as perforating fibres. The lacunae are occupied by nucleated corpuscles, which send branches along the canaliculi (fig. 70). The Haversian canals contain one or two blood-capillaries and nervous filaments, besides a little connective tissue; and the larger Fig. 71.—Section op a haversian canal, showing ITS CONTENTS. (Highly- magnified.) Fig. 70.—A bone-cell isolated and HIGHLY MAGNIFIED. (Joseph.) a, proper wall of the lacuna, where the cor- puscle has shrunken away from it. a, small arterial capillary vessel: v, large venous capillary; n, pale nerve-fibres cut across; l, cleft-like lymphatic vessel; one of the cells forming its wall communicates by fine branches with the branches of a bone-corpuscle. The substance in which the vessels run is connec- tive tissue with ramified cells; its finely granular appearance is probably due to the cross-section of fibrils. The canal is sur- rounded by several concentric lamellae. ones may also contain a few mar- row-cells. There are also cleft- like lymphatic spaces running with the vessels and connected through canaliculi with branches from cor- puscles within the neighbouring lacunae of the osseous substance (fig. 71). The periosteum (which is studied in torn-off shreds, in preparations stained in situ with silver nitrate, and in logwood-stained sections from a bone which has been decalcified in chromic or picric acid) is a fibrous membrane composed of two layers, the inner of which contains many elastic fibres. In the outer layer numerous blood-vessels ramify and send from it branches to the Haversian canals of the bone. The periosteum ministers to the nutrition of the bone, partly on account of the blood-vessels and lymphatics it contains, partly, especially in young 62 THE ESSENTIALS OF HISTOLOGY. animals, on account of the existence between it and the bone of a layer of osteoblasts or bone-forming cells, a remainder of those which originally produced the bone. The marrow of bone is of a yellow colour in the shafts of the long bones, and is there largely composed of adipose tissue, but in the can- cellated tissue it is usually red, the colour being partly due to the large amount of blood in its vessels. This red marrow is chiefly composed of round nucleated cells—the marrow cells (fig. 72, e-i)—which resemble large lymph-corpuscles, and, like these, are amoeboid. There are also to be seen mingled with them a number of corpuscles somewhat smaller in size, but nucleated and amoeboid, and of a reddish tint (fig. 72, j-t). These cells, which are termed erythroblasts, resemble the nucleated Fig. 72.—Cells of the red marrow of the guinea-pig. (Highly magnified.)