ROPERTY OP MEMCAL HE PROPERTY OP al College of tie Pacific. DONATED BY STUDENTS HISTOLOGY A COURSE OF NORMAL HISTOLOGY FOE STUDENTS AND PEACTITIONEES OF MEDICINE BY MAURICE N. MILLER, M.D. .^' Late Director of the Department of Normal Histology in Loomis1 Laboratory, University of the City of New York REVISED BY HERBERT U. WILLIAMS, M.D. Professor of Pathology and Bacteriology, Medical Department, University of Buffalo THIRD REVISED EDITION PROFUSELY ILLUSTRATED NEYvT YORK WILLIAM WOOD & COMPANY 1898 COPYRIGHT BY WILLIAM WOOD & COMPANY 1898 J. Horace McFarland Company Harrisburg, Pa. 1*59 f PREFACE This volume has been prepared with a view of aiding the instructors and students of the laboratory classes which are under my direction. It is also presented with the hope that it may be useful to other instructors. Again, students often wish to continue microscopical work during the interim of college attendance ; to such, it is my belief, these pages will have some value. Still again, very many practitioners, not having had, during pupilage, advantages equal to those provided by the modern laboratory equipment, wish to acquire more knowledge of microscopy, for its value in practical medicine. To such workers, also, I desire to be useful. So much technique has been introduced as has been found to be of absolute necessity, and no more. The processes for the preparation and exhibition of tissues are generally simple and always practicable. In the description of organs, I assume that the student has a fair knowledge of gross anatomy, but knows nothing of his- tology. The scheme or plan of the structure is first described — using diagrams where requisite to clearness — after which the mode of preparing the sections is indicated, and, under prac- tical demonstration, every histological detail tabulated in proper order. The ^drawings will, I believe, aid in the recognition of such elements in the field of the microscope. The illustrations are exact reproductions, by photography, of my own pen - pictures ; and distinction must always be made between the drawings which are schematic — used to emphasize ("i) IV PREFACE the plan of structures — and those drawn from the tissue as seen in the microscope. Our literature abounds in excellent works for the advanced student, and this volume is designed to pave the way for their appreciation. I desire to record my high appreciation of the aid of Drs. Charles T. Jewett, Egbert Le Fevre, E. Eliot Harris, Milton Turnure, H. Pereira Mendes, J. Gorman, A. M. Lesser, J. Alex- ander Moore, Robert Eoberts, Esq., Warden, and Mr. John Burns, Clerk of Charity Hospital, in facilitating my access to valuable tissue for the illustrations and for my own studies. My thanks are due my First Assistant, Dr. F. T. Reyling, for his indefatigable efforts in furthering the work ; and to Mr. A. J. Drummond, for photographical favors. MAURICE N. MILLER. NEW YORK, June 1st, 1887. PREFACE TO THIRD REVISED EDITION A revision of Miller's Microscopy became necessary, partly on account of the advances made in histology during the last ten years, and partly because of the increasing tendency in medical schools to devote more time to laboratory studies. Substantially all of the original matter has been retained, although somewhat rearranged ; and where new matter has been inserted, the attempt has been made to 'give it the form which was the peculiarity of the original, namely, in being written from the point of view of the student, and not of the teacher. In pre- paring the various additions, the standard text -books on his- tology have been constantly consulted. BUFFALO, N. Y., August, 1898. CONTENTS PART FIRST TECHNOLOGY THE LABORATORY MICROSCOPE PAGE Description of the Stand 1 Lenses 2 General Adjustment 4 Adjustment for Illumination 4 Adjustment for Focus 5 Method in Observation • 6 Conservation of the Eyesight 7 Magnifying Power 7 Measurement of Objects 8 Sketching from the Instrument 9 PREPARATION OF TISSUES FOR MICROSCOPICAL PURPOSES Teasing of Tissues 9 Section Cutting 10 Free-hand Section Cutting 10 Section Cutting with the Stirling Microtome 12 Section Cutting with the Larger Microtomes 15 Sharpening Knives — Honing and Stropping 16 Supporting Tissues for Cutting 18 Paraffin Soldering 18 The Freezing Microtome 19 Fixing or Hardening Fluids 20 Alcohol Hardening 21 Mailer's Fluid • 22 Orth's Fluid 22 Formaldehyde 22 Picric Alcohol 23 Osmic Acid 23 Flemming's Solution 23 Chromic Acid 24 Erlicki's Fluid 24 (v) VI CONTENTS PAGE Decalcifying Fluid 24 Dissociating Fluid 25 Imbedding— Paraffin 25 " — Celloidin 26 Staining Methods in General 27 Hsematoxylin 28 Eosin 28 Carmine . 29 Weigert-Pal Method 30 Aniline Dyes 31 Van Gieson's Stain 32 Ehrlich Tricolor Stain . . 32 Metallic Impregnations — Nitrate of Silver 33 Golgi's Method . 33 Injection Methods 34 Clearing Agents 35 Mounting Media 35 Hsematoxylin Staining Process 36 Heematoxylin and Eosin Double Staining 38 Borax -Carmine Staining Process 39 Cleaning Slides and Cover-glasses 41 Mounting Methods 41 Labeling Slides . . 42 Care of the Microscope 43 PAKT SECOND STRUCTURAL ELEMENTS PRELIMINARY STUDY Form of Objects 45 Movement of Objects 46 Extraneous Substances . 47 STRUCTURAL ELEMENTS Cells 49 Cell Distribution 50 Cell Division— Karyokinesis 51 Classification of Tissues 53 Embryonic Derivation of Tissues 54 Epithelium 54 Distribution of Epithelium 55 Squamous, Stratified, and Transitional Epithelium 55 Pavement Epithelium . 56 Columnar Epithelium . . 57 Ciliated Columnar Epithelium 58 CONTENTS Vii PAGE Glandular Epithelium 59 Endothelium— Serous Membranes 60 CONNECTIVE (FIBEOUS) TISSUES White Fibrous Tissue —62- Yellow Elastic Tissue 63 Adipose Tissue 65 CARTILAGE Hyaline Cartilage 67 White Fibro- Cartilage ... . . . 68 Elastic Cartilage 68 BONE Bone 70 Periosteum .- 73 Marrow 73 Development of Bone 73 SPECIAL CONNECTIVE TISSUES (page 76) MUSCULAR TISSUE Non-striated Muscle 76 Striated Muscle 78 Cardiac Muscle 80 BLOOD Red Corpuscles 81 Blood-plates 82 White Corpuscles -83 Enumeration of Blood-corpuscles 87 Haemoglobin 89 Fibrin 90 Effect of Reagents 90 Development of Red Blood -corpuscles 91 PART THIRD ORGANS THE SKIN Layers or Strata 92 Hairs 95 Sudoriferous Glands 97 viii CONTENTS PAGE Sebaceous Glands 98 Nails 98 Practical Demonstration 98 THE CIRCULATORY SYSTEM The Heart 102 Blood-vessels : 102 Development of Capillaries 105 THE LYMPHATIC SYSTEM General Description 10G Lymph-channels 107 Practical Demonstration, Lymph-channels of Central Tendon of the Diaphragm f 108 Lymphatic Nodes or Glands . P Ill Practical Demonstration, Mesenteric Lymph-node 113 THE SPLEEN Scheme of Organ 117 Practical Demonstration 118 THE THYMUS BODY General Description 121 Practical Demonstration . 121 THE RESPIRATORY ORGANS The Larynx and Trachea ] 23 The Lungs .••••' 123 Bronchial Tubes 123 Practical Demonstration 125 Pulmonary Blood-vessels 128 Pleura .- 128 Pulmonary Alveoli 128 Practical Demonstration, Lung of Pig 131 Practical Demonstration, Human Lung 133 Foetal Lung . 134 THE TEETH The Pulp 135 Dentine 135 Enamel 137 Cementum 137 Practical Demonstration . 137 CONTENTS ix GLANDS PAGE Typical Glandular Histology 140 Tubular Glands 140 Coiled Tubular Glands 140 Branched Tubular Glands ~143~ Acinous Glands * 143 Parotid Gland 143 Submaxillary Gland 144 Pancreas 145 Practical Demonstration, Parotid Gland, Submaxillary Gland, Pancreas . 146 Thyroid Gland 148 THE ALIMENTARY CANAL The Mouth and Pharynx . 149 The (Esophagus 150 Practical Demonstrations" 150 General Histology of the Stomach and Intestines 151 The Stomach 152 Practical Demonstration . 155 Small Intestine 156 Practical Demonstration 160 Duodenum, Vermiform Appendix, Large Intestine 162 THE LIVER General Scheme 163 The Portal Canals 165 The Lobular Parenchyma 165 Practical Demonstration, Liver of Pig 167 Practical Demonstration, Human Liver 170 Practical Demonstration, The Portal Canals 172 Practical Demonstration, The Lobular Parenchyma 173 Practical Demonstration, Origin of Bile -ducts 176 Gall-bladder 177 THE KIDNEY General Description 178 The Tubuli Uriniferi 180 Blood-vessels '. 183 Practical Demonstration, with Low-power 186 Practical Demonstration, The Cortical Portion 187 Practical Demonstration, The Medullary Portion 192 Pelvis of the Kidney and Ureter 193 Urinary Bladder 19 1 Urethra". . 196 X CONTENTS FEMALE GENERATIVE ORGANS PAGE Vagina and Uterus— Practical Demonstration ] 97 Fallopian Tube — Practical Demonstration ... • . . . 202 Ovary 203 Adult Human Ovary— Practical Demonstration 203 Formation of the Ovum 206 Ovary of Human Infant— Practical Demonstration 206 Mammary Gland 208 MALE GENERATIVE ORGANS Testicle — General Description 209 Practical Demonstration 211 Spermatozoa 211 Prostate Gland 212 Erectile Tissue 212 SUPRARENAL BODY Practical Demonstration 213 THE NERVOUS SYSTEM Structural Elements 216 Nerve-fibers 216 Nerve-trunks 217 Practical Demonstration 219 Nerve-cells 219 Neuroglia 222 Peripheral Nerve-endings 223 THE SPINAL CORD General Description 226 Division into Tracts 228 Practical Demonstration . . 229 Relation of Ganglion- cells and Nerve -fibers 233 THE BRAIN Membranes 235 Cerebrum — Practical Demonstration 237 Layers of the Cerebral Cortex 238 Paths followed by Nerve -fibers in the White Matter 239 Cerebellum— Practical Demonstration 240 INDEX (page 245) LIST OF ILLUSTRATIONS FIG. PAGE 1. Microscope 2 2. Relation of Objective to Eye-piece 3 3. English and Metric Scales 8 4. Free-hand Section Cutting 11 5. Stirling's Microtome 12 6. Method of Imbedding with Pith, Turnip, etc 13 7. Section Cutting with Stirling Microtome . 14 8. Thoma Microtome 14 9. Schanze Microtome 15 10. Method of Honing Razor 16 11. Turning the Razor on the Hone 16 12. Paraffin Soldering Wire 18 13. Cementing Hardened Tissue to Cork 18 14. Freezing Microtome 19 15. Using Turn-table 36 16. Needle for Lifting Sections 36 17. Diagram Illustrating Steps in Staining with Hgematoxylin 37 18. Diagram Illustrating Steps in Staining with Haeinatoxylin and Eosin. 39 19. Diagram Illustrating Steps in Staining with Borax-Carmine .... 40 20. Section -lifters 41 21. Appearance of Balsam-mounted Specimen 42 22. Mode of Handling Cover-glass 42 23. Diagram Showing Effect of Oil and Air Globules 46 24. Extraneous Substances— Fibers, etc 47 25. Extraneous Substances — Starch, etc 48 26. Elements of a Typical Cell 49 27. Structure of a Cell-nucleus 50 28. Indirect Cell Division 51 29. Karyokinesis 52 30. Squamous Epithelial Cells from Mouth 56 31. Pavement Epithelium 57 32. Columnar Cells from Intestine 58 33. Ciliated Columnar Cells from Bronchus 58 34. Diagram Showing Organs of the Oyster 59 35. Glandular Cells from Liver 60 36. Frog's Mesentery — Silver- staining 61 37. White Fibrous Tissue 63 38. Yellow Elastic Tissue 64 39. Transverse Section of Ligamentum Nuchse 64 (xi) xii LIST OF ILLUSTRATIONS FIO. PAGE 40. Cells containing Fat 6G 41. Adipose Tissue from Omentum 66 42. Hyaline Cartilage from Bronchus 67 43. Fibro- Cartilage from Intervertebral Disc 68 44. Elastic Cartilage from Ear of Bullock 69 45. Bone — Showing Laminated Structure 69 46. Bone — Showing Haversian Systems 70 47. Bone — Showing Sharpey's Fibers 71 48. Contents of Haversian Canals 71 49. Contents of Bone Lacuna 72 50. Cells from Bed Marrow 74 51. Developing Bone 75 52. Non-striated Muscular Fiber 77 53. Striated Muscular Fiber '.'..' 78 54. Striated Muscular Fiber from Tongue 79 55. Cardiac Muscular Fiber .' 80 56. Corpuscular Elements of Human Blood 81 57. Diagram of Red Blood-corpuscle 82 58. Blood Showing Blood-plates 83 59. Cover- glass, Preparations of Blood 84 60. Varieties of Leucocytes . . . 85 61. Pipettes for Hsemocytometer 86 62. Disk of Haamocytometer 87 63. Magnified Field of Haemocytometer 88 64. Crystals of Haemoglobin 89 65. Blood-corpuscles of the Frog 90 66. Layers of the Epidermis 93 67. Structure of the Derma — Injected 94 68. Hair in Transverse Section 95 69. Hair Follicle ..... 96 70. Sudoriferous Gland 97 71. Sebaceous Gland ... 98 72. Skin in Vertical Section. . . 100 73. Diagrammatic Section of Artery ... 103 74. Blood-capillaries 104 75. Perivascular Lymph-spaces ... ... 107 76. Lymphatics of Central Tendon of the Diaphragm — Low-power . . 110 77. Lymphatics of Central Tendon of the Diaphragm — High-power . . Ill 78. Lymph-node — Diagrammatic .112 79. Mesenteric Lymph-node — Low-power 114 80. Mesenteric Lymph-node — High-power 115 81. Blood-vessel Arrangement in the Spleen 117 82. Spleen 119 83. Thymus Body . 122 84. Bronchial Tube— Small 124 85. Bronchial Tube— Medium 127 86. Pulmonary Lobule— Perspective 129 87. Pulmonary Lobule — Longitudinal Section 129 88. Pulmonary Alveolus — Capillaries Injected 130 LIST OF ILLUSTRATIONS Xlll FIG. PAGE 89. Lung of Pig 132 90. Pulmonary Alveolus Showing Lining 133 91. Diagrammatic Section of Tooth 136 92. Section of Part of Tooth— High -power 139 93. Simple Tubular Gland _141_ 94. Coiled Tubular Gland .... 141 95. Branched Tubular Gland 142 96. Acinous Gland 142 97. Parotid Gland 144 98. Submaxillary Gland 145 99. Pancreas 146 100. Stomach — Diagrammatic Section 152 101. Cardiac Gastric Gland 153 102. Pyloric Gastric Gland .... 154 103. Stomach of Dog • 155 104. Diagram Illustrating Intestinal Secretion 158 105. Diagram of Intestinal Absorption . . 159 106. Small Intestine with Peyer's Patch 161 107. Liver — Diagram Illustrating Plan of Structure 164 108. Glandular Cells in Connection with Blood-vessels and Ducts . . . 166 109. Liver of Pig 168 110. Human Liver — Low-power 171 111. Portal Canal 173 112. Hepatic Cells— Detached 174 113. Hepatic Lobule in Transverse Section 175 114. Bile-capillaries — Origin of Bile-duct 176 115. Kidney — Diagram Illustrating Plan of Structure 179 116. Kidney Tubules— Isolated. ' .... .181 117. Blood-vessels — Arrangement in Kidney . 183 118. Kidney — Low-power 186 119. Kidney— Cortex in Vertical Section ....... . 188 120. Kidney — Medulla in Longitudinal Section 191 121. Kidney— Medulla in Transverse Section . . 192 122. Epithelium of Ureter 194 123. Epithelium of Urinary Bladder .... 195 124. Uterus with Vaginal Cul-de-sac 198 125. External Os Uteri . ....... 200 126. Vaginal Epithelium 201 127. Fallopian Tube 202 128. Ovary— Adult 204 129. Ovary— Child's 207 130. Mammary Gland— Dog ... 208 131. Mammary Gland — Dog .... 208 132. Testicle, Diagram 209 133. Seminiferous Tubules . 210 134. Spermatozoa r 211 135. Suprarenal Body — Low-power . 214 136. Suprarenal Body— High-power 215 137. Nerve -fibers .216 XIV LIST OF ILLUSTRATIONS FIG. PAGK 138. Nerve -trunk — Transverse Section 218 139. Two Types of Ganglion -cells ....... 220 140. Diagram of a Neurone ... 221 141. Neuroglia 222 142. Nerve-endings in the Cornea 223 143. Tactile Corpuscle 224 144. Pacinian Corpuscle 224 145. Nerve-ending in Striated Muscle 225 146. Diagram, Spinal Cord 226 147. Dorsal Spinal Cord 227 148. Lumbar Spinal Cord 228 149. Cervical Spinal Cord . 230 150. Anterior Horn — Gray Matter— Cervical Spinal Cord 231 151. Ganglion cells, Anterior Horn 233 152. Diagram, Relations of Cells -and Nerve-fibers in the Spinal Cord. . 234 153. Layers of the Cerebral Cortex 236 154. Section of Cerebrum 237 155. Ganglion-cell and Neuroglia-cell — Cerebral Cortex 239 156. Cerebellum — Low-power 240 157. Cerebellum — High-power 241 158. Cell of Purkinje 242 STUDENTS HISTOLOGY PART FIRST TECHNOLOGY THE LABORATORY MICROSCOPE The histologist should be provided with a microscope, in which the principal features of the laboratory instrument, Fig. 1, are embraced. The body A, which carries the optical parts, is made of two pieces of brass tubing, one sliding within the other and providing for alterations in length. The objectives, B, C, D, are attached to the body by means of the triple nose-piece, E. The nose-piece is so pivoted that either objective may be turned into the optical axis at will. The eye-piece, F, slips into the upper part of the body with but little friction, so that it may be quickly and easily removed. The coarse or quick adjustment for focusing consists of a rack, G, which is attached to the body, and into this gears a small (concealed) pinion turned by the milled heads, H. The more delicate adjustments are accomplished by means of a micrometer screw acting by a simple mechanical device upon an enclosed spring in connection with a prism slide. By turning the milled head, L, the body of the instrument is raised or lowered, as desired, and with extreme delicacy. The stage, M, upon which objects are placed for examination, is perforated, and an iris diaphragm and Abbe condenser, K, may be inserted. The iris diaphragm enables one to alter the size of the opening at will. Below the stage an arm may be seen which carries a sliding fork supporting the mirror, N, one side of which is plane and the other concave. STUDENTS HISTOLOGY FIG. 1. THE MICROSCOPE. The whole is supported on a short, stout pillar rising from the base, O. LENSES OF THE MICROSCOPE Fig. 2 shows the arrangement of lenses, including a high- power objective. The objective, A, is provided with one simple and two compound lenses. The lens, B, nearest the object, and the one upon which the magnifying power mainly depends, is a hemisphere of crown glass. A lens of such form possesses both chromatic and spherical aberration in high degree. These faults are corrected by the com- pound flint and crown lenses, C and D, placed above the hemi- spherical glass. The eye -piece consists of two crown glass, plano-convex lenses, LENSES OF THE MICROSCOPE with their plane surfaces upward. The lower, E, is known as the field -lens ; the upper, F, as the eye -lens. Eye -pieces add very materially to the magnifying power of the instrument, and are FIG. 2. DIAGRAM SHOWING THE RELATION OP THE OBJECTIVE TO THE EYE-PIECE. constructed of various strengths depending upon the curvature of the lenses. They are named according to power, A, B, C, or 1, 2, and 3, or according to their focal distances. The medium power is more commonly employed. The microscope previously described stands, with the draw- tube 4 STUDENTS HISTOLOGY in place, about twelve inches high. If the height of the table upon which it is placed and the chair of the observer be in a proper relation, no discomfort need be experienced in using the microscope in the vertical position. The form of condenser invented by Abbe may be placed directly below the stage (Fig. 1, K) . It consists of two or three lenses combined so as to focus the rays coming from the plane mirror upon the object. The condenser gives a very intense illumination over a small field, and is adapted to bacteriological work, where a high -power oil -immersion objective is required. An oil -immersion objective is a specially constructed system of lenses, with which a layer of thickened oil of cedar wood is placed between the lower surface of the objective and the upper surface of the glass covering the object under examination. The oil -immersion lens in general use has an equivalent focal length of one -twelfth of an inch, and is usually designated as the iV-inch oil -immersion. For similar reasons, the low -power is a f- or f-inch, and the ordinary high-power is a i-, i-, or i-inch objective, as the case may be. The condenser is not necessary, except with the high-power oil-immersion objective. If used with the other objectives, the illumination must be regulated by lowering the condenser, closing the diaphragm, and substituting the concave for the plane mirror, till a clear and satisfactory picture is secured. ADJUSTMENT OF THE MICROSCOPE The microscope should be placed in front of the observer, on a table of such height that, when seated, he may, by slightly inclin- ing the head, and without bending the body, bring the eye easily over the eye -piece. The slightest straining of the body or neck should be avoided. The light should always be taken from the side, and it matters little which side. Clouds or clear sky serve as the best source of light for our present work. Always avoid direct sunlight. If artificial illumination be employed — though it is not advised for prolonged investigation — a small coal -oil flame may be tempered by blue glass, or better a Welsbach gas-burner with a blue glass globe, or an incandescent electric light. ADJUSTMENT FOR ILLUMINATION It will be observed that there are two mirrors in the circular frame below the stage — one plane and the other concave, The ADJUSTMENT FOR FOCUS O latter will be employed almost exclusively in the work of this volume, and its curvature is such that parallel rays, impinging upon its surface, are focused about two inches from the mirror. It will also be noticed that the bar, carrying the mirror -fork, may be made to swing the mirror from side to side. The work whieh we are about to undertake is of such a character as to require the avoidance of oblique illumination. We must, therefore, keep our mirror -bar strictly in the vertical position. If — the mirror -bar being vertical — a line be drawn from the center of the face of the mirror, through the opening (diaphragm) in the stage, passing on through the objective, and so continued upward through the body and the eye -piece, such a line would pass through the optical axis. The center of the face of the mirror must be in this axis. If, then, having gotten the mirror -bar properly fixed once for all, the light from the adjacent right or left hand window impinges upon the concave surface of the mirror, and the latter be properly inclined, the rays will pass through the diaphragm in the stage, and become focused a little above the same. The light rays will afterward diverge, enter the objective, and finally reach the eye of the observer. The field of view (as the area seen in the microscope is termed) we will suppose to have been properly illuminated — and by this we mean that it presents us a clear, evenly lighted area. Turn all the factors spoken of out of adjustment, and proceed to readjust. Observe that, if the mirror be turned— not swung— slightly out of proper position, one side of the field will appear dim or cloudy. This must be corrected, and the student must practice until this adjustment becomes easy of accomplishment. Then proceed to the ADJUSTMENT FOR FOCUS Swing the low -power objective into use, and rack the tube up or down until it is about one -fourth of an inch from the stage. Place a mounted object upon the stage (a stained section of some organ— say kidney— will be preferable). Examine the field through the eye -piece, and it will be found obscured by the stained object, and perhaps a dim notion of figure may be made out. Rack the body up carefully, watching the effect. The image becomes more and more distinct until, at a certain point, the best effect is secured. The object is in focus. Note carefully the distance between the object and the objective 6 STUDENTS HISTOLOGY (with the low -power this will be less than one -half of an inch), and hereafter you will be able to focus more quickly. Having observed the details of structure as shown with the low- power, swing the high -power into use. Rack the tube down until the objective is very near to the glass covering the object. The field is much obscured. Watching the effect through the eye- piece, rack the tube up with great care until the image appears sharp. Note the distance with this objective, as before with the low -power, from one -twelfth of an inch to considerably less. Then endeavor, by slight alterations in the inclination of the mirror, to increase the illumination. Turn the diaphragm so that the light passes through a small opening, and note the improve- ment in definition. The rule is: The higher the power, the smaller the diaphragm. You have doubtless observed, before this, that you cannot con- trol the focusing as easily as when the low -power was in use. Slight movements of the rack -work produce marked changes in definition; and it is difficult, with the coarse adjustment alone, to make as slight movements as you may desire. Recourse must be had to the fine adjustment. Place the tip of the forefinger (either) upon the milled head of the fine focusing -screw, and the ball of the thumb against its side, so that the hand is in an easy position. By a little lateral pressure the milled head may be turned slightly either way. Note the effect on the image. You thus have the focusing under the most perfect control. Remember that the fine adjustment is only necessary with high- powers, and then only after the image has been found with the coarse adjustment. METHOD IN OBSERVATION The study of objects under the microscope should be conducted with order and method. The body being in the position before advised, so that the sitting may be prolonged without fatigue, let one hand be occupied in the maintenance of the focal adjustment. It will be found, however flat an object may seem to the unaided eye, that as it is moved so as to present different areas for examination (and with the higher- powers only a small area can be seen at once), constant manipulation with the fine adjustment will be required. It will MAGNIFYING POWER AND MEASUREMENT OF OBJECTS 1 also be found that even the various parts of a simple histological element — like a cell — cannot be seen sharply with a single focal adjustment. The forefinger and thumb of one hand must be kept constantly on the milled head of the fine focusing -screw. Sup- posing the light to be 011 our right, we devote the right hand to the focusing. The left hand will be engaged with the glass slip upon which the object has been mounted. The forearm resting upon the table, let the thumb and forefinger rest on the left upper side of the stage, just touching the edges of the glass slip. The slightest pressure will then enable you to move the slip smoothly, steadily, and delicately. Proceed to examine the object with method. Suppose a section of some tissue to be under examination — say one -fourth of an inch square. With the high -power you will be able to see only a small fraction of the area at once. Commence at one corner to observe, and, with the left hand, move tfre object slowly in successive parallel lines, preserving the focus with the right hand, until the whole area of the section has been traversed. Practice will soon establish perfect co-ordination of the move- ments involved, and will result in the ability to work with ease, celerity, and profit. CONSERVATION OF THE EYESIGHT The beginner should not become accustomed to the use of one eye alone, or of closing either in microscopical work. It will require but little practice to use the eyes alternately, and the retinal image of the unemployed eye will soon be ignored and unnoticed. MAGNIFYING POWER AND MEASUREMENT OF OBJECTS The microscope is not, as the beginner usually supposes, to be valued according to its power of enlargement or magnification, but rather according to the clearness and sharpness of the image afforded. Magnifying power is generally expressed in diameters. A certain area is by the instrument made to appear, say, ten times as large as it appears to the naked eye. This object, then, has its apparent area increased one hundred times; but reference is made 8 STUDENTS HISTOLOGY in describing such phenomena only to amplification in a single direction. The diameter of the object under examination has been increased ten times and would be expressed by prefixing the sign of multiplication: e.g., X 10. A convenient unit of approximate measurement for the histolo- gist is the apparent size of a human red blood -corpuscle with a given objective. Thirty -two hundred corpuscles, placed side by side, would measure one inch; or, we say, the diameter of a single corpuscle is the thirty -two -hundredth of an inch. After consider- able practice, you will become accustomed to the apparent size of this object with a certain objective and eye -piece. This will aid in an approximate measurement of objects by comparison, and will further give the magnifying power of the microscope. If a cor- puscle appears magnified to one inch in diameter, it is evident that the instrument magnifies thirty -two hundred times. Should the 1 Inches. 2 3 4 I 1 1 II 1 1 1 1 11 1 1 1 1 1 1 1 1 MM 1 1 1 I i MM II II II 1 1 1 1 II 1 1 1 i 1 II 1 Illl Illllllll Illl1 U ! MM || lIlMI mi mi Illl Illl IIU 3 10 4567 Centimeters. FIG. 3. ENGLISH AND METRIC SCALES. diameter appear one -quarter of an inch, the power is eight hun- dred ; one-eighth of an inch, four hundred, etc. The instrument which we have heretofore described, with the high- power in use and the tube withdrawn, will present the corpuscle as averaging very nearly one -eighth of an inch in diameter — X 400. While this gives a gross idea of amplification, the method will often prove to be inaccurate because of individual errors in the estimation of proportions. Measurements made with the microscope are usually based on the metric system. The unit taken is one -thousandth part of a millimeter, or a micro -millimeter, called also a micron, for which the Greek letter p has been taken as the symbol. Roughly, 1 p equals 25000 inch. A convenient instrument for measuring is an eye -piece micrometer, a ruled disk of glass, which may be placed within the eye -piece, and the diameters of objects read off by means of the ruled scale. PREPARATION OF TISSUES 9 SKETCHING FROM THE MICROSCOPE Let us most emphatically urge the practice of sketching in connection with microscopy. "I am no artist," or "I have no skill in drawing," is often the reply to our advice in this matter. We- then suggest that no special skill is needed to begin with, only patience and a dogged determination to succeed. The pictures in the microscopic field have no perspective, and may be reproduced in outline merely. Begin with simple tissues, reserving intricate detail until a short period of practice gives the technique needed. We do not recommend the camera lucida, as our experience strongly impresses us with this as a fact, that he who cannot sketch without a camera will never sketch with one. Pencil drawings may be very effectively colored with our staining fluids, diluted if necessary. PREPARATION OF TISSUES FOR MICROSCOPICAL PURPOSES TISSUES ARE STUDIED BY TRANSMITTED LIGHT The microscopical study of both normal and pathological tis- sues is invariably conducted by the aid of transmitted light. Tissues, if not naturally of sufficient delicacy to transmit light, must in some way be made translucent. Delicate tissues like omenta, desquamating epithelia, fluids containing morphological elements, certain fibers, etc., are suffi- ciently diaphanous, and require no preparation. Such objects are simply placed upon the glass slip, a drop of some liquid added, and, when protected by a thin covering glass, are ready for the stage of the microscope. PREPARATION BY TEASING The elements of structures mainly fibrous — e.g., muscle, nerve, ligament, etc. — are well studied after a process of separation, by means of needles, known as teasing. A minute fragment of the organ or part, having been isolated by the knife or scissors, is placed upon a glass slip, and a drop of some fluid which will not alter the tissue added. Stout sewing -needles, stuck in slender wood handles, are commonly employed in the teasing process. 10 STUDENTS HISTOLOGY The separation of tissues is frequently facilitated by means of dissociating fluids, which remove the cement substance. SECTION CUTTING After having become familiar with the various elementary structures of animal tissues, we proceed to the study of their relation to organs. As the teasing process is not available with such complicated structures as lung, liver, kidney, brain, etc., we resort to methods of slicing — i.e., section cutting. Sections must be made of extreme tenuity, in order that the naturally opaque structures may be illuminated ~by transmitted light. This becomes an easy matter with such tissues as cartilage; but some, like bone, are much too hard to admit of cutting, and others are as much too soft ; so that while certain tissues must be softened, the majority must be hardened. Fortunately, both of these conditions may be secured without in any way altering the appearance or relations of the structures. Hardening processes, from necessity, become a prominent feature in histological work ; but we propose here to indicate some of the more useful methods of section cutting, reserving the hardening processes for another place. FREE-HAND SECTION CUTTING The students, when ready for this work, are provided with some tissue which has been previously hardened. We will take, for example, a piece of liver which has been rendered sufficiently firm for our work by immersion in alcohol, and proceed to direct the steps in obtaining suitable sections by the simple free-hand method. We wish to strongly emphasize the importance of this mode of cutting. A moderate amount of practice will render the micros- copist independent of all appliances, save those of the most simple character and which are always obtainable. An ordinary razor with keen edge, and a shallow dish, prefer- ably a saucer, partly filled with alcohol, are required. The razor best adapted to the work is concave on one side (the upper side, as seen in Fig., 4) and nearly flat on the other, although this is largely a matter of personal preference. Fig. 4 indicates the proper position of the hands in commenc- FREE -H AS 'D SECTION CUTTING 11 ing the cut. The beginner must follow directions closely until he acquires skill with practice. The student should be seated at a table of such height as to afford a convenient rest for the fore- arms. A small piece of tissue is held between the thumb and forefinger of the left hand, so that it projects slightly above both (In the cut, a cube of tissue too small to handle in this way has been cemented to a cork with paraffin in the manner hereafter described, and the cork held as just mentioned.) The hand carry- ing the tissue is held over the saucer of alcohol. The razor, held lightly in the right hand, as seen in the figure, is, previous to FIG. 4. FREE-HAND SECTION CUTTING. making every cut, dipped flatwise into the alcohol so as to wet it thoroughly, and is then lifted horizontally, carrying several drops— perhaps half a drachm— of the fluid on the concave upper surface. The alcohol serves to prevent the section from adhering to the knife, and to moisten the tissue. If allowed to become dry, the latter would be ruined by alterations of structure. Now, as to the manner of moving the knife. Resting the under surface upon the forefinger for steadiness, bring the edge of the blade nearest the heel to the margin of the tissue furthest from you. Then, entering the edge just below the upper surface of the tissue, with a light but steady hold draw the knife toward the right, at the same time advancing the edge toward the body. This passes the knife through the tissue diagonally, and leaves the upper surface of the latter perfectly flat or level. Remove the 12 STUDENTS HISTOLOGY piece which has been cut, and repeat the operation. Do not attempt to cut large or very thin sections at first. A minute fragment, if thin, is valuable. As the razor is drawn through the tissue, the section floats in the alcohol; depress the point of the knife, and the section will slide into the saucer of spirit, and thus prevent its injury. If it does not leave the knife readily, brush it along with a camel' s- hair pencil which has been well wet with the alcohol. Proceed in the above manner until the tissue is exhausted, when you will have a great number of sections, large and small, FIG. 5. STIRLING'S MICROTOME. thick and thin. Selecting the thinnest, lift them carefully with a needle, one at a time, into a small, wide -mouthed bottle of alcohol; cork and label for future use. When the work is finished, and before the spirit has evaporated from your fingers — it is impossible to avoid wetting the skin more or less— wash them thoroughly and wipe dry. This saves the roughening of the hands which is apt to result when alcohol has been allowed to dry upon them repeatedly. SECTION CUTTING WITH THE STIRLING MICROTOME Of the numerous mechanical aids to section cutting, we shall mention only two or three. One of the earlier and better known SECTION CUTTING WITH THE MICROTOME 13 instruments is seen in Fig. 5. The Stirling microtome consists essentially of a short brass tube, into which the tissue is fixed either by pressure or by imbedding in wax. A screw enters below, which, acting on a plug, raises the contents of the tube. As the material to be cut is raised from time to time by the screw, it appears above the plate which surrounds the top of the tube. This plate steadies and guides the razor ; and it is evident that more uniform sections may be cut with this little apparatus than 'would be possible with nothing to support the knife, or to regulate thickness, beyond the unaided skill, of the operator. Much depends upon the manner in which the material is fixed in the tube or well of the microtome. If the tissue be of a solid character, like liver, kidney, spleen, many tumors, etc., it may be surrounded with some carefully fitted pieces of elder-pith,* carrot, FIG. 6. MANNER OF CUTTING AND ARRANGING PIECES OF PITH, TURNIP, ETC., FOR SUPPORTING HARDENED TISSUE IN THE WELL OF A MICROTOME. etc., and the whole pressed evenly and quite firmly into the well. A small piece of tissue which, by cutting, can be made somewhat cubical in shape, may be surrounded by slabs of pith, carrot, or turnip, shaped as in Fig. 6. Indeed, the fragments of imbedding material can be shaped so as to fit tissue of almost any form. Before the whole is pressed into the well of the microtome, the bottom, against which the brass plug fits, should be cut off square. The wax method of imbedding is employed with tissues such as brain, lung, soft tumors, etc., which might be injured by the previous treatment. To three parts of paraffin wax (a paraffin candle answers perfectly) add one part of vaselin, and heat until thoroughly mixed. The microtome having been previously warmed — standing upright — is filled with the imbedding mixture. The "The pith from the young shoots of Ailantus glandulosus (improperly called "Alanthus"), gathered in early autumn, is the best material for this method of imbedding with which I am. acquainted. The wood is easily cut from the pith, and the latter is very large and firm. 14 STUDENTS HISTOLOGY piece of tissue is then carefully wiped dry with the blotting-paper, and, just as the imbedding begins to congeal around the edges, is pressed below the surface with a needle and held until the cool mixture fixes it in position. The whole is now allowed to become thoroughly cold. By turning the screw the plug of wax is raised; FIG. 7. METHOD OF CUTTING SECTIONS WITH THE STIRLING MICROTOME. and it must be gradually cut away, by sliding the knife across the plate, until the upper part of the tissue appears. Before commencing to cut sections — however the tissue may have been imbedded — provide yourself with a saucer of alcohol and a camel's -hair pencil. Having wet the knife, turn the screw so that the tissue, with its imbedding, appears slightly above the FIG. 8. THOMA MICROTOME. plate of the microtome, and then, resting the blade of the razor on the plate (vide Fig. 7), make the cut precisely as in free-hand cutting. The section is then brushed off into the saucer, the screw turned up slightly, the razor wet, and a second cut made. These steps are repeated until the required number of sections has been obtained. SECTION CUTTING WITH THE MICROTOME 15 The imbedding material will separate from the cuts as they are floated in the alcohol. They may now be selected, lifted with the needle into clean spirit, and preserved, as before indi- cated, for future operations.- The finest section cutting can only be done with one of the more elaborate and expensive microtomes (Figs. 8 and 9). Each of these instruments consists of a heavy sliding knife -carrier, which moves on a level and with great precision, and of a device FIG. 9. SCHANZE MICROTOME. (screw or inclined plane) for elevating the object that is cut the desired distance after each excursion of the knife. The distance that the object is raised is, of course, the thickness of the sec- tion. Such microtomes are especially adapted to cutting frozen tissues or those imbedded in celloidin, but they may be used with paraffin imbedding. The Minot microtome, which is intended for cutting objects in paraffin, is preferable for the latter purpose, especially if a number of sections are to be kept in the order in which they were cut, — "serial sections." 16 STUDENTS HISTOLOGY SHARPENING KNIVES In the majority of instances of failure to produce suitable sec- tions for microscopical work, the cause can be set down to dull knives, and we would urge the student to practice honing until able to put cutting instruments in good condition. If he will but start properly, success is sure. Nine -tenths of the microtomes are pur- chased because of failure in free-hand work with a dull knife ; but no advantage will ~be gained by a machine, if the student be incapa- ble of keeping the knife up to a proper degree of keenness. A knife is a wedge, and for our purpose the edge must be of more than microscopical tenuity — it being impossible with the microscope to discover notches and nicks if properly sharpened. It is impossible to secure the best results with indifferent tools. FIGS. 10 AND 11. METHOD OF HONING. The knife is first brought with its heel in the position shown at A, Fig. 10. It is then drawn forward as indicated by the curved dotted line until, at the end of the stroke, the position C is attained. Fig. 11 indicates the method of turning the blade before reversing and between each stroke. The knife should be hard enough to support an edge, but not so hard as to be brittle. The proper temper is about that given a good razor. We need at least two hones — one comparatively coarse, for removing slight nicks, and another for finishing. The first part of the work is best done by means of a sort of artificial hone made of ground corundum. These are kept in stock by dealers in mechanics' supplies, of great variety 'in size and fineness. For razors a "00" corundum slip will best answer. This will very SHARPENING KNIVES 17 rapidly remove the inequalities from an exceedingly dull razor. A Turkish hone will be best for. finishing. For large knives we use a third very soft and fine stone. Let the corundum slip be placed on a level support (or be fitted into a block like the carpenter's oil-stone), and cover the surface liberally with water.* The hones should always be worked wet. Place the knife flat on the stone near the right hand, as at A, Fig. 10. Draw steadily in the direction of the curved dotted line — i.e., from right to left — holding the blade firmly on the stone, B, with slight pressure until the position C is attained. Rotate the razor on its back — vide Fig. 11 — so as so bring the other side on the stone, and draw from left to right. Observe that as the knife is drawn from side to side (the edge invariably looking toward the draw) it is always worked from heel to point. The amount of pressure may be proportioned to the condition of the edge. If it be badly nicked, considerable pressure may be employed ; while, as it approaches keenness, the pressure is to be lessened, until the weight of the blade alone gives sufficient friction. Repeat the process fifteen or twenty times, and examine the blade. If the nicks are yet visible, continue honing until they can no longer be seen. Then draw the edge across the thumb-nail. Do this lightly, and the sense of touch will reveal indentions which the eye failed to recognize. Continue the use of the coarse stone until the edge is perfect, so far as the thumb-nail test indicates. The knife is then to be carefully wiped, so as to remove any coarse particles of corundum, and applied to the wetted Turkish hone with precisely the same motions as were employed in the first process. After a dozen or two strokes, examine the edge by applying the palmar aspect of the thumb, with repeated light touches, from heel to point. This looks slightly dangerous to the novice, but it is an excellent method of determining the con- dition. Of course actual trial with a piece of hardened tissue is the best test. A fine water -stone or the Belgian hone of the hardware shops may be used instead of the corundum hone. It is best to finish with stropping, and often a knife may be sharpened by stropping alone. The leather of the strop should be glued to a support of wood to keep it flat. The movement is *A few drops of glycerin added to the water retard evaporation, and appear to keep the surface of the hone in good condition. 18 STUDENTS HISTOLOGY the reverse of that employed for honing. The motion is from toe to heel, the back of the knife preceding the edge. Fig. 10, with the arrow reversed, illustrates the movement. SUPPORTING TISSUES FOR CUTTING Frequently small bits of tissue are required to be cut — pieces too small to be held with the fingers. We are in the habit of FIG. 12. INSTRUMENT FOR SOLDERING TISSUE TO CORK SUPPORTS WITH PARAFFIN. It consists of an awl-handle of wood into which a short piece of wire, preferably copper, is driven and bent as shown. cementing such tissues into a hole in a bit of ailantus- or elder - pith, when the whole may be cut as one mass. Tissue is fre- quently cemented to cork for convenience of holding in free-hand FIG. 13. METHOD OF CEMENTING TISSUE TO A CORK SUPPORT WITH PARAFFIN. cutting ; or the cork may be held in the vise of the microtome. The edge of the knife should not be allowed to touch the cork. Fig.. 12 shows a simple little instrument, very convenient for THE FREEZING MICROTOME 19 using paraffin as a cement. A piece of stout copper or brass wire is bent as indicated, pointed, and driven into an ordinary awl- handle. Paraffin wax possesses the very valuable property of remaining solid at ordinary temperatures, not cracking in the cold, of winter nor softening in summer. It is unaltered by most re- agents, is easily rendered fluid, and quickly solidifies. As a cement, it is invaluable to the microscopical technologist. Fig. 13 indicates the method of cementing a piece of tissue to a cork or other support. The tissue having been properly placed, the wire tool is heated for a moment in the alcohol flame, and then FIG. 14. CATHCART'S ETHER FREEZING MICROTOME. touched to a cake of paraffin. The paraffin is melted in the vicinity of the hot wire, a drop adheres to the latter and is carried to the edge of the tissue. In the cut the wire tool is seen in the position necessary for cementing one edge. The wire being removed, the wax immediately cools and becomes solid. The other sides are afterward cemented in like manner. The whole is done in less time than is necessary to the description of the process. THE FREEZING MICROTOME After freezing a piece of tissue, sections may be cut without the delay required for imbedding in paraffin or celloidin. Fresh tissues may be used, or those hardened by some of the various fixing 20 STUDENTS HISTOLOGY fluids, preferably formaldehyde, may be taken. Small pieces that have been hardened for a few hours in formaldehyde may be cut very thin after freezing, and this plan is most useful in pathological work. Sections of fresh tissues may be examined with the micro- scope directly, or they may be placed in formaldehyde, and after- wards stained and mounted as directed in the succeeding pages. Sections of hardened tissues may be stained and mounted in balsam. Freezing of the specimen is done upon a metal box or plate, which may be attached to the ordinary microtome. Microtomes designed especially for freezing are also made (Fig. 14). Freezing is best accomplished by the escaping stream of carbon dioxide gas, derived from a cylinder of the compressed gas. An ether -spray may be used for the same purpose, or the water running from a salt and ice freezing mixture through a small metal case which may be placed on the microtome. The pieces of tissue should be not more than five millimeters thick. FIXING OR HARDENING FLUIDS We have already seen that most animal tissues are unsuitable for the production of thin sections until hardened. It is also a fact, paradoxical though it may seem, that fresh tissues do not present truthful appearances of structural elements. The old -school histologists insisted upon the presentation of struc- tures unaltered by chemical substances, while the modern worker has discarded such tissue with very few exceptions. Many descrip- tions for structure and growth, the result of study upon fresh material, have been proved by later methods grossly inaccurate. It is impossible to remove tissues from the living animal and to subject them to microscopical observation without, at the same time, exposing them to such radical changes of environment as to produce structural alterations. Certain tissues presenting in the living condition stellate cells with the most delicate, though well- defined, branching processes, when removed from contact with the body, however expeditiously, afford no hint of anything resem- bling such elements, as they are quickly reduced to simple spherical outlines. In short, it is impossible to study fresh material, as such, with- out constant danger of erroneous conclusions, as retrograde altera- tions of structure commence with surprising rapidity the moment ALCOHOL HARDENING 21 a part is severed from the influences which control the complete organism. From what has been said, we appreciate the necessity of agents which, when applied to portions freshly removed from the animal, or even before removal, shall instantly stop all physiological pro- cesses and retain the elements in permanent fixity. Very much of the human structure which is available will be secured only after functional activities have long ceased, and the structure essentially altered. We are, therefore, compelled to resort to the use of material from the lower animals in very many instances. ALCOHOL HARDENING The tissue, whatever process may be in contemplation, having been removed from the body as quickly after death as possible, should, with a sharp scalpel, be divided into small pieces. Of the more solid organs, pieces not more than one -half to one centimeter thick will be sufficiently small, and they will harden rapidly. The smaller the pieces and the larger the quantity of hardening fluid, the more quickly will the process be completed. The volume of fluid should exceed that of the tissue at least twenty times. Wide- mouthed, well stoppered bottles, from one ounce to a pint, or even larger, are best, and they should be carefully labelled and kept in a cool place, with occasional agitation. Quick Method. — A piece of any solid organ, say liver, spleen, pancreas, kidney, uterus, lymph -node, etc., not more than one -half centimeter thick, may be perfectly hardened in twelve hours by immersion in one ounce of ninety -five per cent, alcohol. No more should be thus prepared than is to be cut within twenty -four hours, on account of the shrinkage which results after the pro- longed immersion of solid structures in strong spirit. After the tissue has been one hour in the above, it may be hardened in one or two hours more if transferred to absolute alcohol. This method is of frequent advantage in pathological histology. Ordinary Method. — Generally it is better to place the pieces of tissue first in eighty per cent, alcohol, and to transfer them after a few hours to ninety -five per cent, alcohol, in which the hardening is completed. In this manner the shrinkage and the extreme hardening of the surface, which result when strong alcohol alone is used, are avoided. The jar needs to be shaken occasionally. 22 STUDENTS HISTOLOGY The portions of tissue should be not more than one -half to one centimeter thick. Alcohol is the most generally used of all hardening and fixing fluids. Tissues preserved in it will keep indefinitely, especially if the strong alcohol be replaced by eighty per cent, alcohol after a time. When bacteria are to be demonstrated in the organs, alcohol is as good as any agent that can be used. But when the blood, the nuclear figures, the elements of the nervous system, and the finer points in histological structure are to be studied, other fluids are more suitable. MULLER'S FLUID Bichromate of potassium 2.5 grams. Sulphate of sodium 1 gram. Water 100 c.c. Miiller's fluid is one of the most valuable of all fixing agents. The time required for hardening is six weeks or more, but harden- ing may be hastened by placing the jar in an incubator. The pieces of tissue must be small. The fluid must not become dis- colored, and must be changed frequently at first. It is most valuable for nervous tissues, which harden only after months. After hardening, the tissues must be washed in running water for twenty -four hours, and are then placed in alcohol, except nervous tissues, which are placed in strong alcohol without washing, when the Weigert haematoxylin stain is to be used. Miiller's fluid preserves the blood - corpuscles in the organs admirably. The hardening can be hastened by adding ten per cent, of formaldehyde (forty per cent, solution). This mixture is known as ORTH'S FLUID. ' It has the advantages of Miiller's fluid, while the hardening is completed in a week or less. FORMALDEHYDE is a gas which is manufactured in a forty per •cent, solution in water. The solution has a very pungent and irritating odor, and is a powerful germicide. It is most valuable for preserving large anatomical and pathological specimens, which keep their natural colors in it much better than in alcohol. It is also useful in histological work as a fixing agent. Pieces of tissue one centimeter thick are hardened in twenty -four hours in ten parts of the forty per cent, formaldehyde solution of commerce, and ninety parts of water. Very small pieces of tissue may be hardened in formaldehyde for a few hours, and excellent sections FLEMMING'S SOLUTION 23 may then be cut directly after freezing. The staining properties of tissues hardened in this fluid are well preserved. The speci- mens may be kept in it indefinitely. PICRIC ALCOHOL (GAGE) Picric acid 2 grams. Alcohol 500 c.c. Water 500 c.c. This is an excellent fixing agent for most tissues. Hardening is completed in one to three days. OSMIC ACID is sold in hermetically sealed capsules. It is used in one per cent, solution in distilled water, The solution should be freshly made when possible. It deteriorates in the light, and must be kept in a dark closet. Its vapor is very irritating. The use- fulness of osmic acid depends largely upon its property of staining fat black. It is often employed in pathology. In histology it is most valuable for nervous tissues. It stains the medullary sheaths of medullated nerve-fibers black. The pieces of tissue must be not more than four millimeters thick, as osmic acid has very feeble penetrating power. They are left in the solution twenty -four hours, washed thoroughly, and transferred to eighty per cent, alcohol. In preparing specimens for microscopical study, it is best to avoid using turpentine or xylol, which dissolve the stained fat, if that is to be preserved. FLEMMING'S SOLUTION Two per cent, osmic acid solution 4 parts. One per cent, chromic acid solution 15 parts. Glacial acetic acid 1 part. The pieces of tissue should be four millimeters or less in thick- ness. They are left in the fluid twenty-four hours, and, after thorough washing, are transferred to alcohol. This fluid is designed to preserve the karyokinetic figures, using safranin or hasmatoxylin to stain them. It is also admirable to fix cellular structures in general, and it stains fat black as well as osmic acid alone. Another formula proposed by Flemming for fixing the karyo- kinetic figures consists of Chromic acid 2 grams. Glacial acetic acid 1 c.c. Water . 100.0 c.c. 24 STUDENTS HISTOLOGY It serves very well all the purposes of hardening agents. Using small pieces, leave them in the solution twenty-four hours. After washing, place in eighty per cent, alcohol, and subsequently in strong alcohol. Stain with safranin or haematoxylin . CHROMIC ACID FIXING AND HARDENING Chromic acid is a very deliquescent salt, and is best preserved by making a strong solution at once, and then diluting it as may be needed. A stock solution may be made as follows: Chromic acid (crystals) 25 grams. Water 75 c.c. For general use, dilute three parts with six hundred parts of water, which gives a strength of nearly one -sixth of one per cent. The tissue, as soon as secured and properly divided, is placed in the above, remembering the rule regarding quantity. Change in twenty -four hours to fresh solution, and again on the third day. In seven days, or thereabout, change the fluid again. The tissue must now be watched carefully; and when, on cutting through a piece, the fluid is found to have stained the blocks completely, taking from two to three, or even four weeks, remove to a large jar of clear water and wash, preferably with running water, for twenty- four hours. The wrashing having removed the chromic acid, the tissue is further hardened in alcohol. Very small pieces of tissue maybe hardened in one or two days. Chromic acid is useful to preserve the nuclear figures. ERLICKl'S FLUID Bichromate of potassium 25 grams. Sulphate of copper 10 " Water 1,000 c.c. This may be employed in precisely the same manner as the dilute chromic acid solution. DECALCIFYING PROCESS Six per cent, chromic acid solution 9 parts. Nitric acid, C. P 1 part. Water 90 parts. The earthy salts may be removed from teeth and small pieces of IMBEDDING 25 bone with a liberal supply of the above in about twenty days. A frequent change of the solution will greatly facilitate the process, and an occasional addition of a few drops of the nitric acid may be made with very dense bone. After the 'removal of the lime salts, the pieces may be preserved in alcohol until such time as sectioi are needed, when they may be cut with the microtome without injury to the knife. DISSOCIATING PROCESS (w. STIRLING) Artificial Gastric Fluid Pepsin 1 gram. Hydrochloric acid 1 c.c. Water 500 c.c. This process depends for its value upon the fact that certain connective tissues are more rapidly dissolved by the fluid than others. IMBEDDING The best and thinnest sections can only be cut when properly hardened tissues are used, and when the meshes of the tissue have been filled with some substance which supports the delicate elements. The substances used for this purpose are paraffin and celloidin, or collodion. THE PARAFFIN METHOD a. Pieces of tissue 2-3 mm. thick are to be placed in ninety- five per cent, alcohol for twenty -four hours. 1). In pure chloroform six to eight hours. c. In a saturated solution of paraffin in choloroform two to three hours. d. In melted paraffin, having a melting point of 50° C., which requires the use of a water bath or oven, one to six hours. The chloroform must be entirely driven off, and the tissue thoroughly infiltrated. e. Change to fresh paraffin for a few minutes. /. Finally jplace the tissue in a small paper box and pour the melted paraffin about it. Harden as quickly as possible with run- ning water. It is important to fix the piece of tissue in a suitable position, if the position is of importance, before pouring in the melted paraffin. 26 STUDENTS HISTOLOGY Sections of exquisite thinness may now be cut. The knife need not be wet. Paraffin imbedding is especially adapted to making serial sections. In order to mount the sections, proceed as follows: a. Place the sections on a slide. Add a thin solution of gum arabic, upon which they float. Warm slightly, when the sections will flatten nicely. Drain off the superfluous gum solution, leaving the sections in their proper positions. Let them dry for some hours, and they will be firmly fastened to the slide. b. Dissolve out the paraffin in one of the numerous solvents (xylol, half an hour or less). c. At this point, unless the piece of tissue was stained in bulk before imbedding, the xylol should be washed off with alcohol and d. The section stained with one of the dyes described here- after. e. Dehydrate in alcohol. /. Clear in some suitable agent, as xylol or oil of cloves. g. Mount in balsam. CELLOIDIN INFILTRATION Certain structures require permanent support — i.e., not only while being cut, but during the subsequent handling of the sec- tions. The celloidin infiltrating process is best adapted to such material. Considerable time is needed for the successful employ- ment of the process, but results can be secured that cannot be equaled with any other method. Celloidin is the proprietary name of a sort of pyroxylin, very soluble in a mixture of ether and alcohol, producing a collodion. If thick collodion be exposed for a few moments to the air it becomes semi -solid — not unlike boiled egg -albumen; and to this property is due the value of a solution of celloidin in histology. It may be used as follows: To a mixture of equal parts of ether and alcohol add celloidin* until the thickest possible solution has been obtained. A piece of alcohol -hardened tissue, having been selected and kept for the preceding twenty -four hours in a mixture of equal parts of alcohol and ether, is placed in about an ounce of the solu- *We find, after repeated trial, that the ordinary soluble gun-cotton, such as is employed by photographers, is in no way inferior to the celloidin. STAINING AGENTS AND METHODS 27 tion and allowed to remain twenty -four hours. The bottle con- taining the whole should be well corked, to prevent evaporation. The tissue after infiltration is to be placed on a wooden block, and allowed to remain in the open air for a few minutes, after which it should be plunged into a mixture of alcohol two parts7 water one part. Here it may remain for twenty -four hours, or until wanted. Cut in the usual way, using a mixture of alcohol two parts, water one part, for flooding the knife. The section should be finally preserved in the same instead of pure alcohol, which would dissolve the celloidin. In infiltrating the tissue with the collodion it is best, especially if it be very dense in parts, to use first a thin and subsequently the thick solution. A more perfect infiltration is often obtained in this way. In some cases we have been obliged to continue the maceration for several days. The solution should be kept in well stoppered bottles, as the ether is exceedingly volatile. Should the collodion at any time become solid from evaporation, it may be easily dissolved by adding the ether and alcohol mixture. The process is of inestimable value where delicate parts are weakly supported, and where it is important to preserve the normal relations. The gelatin -like collodion permeates every space, and as it is not to be removed in the future handling of the sections, it affords a support to portions that would otherwise be lost or distorted. It offers no obstruction to the light, being perfectly translucent and nearly colorless. STAINING AGENTS AND METHODS STAINING FLUIDS It is a very interesting fact (and one upon which our present knowledge of histology largely depends) that, on examination of tissues which have been dyed with special colored fluids, the dye will be found to have colored certain anatomical elements very deeply, others slightly, while others still remain unstained. Certain dyes are called general or ground stains, because they stain all parts of a tissue alike, or nearly so. Others, which are entitled selective, exhibit an affinity for some particular struc- ture, usually the nucleus of the cell. Haematoxylin, or logwood, for instance, has such an affinity for nuclei. The whole nucleus is 28 STUDENTS HISTOLOGY not stained, but certain threads in it, which ordinarily appear as granules. This part of the nucleus is called chromatin, on account of its affinity for dyes. In a tissue colored with haBmatoxylin, the minute granules of the nuclei are so deeply stained in the logwood dye as to appear almost black. The nuclei are plainly stained, while the limiting membrane of cells is usually but slightly colored. Old, dense connective tissues stain feebly, or fail entirely to take color. The differentiation is, without doubt, due to chemical action between the elements of the dye and those of the tissue. A very great number and variety of materials have been used for histological differentiation, and a simple enumeration of them all would very nearly fill the remainder of our pages. It will be found, however, that leading histologists confine themselves to two or three standard formulas for general work. We shall notice only those methods that have been thoroughly demonstrated by years of employment as best for the purpose suggested. Special cases will require special treatment, which will be indicated in proper connection. It is important in all cases to secure the purest and most con- centrated dyes obtainable. It is better to make your own solutions than to buy them already prepared. DELAFIELD ' S H^M ATOXYLIN Haematoxylin crystals 4 grams. Alcohol 25 c.c. Ammonia alum . 50 grams. Water 400 c.c. Glycerin 100 c.c. Methyl alcohol 100 c.c. Dissolve the haematoxylin in the alcohol, and the ammonia alum in the water. Mix the two solutions. Let the mixture stand four or five days uncovered ; it should have become a deep purple. Filter and add the glycerin and the methyl alcohol. After it has become dark enough filter again. Keep it a month or longer * before using ; the solution improves with age. At the time of using, filter and dilute with water as, desired. EOSIN Eosin is an aniline dye, sold in the form of a red powder. It is best to keep on hand a saturated solution in alcohol. A few drops STAINING FLUIDS 29 i of this stock solution may be added to a small dish full of water at the time of using. BORAX - CARMINE (GRENACHER) Carmine 2.5 grams. Borax 4.0 grams. Alcohol (70%) 100.0 c.e. Water 100.0 c.c. Rub the carmine and borax together. Dissolve them in the water, which should be hot. The alcohol may be added when the mixture is cold. The stain will be available after two weeks, but improves with age. This solution is especially suitable for the staining of whole masses of tissue before imbedding, i.e., in bulk. For staining in bulk, leave the tissue in the carmine for twenty- four hours. It may be used for sections, however, which are to be left in the stain fifteen minutes or longer. In all cases carmine staining is to be finished with the use of acid alcohol, which diifer- entiates the elements. ACID ALCOHOL Alcohol (70%} 100 c.c. Hydrochloric acid (strong) 1 c.c. Sections stained in carmine are placed in acid alcohol for a few minutes. They acquire a brilliant scarlet color. For specimens stained in bulk, dilute the acid alcohol with twice as much 70 per cent, alcohol, and leave the tissue in the mixture twenty-four hours. PICRO- CARMINE Carmine 1 gram. Aqua ammonia (strong) 5 c.c. Water 50 c.c. Saturated watery solution of picric acid . . . . 50 c.c. Add the picric acid solution after dissolving the carmine in the diluted ammonia. Let the mixture stand uncorked for two days, and filter. The carmine gives a nuclear stain, while the picric acid serves as a counter stain. IODINE SOLUTION Iodine 1 gram. Potassium iodide 2 grams. Water . . 300 c.c. 30 STUDENTS HISTOLOGY i WEIGERT-PAL H^MATOXYLIN METHOD This stain is employed for nervous tissues containing medullated nerve -fibers. The medullary sheaths of these fibers are stained intensely black, while the other elements of the tissue remain pale. It is used chiefly for the spinal cord and the brain. The tissue is first hardened in Miiller's fluid. Unless the pieces of tissue are very small, the hardening requires months. The fluid must be changed frequently. Hardening is completed in strong alcohol, without washing in water. Imbed in celloidin. The sections are overstained with haBmatoxylin, and subse- quently are partly decolorized. The medullary sheaths retain the stain with greater tenacity than the other elements of the tissue. The solutions used are as follows: Hsematoxylin crystals 1 gram. Alcohol 10 c.c. Water 90 c.c. Boil and filter. Allow the solution to stand a week or two. If used sooner, add a drop of saturated solution of lithium carbonate to a part of the stain in a small dish. Permanganate of potassium 1 gram. Water 400 c.c. One per cent, solution of oxalic acid. One per cent, solution of sodium or potassium sulphite. Mix the oxalic acid and sodium sulphite in equal parts at the time of using. Sulphurous acid is formed. The steps in staining are as follows: a. Sections are placed in the haematoxylin for twenty -four hours, and are intensely stained, becoming nearly black. The pro- cess may be hastened by letting them stand in an incubator, where they may be sufficiently stairned in a few hours. 6. Wash in water. c. Place the sections in the permanganate of potassium until they acquire a dark brown color (one -half to five minutes). d. Wash in water. e. Place the sections in the mixture of oxalic acid and sodium sulphite. The brown color should give way to a blue black in the white matter, while the gray matter becomes nearly white. If the sections remain too dark they may be returned to the permanganate ANILINE DYES 31 for a few minutes, and then to the sulphurous acid again, always washing in water between the two changes. /. Wash thoroughly in water, and mount in the usual way in balsam. ANILINE DYES The substances known as aniline dyes are derivatives of coal tar, but not always of aniline. These dyes have become of great importance in all kinds of biological work. The number of different compounds is very large, and only a few of the most common can be mentioned. None but the purest dyes should be used, and those manufactured by Grubler can be recommended. They are sold in the form of powders. American agents for Grubler 's dyes are Eimer & Amend, of New York city. It is simplest to classify the dyes as basic or acid. Fuchsin, methylene blue, gentian violet, and safranin are basic dyes. They have an affinity for nuclei and for bacteria. Eosin, picric acid, and acid fuchsin are acid dyes, tending to stain tissues diffusely. The use of eosin and picric acid has been described on pages 28 and 29. Certain cells have granules in their protoplasm. The granules of some cells manifest an affinity for basic dyes (basophile, S and y) , others for acid dyes (acidophile or eosinophile, «), and others for a mixture of the two (neutrophile, *), and others still for both the acid and the basic (amphophile, ft). It is best to keep on hand saturated alcoholic solutions of these dyes, from which waterjr solutions may be made when needed, by adding a few drops of the alcoholic solution to a small dish filled with water. The basic dyes may be used as nuclear stains as follows : a. Stain sections in a strong watery solution of the dye five minutes or more. The sections will be somewhat overstained. 5. Wash in one per cent, acetic acid a few seconds. c. Alcohol. Dehydration must be done rapidly, as alcohol extracts the color from the tissues. It must, nevertheless, be thorough, as xylol, which is used in the next step, only mixes with strong alcohol. d. Xylol. This agent is the one used to clear specimens after staining with basic aniline dyes, because most of the oils slowly dissolve out the aniline colors. e. Balsam. Among the dyes used in this manner, SAFRANIN is to be espe- 32 STUDENTS HISTOLOGY cially recommended as a nuclear staiii. The sections are treated according to the programme just given, except that they should remain in the safranin solution (one per cent.) some hours. Safranin has been employed largely in staining karyokinetic figures, after hardening with Flemming's solution (page 23). NIGROSIN is an aniline color used mostly for nervous tissues. It is valuable when results are desired at once, without waiting for the tedious hardening process required for the Weigert-Pal method. a. Stain sections in strong watery solution of nigrosin five to ten minutes. &. Wash and mount in the usual way in balsam, clearing in xylol. VAN GIBSON'S STAIN Acid fuchsin (one per cent, watery solution) . . . 15 c.c. Picric acid (saturated watery solution) 50 c.c. Water 50 c.c. a. Stain sections in haematoxylin. &. Wash in water. c. Stain in the picric acid and acid fuchsin from three to five minutes. d. Wash in water and mount in the usual way in balsam. The Van Gieson stain is used chiefly for connective and nervous tissues. EHRLICH TRICOLOR STAIN Saturated watery solution orange G. . . . " acid fuchsin . . " " ll methyl green . Glycerin • . 120-135 c.c. . 80-165 c.c. 125 c.c. 100 c c Absolute alcohol Distilled water . , . 200 c.c. 300 c.c. This formula is only one of a number with which Ehrlich's name is associated, as well as those of Biondi and Heidenhain. A powder containing the dyes already mixed is sold by dealers, and usually works very well. It may be used to stain sections of tissues, but is employed mostly with preparations of blood, dried on cover -glasses and fixed lay heating. Stain five minutes or less. It is designed to stain the neutrophile granules of certain leucocytes, METALLIC STAINS— GOLGI METHODS 33 which become colored reddish brown. Eosinophile granules become brilliant red. Nuclei are stained green by it, while the red corpuscles are orange -red or brown. Its use will be referred to in the chapter on blood. METALLIC STAINS OR IMPREGNATIONS Compounds of silver, gold, mercury, and osmium are used to color particular elements in the tissue, or they are precipitated in certain structures as opaque deposits. Osmic acid has already been described on page 23. Of the other substances, NITRATE OP SILVER is the most important. This salt is used in dilute solutions (1-300, 1-500) made with distilled water. It is most valuable to stain the cement-sub- stance between the endothelial cells of membranes, like the peri- toneum. The membrane is stained while fresh, and must be washed free of all albuminous substances which might precipitate the silver. It should also be stretched over the rim of a dish. The solution of nitrate of silver should be allowed to act upon the membrane, care being taken to have it reach all parts of the surface. It has little penetrating power. After five to ten minutes wash in water, and expose to the sunlight, in which the color becomes brown, owing to dark lines which develop between the cells. Mount in glycerin or balsam. GOLGI METHOD FOR STAINING BRAIN AND SPINAL CORD There are many formulas which different investigators have used with more or less success. All of them seek to impregnate the tissue with silver or mercury salts, which become precipitated on £ome of the nerve-elements and render them visible. At best, the Golgi method is uncertain, but when it is successful, the prep- arations obtained are of great beauty and value. The following procedure can be recommended : a. Small pieces of fresh tissue are hardened from two days to a week in Osmic acid, one per cent 10 c.c. Potassium bichromate, three and one -half per cent. 40 c.c. The time required varies according to the part of the tissue to be stained. Neuroglia requires the shortest and nerve-fibers the c 34 STUDENTS HISTOLOGY. longest time. For ganglion-cells the time should be intermediate between these. b. The tissue is placed in a three -fourths per cent, solution of nitrate of silver for one to six days. c. Cut rather thick sections, about 50 p. Alcohol is to be used as little as possible. Sections may be cut free-hand or between pieces of elder -pith, or by fastening on the block with paraffin (page 18); or by imbedding rapidly in celloidin, taking about twenty minutes for all the steps. d. Alcohol, xylol, and balsam, as usual. e. Mount without a cover -glass, and keep in the dark. COX'S MODIFICATION OF THE GOLGI METHOD Golgi found that the bichloride of mercury could be used for the impregnation of nervous tissues in much the same manner as the nitrate of silver. The formula proposed by Cox has been highly recomended. Bichromate of potassium (five per. cent. solution) 20 parts. Bichloride of mercury (five per cent, solution) . . 20 parts. Simple chromate of potassium (five per cent, sol.) 16 parts. Distilled water 30 to 40 parts. Specimens should be left in the mixture for one month in sum- mer, and for two to three months in winter. The impregnation should take place uniformly throughout the preparation. INJECTION OF BLOOD-VESSELS To make the small blood-vessels appear in microscopical prep- arations they may be filled with some colored substance. An entire animal recently killed, or merely one organ, may be injected. A canula is tied in the proper vessel and injected with a syringe, or from a flask holding the coloring substance, which is emptied by mercurial or water pressure. Among many formulas for injecting fluids, the following may be used: Soluble Berlin blue . . 3 grams. Water 600 c.c. This mixture has the advantage that it may be used cold. CARMINE-GELATIN must be kept warm while injection is pro- ceeding, as must the object which is being injected. Two and one- CLEARING AGENTS— MOUNTING MEDIA 35 half grams of carmine are rubbed up with a little water, and strong ammonia added, a drop at a time, till the carmine is dissolved. Then filter. Five grams of gelatin having previously been dis- solved in water, add the carmine solution. Now neutralize the carmine -gelatin exactly with acetic acid. As the neutral point is approached, the acid must be diluted and added cautiously; filter. CLEARING AGENTS The commonest method of preparing sections is to mount them finally in Canada balsam. Staining is usually performed in watery solutions. Water does not mix with balsam. After staining, therefore, the water is to be removed with alcohol, which has an affinity for water. The alcohol must in turn be removed with some substance which will mix with balsam. The various fluids used for clearing have this property, and also make the tissue trans- parent. ANILINE OIL is a clearing agent which will itself remove water from the tissues without the use of alcohol. It does not dis- solve celloidin. It extracts the aniline colors, however. XYLOL can only be used when dehydration is perfect. It does not dissolve celloidin, nor extract the aniline dyes. It is often used mixed with one -third of its volume of carbolic acid — CARBOL-XYLOL. When an agent that will not dissolve celloidin is desired, a cheap and excel- lent substitute for the last is the CARBOL- TURPENTINE of Gage: Carbolic acid crystals (melted) 40 c.c. Oil of turpentine CO c.c. The essential oils and CREOSOTE are used frequently for clear- ing, for example, the OILS of ORIGANUM, of BERGAMOT, and of THYME. OIL OP CLOVES is used very extensively, but it has the property of removing the aniline dyes quite rapidly, and it dissolves celloidin. It may be used to clear celloidin sections, except the most delicate, if care be used. Delicate sections should be cleared on the slide. Other sections may be placed in a small dish of the clearing fluid. MOUNTING MEDIA CANADA BALSAM is the medium most used for the permanent preservation of microscopical preparations. It should be dissolved in xylol, which does not affect the aniline stains. 36 STUDENTS HISTOLOGY DAMMAR is sometimes used in the same manner as balsam. Balsam and dammar may be kept in large-mouthed bottles, from which they are removed with glass rods. They are also sold in flexible lead tubes, which make a convenient way of handling them. Fresh tissues are often studied in a six -tenths Of one per cent, solution of sodium chloride in water — NORMAL SALT SOLUTION. FIG. 15. USING TURN-TABLE— AFTER FREEBORN. Objects are often mounted in GLYCERIN, especially those that would be injured by alcohol. It is best to use a circular cover- glass, and to surround the edge with some soluble cement, using a turn-table for this purpose. Zinc cement, asphalt varnish, and other suitable cements may be purchased from dealers in microscopical supplies. STAINING METHODS H^MATOXYLIN STAINING PROCESS You will require for future work a needle like Fig. 16, several saucers, preferably of white ware; a few watch-glasses (large, odd sizes are usually cheaply obtainable at a jeweler's) ; half a dozen FIG. 16. NEEDLE FOR LIFTING SECTIONS, ETC. glass saltcellars— small ones known as "individual salts," — and a two -ounce, shallow, covered porcelain box, such as druggists use for ointments, dentifrices, etc. Place on the work-table (best located so as to be lighted from your side and not from the front) in order, as in Fig. 17: H&MATOXYLIN STAINING PROCESS 37 1. Watch-glass, containing say 10 c.c. diluted haematoxylin. 2. Saucer, filled with water. 3. Saltcellar, filled with alcohol. 4. The covered porcelain box, containing about 20 c.c. oil of cloves* or carbol- turpentine. Select a section from some one of your stock bottles, lifting it out with the needle, and place it in the haematoxylin solution. The FIG. 17. DIAGRAM INDICATING THE SUCCESSIVE STEPS IN STAINING WITH THE HAEMATOXYLIN SOLUTION. section, having been taken from alcohol and transferred to a watery staining fluid, will twirl about on the surface of the latter, inasmuch as currents are formed by the union of the water and the spirit. "How long shall I let the section remain in the haematoxylin ? " The only answer we can give is, "Until properly stained." Nothing but experience will give you any more definite information. Much depends upon some peculiar property in the tissues: some stain rapidly, others stain very slowly. The strength of the dye is another determining factor. Usually with the haematoxylin form- ula, as given, from two to three minutes will suffice. Place the needle under the section (if the fluid be so opaque as to hide the tissue, place the watch-glass over a piece of white paper or a bit of mirror) and gently lift it out; drain off the adhering drop of dye on the edge of the glass, and drop into the saucer of water. Here we can judge as to the color, and we, perhaps, find it to be of a light purple — too light, so you may return it to the haematoxylin for another period of two or three minutes, which will probably give sufficient depth. As the section floats on the washing water, you will notice that the latter will be colored by the dye, some of which leaves the *Although oil of cloves is the clearing agent mentioned throughout this book, it is to be remembered that it dissolves celloidin, and that carbol -turpentine is preferable for sections if they are delicate. 38 STUDENTS HISTOLOGY tissue. Allow the water to act until no more color comes out. The tint of the section changes from purple to violet, and the water must be allowed to act until the change is complete. If you were to examine your section at this stage, you would find it opaque, and as we are obliged to study our objects mainly by transmitted light, we must find some means of securing trans- lucency. The essential oils are used for this purpose, oil of cloves being commonly employed. Lift the section from the water with the needle, let it drain a moment, and then drop it into the alcohol with which the saltcellar was filled. The object of this bath is the removal of the water from the tissue, and this will be accomplished in from five to ten minutes. Again lift the section and place it in the oil of cloves. The tissue floats out flat, and in a few minutes sinks in the oil. We might proceed to the examination of the stained section; but we shall ask you to let it remain in the oil, covering the box carefully to exclude our great enemy, dust, until we have learned more about staining. To recapitulate: The essential steps in the haematoxylin pro- cess are: 1. Staining the tissue — haematoxylin. 2. Washing — water. 3. Dehydrating — alcohol. 4. Rendering transparent — oil of cloves. As the section is put in the dye, care should be taken to so float it out that it may not be curled. This is easily done with the needle. After the alcohol bath, however, this becomes difficult, as the tissue is rendered stiff by the removal of the water. This is the simplest and best of all methods for general work, and you are advised to master every detail of the process. After reading the directions which we have given, and having never seen the work actually done, it will not be singular if you conclude that the staining of tissues is a tedious and slow process; but after a month's work you will be able to stain fifty different sections in half an hour, and have them ready for mounting. HJEMATOXYLIN AND EOSIN— DOUBLE STAINING Very beautiful and valuable results in differentiation are obtained by staining first with haematoxylin and subsequently with eosin. Eosin, a coal-tar derivative, stains most animal tissues BORAX- CARMINE STAINING PROCESS 39 pink, and it affords with the ha3matoxylin a good contrasting color. The tissue is to be stained in haBmatoxylin and washed in water as usual ; then it is placed in the eosin solution, and afterward washed again. The subsequent treatment is as with the plain hromatoxyliii process; viz., dehydration with alcohol, after whicji the oil of cloves. The diagram, Fig. 18, shows the process complete: 1. Watch-glass with ha3matoxylin. 2. Saucer with water. 3. Watch-glass two -thirds filled with water, with five drops of eosin solution added. 4. Saucer containing water. 5. Saltcellar filled with alcohol. 6. Covered oil -dish. The eosin stains very quickly, generally in about a minute. Care should be taken not to overstain with it, as it cannot be FIG. 18. DIAGRAM INDICATING THE SUCCESSIVE STEPS IN DOUBLE STAINING WITH H^EMATOXYI.IN AND EOSIN. washed out. If the sections are found at any time to be over- stained with haematoxylin the color may be removed to any desired extent by floating them in a weak solution of acetic acid. They must afterward be washed very thoroughly in clean water. BORAX -CARMINE STAINING PROCESS Arrange your materials as in the diagram, Fig. 19. 1. Watch-glass two -thirds filled with the carmine fluid. 2. Saucer containing about an ounce of alcohol. 3. Saltcellar filled with alcohol containing one per cent, of hydrochloric acid. 4. Saltcellar with alcohol. 5. Porcelain dish containing oil of cloves. The carmine solution will stain ordinarily in fifteen or twenty minutes. After the section has been in the dye for a few minutes, 40 STUDENTS HISTOLOGY lift it with the needle, drain, and transfer to the saucer containing alcohol. You will then be enabled to determine whether the section is sufficiently stained; it should be a deep, opaque red. The alcohol washes off the section, removing the adhering solution of carmine. The carmine must now be fixed in the tissue, or mordanted; and this you proceed to do by transferring the section to the watch - PIG. 19. DIAGRAM INDICATING THE SUCCESSIVE STEPS ix STAINING WITH BORAX-CARMINE . glass containing acid alcohol. Notice the change in color, from a dull red to a bright crimson, and when the change is complete, lift it into the saltcellar containing clean alcohol. This dissolves out the acid. Five minutes suffice for this washing, after which trans- fer to the oil of cloves. This process does not give as sharp contrasts as the hgematoxy- lin and eosin, but it is simpler and very permanent. It is best to select some one process for general work, and adhere to it. The acid of the carmine process must be guarded with extreme care, as the smallest particle is sufficient to spoil the haematoxylin solution. Look to it that the dishes are kept scrupulously clean, and the sanje care must be bestowed upon the needles, forceps, and all other instruments. Picro- carmine may be used instead of borax -carmine. The sections may need to remain in picro- carmine as much as an hour to become thoroughly stained. Acid alcohol is not to be em- ployed after staining. In dehydrating, it is well to add some picric acid to the alcohol, in order to prevent extracting the yel- low stain from the specimen. You may, of course, stain several sections at once, providing you take care to keep them from rolling up or sticking together. While the vessels which we have recommended will be found of convenient, proportionate, and economical size for general work, larger ones are sometimes needed ; and almost any glass or porce- lain vessel may be impressed for duty. CLEANING SLIDES— TRANSFERRING SECTIONS 41 MOUNTING OBJECTS CLEANING SLIDES AND COVERS When purchasing slides, let us urge you to get them of goad quality. The regular size is one by three inches, and the edges should be smoothed. As furnished by the dealers they are usually quite clean, and only require rubbing with a piece of old linen to prepare them for use. The cover -glasses should be thin, not over TO~O of an inch, called in the trade -lists "No. 1." Circles or squares three-quarters of an inch in diameter are generally convenient. They must be thoroughly cleaned. Drop them singly into a saucer containing hydrochloric acid. Then pour off the acid, and let clean water run into the dish for several minutes. Drain off the water and pour a little alcohol on the covers. Remove them one at a time with the forceps or needle, and wipe dry with old linen.* The glass may be held between the thumb and forefinger, the linen being inter- posed. Very slight pressure and rubbing will complete the process. The surface of the glasses should be brilliant, and they should be preserved for future use in a dust -tight box. TRANSFERRING THE SECTIONS TO THE SLIDE The section is to be taken from the oil with a section -lifter or spatula, Fig. 20. Smooth, stiff paper, cut in strips, may be used in the same wav. FIG. 20. SECTION-LIFTERS. In changing the sections on the needle or spatula from one fluid to another, as from alcohol to oil of cloves, it is well to touch the *We are indebted to Professor Gage, of Cornell University, for suggesting the use of Japanese tissue-paper for wiping cover-glasses, lenses, etc. Ordinary manilla toilet-paper is also an excel- lent material for such work. 42 STUDENTS HISTOLOGY edge of the section to a sheet of blotting-paper or filter- paper, to remove as much as possible of the first fluid and prevent its dilut- ing the second. Place a clean slide on the table before you, and, with the section- lifter used like a spoon, dip up one of the sections from the clove oil. By inclining the lifter, the section may be made to float to FIG. 21. METHOD OF LABELING A MOUNTED SPECIMEN. the center of the slide. A small sable brush is often convenient for coaxing the section off the lifter. If it were our present object to simply examine the section, we could drop a thin cover -glass on the specimen, and it would be ready for study. Such an object would afford every requirement for present observation, but would not be permanent. The oil of cloves would evaporate after a few days, and the section be ruined. We proceed to make a permanent mounting of our object. The clove oil surrounding the section on the slide is first to be removed, and it can easily be done by means of blotting-paper. With a narrow slip of thin filter -paper wipe up the oil, exercising care not to touch the section, or it will become torn. Proceed care- fully, taking fresh paper until the oil will no longer drain from the FIG. 22. MODE OF HANDLING THE COVER-GLASS IN MOUNTING TISSUES— FREEBORN. section when the slide is held vertically. With a glass rod remove a little of the xylol balsam (vide formulae) from the bottle, and allow a drop of this balsam to fall upon the section. Pick up a clean cover- glass with the forceps, and place it on the drop of balsam. This operation is seen in Fig. 22. The point CARE OF THE MICROSCOPE 43 of the forceps may be placed beneath the cover -glass, the tip of the forefinger pressing lightly over it, and you will be enabled to carry the thin glass wherever desired. As the cover settles down the air is pressed out, until finally the section appears imbedded in the varnish — the latter filling the space between the cover and the slide. The object is "mounted." You have a permanent specimen. The slide must be kept flat, as the balsam is soft. After some weeks, the varnish around the edges of the cover will stiffen, and eventually become solid. Do not paint colored rings around the specimen. Nothing can present a neater appearance than the simple mount, as we have described it, after having been properly labeled. Labels seven -eighths of an inch square may be put on one or both ends, with the name of the object, date, method of staining, or whatever particulars you may prefer. Specimens should be kept in trays or boxes in such manner that they may always lie flat. CARE OF THE MICROSCOPE The objectives constitute the most valuable part of the instru- ment. The lenses should never be touched with the fingers; indeed, the same rule applies to all optical surfaces. When the glasses become soiled they may be cleaned, but it should be done with great care. While the effect of a single cleaning would probably not be of the slightest appreciable injury to the glass, repeated wiping writh any material, however soft, will destroy the perfect polish, and result in obstruction of light and consequent dimness in the field. Never use a chamois leather on an optical surface, as these skins contain gritty particles. Old, well worn linen and Japanese paper are by far the best materials for wiping glasses. If a lens be covered with dust, brush it off, breathe on the surface, and wipe gently with the linen or paper. Should you get clove oil on the front lens of the objective (as frequently happens when examining temporary mounts) wipe it dry, and then clean with the linen moistened with a drop of alcohol. Canada balsam can be very readily removed from any surface after having softened it with oil of cloves. The front lens of the objective, being the only one exposed, is the one usually soiled. Particles of dirt on the objective, as I have said, cause a dim- ness in the field — the image is blurred. Dust on the lenses of the 44 STUDENTS HISTOLOGY eye -piece, however, appears in the field. These lenses are readily cleaned by dusting and wiping with the linen, after having breathed on the surface. Never wipe a lens when dusting with a cam el' s- hair brush will answer the purpose. The microscope should either be covered with a shade or cloth, or put away in its case when not in use. The delicate mechanism of the fine adjustment becomes worn and shaky if not kept free from dirt. PAKT SECOND STRUCTURAL ELEMENTS PRELIMINARY STUDY FORM OF OBJECTS From a single and hasty view of bodies under the microscope, we are liable to form erroneous ideas of form. Either a sphere, disc, ellipsoid, ovoid, or cone may be so viewed as to present a circular outline. It therefore becomes important to view objects in more than a single position. This can easily be accomplished with isolated particles by suspension in a liquid. In this way the true shape of a blood -corpuscle, e.g., may be determined. Again, much information concerning the actual form of bodies may be gained by a proper adjustment of the fine focusing screw. You maj' remember that the depth of the field of view in the micro- scope is exceedingly slight. Speaking accurately, only a single plane can be seen with a single focal adjustment ; but by gradually raising or lowering the t tube of the microscope the different parts of a body may be focused and studied, and an accurate idea of form secured. With a glass rod place a drop of milk, which has been pre- viously diluted with three parts of water, on a slide, and put a cover- glass thereon, as in Fig. 23. Focus first with the low-power (L). A multitude of minute dots are observed. Then change to the high- power (H), and the dots will resolve into circular figures. Select one of the smaller particles, and, as you raise the focus, the center of the figure retains its brilliancy, while the edges become dark or blurred, showing convexity. Reverse the focus, and the center again retains its sharpness long after the edge has become blurred. The figure, then, is a spheroid. These bodies are fat -globules. Particles of free fat always assume the spheroidal form when sus- pended in a liquid. (45) 46 STUDENTS HISTOLOGY Note the larger globules: they have become flattened by the pressure of the cover -glass. Clean the slide, and make a second preparation from the diluted milk — first, however, shaking it violently in a bottle. Note the flattened air -bubbles among the oil -globules. Observe that these air -bubbles have no intrinsic color, while the fat -globules are A | FIG. 23. DIAGRAM SHOWING THE EFFECT OF AIR-BUBBLES AND OIL-GLOBULES IN A MOUNTED SPECIMEN UPON THE RAYS OF LIGHT. The lines A, B show the refraction of the rays (so as to produce a ring of color) by the action of two plano-concave water-lenses which are formed by the air-bubble. The oil is seen to correct the refraction of C, D, thus giving but little color to the margin of this globule. faintly yellow. Observe the change in the ring of prismatic color about the edge of the air -bubble, as the focus is altered. No such color will be seen in connection with the oil -globule. The bubbles assume various figures from the pressure of the cover- glass. MOVEMENT OF OBJECTS Objects are frequently seen moving in the field of the micro- scope, the movement being magnified equally with their dimen- sions. Thermal Currents. — When, with the previous specimen or any other fluid mount, the warm hand is brought close to one side of the stage, the globules in the field will be seen swimming more or less rapidly. These currents are due to the unequal heating of the liquid under observation. The direction of the current is in the reverse of its apparent motion. Brownian Movement. — Place a fragment of dry carmine on a slide, add a drop of water, and with a needle stir until a paste is formed. Add another drop of water, and immediately put on the cover -glass. With H, note the most minute particles, and observe their peculiar, dancing motion. This occurs when almost any finely divided and generally insoluble solid is mixed with water. It EXTRANEOUS SUBSTANCES 47 ceases after a short time. The movement has been attributed to several causes. Vital Movements. — Place a drop of decomposing urine on a slide, cover, and focus with H. The field contains innumerable minute spherules and rods (bacteria) which are in active motion, resembling somewhat the Brownian movement, although sufficiently distinctive after close observation. After having rubbed the tongue for a moment against the inner surface of the cheek, put a drop of saliva on a slide, cover, and focus (H) . Among the numerous thin, nucleated scales and debris, small granular spherules — the salivary corpuscles — will be found. Select one of the last, center, and focus (H) with extreme care. The minute granules within the cells are in active motion, resembling the Brownian movement, but with proper conditions the motion may continue for many hours. EXTRANEOUS SUBSTANCES Before we begin the study of animal tissues, we wish to have you become somewhat familiar with the appearance of certain objects FIG. 24. EXTRANEOUS SUBSTANCES. A. Cotton fibers, showing the characteristic twist. B. Linen fibers, with transverse markings, indicating segments. C. Wool. The irregular markings are produced by the overlapping of flattened cells. Wool may be distinguished from other hairs by the swellings which appear at irregular intervals in the course of the former. D. Silk. Smooth and cylindrical. 48 STUDENTS HISTOLOGY which are frequently, through accident or carelessness, and often in spite of the utmost care, found mixed with our microscopical specimens. Among the more common objects floating in the air and gaining access to reagents, to subsequently appear in our mounted specimens, are the following : Fibers. — Procure minute pieces of uncolored linen, cotton, wool, and silk. With a needle in either hand, tease out or separate a few fibers on slides, add a drop of water, and cover.* FIG. 25. EXTRANEOUS SUBSTANCES. A. Granules of potato starch. B. Corn starch. C. Wood fibers. The circular dots are peculiar to the tissue of cone-bearing trees. D. Spiral thread from a tea leaf. E. Fragment of feather. F. Cells of yeast and mould. Starch. — Procure samples of wheat, corn, potato, and arrow-root starch, or scrape materials containing any one of these substances with a sharp knife. To a minute portion on the slide add a drop of water, cover, and examine with L and H. Wood Shavings, Feathers, Minnie Insects, Portions of Larger Insects, Pollen, etc., are easily mounted temporarily or permanently, These substances, as well as most of those which follow under the same heading, may be mounted permanently as follows: Put the dry material in clean turpentine for a day or two, to remove the contained air. Transfer to the slide, tease, separate, or arrange the elements, after which wipe away the turpentine with strips of blotting-paper. Add a drop of balsam, and place the cover-glass thereon. The weight of the cover will be sufficient to press the object flat, if it be properly teased or separated. STRUCTURAL ELEMENTS— CELLS 49 as before noted. They are very commonly found in urine after it has been exposed to the air, and their recognition is very important. Let me urge you to become familiar with the microscopical^ appearance of the commoner objects which surround us in every- day life. The most serious mistakes have resulted from ignorance of this subject. Vegetable fibers have been mistaken for nerves and urinary casts, starch granules for cells, vegetable spores for parasitic ova, etc. STRUCTURAL ELEMENTS Certain anatomical structures, of a more or less elementary nature, are united in the composition of organs. These structural elements will, with propriety, first claim notice from us. CELLS A typical cell is a microscopical sphere of protoplasm, consti- tuted as follows (vide Fig. 26) : A. Limiting membrane. B. Cell-body. C. Nucleus. D. Nucleolus. D FIG. 20. ELEMENTS OF A TYPICAL CELL. The wall consists of an apparently structureless membrane of extreme tenuity. The cell-body may be either clear (jelly-like), granular, or fibrilliated. It contains an albuminous substance called protoplasm. The nucleus is a minute spherical vesicle, with a limiting mem- D 50 STUDENTS HISTOLOGY brane inclosing a clear gelatinous material, traversed by a reticulum of fibrillae. The nucleolus consists of a spherical, highly refracting body lying inside of the nucleus, sometimes appearing to be a granular enlargement upon the fibrillas of the nucleus. Deviations from the type are most frequent, and vary greatly as to form, number of elements, and chemical composition. FIG. 27. A CELL, NUCLEUS, WITH NETWORK AND NUCLEOLUS. DIAGRAMMATIC. The typically perfect cell is rarely seen in. human tissue on account of the length of time which commonly elapses between death and observation of the structure, the delicate fibrillae of the nuclei usually appearing as a mass of granules. CELL DISTRIBUTION The complex mechanism of the body had its origin in a single cell. This preliminary structure, endowed with the power of pro- liferation, became two cells. Two having been produced, they became four: the four, eight ; and thus progression advanced until they became countless. Some of these cells remained as such; others, altered in form and composition, gave birth to muscle, bone, etc. The study of these processes belongs to physiology. The adult body is composed largely of cells of various forms. The different physiological processes, as secretion, absorption, res- piration, etc., are effected through the intervention of these anatomical elements. All free surfaces, within or without the body, are covered with cells. The entire skin, the outside of organs, as the lungs, liver, stomach, intestines, brain, etc.; all cavities, as the alimen- tary tract, heart, ventricles of the brain, blood-vessels and ducts, present a superficial layer of cells. The cells are held together by an intercellular substance, which may be so abundant that the cells form a comparatively small part of the tissue. The intercellular material is to be regarded as hav- KAETOKINESIS 51 ing been made by the cells. In the case of the connective tissues, it has a more important function than the cells; while the amount of intercellular substance in epithelial structures is trifling. CELL -DIVISION The increase in the number of cells in the body may be accom- plished in two ways: a. By direct division. b. By indirect division. Direct division is a process in which the cell becomes con- stricted and a portion of it separated from the remainder. It is FIG. 28. INDIRECT CELL-DIVISION— AFTER FLEMMING. now believed that in most instances the separation of the proto- plasm is preceded by a series of changes in the nucleus. This mode of division is called indirect. The changes in the nucleus go by the name of karyokinesis, or mitosis. KARYOKINESIS The reticulum of fibers already mentioned as traversing the substance of the nucleus has an affinity for nuclear stains, and its substance is therefore called chromatin. During karyokinesis the chromatin of the nucleus undergoes a series of complicated changes, resulting in its division into two equal parts. This is followed by the division of the rest of the cell. During the process the chromatin presents figures of great variety and intricacy. 52 STUDENTS HISTOLOGY The effect of these changes is to separate the chromatin into masses called chromosomes. The number of chromosomes varies in different species, but is probably constant in the same species. Fig. 29 shows the main events in karyokinesis in a diagrammatic manner. It represents the process as it occurs in the starfish, where the process is less complicated than in some cases. Each C D FIG. 29. KARYOKINESIS— AFTER WILSON. chromosome becomes split lengthwise into two halves. One group of halves moves to one end or pole of the cell, the other group to the other pole. The two groups of chromosomes give rise to two new nuclei. The separation of the groups of chromosomes is accomplished by delicate filaments, which radiate from the two poles to which the chromosomes are to travel. Some of these filaments meet at the equator of the cell to form what is called the nuclear spindle. The CLASSIFICATION OF TISSUES 53 radiating filaments at the poles of the cells present figures which have been compared by Wilson to the arrangement of iron filings about the poles of a horseshoe magnet. The circle of radiating filaments at each pole is called the attraction- sphere, the center of which is the centrosome. It is possible that the substance of the filaments serves to draw the chromosomes apart. Apparently the centrosome is to be regarded as a permanent organ of the cell. Its division must precede the division of the nucleus. The time required for cell division in man is about half an hour (Stohr). Most of the stages in karyokinesis may be demonstrated in the epithelial cells in the tail of a young newt or salamander tadpole. Fix in Flemming's osmie acid or chromic -acetic solution for twenty-four hours; wash; stain with safranin or hsematoxylin ; and, after dehydrating and clearing, mount in balsam. Examine with oil -immersion lens, if possible. CLASSIFICATION OF TISSUES Histology, or microscopical anatomy, treats of the minute structure of the tissues and organs of the body. A tissue is a collection of similar cells and intercellular sub- stances. The principal tissues are epithelial, connective, muscular, and nervous. Blood is sometimes considered as a tissue. Most organs are made of several different tissues. /The following table shows the varieties of tissues: / Epiilielial Tissue { <• Connective Tissue Columnar. Endothelium. Blood. Mucoid. White Fibrous. Yellow Elastic. Adipose. Retiform (of lymphoid tissue), Cartilaginous. Osseous. L Dentine. {Unstriated. Striated. Cardiac. Nervous Tissue. 54 STUDENTS HISTOLOGY Ectoderm or Epiblast EMBRYONIC DERIVATION OP TISSUES In correlating the work of histology with that of embryology, the student will find the following table serviceable. The table indicates from which layer of the blastoderm each of the prin- cipal tissues is derived: The epithelium of the skin and all its appendages and glands. The epithelium covering the front of the eye, the crystalline lens, and the retina. The epithelium of the external auditory canal and of the mem- braneous labyrinth. The epithelium of the nasal cavity and its diverticula. The epithelium of the mouth and its glands, the salivary glands, and the enamel of the teeth. The epithelium of the anal end of the alimentary canal. The tissues of the nervous system, the lining of the central canal of the spinal cord and of the cerebral ventricles, the pituitary and pineal bodies. All connective tissues. Muscular tissue. The blood- and lymph-vessels and their endothelium, and the endo- Mesoderm thelium of the serous membranes. or < Blood -and lymph-'corpuscles. Mesollast The spleen and lymph -nodes. The kidney and ureter. The ovary and testicle and their ducts, except the external ends of the ducts. The epithelium of the alimentary canal (except at the upper and lower extremities), and all the glands opening into it, includ- Entoderm ing the liver, pancreas, thyroid, and thymus. or -s The epithelium of the respiratory tract which originates as a Jlypoblast diverticulum from the alimentary canal. The epithelium of the Eustachian tube and tympanum. The epithelium of the urinary bladder and urethra. EPITHELIUM Epithelium is the tissue covering the surfaces of the body that V/ communicate with the external world, and lining the glands.* It is made of cells, held together by only a small amount of intercellular substance. *Note the exceptions in the case of the peritoneum, which is lined by endothelium, and which opens externally by means of the Fallopian tube; the thymus and thyroid glands, and the cavity of the central nervous system, which contain or are lined by epithelium, but do not open externally. SQUAMOUS, STRATIFIED, AND TRANSITIONAL EPITHELIUM 55 Where a layer of epithelial cells comes in contact with the underlying connective tissue, the deepest epithelial cells often rest upon a thin basement membrane, which is modified connective tissue, either structureless or made of flattened cells. The succeeding pages will show that epithelial cells may £e squamous or columnar, ciliated or otherwise, simple or stratified. These varieties of epithelium are distributed as follows, giving cinly their most important locations: Simple Squamous Epithelium Stratified Squamous Epithelium Simple Columnar Epithelium Stratified Columnar Epithelium The alveoli of the lungs. The capsule of the Malpighian body and the descending limb of the loop of Henle in the kidney. The covering of the skin, of the eye, mouth and tongue, pharynx, oesophagus, epiglottis, and of the upper part of the larynx. The lining of the urinary tract from the pelvis of the kidney down (except part of the male urethra), most of it being of the special variety of stratified squamous epithelium known as transitional. The lining of the vagina. The alimentary canal from the beginning of the stomach to the lower part of the rectum; the ducts of its glands and of many other glands; the seminal vesicles, ejaculatory ducts, and part of the male urethra; the surface of the ovary. It is ciliated in the uterus and Fallopian tube, the central canal of the spinal cord, and the cerebral ventricles. It is ciliated in the most important situations: The trachea and bronchial tubes, the Eustachian tube, the upper part of the pharynx, the lower part of the nasal cavity, the vas deferens. SQUAMOUS, STRATIFIED, AND TRANSITIONAL EPITHELIUM The simplest method of tissue production by means of flat cells is that of superposition, constituting squamous epithelium. Cells are placed one over the other, generally without great regularity. If regular, and in several layers, the structure is called stratified epithelium; if only in a few layers, it is termed transitional epithe- lium. The superficial layer of the skin affords an example of squamous, stratified epithelium. The bladder and pelvis of the kidney are lined with transitional epithelium. The thin, flat .scales from the mouth may be demonstrated by scraping a drop of saliva from the tongue with the handle of a scalpel, transferring it to the slide, and applying the cover. The 56 STUDENTS HISTOLOGY size of the drop of saliva should be carefully adjusted so as to fill the space between the cover- glass and slide. Too little will cause the cover to adhere so tightly to the slide as to press the cells out of form; too much, and the saliva flows over the cover and soils the objective. With a glass rod place a drop of the dilute eosin solution on the slide, and with a needle lead it to the edge of the saliva. The dye will pass under the cover slowly, and whatever anatomical elements there may be present will be gradually stained. Observe that the nuclei of the flat scales first take the dye and appear of a deep pink; while the other portions are either colorless or very lightly stained. Find a typical field and sketch it with a pencil, afterward tint- ing with dilute eosin. An admirable view of the surface of a squamous epithelium may be had by using the superficial layer of the skin of a frog, which is FIG. 30. SQUAMOUS EPITHELIAL, CELLS FROM THE MOUTH. often shed when the animal is kept in confinement. It may be stained with haematoxylin; and small pieces, after alcohol and clearing, may be mounted in balsam. PAVEMENT EPITHELIUM When thin, flat cells are disposed in a single layer, like tiles, the epithelium is termed simple squamous, pavement, or tessellated. These cells are often quite regularly polygonal (although this COLUMNAR EPITHELIUM 57 obtains more frequently with tissue from the lower animals), and they are always connected by their edges by means of an albumi- nous cement. Such a simple squamous epithelium is found in only a few locations. The most important are the alveoli of the lungs, the FIG. 31. PAVEMENT EPITHELIUM. DIAGRAMMATIC. capsule of the Malpighian body of the kidney, and the descending limb of the loop of Henle in the kidney. Seen from the surface, a stratified or columnar epithelium appears like pavement epithe- lium. COLUMNAR EPITHELIUM Columnar cells are found, generally, throughout the alimentary and respiratory tracts, except near their external openings. They also line the cerebral ventricles, most of the uriniferous tubules, Fallopian tubes, the uterus, etc. This epithelium is quickly de- stroyed after death, and is difficult of perfect demonstration, except in an animal recently killed. Procure from the abattoir a portion of the small intestine and bronchus of a pig, and with the curved scissors snip out small pieces from the mucous surface of each. Macerate in one-sixth per cent, of chromic acid for twenty -four hours. Place a piece of the gut on a slide, and, after having added a drop of the acid solution, scrape off the mucous surface with a knife and remove the remainder of the gut. Add a cover -glass, and focus (H). You will find cells in various conditions, from isolated examples to small groups like Fig. 32. Observe that the attached ends of the cells are often small and 58 STUDENTS HISTOLOGY pointed, and that spheroidal and ovoidal cells are frequently wedged in between them. Note the free border: it consists of striae, and PIG. 32. COLUMNAR CELLS FROM SMALL INTESTINE OF RABBIT. A. Tapering attached extremity. B. A swollen goblet cell. C. Finely striated free border. D. Transparent line of union between the striated portion and the body of the cell (X 400). is separated from the body of the cell by a translucent line. This appearance is also that of the epithelium in the human intestine. PIG. 33. CILIATED COLUMNAR CELLS FROM BRONCHUS OF PIG (X 400). Ciliated Columnar Epithelium Prepare, by scraping, a slide from the mucous surface of the pig's bronchus (which has been macerating in the chromic acid) GLANDULAR EPITHELIUM 59 Observe the cilia on the free border of the cells. Interspersed between ciliated cells, much -enlarged individuals may be found — the so-called beaker, goblet, or mucous cells. The motion of the cilia may be demonstrated as follows: Carefully open an oyster so as to preserve the fluid. On exam- ination you will notice the gills, shown in Fig.. 34, commonly FIG. 34. OYSTER, OPENED TO SHOW METHOD OF PROCURING LIVING CILIATED CELLS. A. The divided muscle. This must be sectioned before the shell can be opened B. The heart. 0. Liver. D, D. The so-called "beard." These laminae are covered with cells provided with cilia; and a fragment of the free border of one of the leaflets may be snipped off with the scissors and examined as described in the text. called the beard. With the scissors snip off a fragment of the free border of this beard, add a drop of the liquid from the oyster, and tease with a pair of needles. Apply the cover, and focus (H). At first the individual cilia cannot be demonstrated on account of their rapid vibration. After a few moments, however, the action becomes less energetic, and the hair -like appendages of the cells are to be plainly seen. GLANDULAR EPITHELIUM Scrape the cut surface of a piece of liver; place the scrapings on a slide; add a drop of normal salt solution (vide formulae) ; mix with a needle, and put on the cover -glass. With H observe, among the numerous blood -corpuscles, fat- globules, etc., the polyhedral liver -cells, about twice or three times the diameter of a white blood -corpuscle (Fig. 35). Notice the large spherical nuclei, with nucleoli. Note, also, the yellow 60 STUDENTS HISTOLOGY FIG. 35. GLANDULAK EPITHELIUM. A. A. Polyhedral cells from human liver. B. Double nuclei. C. Cells from same showing connection with a capillary. D. Same cells infiltrated with globules of fat. E. Cells from liver of pig showing intracellular network (X 400). pigment -granules and the fat -globules in the body of the cells. Masses of these cells resemble somewhat pavement epithelium ; they are not flat, but polyhedral. ENDOTHELIUM Endothelium resembles simple squamous epithelium in appear- ance, and it is called epithelium by some histologists. Endothelium forms the superficial covering of the pleural, peri- cardial, and peritoneal cavities, the tunica vaginalis, and the joints. It covers the membranes of the brain and spinal cord, the inner surfaces of the heart, and the blood- and lymphatic vessels. Therefore endothelium lines the cavities that do not communicate with the external world. * It consists of a single layer of thin, flat cells, held together by a small amount of intercellular substance. It is demonstrated best on fresh tissues, hence the human subject is seldom available. The mesentery of the frog affords a good example of endothelial *Remember that the peritoneum may be said to have an opening by way of the Fallopian tube, and that the central canal of the spinal cord and brain is lined by epithelium but has no opening that connects it with the external world. ENDOTHELIUM 61 structure, and differs but little from the arrangement on human serous surfaces. Kill a large frog by decapitation, and open the abdomen freely by an incision along the median line. Pull out the intestines grasping the stomach with the forceps. This will expose the small intestine, which you will remove, together with the attached mes- entery, by means of quick snips of the scissors. Work as rapidly as possible and avoid soiling the tissue with blood. Throw the gut into a saltcellar filled with silver solution (vide formula), where it must remain for ten minutes covered from the light. Lift the tissue from the solution by means of a strip of glass (or a platinum wi#e), and throw into a saucer of clean (preferably dis- tilled) water, changing the latter repeatedly for some minutes. FIG. 36. ENDOTHELIUM PROM FROG'S MESENTERY. SILVER STAINING. A. Area showing the outlining of the cells by the silver stained cement-substance. The nuclei have been brought out by the carmine. Minute stomata may be seen between certain cells. B. A blood-capillary terminating below in an arteriole. The silver has outlined the endo- thelial cells of the vessels. C. An area showing both layers of the cells. The deeper cells are faintly outlined, being out of focus. The silver has been deposited over the lower portion of the specimen, nearly obscuring the cement lines (X 250). 62 STUDENTS HISTOLOGY After washing, and while yet in the water, expose to sunlight (perhaps fifteen minutes) until a brown tint is acquired, which indicates the proper staining. The mesentery has been left connected with the intestine, so that the former might not curl. The preparation having been dehydrated with alcohol, and having reached the oil of cloves, pro- ceed with a pair of scissors to snip off a small, flat piece of mesen- tery. Remove it to a slide and mount in balsam. The omen turn of a rabbit, cat, or dog may be prepared in a similar manner. The outlines of the endothelial cells appear as dark lines where the silver has stained the cement-substance. These lines are often tortuous and serrated. The nuclei are not seen, unless they have been separately stained with carmine or some other nuclear dye. Openings called stomata occur on the serous surfaces, which connect the cavities with underlying lymphatic vessels. The stomata occur at the points where several endothelial cells meet. The opening is sometimes surrounded by several small, granular cells, called "guard cells." Changes in the size of these cells modify the size of the stomata. CONNECTIVE TISSUES Certain elementary structures of similar origin and mode of development, and serving alike to unite the various parts of the body, have been termed connective tissues. Custom has restricted the term, in its everyday employment, so as to apply to white fibrous tissue, or, at least, to tissue which always resembles this more closely than any other. WHITE FIBROUS TISSUE This, the connective tissue par excellence, is composed of exceed- ingly fine fibrillse (only 0.6 /* in diameter), which are aggregated in irregularly sized and variously disposed bundles. It forms long and exceedingly strong tendons connecting muscle and bone; its fibers interlace, forming the delicate network of areolar tissue; it forms thin sheets of protecting and connecting aponeuroses ; or, supporting vessels, it permeates organs and sustains the paren- chyma of glands. The fibers are held together by means of a transparent cement, CONNECTIVE TISSUES 63 which may be softened or dissolved in acetic acid. They may exist, as in dense tendons, without admixture. Cells are found between the bundles of fibers, known as con- nective tissue corpuscles. The older and more dense the structure, the less frequent are these cells; while in young connective tissue, stained, the nuclei of the corpuscles constitute a promi- nent feature of the specimen under the microscope. Having obtained a piece of tendon from a recently killed bul- lock, tease a fragment on a slide in a few drops of water, Select a FIG. 37. CONNECTIVE TISSUE. A. Teased fibers. C. Fibrillse. portion which splits easily and separate the fibrils as much as possible. Cover, and examine (H). Fine, wavy fibers are seen composing the fasciculi. If the dissection has been sufficiently minute, you may succeed in demon- strating ultimate fibrillaa. These are best made out, as at C in Fig. 37, where the parts of a bundle have been separated for some dis- tance, leaving the finer elements stretching across the interval. YELLOW ELASTIC TISSUE This tissue consists of coarse, shining fibers (averaging about 8f- in diameter) which frequently branch and anastomose. They are 64 STUDENTS HISTOLOGY FIG. 38. TEASED YELLOW ELASTIC TISSUE FROM THE LIGAMENTUM (X 250.) FIG. 39. TRANSVERSE SECTION OF PART OF THE LIGAMENTUM NUCH^E. S. Sheath of the ligament, sending prolongations within— as at T, T— dividing the structure into irregular bundles or fasciculi. L. Lymph-spaces in the connective tissue. A. Adipose tissue in the sheath. . V. Blood-vessels in transverse section. E, E. Primitive fasciculi of yellow elastic tissue fibers. ADIPOSE TISSUE 65 highly elastic. Under the microscope the fibers are colorless, but when aggregated, as in a ligament, the mass is yellow. Procure a small piece of the ligamentum nuchce of the ox, and tease it on the slide after it has been macerated in acetic acid f OIL a few moments. The acid softens the fibrous connective tissue and facilitates the teasing process. The individual fibers having been isolated, they appear as in Fig. 38. When broken, they curl upon themselves, like threads of India rubber. This tissue is variously disposed throughout the body where great strength with elasticity becomes necessary. The large arteries are abundantly supplied with elastic fiber, arranged in plates, in alternation with muscle. As a network, it is mixed with connective tissue in the skin, and in membranes generally. It con- . tributes elasticity to cartilage where the fibers form an intricate network. Ligaments are composed largely of yellow elastic tissue. Fig. 39 is drawn from a portion of a stained transverse section of part of the II (j amenta tsttbflava. A strong sheath of fibrous tissue is thrown around the whole ligament, a portion of which is seen at S. This sheath sends pro- longations, T, T, into the structure, dividing it into irregular bundles, which support nutrient vessels. The elastic fibers seen in transverse section, as at E, E, are observed strongly bound together with fibrous tissue, which penetrates the smaller fasciculi, dividing them into the ultimate fibrillce. ADIPOSE TISSUE Adipose or fat tissue is a modification of and development from ordinary connective tissue. It originates in certain contiguous connective tissue corpuscles becoming filled with minute fat -globules. These ultimately coa- lesce and form single large globules, which bulge out the cell -bodies until they become spheroids; the nuclei at the same time are dis- placed to the periphery. An aggregation of such cells forms a lobule of adipose tissue. The cells are often so closely packed as to assume a polyhedral form. From malnutrition, this fat may be absorbed, ordinary connective tissue remaining. You will bear in mind the fact that whenever fat exists in a condition of minute subdivision, the particles always assume the 66 STUDENTS HISTOLOGY FIG. 40. CONNECTIVE TISSUE CELLS CONTAINING FAT — INDICATING THE MODE OF FORMATION OF ADIPOSE TISSUE (X 400). A. Ordinary elongate connective tissue cells. B. Same containing minute globules of fat. C. Coalescence of the fat-globules and displacement of the nucleus. D. Still greater increase of the fat. FIG. 41. ADIPOSE TISSUE FROM TEASED HUMAN OMENTUM. STAINED WITH HJEMATOXYLIN (X 400). A. Connective tissue framework. B. Just below the letter is a cell containing crystallized fat CARTILAGE 67 globular form ; and that while adipose tissue contains fat, fat alone is not adipose tissue. CARTILAGE Cartilage consists of a dense basis substance, in which cells or corpuscles are imbedded. It presents three forms: HYALINE CARTILAGE The matrix of hyaline cartilage is translucent, dense, and apparently structureless. Minute channels in certain instances and delicate fibrilla? in others have been demonstrated. The sur- face of the cartilage is surrounded by a fibrous envelope, called the perichondrium . The basis material contains excavations, generally spherical, called lacunce. They appear to be lined with a membrane, ,and Fig. 42. SECTION OF HYALINE CARTILAGE FROM A HUMAN BRONCHUS. The ground-substance is apparently structureless, and it contains the lacunae or exca- vations in which one, two, three, or more cartilage cells appear. These cells show a well marked intracellular network (X 400). contain one, two, three, and perhaps as many as eight cells — the cartilage -corpuscles. The matrix around the lacuna? is called the capsule. It differs from the rest of the matrix and creates the appearance of a membrane surrounding the lacuna?. Hyaline cartilage is found covering joints generally, where it 68 STUDENTS HISTOLOGY is termed articular cartilage. It is also found in the trachea, the bronchi, the septum narium, etc. Fig. 42 shows a section of hyaline cartilage from one of the rings of a large bronchus. FIBRO - CARTILAGE Fibrous connective tissue, predominating largely in the basis substance, produces a structure of great strength— fibro- cartilage. The intervertebral disks afford an example of this variety, from a FlG. 43. FlBRO-CARTILAGE FROM AN INTERVERTEBRAL PLATE OR DlSK. The ground-substance, unlike that of the hyaline varisty, consists of dense fibrous tissue with little calcareous matter (X400). section of which Fig. 43 has been drawn. The fibrous tissue is a very prominent feature of the ground substance. ELASTIC OR RETICULAR CARTILAGE As the name implies, yellow elastic tissue is an important ele- ment of the ground - substance of elastic cartilage. It presents the form of a reticulum, as shown in Fig. 44. It is not exten- sively distributed in the human being, although the cartilages of the external ear, Eustachian tube, epiglottis, etc., are of this variety. Cartilage should be hardened by the chromic acid and alcohol process. ' The sections from which the illustrations have been •drawn were cut without the microtome. They should be cut CARTILA GE—BONE FIG. 44. ELASTIC CARTILAGE FROM EAR OF BULLOCK. The ground-substance consists largely of a network of coarse, yellow elastic tissue (X400K FIG. 45. PORTION OF A TRANSVERSE SECTION FROM A DRIED FEMUR, SHOWING PART OF THE WALL OF A HAVERSIAN SYSTEM. (See pages 70 and 71.) A, A. Bony lamellae. B, B. Lacunae. C, C. Canaliculi (X400). 70 STUDENTS HISTOLOGY extremely thin, but not necessarily large. We frequently succeed in getting good fields from the thin edges of sections which may be elsewhere too thick. Stain with hasmatoxylhi and eosin. The differentiation will be excellent. The delicate nutritive channels in the matrix connecting the lacunae may be demonstrated in the cartilage of the sternum of the newt ; the xiphoid appendix is sufficiently thin without sectioning. FIG. 46. TRANSVERSE SECTION OP PORTION OP A DRIED LONG BONE, SHOWING THE HAVERSIAN SYSTEMS. A, A, A. A Haversian system. B. Haversian canal. The lacunas, canaliculi, and Haversian canals all appear black in the section, as they are filled with air and the bony fragments resulting from grinding of the section (X60). BONE Bone consists of an osseous, lamellated matrix, in which occur irregularly - shaped cavities — lacunae. The latter are connected by means of exceedingly fine channels — canaliculi. The lacunaB con- tain the bone -corpuscles, the bodies of which are projected into the canaliculi. BONE 71 In compact bone, the blood-vessels run in a line parallel with the long axis of the bone, in branching inosculating channels (averaging about 50 /><< in diameter) — the Haversian canal*. The lamellae of osseous tissue are arranged concentrically around these FIG. 47. SECTION OF BONE SHOWING SHARPEY'S FIBERS PULLEK OUT ov POSITION. AFTER H. M TILLER. canals. A single Haversian canal, and the lamella? surrounding and belonging to it, constitute a Haversian system. The lamellae beneath the periosteum are not arranged as above, but parallel with the surface of the bone. These plates are per- forated at a right angle and obliquely by blood-vessels from the FIG. 48. DIAGRAM OF A HAVERSIAN CANAL. A. Artery. B. Vein. C. Nerve. D. D, D. Lymph-channels. periosteum, as they pass on their way to the Haversian canals. These lamellae are also perforated by partly calcined connective tissue — the perforating fibers of Sharpey. (Fig. 47.) A Haversian canal contains (Fig. 48) an artery, a venule, lymph -channels, and a nerve -filament. The whole is supported by connective tissue cells with delicate processes. The walls 72 STUDENTS HISTOLOGY of the lymph -spaces are prolonged into the eanaliculi, and1 thus placed in connection with the elements of the surrounding1 lacunae. Each lacuna contains a bone-corpuscle, the protoplasmic body of which sends prolongations into the contiguous eanaliculi. In the adult bone the cell is shrunken, and the processes just .men- tioned are» not readily demonstrable. The eanaliculi of any Haversian system communicate with one another, but not with those of different systems. The concentrically placed lamellae around the Haversian canals are. called Haversian lamellce. The angular area formed where several Haversian systems join is filled out by interstitial lamella., FIG. 49. DIAGRAM OF A BONE LACUNA. A, A. Ground-substance of the bone. B, B. Limiting membrane of the bone-corpuscle within the lacuna. C, Nucleus and nucleolus of the corpuscle. D, D. Projections of the cell-body into the eanaliculi. while those lamellae mentioned above as occurring just below the periosteum are the circumferential or fundamental lamellce. The arrangement of Haversian canals and their concentric- lamellae is confined to compact bone. Compact bone is formed on the surface of all bones, and makes up the bulk of the shaft in long bones. The other variety of bone, called cancellous or spongy, occurs extensively at the ends of long bones, and in short and flat bones. It consists of lamellae containing lacunae and eanaliculi, lying between good sized cavities, which are occupied by blood- vessels and marrow, and which correspond to Haversian canals. Bone is to be regarded as dense connective tissue, arranged in lamellae, of which the ground-substance is impregnated with salts of calcium, chiefly the phosphate, to which its hardness is due. At the same time, bone retains the elasticity and strength of DEVELOPMENT OF BONE 73 connective tissue, which distinguish it from a structure that has merely been calcined, as may happen in certain disease processes. PERIOSTEUM The surface of bone is covered by an envelope called periosteum r which has two layers — a dense, outer, fibrous layer and a looser, inner, vascular layer. The inner layer contains the cells, osteoblasts, which form osseous tissue, hence this layer is called the osteogenetic layer of the periosteum. In operating on bone, surgeons guard the periosteum very carefully, on account of its blood-vessels and its- osteogenetic elements. MARROW The spaces of bones are everywhere filled with marrow. The largest of these spaces are the medullary cavities of long bones. ID the medullary cavities the marrow is yellow, owing to the deposit of fat in the cells. The smaller spaces of bone are filled with red marrow. Red marrow contains marrow -cells, which are similar to connective tissue cells, supported in a very vascular connective tissue framework. The marrow cells are identical with the osteo- blasts of the periosteum and with bone-corpuscles. Immense cells, giant cells, containing many nuclei, also occur in red marrow. On account of their function of absorbing superfluous bone, they are called osteoclasts (Fig. 50). Red marrow also contains nucleated cells, tinged with hemoglobin, that are connected with the forma- tion of red blood -corpuscles, which probably takes place exten- sively in the red marrow. DEVELOPMENT OF BONE The majority of the bones of the body are first formed in the embryo as hyaline cartilage, which is subsequently replaced by true bone — endochondral ossification . 'The bones of the face and of the vault of the cranium and a portion of the lower jaw are the principal exceptions — intramem- branous ossification. In the latter case, the basis of the forming bone is an embryonic fibrous tissue, and this form of ossification differs from the endochondral in not being carried on in a carti- laginous basis, which is, however, a temporary structure. 74 STUDENTS HISTOLOGY (a) In the case of endochondral bone, the commencement of ossification is indicated by the enlargement of the cartilage -cells and their arrangement into vertical rows. This takes place at a point called the center of ossification. (?>) The- matrix between them becomes calcified, (c) From the surface of the bone, which is covered by a membrane corresponding to the periosteum, processes extend into the cartilage, (d) The cartilage is absorbed to make room for these processes through the agency of the osteoclasts (see Fig. 50). (e) The absorption proceeds until it includes the region of calcified cartilage. (/) The osteoblasts, which have accompanied the periostea! ingrowth, arrange themselves .on the surface of the spaces resulting from absorption of the cartilage, and form layers of bone, (g) At the same time the osteoblasts of the osteogenetic layer of the periosteum form layers of bone beneath the periosteum — periosteal ossification. The first bone formed is soft and spongy. It will be observed that as ossification proceeds the whole of the cartilage will be absorbed, except that at the epiphyses. In the course of the growth of the individual, the network and spaces of the origi- nal spongy bone undergo considerable rearrangement. Haver- FIG. 50. CELLS FROM RED MARROW OF RABBIT (PRUDDEX). A. Marrow cells proper. B. Giant cells. sian systems result from deposition of successive layers of lamellae around the spaces of spongy bone, from without inward, leaving a channel at the center, which is the Haversian canal. The medullary cavity is produced by the absorption of the central part of the bone, while new layers continue to form under the periosteum. SECTIONS OF BONE 75 PIG. 51. DEVELOPING BONE, FROM THE PIG (PRUDDEN). Fig. 46 lias been drawn from a section of dry bone which has been sawn as thin as possible, and afterward rubbed down on a hone with water. It is a tedious process, and shows little but the osseous matrix. Bone should be decalcified for microscopical work, and it may then be readily cut in thin sections with a razor. The process* is as follows: To 100 c.c. of the dilute chromic acid solution add 3 c.c. of 0. P. nitric acid. The bone, previously divided into slices not over one -half centimeter in thickness, is then placed in the fluid, and should be completely decalcified in a week or ten days. Examine the pieces after twenty -four hours by puncturing with a needle. Should the action proceed too slowly, add a few drops more of the nitric acid from time to time. The bone eventually takes on a green color. After complete decalcification, wash the pieces for twenty-four hours in clean water, and preserve them, until required, in 80 per cent, alcohol. Small pieces of young bone may be decalcified in a saturated aqueous solution of picric 76 STUDENTS HISTOLOGY acid. The process is slow, but it leaves the tissue in excellent condition . Sections cut in the usual way may be stained with carmine and picric acid, and examined in a drop of glycerin. They should not, after the staining, be placed in the oil of cloves, as they would curl and become hard. Transfer them to equal parts of glycerin and water, from which they are to be carried to the slide. Add a drop more of the dilute glycerin if necessary and put on the cover -glass, carefully avoiding air -bubbles. If you desire to make a perma- nent mounting, the edge of the cover must be cemented to the slide. Thoroughly wipe the slide around the cover with moistened paper, until every trace of glycerin is removed. Then with a sable brush, paint a ring of zinc cement (vide formulae) around the slide just touching the edge of the cover -glass. Repeat the cementing in twenty -four hours. A turn-table will be a useful aid in this work. SPECIAL CONNECTIVE TISSUES Connective Tissue of the Lymphatic System. — The matrix of lymphoid or adenoid tissue consists of a network of fibers and cells, which support the lymph -corpuscles. It is distributed exten- sively in organs, and where it appears in stained sections, the lymphoid cells are so numerous as to obscure the reticulum almost entirely. The structure will be minutely described in connection with the lymphatic system. Embryonic Connective Tissue presents a homogenous, mucoid matrix containing branched cells. It is not found normally in the adult. The jelly of Wharton of the umbilical cord is mucoid tissue. i MUSCULAR TISSUE This tissue is found in three varieties: 1. Non-striated, smooth or in voluntary. 2. Striated, skeletal, or voluntary. 3. Cardiac. NON- STRIATED MUSCLE The histological element of non- striated muscle is a spindle- shaped cell from 45-225 p long and 4-7 p broad. The cell body presents longitudinal striae, and contains an ovoid nucleus. The nucleus contains a reticulum which is probably in connection with NON- STRIATED MUSCLE 77 the fibrillae, which produce the longitudinal striation of the body. The cells are not infrequently bifid at one or both extremities. A transparent cement substance serves to unite these cells in form- ing, with connecting tissue, broad membranous plates, bundles, A| FIG, 52. NON-STRIATED MUSCLE. (SCHAFER.) A. Complete cell. B. Broken cell. plexuses, etc. It serves to afford contractility, especially to the organs of vegetative life. Kill a good -sized frog by decapitation, and open the abdomen on the median line. Fill the bladder with air, after the introduc- tion of a blow -pipe into the vent. Remove the inflated bladder 78 STUDENTS HISTOLOGY with a single cut with the curved scissors, and place it in a saucer of water. Proceed to brush it, under the water, with two camel's- hair pencils, so as to remove all of the cells from the inner surface. It will bear vigorous rubbing with one of the brushes, holding it at the same time with the other. Transfer to alcohol for ten minutes, and afterward stain with hsematoxylin and eosin. While in the oil, cut the tissue into small pieces, and mount flat in balsam. Examine with L and H. Observe the bands of involuntary muscle crossing in various directions. You will distinguish between the muscle and the con- nective tissue cells by their nuclei. STRIATED MUSCULAR TISSUE A skeletal or striated muscle consists of cylindrical fibers, vary- ing from 10 p to 100 p- in diameter and 5 to 12 cm. long. These primitive fibers are supported by a delicate, transparent sheath— the sarcolemma. They are aggregated, forming primitive fasciculi, which are again united to form the larger bundles of a complete FIG. 53. PART OF A MUSCLE FIBER. (RANVIKR.) A. Dark disk. B. Membrane of Krause. •0. Light disk. N. Muscle nucleus. muscle. The connective tissue uniting the primitive fibers is termed endomysium; while that uniting the primitive bundles is the perimysium. The primitive muscular fibers exhibit marked cross striations with faint longitudinal markings, the former being produced by alternate dark and light spaces. STRIATED MUSCLE 79 Under very high magnification each light band appears to be crossed by a dark line, called the intermediate disk or membrane of Kranse. The dark band consists of rows of spindle-shaped bodies. FIG. 54. STRIATED MUSCULAR FIBERS FROM THE TONGUE, TEASED AND STAINED WITH H^EMATOXYLIN (X 400). A. A fiber, with the muscle substance wanting, from stretching during the teasing, the sar- colemma alone remaining. B. Partly separated disk of Bowman. C. Ultimate fibrillae. D. A blood-capillary. Those of the different dark bands are placed end to end, forming- continuous elements. They constitute the contractile fibrillce. The light bands correspond to the attenuated ends of the spindle- shaped bodies. Little knobs on the ends of the spindles make the dark line called the intermediate disk. The fibrilla? are held in bundles by a pale sarcoplasm. In transverse sec- tions of muscle these bundles show as polygonal areas — Colin- lie im1 s fields. Macerate human muscle, preferably that from the tongue, in dilute chromic acid for twenty -four hours, wash, tease in water, .80 STUDENTS HISTOLOGY -cover, and focus with H. Fig. 54 was drawn from such a preparation. The sarcolemma is best seen where the contractile substance las been broken. The muscle nuclei are seen at various points beneath the sarcolemma. Portions of a fiber have been split off transversely in places, indicating the disk of Bowman. The fibrillae are indicated where the fiber has been split longitudinally during the teasing. The capillaries are arranged in a direction parallel to the fibers, with frequent transverse connections. CARDIAC MUSCULAR FIBER It presents the following characteristics: 1. The fibers are smaller than those of ordinary skeletal muscle. 2. They are striated both transversely and longitudinally. 3. They branch, forming frequent inosculations. FIG. 55. TEASED CARDIAC MUSCULAR FIBERS. Stained with haematoxylin, X 400 and reduced. 4. They are divided by distinct transverse lines into short cells. 5. Their nuclei are situated within the fiber. 6. They present no distinct sarcolemma. Notice the groups of yellowish brown pigment -granules within the muscle-cells close to the nuclei. Fig. 55 has been drawn from fresh cardiac muscle, teased in normal salt solution and tinted with eosin. NERVOUS TISSUES— BLOOD 81 NERVOUS TISSUES Following the order given in the classification of tissues, the nervous tissues should be studied at this point. But in laboratory work it will be found more satisfactory to consider them in con- nection with the histology of the central nervous system (see page 216). BLOOD The human red blood -corpuscle is a flattened, bi- concave disk, circular in outline, and from 7 p to 8 M (Woro inch) in diameter. It FIG. 50. CORPUSCULAR ELEMENTS OF HUMAN BLOOD (X 400). A. Colored corpuscles adhering by their sides — rouleaux. B. The same crenated. C. The same shrunken. I). The same having absorbed water. E. The same still more swollen. F. The same with the plane C D, Fig. 57, in focus. G. The same with the plane A B, Fig. 57 in focus. H. Colorless corpuscles. presents a mass of protoplasm destitute, as far as the microscope shows, of nuclei, cell -wall, or any structure whatsoever. Certain changes in form result, after removal from the circula- tion, viz.: 1. They may adhere by their broad surfaces forming 82 STUDENTS HISTOLOGY columns. 2. From shrinkage they may become crenated. 3. Still further shrinkage produces the chestnut -burr appearance. 4. From absorption of water they may swell irregularly, obliterating the concavity of one side. 5. From continuous absorption they swell, forming spheres which are finally dissolved. Wind a twisted handkerchief tightly around the left ring-finger, FIG. 57. DIAGRAM OF A COLORED BLOOD-CORPUSCLE, SIDE VIEW, SHOWING THE Bi -CONCAVITY. (The thickness is exaggerated.) A, B. Upper plane, which, in focus, gives the appearance shown at G, Fig. 56. C, D. Plane giving the appearance shown at F, Fig. 56. prick the end with a clean needle, and squeeze a minute drop of blood on a slide, add a drop of salt solution, cover, and focus with H. Observe: 1. That considerable variation in size of the red blood -corpuscles exists. 2. The color— a delicate straw tint. 3. That the concave centers of the corpuscles which lie flat can be made to appear alternately dark and light according to the focal adjustment. 4. That the concavity is also demonstrated as the corpuscles are turned over by the thermal currents.* BLOOD -PLATES Minute corpuscular elements in the blood, about one-fourth the size of the red disks, exist in the proportion of about one of the former to twenty of the latter. They are colorless ovoid disks, and are regarded by Osier as an essential factor in the coagulation of the blood. Prick the thoroughly clean finger with a needle. Over the puncture place a drop of solution of osmic acid (one per cent.), and squeeze out a minute drop of blood, so that, as it flows, it is covered by the acid solution. This fixes the anatomical elements, provid- ing against further change. The blood, as soon as drawn, must, with the acid, be immediately transferred to a slide and covered. "The student is at this time advised to study the corpuscular elements of the blood of such animals as he may be able to command. The red corpuscles of mammals (excepting the camelidae) do not vary in appearance from those of man, excepting in size. Those of birds, fishes, and reptiles are elliptical, with oval nuclei. Corpuscles of the blood of invertebrates are not colored. WHITE BLOOD- CORPUSCLES 83 To provide against evaporation, run a drop of sweet oil around the edge of the cover. The blood -plates may be found, after careful search, bearing the relation to the red corpuscles seen in Fig. 58. WHITE OR COLORLESS BLOOD -CORPUSCLES The white blood -corpuscles are also called leucocytes. In fresh preparations they are seen to be perfectly colorless, nearly spherical cells, often slightly granular. The nucleus is distinguished with difficulty unless reagents are used. The leucocytes are not all of the same size. The larger ones are the more numerous. If they are watched carefully, the larger ones may be observed to change FIG. 58. HUMAN BLOOD PRESERVED WITH OSMIC ACID. A. Colored corpuscles. B. Colorless corpuscle. C. C, C. Groups of plaques. (X 400 and reduced.) their shapes slowly. This movement is a property belonging to their protoplasm called amoeboid movement.* Portions of the protoplasm are slowly extended outwards, making projections called pseudopodia, which may be 'drawn in again. By means of this movement the leucocytes can travel slowly from one part of the field to another. They are more active when the slide is warmed slightly. *The Amoeba is an exti-emely simple unicellular animal (Protozoon), which is found in the water of ponds. It is usually much larger than the white blood -corpuscle, and its move- ment is ordinarily very active and easily seen. The student should examine specimens of water and watch the movement of the Amoeba. $4 STUDENTS HISTOLOGY The leucocytes, especially the larger ones, are of great impor- tance in pathology in connection with inflammation and the forma- tion of pus. The large leucocytes furnish the great majority of the cells in ordinary pus. The smaller leucocytes are about the size of the red corpuscles; the larger ones are about 13 ^ in diam- eter. The leucocytes are very much less numerous than the red •corpuscles. The ratio to the red corpuscles varies from 1 to 500 to 1 to 1,000. The leucocytes become more numerous a few hours after eating. In order to study the leucocytes more carefully, they should be examined in dried and stained preparations, with an oil- immersion lens if possible. .Square cover-glasses are used, which need to be clean and perfectly free from dust. They should be handled with forceps. Having cleaned and dried the skin of the finger, puncture it quickly with a clean, sharp needle, using no pressure. A drop of blood should be allowed to issue. Wipe away the first •drop, and use the next, which should be no larger than a pin's head. Apply the surface of one cover-glass to the summit of the drop. Let this cover- glass fall on the other at the angle shown in Fig. 59. The blood is spread between the cover-glasses in a thin film. Quickly draw them apart, without lifting. The film of blood dries immediately. The object is to spread the t>lood on the cover-glass in a thin film within a few seconds after it leaves the capillaries, before coagulation or changes in the shapes of the cells can occur. To fix the preparations they should be placed in a mixture of alcohol and ether -(equal parts) for half an hour; or they may be subjected to dry heat (110° C.) preferably for half an hour or even longer. There are many methods of staining. Much can be done with the ordi- nary hsematoxylin and eosin stain. Very beautiful results can be obtained FIG. 59. MANNER OF PLACING COVER-GLASSES. (CABOT.) "with combinations of aniline dyes; for instance, eosin and methylene blue: one- half per cent, solution of eosin in sixty per cent, alcohol three minutes ; wash ; dry, by pressing between two pieces of filter paper; strong watery solution of methylene blue, one minute; wash; dry; balsam. The very large nucleated red blood-corpuscles of the frog and newt should Ibe stained in this manner. WHITE BLOOD-CORPUSCLES 85- To study the leucocytes of human blood after fixation, preferably with heat, use the Ehrlich tricolor stain (p. 32) for five minutes ; wash, dry, mount in balsam. The red corpuscles are stained orange -yellow to brown. The nuclei of the leucocytes are stained green. The Ehrlich method of staining shows us that the leucocytes, are of several kinds. Some have large, round nuclei; others- A B FIG. GO. VARIETIES OF LEUCOCYTES. A. Small Lymphocytes. B. Large Lymphocytes. C. Polymorpho-nuclear Neutrophiles. D. Eosinophile. have nuclei that are distorted or that appear to be in several parts. Some have stained granules in their protoplasm; others have no granules. The nature of the granules and their affinities for the aniline dyes have been described in the chapter on staining. The granules in the leucocytes of man are of two sorts: (a) Neu- trophile granules, very small and numerous dust -like granules of a reddish brown color; the leucocytes containing them look as though they had been sprinkled with red pepper, (b) Eosinophila granules — good sized, round, shining granules, not so numerous in the cells as the last. These characters enable us to classify leucocytes as follows: 1. Small lymphocytes, which have a large, round nucleus and a thin band of protoplasm with no granules. They are the same as the lymphoid cells of the lymph- nodes and lymph from which they originate. They make twenty per cent, to thirty per cent, of all leucocytes. 2. Large lymphocytes, or large mononuclear leucocytes. They have a round or indented nucleus, and a considerable amount of protoplasm, without granules. Those with indented nuclei are often known as transitional forms. The large lymphocytes are not numerous — four to eight per cent. 86 STUDENTS HISTOLOGY 3. Polymorphonuclear neutrophiles (often called polynuclear) . The nuclei are much indented, or even seem to exist as several different parts. They contain immense numbers of fine neutro- phile granules. They constitute the majority of leucocytes — sixty- two to seventy per cent. 4. Eosinophiles, which have polymorphous nuclei and eosino- B \7 FIG. 61. MIXING PIPETTES. (CABOT.) A. For red blood-corpuscles. It is the one referred to in the text. B. For white blood-corpuscles, where the dilution is not so great. Weak acetic acid is used as a diluting fluid, which decolorizes the red corpuscles so that the white corpuscles alone are phile granules. They are of great importance in some of the diseases of the blood. The proportion of eosinophiles is from one- half to four per cent.* *The percentages given are quoted from Cabot: Clinical Examination of the Blood, to which the student is referred for further information on this subject. ENUMERATION OF BLOOD-CORPUSCLES 87 In disease the percentages above given are subject to much variation. Cells that contain basophile granules are occasionally seen in normal human blood, as well as in disease. Their significance js not understood. They are not demonstrated by the Ehrlich tri- color stain. They are easily found in the blood of the Amphibia, by staining with basic dyes. There is some reason for believing that the lymphocyte is the youngest form of leucocyte, and that the other varieties are developed from it in succession while circulating in the blood- channels. ENUMERATION OF BLOOD - CORPUSCLES The number of blood -corpuscles in a cubic millimeter of blood may be determined quite accurately by means of the haemocyto- meter. To lessen the labor of counting, the blood is diluted with normal salt solution or Toison's fluid.* The blood is drawn into the pipette, Fig. 61 A, to the point marked 0.5, or to 1.0, and then the fluid is drawn in till the bulb is filled to 101.0. With the finger on the end of the pipette it is shaken to mix the blood with the solution. Discarding the first drop, a small quantity is placed in the center of the small FIG. 62. PLATE AND RULED DISK OF THE H^MOCYTOMETEK. disk in the middle of the slide, Fig. 62. The cover-glass, which goes with the instrument, is placed. over the drop. Air bubbles are to be avoided, and no dust *Toison's fluid — Methyl violet. 5B 025 grams. Sodium chloride 1. Sodium sulphate 8. Glycerin 30 c.c. Water 160 c.c. The leucocytes are stained faintly purple. The red blood-cor-puscles retain their normal color and form. 88 STUDENTS HISTOLOGY particles or fluid should separate the cover-glass from the surface of the square of glass surrounding the disk. The disk is depressed -n> mm. below the upper surface of the square. It is also ruled into 400 squares -^ mm. on each side. When the blood- corpuscles fall to the surface of the di^k, as they do after a few minutes, the number counted in one square represents those present in Tcfoo" cu.mm. of the diluted blood. UVX a^X TO-) Every fifth square is crossed by a second ruled line, which encloses the small squares into groups of sixteens and assists in counting. Using the high-power, tne corpuscles in a given number of squares in various parts of the plate are counted. In counting, discard the corpuscles that touch the lines on two sides (above and at the right), and FIG. 63. APPEARANCE OF FIELD OF HJEMOCYTOMETER UNDER HIGH-POWER. (FREEBORN.) count those that touch the other two (below and at the left); the average for one small square is thus taken. It is best to wipe away the first drop after a number of squares have been counted, replacing it with another, after thoroughly shaking the pipette. In all, count the number in about four hundred small squares. Multiply the average number for one square by 4,000 and by the dilution (100 or 200), and the number of red (or white) corpuscles in a cubic millimeter of blood is obtained. The number of red blood -corpuscles in a cu.mm. of human blood is 5,000,000, or somewhat more, for men, and about half a million less for women. The normal number of white corpuscles is from 5,000 to 10,000 in a cu.mm. HEMOGLOBIN HAEMOGLOBIN The substance that gives to the red blood -corpuscles their char- acteristic color is haemoglobin, which has the important function^ of being the oxygen -carrier of the corpuscle. The haemoglobin of most mammals crystallizes in the form of rhombic prisms of a red color. That of the rat crystallizes quite readily. FIG. 64. CRYSTALS OP HEMOGLOBIN. (RANVIER.) A, B. Of man. C. Of cat. D. Of guinea pig. E. Of hamster. F. Of squirrel. Haematoidin and haemosiderin are substances derived from haemoglobin often found in pathological tissues, as after haemor- rhages. Haemosiderin contains iron, and occurs as yellow or brown granules. Haematoidin, which is the same as bilirubin, contains no iron, and occurs as granules or rhombic plates of a yellow to brown color. 90 STUDENTS HISTOLOGY H^EMIN CRYSTALS Let a drop of blood dry on a slide. Add a few drops of glacial acetic acid, and heat over a flame until bubbles appear. Dry and mount in balsam. Dark brown rhombic prisms will be seen, which are crystals of hsemin, or the crystals of Teichmann. They are proof of the presence of blood, but do not indicate its source. They may be of importance in medico -legal cases. FIBRIN The delicate network of straight fibrin filaments is easily demonstrated by the method recommended by Gage. A large drop of blood is placed on a slide, and is covered with a cover-glass. The slide is laid on a piece of wet blotting paper, and covered with a saucer to prevent evaporation. After half an hour coagulation will have occurred. Draw a drop of water around the edge of the coVer-glass, and float it carefully from the slide, endeavoring to keep the coagulum of fibrin on the cover-glass. Wash carefully in water, stain in hsematoxylin and eosin; dry; mount in balsam. (Gage recommends mounting •without balsam over a hard-rubber cell.) EFFECT OF REAGENTS Reagents produce characteristic changes in the blood-corpuscles. A strong saline solution leads to the formation of projections on FIG. 65. BLOOD-CORPUSCLES OF FROG. (RANVIER.) the red corpuscles, known as crenation; if sufficiently concentrated, the corpuscle becomes a shrunken, shapeless mass. Water causes the red corpuscles to swell ; the haBmoglobin is finally dissolved out, leaving the colorless, barely visible outline of the stroma called DEVELOPMENT OF THE BED BLOOD-CORPUSCLES 91 the "ghost." After the addition of water the white corpuscles become' spherical ; their granules display the dancing " Brownian motion; " they swell, and finally burst. Weak acetic acid decolorizes the red corpuscles, and clears the granules of the white corpuscles so that their nuclei become visible. Weak solutions of tannic acid coagulate the coloring matter of the red corpuscles, which escapes from the cell, clinging as a minute particle to one edge. DEVELOPMENT OF THE RED BLOOD -CORPUSCLES The red blood -corpuscles of the mammalian embryo possess nuclei, and in this respect resemble those of birds, reptiles, amphib- ians, and fishes.* The nucleated red corpuscles of the mammalian embryo and of the young forms of the lower vertebrates multiply by karyokinesis. At birth, however, in mammals the nucleated corpuscles are found to have been replaced by the ordinary, non- nucleated, discoidal forms. The origin of the non -nucleated corpuscles and the man- ner of their renewal throughout life are uncertain. It has been- suggested that they are developed from leucocytes and also from the blood -plates, but both of these theories lack confirmation. It seems probable that the nucleated cells colored with haemoglobin, found in the red marrow of the bones, are the most important source of the red corpuscles. According to Ho well, the nucleated red corpuscles of the marrow lose their nuclei by extruding them. *The red blood-corpuscles of the order of fishes known as Cyclostomi, of which the lamprey is a member, are circular, nucleated disks. Amphioxus has no red blood-corpuscles. PART THIRD ORGANS THE SKIN The skin consists of (1) the epidermis (or scarf skin), which everywhere covers and protects (2) the derma (corium or true skin ) . The epidermis varies greatly in thickness in different locations; and in the thicker portions several layers may be differentiated. It is composed entirely of cells, while the derma is fibrous. 1. Stratum Corneum, ) T 2. Stratum Lucidum, j Horny Lftyer- i & r^ li \ 3. Stratum Granulosum, 1 AT , . , . T 'M 4. Stratum of Prickle Cells, Malpighian Layer or t 1 5. Stratum of Columnar Cells. } Eete Mucosnm. J f The stratum corneum consists of old, exhausted, flattened, and desiccated cells, which are constantly falling from the entire sur- face of the body. Dandruff consists of impacted cells from this source. Those portions most frequently exposed to friction — e.g., the palms of the hands and the soles of the feet — are protected ~by a corneous epidermal layer of great thickness. The stratum lucidum, or clear layer, presents cells in form not unlike those in the preceding stratum ; they are, however, trans- lucent. This is properly a part of the previous stratum, is often absent, and frequently very difficult of demonstration. The stratum lucidum and stratum corneum owe their characteristic properties largely to the development in their cells of a substance called keratin. The stratum granulosum, or granular layer, is composed of flattened cells containing opaque granules of eleidin, which is related to the keratin of the horny layers. Immediately beneath the last-named layer, the cells become strikingly altered in form and appearance. The pricUle cells are (92) THE SKIN 93 polygons or compressed spheroids, with large, oval nuclei, and minute, projecting spines. By means of these processes they are connected with one another. The fifth and last (deepest) layer of the epidermis is composed of a single rank of elongated cells, placed with their long axes lit a right angle to the surface of the skin. These cells contain the FIG. 66. VERTICAL SECTION OP THE EPIDERMIS FROM THE PALM OP THE HAND. STAINED WITH H^EMATOXYLIN AND EOSIN. (X 400.) A. Stratum corneum. B. Stratum lucidum. C. Stratum granulosum. D. Prickle cells of rete mucosum or rete Malpighii. E. Stratum of elongated cells, the lower limit of the epidermis. F. F. Indicate the position of two papillae of the true skin or derma. pigment which gives the hue peculiar to the skin of colored individuals. The first two layers of the epidermis constitute, properly, the horny layer ; while the remaining three strata compose the rete mucosum or rete Malpigliii. The derma, corium or true skin, is composed of dense, fibril- lated connective tissue, so formed as to present minute elevations or papillae over the entire surface of the body. These papillae are 94 HJSTOLOG Y covered with a basement membrane, and are protected from undue irritation by the epidermal layers. The subcutaneous cellular tissue (upon which the true skin rests) consists of fibrillated connective tissue with elastic ele- ments, from which strong interlacing bands are formed. These, in the deeper parts, form septa which support lobules of adipose tissue. These isolated collections of adipose tissue, when elon- gated and placed vertically to the surface, constitute the fat- columns. FIG. 67. VERTICAL SECTION SHOWING THE DERMA, OR TRUE SKIN. INJECTED— PARTLY DIAGRAMMATIC. A. Line of junction of derma with epidermis. B. Capillaries distributed to papillae. The blood-vessels supplying the skin may be seen in vertical sections, in the subcutaneous tissue. Branches from these are sent to the papillae, where they terminate in delicate, interlacing loops of capillaries. Medullated nerves are also sent to the papillas ; and in certain locations they may be seen to terminate in tortuous structures — the tactile corpuscles. Varicose nerve- fibrils have been traced between the cells in the rete mucosum of the epidermis. SKIN— HAIR 95 APPENDAGES OF THE SKIN The appendages of the skin are the hairs, sebaceous glands, sudoriferous glands, and the nails. t THE HAIR A hair, consisting of a root and shaft, is constructed from elongated, often pigmented cells, which are cemented together and overlapped with cell -plates, which form the cuticle. The central part of medullated hairs is composed of cubical cells and occasional minute air -bubbles. The root penetrates the stratum corneum and (appearing to have pushed the rete mucosum before it) passes through the true skin and terminates in a bulb usually in the subcutaneous tissue, where it rests upon a papilla composed of an extremely delicate plexus of blood -capillaries. The Hair- Follicle. — The root of the hair, in its passage to the papilla, is invested with sheaths derived from the skin. The hair, E FIG. 68. TRANSVERSE SECTION OF HAIR AND HAIR-FOLLICLE. PARTLY DIAGRAMMATIC. A. Medulla of hair. B. Cortex of same. C. Root-sheath. D. Glassy membrane. E. Fibrous wall of the follicle. with its follicle, is indicated in transverse section in Fig. 68. A represents the medulla, and B the cortex of the hair. Outside the root-sheath, C, and derived from the rete mucosum of the epider- mis, is a thin layer, the glassy membrane, D. This is projected from the basement membrane covering the surface of the corium, 96 STUDENTS HISTOLOGY or true skin. The whole is surrounded by a fibrous coat, E, de rived from the connective tissue of the derma. A vertical section of the follicle is indicated in Fig. 69. A, B, and C represent the epidermal layers, which do not enter into its FIG. 69. DIAGRAM SHOWING MODE OF FORMATION OF HAIR-FOLLICLE A'. Epidermal layers. B'. Derma, or true sftin. A. Horny layer of epidermis. B. Stratum lucidum. C. Stratum granulosum. The three last mentioned form no part of the follicle. D. Rete Malpighii. This will be seen projected into the depths of -the true skin to form Ihe root-sheath, G. E. Hyaline membrane covering the derma. This is projected into the follicle, forming the glassy membrane, H. F. Fibrous tissue of the derma, forming the fibrous sheath of the hair-follicle, I. G. Root-sheath of the hair-follicle. H. Glassy membrane of the follicle. I. Fibrous sheath of the follicle. J. The hair-follicle. composition. The rete mucosum, D, forms the root-sheath at G. The basement membrane of the corium, E, forms the glassy mem- brane, H, while the connective tissue, F, constitutes the fibrous layer of the hair -follicle, J. The scales lining the hair-follicle are imbricated, and are directed downwards, fitting over the scales covering the surface of the hair, which are directed up- wards, and also imbricated. MUSCLES OF THE HAIR -FOLLICLES Attached to the fibrous layer of each hair -follicle is a small band of involuntary or smooth muscular fiber — the arrector pili. This passes obliquely toward the surface of the skin; and when contraction takes place, the follicle and hair are elevated, producing the phenomenon known as goose-flesh. SUDORIFEROUS GLANDS SUDORIFEROUS OR SWEAT-GLANDS 97 A sweat-gland (Figs. 67 and 70) consists of a tube or duct which, from the opening upon the surface, passes in a spiral course through the several layers of the skin to the deeper part of the corium, where it becomes coiled in a bunch, as at D, Fig. 70. The coiled or glandular part of the tube is surrounded by a net -work of capillaries. At B the tube is seen in transverse section. The gland -tube, D, is provided with a wall of connec- tive tissue and smooth or involuntary muscle, lined with conical FIG. 70. SUDORIFEROUS TUBULAR GLAND. A. Diagrammatic sweat-gland. C. Its duct. D. Coiled, glandular part. B. The same, showing a transverse section of both parts (X 400). C'. The duct lined with several layers of cells. D'. The coiled glandular part lined with columnar cells in a single layer, resting on a basement membrane. cells. The duct, C, is lined with granular epithelium covered with a thin cuticular membrane. Near the surface of the epider- mis the lining cells disappear. Krause estimated the number of sweat-glands at over two million. 98 STUDENTS HISTOLOGY SEBACEOUS GLANDS These glands are little sacs or lobules, one or more of which open into each hair -follicle. These sacs are entirely filled with polyhedral epithelial cells (vide Fig. 71) . At the neck of the gland FIG. 71. SINGLE LOBULE OF A SEBACEOUS GLAND (X 400). A. The fibrous wall of the sac. B. Membrane propria. C. Polyhedral cells filling the sac completely. D. Fatty degeneration of the parenchyma at the neck of the gland, formation of sebum. the cells become granular, fatty, and disintegrated, producing the sebum. THE NAILS The peculiar tissue of the nails corresponds to the stratum lucidum of the epidermis developed to an extreme degree. The nail rests upon a nail -bed, which represents the corium and the Malpighian layer of the epidermis. Minute longitudinal ridges take the place of papillae. The root of the nails is imbedded in a part of the nail -bed called the matrix, from which its growth occurs. PRACTICAL DEMONSTRATION Remove the skin from the parts below as soon after death as practicable. Tissue may frequently be secured after surgical operations from stumps, etc. Dissect deeply, so as to preserve the subcutaneous tissue. Small cubes from the finger-tips, the palm of the hand, the scalp, and the groin may be hardened quickly in strong alcohol; and vertical sections should be made as soon as the THE SKIN 99 tissue has become sufficiently firm. Stain with haBmatoxylin and eosin, and mount in balsam. The structure of hairs may be best demonstrated by washing the soap from lather, after shaving, with several changes of water. When clean, decant the water and add alcohol. After twenty-four hours again decant and add oil~of cloves. With a pipette carry a drop of the oil with the deposited hair-cuttings to a slide, remove as much of the oil as possible with slips of blotting-paper, and mount in balsam. Oblique, vertical, and transverse sections maybe readily obtained by this method. VERTICAL SECTION OF SKIN FROM THE GROIN (Vide Fig. 72) OBSERVE : (L.)* 1. The horny layer of the epidermis. (The stratum lucidum will hardly be demonstrable on account of the thinness of the epidermis in this region.) 2. The rete mucosum. (The section from which the illustration has been drawn was taken from a negro, and the deep cells were pigmented.) 3. The sharp line of demarcation between the epidermis and the true skin. 4. The papillae of the corium or derma. (Note the absence of any sharp line dividing the corium and subcutaneous tissues.) 5. The larger blood-vessels of the subcutaneous region. (The arteries in transverse sections are plainly indicated by their prominent media, the appearance of the fenestrated membrane as a wavy yellowish line, and by the elliptical or circular outline. The veins are smaller, with thinner walls, and their outline is gen- erally irregular. The smaller veins are commonly overlooked, on account of their lumen having become obliterated by contraction of the tissue in hardening. 6. Coils and ducts of sweat-glands in subcutaneous region. (The tubes are cut in various directions, and the whole is sur- rounded by dense fibrous tissue, forming a kind of capsule.) 7. The subcutaneous collections of adipose tissue beneath the last region. (The septa are dense and strong.) 8. (Having selected a vertical section of a hair-follicle: ) (a) The root of the contained hair. (&) The bulb and the hair- papilla, (c) The medulla of the hair, (d) The root-sheath pro- longed from the rete mucosum. (e) The fibrous (outer) sheath. *Low-power — i. e., from thirty to sixty diameters. 100 STUDENTS HISTOLOGY 9. The sebaceous glands. (The demonstration of the connec- tion between the neck of the gland and the follicle will require a very favorable section.) 10. (Scattered through the corium and upper subcutaneous Fig. 72. VERTICAL SECTION OF SKIN FROM THE GROIN. STAINED WITH H^EMATOXYLIN AND EOSIN. A. Epidermis. B. Deep, elongated cells of the rete mucosum. C. C. Papillae of true skin. D. D. Subcutaneous areolar tissue. E. E. Collections of adipose tissue. F. Shaft of hair (obliquely sectioned). G. Root-sheath of hair. H. Fibrous sheath of hair. I. Hair-papilla (vertical section). J. J. J. Portions of sebaceous glands (one on the extreme right of the cut is seen in connection with the hair- follicle.) K. K. Arrectores pili. L. Hair- follicle with contained shaft of hair in very oblique section. M. M. Coils of sudoriferous glands. N. Spiral duct of last. O. O. Arteries of subcutaneous plane. region:) (a) Small portions of sebaceous glands. (&) Ducts of sudoriferous glands, (c) Oblique sections at various angles of hair-follicles, (d) Small vessels. THE SKIN 101 11. Arrector pili mtiscle. (Nearly always to be found stand- ing obliquely to the divided hair -follicle. (H.)* 12. (If demonstrable:) (a) The stratum lucidum. (b) Stra- tum granulosum. 13. The columnar cells of the rete, next the corium. 14. (Where the tissue has been torn:) The impacted cells of the horny epidermis. 15. The basement membrane covering the corium. 16. Capillaries of the papillae of the corium. (These may be distinguished, when seen longitudinally, by tortuous lines of elongated and deeply stained nuclei belonging to their endothelium. Arterioles may be differentiated by their long muscle cells, the cir- cular fibers lying transversely to the vessel.) 17. The root-sheath of the hair-follicles. (The cells compos- ing the root -sheath vary in appearance, according to their position relatively to the hair; and this will enable you to demonstrate two layers, or an inner and an outer root -sheath.) 18. The glassy membrane of the hair-follicle. (Appearing simply as a clear space between the root -sheaths and the outer fibrous coat.) 19 . The intra-cellular network in the large polyhedral epithelial cells of the sebaceous glands, and the minute fat- globules in the same. 20. The nuclei of the fat-cells in the adipose tissue. (They appear pressed to one side.) 21. Medullated nerve-bundles in transverse or oblique section. *High-power — i. e., from three hundred to four hundred diameters. 102 . STUDENTS HISTOLOGY THE CIRCULATORY SYSTEM THE HEART The muscle of the heart has already been described. The muscle-cells are supported by a small amount of connective tissue, in which run the blood- and lymphatic vessels and nerve -fibers. Both medullated and non-medullated nerve-fibers are supplied to the heart, and minute ganglia also occur, especially in the auriculo- ventricular and the inter -ventricular furrows. The PERICARDIUM is one of the great serous membranes. Its surface is covered by a single layer of flat endothelial cells, beneath which is a stratum of fibro- elastic connective tissue. This con- nective tissue is continuous with that running between the muscle- fibers. Underneath the pericardium there is usually more or less adipose tissue, especially along the course of the larger blood* vessels. The ENDOCARDIUM is covered with a single layer of flat endo- thelial cells, which rest upon fibro -elastic connective tissue. The connective tissue is continuous with that supporting the muscle- fibers, and also joins with the lining of the blood-vessels that open into the heart. The valves of the heart are duplications of the pericardium, containing abundant connective tissue. The muscle -fibers of the auricle extend a short distance into the auriculo- ventricular valves. A few blood-vessels may be present at the attached borders of the valves. BLOOD- VESSELS Blood-vessels include arteries, arterioles, capillaries, venules, and veins. They are all lined with flattened endothelial cells cemented by their edges; and their walls are constructed from non- striated muscular, yellow elastic, and fibrous connective tissues, in propor- tions varying according to the size and function of the vessel. Arteries are active, while the veins are comparatively passive agents in the circulation of the blood. The large arteries are eminently elastic, from preponderance of yellow elastic tissue; while the arterioles are eminently contractile, from excess of muscular fiber. BLOOD- VESSELS 103 Arteries possess three coats: the intima (internal), media (mid- dle), and adventitia (external). Fig. 73 represents a medium - sized typical artery. The intima, or internal coat, A, B, C, consists of a layer of flattened endo- thelial cells, which rest upon fibrous connective tissue, with a few- elastic fibers. These structures are surrounded by a layer of elastic tissue, the elastic lamina or fenestrated membrane, which is the external limit of the intima. It appears in a transverse section as a wavy (from contraction of the media) shining line, and is an important element, from its relation to certain abnormalities of the blood-vessels. The media, D, consists of alternate layers of FIG. 73. PARTLY TRANSVERSE SECTION OF A MEDIUM-SIZED ARTERY. DIAGRAMMATIC. A. The endothelial cells in profile. B. Elastic and connective tissue supporting the endothelinm. C. The internal elastic lamina or fenestrated membrane. A, B, and C constitute the INTIMA of the artery. D. The MEDIA. It consists of muscular and elastic tissues in alternating layers. E. Points to one of the elastic layers. F. The ADVENTITIA. Loose connective tissue, with few elastic fibers. elastic and muscular tissue. The adventitia, F, is composed of fibrous connective tissue, containing some elastic elements. As we approach the larger arteries, the muscular tissue very distinct differentiation. At this point examine every part of the specimen closely, and en- deavor to detect even the most minute collection of this tissue.) (a) Around arteries, constituting the so-called Malpighian bodies. 120 STUDENTS HISTOLOGY (b) Transverse sections of Malpighian bodies, noting that the vessel is seldom in the center of the nodule. (0). A. A. Capsule which divides the organ into lobes. Portions of six lobes are visible in the section. B. B. Lymph-sp.aces. 0. C. Trabeculze dividing the lobes into imperfect lobules. D. D. Subdivisions of the last into follicles. E. E. Central light portion of the lobules. mononucleated cells. (c) Still larger multinucleated cells. (d) Larger — though varying in size — spherical bodies, Hassall's corpuscles. (These are composed of epithelial cells, arranged concentrically, and are unlike any other structure found in the normal tissues of the body. They resemble the smaller "cell- iiests " of epithelioma. The corpuscles of Hassall are the remains of the epithelial structure, which makes up the bulk of the thy- mus body in its early stages.) (e) Small thin- walled venules. THE RESPIRATORY ORGANS 123 THE RESPIRATORY ORGANS The larynx and trachea have the same general structure as the larger bronchial tubes. The epithelium is stratified columnar and ciliated, except that covering the surfaces of the epiglottis and that of the upper part of the larynx, which is stratified squamous. The cartilages are hyaline, except those of the epiglottis, part of the arytenoids, and the cartilages of Wrisberg and Santorini, which are yellow elastic cartilage. THE LUNGS At the root of each lung the large primary bronchus enters, and divides into two branches, which also divide and branch repeatedly until terminal or capillary bronchial tubes are formed, which are one -fourth to one -eighth of a millimeter in diameter. A typical bronchial tube (Fig. 84) presents four coats, as follows : 1. Epithelial. 2. Internal fibrous or mucosa. 3. Muscular or muscularis mucosce. 4. External fibrous or submiicosa. The lining epithelium is composed of cylindrical cells, provided on their free extremities with delicate hair -like appendages — the cilia. Between the pointed, attached ends of the ciliated cells, small, ovoid cells are wedged, and the whole rests upon a layer of round cells. The epithelium pursues a wavy course, so that the lumen of a tube appears stellate rather than circular in' transverse section. This greatly increases the extent of surface. The internal fibrous coat or mucosa is composed of a small amount of connective tissue, which, just beneath or outside the epithelium, sustains collections of lymphoid (or adenoid) tissue. In the pig, a considerable quantity of yellow elastic tissue is found in the mucosa outside the lymphoid tissue, but the amount is smaller in man. The fibers are, for the most part, disposed longi- tudinally. Many nutrient vessels from the bronchial artery, cap- illaries, venules, and lymph -spaces are also found in this coat. The muscular coat — muscularis mucosae — does not differ from the same layer in other mucous membranes. Its thickness varies 124 STUDENTS HISTOLOGY * in proportion to the size of the bronchus, the smaller tubes possess- ing relatively the thicker walls. The fibers pass circularly, and are of the non- striated or involuntary variety. The external coat, or submucosa, is largely composed of loose connective tissue, the fibers being mostly arranged circularly. A few delicate elastic fibers run longitudinally. The external fibers, like those of all tubes, ducts, and vessels, are for the purpose of establishing connection with the organ or part traversed ; so that it is often difficult to demonstrate the exact external limit of a bronchus. This coat is liberally supplied with nutrient branches from the bronchial artery. The elasticity and strength of the larger and medium -sized FIG. 84. TRANSVERSE SECTION OF A PORTION OF HUMAN LUNG, SHOWING A SMA.L.L BRONCHIAL TUBE (X 60). STAINED WITH H^EMATOXYLIN. A. Lumen of bronchus. B. Ciliated columnar epithelium. C. Internal fibrous layer— mucosa. D. Muscular coat. E. External fibrous layer— submucosa. F. Pulmonary artery. G. Nerve. H, H, H. Pulmonary alveoli surrounding bronchus. bronchial tubes are greatly increased by the presence of cartilage in the form of plates, which are imbedded in the external coat. They are not uniform in size, neither are they placed regularly. They frequently overlap one another, and two or three may be superposed. As the tubes become reduced in size the plates be- come diminished in frequency — disappearing altogether when a THE LUNGS 125 diameter of about one millimeter has been reached. The cartilage is of the hyaline variety; and each plate is covered with a dense fibrous coat, the perichondrium , which unites it with contiguous parts. The principal bronchi are provided with a great number of mucous glands, which are located in the external coat or submu- cosa. They are simple, coiled tubular glands, commencing on the inner surface, penetrating the mucosa and muscularis mucosae, and terminating in the submucosa, generally within the cartilage, where they are coiled in short, close turns in sections resembling somewhat the larger sweat-glands of the skin. The ciliated epi- thelium of the bronchial tube is continued down the beginning of the tube for a short distance, after which the cells are shortened, and lose their cilia. The coiled gland-part of the tube is lined with conical cells, which are so large as to leave the lumen very small. Sometimes, and especially in the aged, an ampulliform dilatation' of the tube may be seen during its passage through the mucosa. The description just given will apply to large and medium- sized bronchial tubes. Very important changes take place as we pass to the terminal tubes. As the tubes decrease in size, the first coat to diminish in thickness is the outer, or submucosa. We have already alluded to the disappearance of the cartilage, and the mucous glands are lost at about the same time. The outer coat becomes, in the small bronchial tubes, so thin as to be no longer distinctly demon- strable. The muscular coat is the last to disappear. It remains a prominent feature of the tube as long as separate coats can be distinguished. The epithelial cells lining the tubes toward the termini become shortened, and, getting lower and lower, at last result in cuboidal cells, without cilia. The walls of terminal bronchial tubes (diameter one -fourth to one -eighth of a millimeter) are composed of a slight amount of connective tissue in which an occasional non- striated muscle -cell and yellow elastic fiber can be distinguished. They are lined with cuboidal or a few flat cells. No definite layers are distin- guishable in these bronchial tubes. In a transverse section the lumen would appear circular. PRACTICAL DEMONSTRATION The histology of the bronchi can be studied to best advantage by using tissue from a freshly killed pig, cat, or dog. Short pieces of tubes, about one •centimeter in diameter, from which most of the lung- substance has been cut 126 STUDENTS HISTOLOGY' away, should be hardened quickly in strong alcohol. Transverse sections can be made free-hand, or the tissue may be infiltrated with paraffin or celloidin, and cut with the microtome. Stain with hsematoxylin and eosin, and mount in balsam. TRANSVERSE SECTION OF PORTION OF BRONCHUS OF PIG (Fig. 85) OBSERVE : (L.) 1. The epithelial lining: (a) The wavy course, (b) Regions occupied by beaker or goblet cells. (The letter E in the drawing leads to such a group, (c) The number of nuclei, indicating the presence of more than a single layer of cells. 2. The mucosa. (a) Deeply stained blue nuclei of the lym- phoid (or adenoid) tissue just beneath the epithelium. (&) Pink portion of the region below the lymphoid tissue. (The longitudi- nal elastic fibers cut transversely.) (c) Blood-vessels. 3. The muscular coat, (a) Apparent solution of continuity in places caused by tubes of mucous glands. (&) The absence of large vessels in this coat. 4. The external layer, (a) Its extent. (It includes the remainder of the section.) (6) Large cartilage plates, C, stained blue, (c) Cartilage-cells. (Note their differing forms and dispo- sition in rows next the surfaces of the plates.) (d) Perichondrium stained pink. (e) Mucous gland -coils. (They are usually between the cartilage and the muscular coat.) (/) Section of bronchial arteries and veins, (g) Collections of adipose tissue on the outer surface, (h) Portion or whole of pulmonary artery and medullated nerve-trunks outside of and accompanying the bronchus. (H.) 5. Epithelial lining, (a) Cilia of columnar cells. (6) The ovoid cells between the tapering columnar cells, (c) The "base- ment membrane," upon which the columnar cells rest, (d) The goblet or beaker cells. 6. The mucosa. (a) The reticulum of the lymphoid tissue. (It will appear only where the lymph -corpuscles have been acci- dentally brushed out.) (&) The transversely divided ends of the elastic fibers. (They appear as a pink mosaic.) (c) Capillaries. (They may frequently be traced for a considerable distance in their tortuous course.) BRONCHIAL TUBES 127 7. The cartilage plates. (a) Several cells in a single cavity. (&) The intracellular network. 8. The mucous glands, (a) That some of the cells are FIG. 85. TRANSVERSE SECTION OF PART OP THE WALL OP A LARGE BRONCHUS. LUNG OF PIG. STAINED WITH H^EMATOXYLIN AND EOSIN (X60). E. Epithelial lining. The line from the letter leads to a part of the lining containing large mucous cells or goblet cells. I. The internal fibrous coat. M. Muscular coat. C. Cartilage plates of external fibrous coat. A. Bronchial artery. The pulmonary artery is not included. V. Bronchial vein. N. Nerve-trunk. < G. Mucous glands. D. Obliquely sectioned duct. stained precisely like the other mucous or goblet cells, along the surface of the membrane. (5) If possible, a gland-tube leading up to tlie lumen of the bronchus. (An ampulliform dilatation is shown in the upper part of the drawing.) 128 STUDENTS HISTOLOGY THE PULMONARY BLOOD-VESSELS The prominent accompaniments of the bronchus, at the root of the lung, are the pulmonary artery (carrying venous blood) and the pulmonary veins. The pulmonary artery enters the lung with the bronchus, fol- lowing its ramifications, to end in capillary plexuses in the walls of the sac -like dilatations, which are in connection with the ultimate bronchial tubes. The blood is then collected in venules, which unite to form the pulmonary veins. The latter pursue an inde- pendent course in their exit, not accompanying the bronchial tubes until the large bronchial tubes have been reached. The bronchial artery (nutrient) enters with the bronchus, sup- plying its walls and the connective tissue framework of the lung. A considerable amount of connective tissue accompanies and supports the structures which enter the lung, and is eventually in connection with the fibrous framework of the organ. The lung will, therefore, be seen to differ from organs gener- ally, in that it contains two distinct vascular supplies, viz. : (1) The pulmonary (of venous blood), entering for the purpose of its own oxygenatiou; (2) The bronchial (arterial), which corresponds to the usual nutrient blood -supply of organs. THE PLEUEA The lung is completely enveloped in a membrane composed externally of endothelium, while the visceral portion is made up of interlacing fibrous and elastic tissue. The deep or visceral layer of the pleura sends prolongations in the form of septa into the substance of the lung, dividing it into rounded polyhedral compart- ments or lobules. The interlobular septa have usually become prominent in the human adult from deposits of inhaled carbon in their lymph -channels. THE PULMONARY ALVEOLI The lung is constantly employed in maintaining the integrity of the blood. This is accomplished by the exposure of the latter to a continual supply of atmospheric air. The air is introduced into little sacs (termed air-vesicles or alveoli), in the walls of which the blood is distributed in a capillary plexus. The air does not reach THE PULMONARY ALVEOLI 129 the capillaries themselves, inasmuch as they are covered with a layer of flat cells. These cells, constituting the parenchyma of the lung, have the power, on the one hand, of selecting such material from the air as may be required, passing it on to the blood in the capillaries; and, on the other, of removing effete material from the FIG. 86. DIAGRAM OP AN ULTIMATE PULMONARY LOBULE. A. A terminal bronchiole. B. The air-sacs or alveoli. blood, transferring it to the atmospheric contents of the air -sacs for exhalation. The air -sacs or alveoli are not unlike minute bladders. Their diameter about equals that of a terminal bronchus; viz., from one- fourth to one -eighth, of a millimeter. A group of these alveoli are associated in the manner shown in Fig. 86, their contiguous walls FIG. 87. DIAGRAM SHOWING AN ULTIMATE PULMONARY LOBULE IN LONGITUDINAL SECTION, SHOWING THE MANNER IN WHICH THE ALVEOLI ARE ASSOCIATED IN CONNECTION WITH A TERMINAL BRONCHIOLE. A. Terminal bronchiole entering. B. The infundibuhim. C. C, C. Alveoli. fusing and all opening into a common cavity, the infundibulum . The whole is in connection with a terminal bronchiole (vide Fig. 87). A primary lobule having been thus constructed, several are associated and united to a slightly larger bronchial twig, and there results one of the polyhedral lobules, previously mentioned as i 130 STUDENTS HISTOLOGY visible, especially on the surface of the lung. By a repetition of such elements the lung is constructed. The wall of a pulmonary alveolus or air -sac is composed of connective tissue, supporting the capillary network, with a con- siderable amount of elastic tissue. The whole, as we have said, is lined with a single layer of flat, pavement epithelium. The capil- lary plexus, when filled with blood, affords the most prominent feature of the wall ; but when the vessels have been emptied of their contents, tHey become very insignificant under the micro- FIG. 88. TRANSVERSE SECTION OF A SINGLE PULMONARY ALVEOLUS. CAPILLARIES INJECTED. STAINED WITH H^MATOLYLIN AND EOSIN (X400). A, A, A. Walls of the alveolus. B, B. Injected capillaries. C, C. Pavement cells lining the alveolus. These cells cover the capillaries, but do not so appear in the drawing, as the latter are filled with an opaque injection. The observer is sup- posed to he above the sectioned alveolus, viewing the cup-shaped cavity. scope, and the fibro- elastic tissue becomes more apparent. You will have observed that, aside from the vascular supply, the his- tology of an alveolar wall resembles very closely that of a terminal bronchiole, and when the vessels are all empty it is frequently diffi- cult to differentiate them in the mounted section. THE LUNGS 131 Fig. 88 shows a single alveolus, the vessels of which have been injected with a solution of colored gelatin. The alveolus has been divided through the middle, and shows as a cup -shaped cavity. The fibrous marginal walls are indicated, with their tortuoua_ capillaries. The epithelial cells lining the bottom are obscured by the opaque capillaries, and shown only between the loops. It is probable that these cells cover the plexus completely, as they line the alveoli. We now encounter an obstacle which will frequently be met in our study of organs. It consists of the difficulty in recognizing in sections the plan of structure which we have learned is peculiar to the organ under consideration. For example: A lung has been compared to a tree. The bronchi are the representatives of the branches, and the air -sacs of the fruit. Well, we make a section from human lung — it matters little as to the direction — with every possible care, and the image in the field of the microscope resem- bles a fragment of ragged lace more nearly than anything else! The arrangement of the tubes and alveoli of the lung has been determined by filling the cavities with melted wax, which, when cold, and the tissue destroyed by acid, gives a perfect mould of the organ. A section gives us but a single plane, and this fact must be always borne in mind. PRACTICAL DEMONSTRATION With a very sharp razor, cut centimeter cubes from pig's lung. Select portions free from large bronchi, with the pleura on one side at least, and harden with strong alcohol. Human lung, as fresh as possible, may be treated in the same manner. The epithelium of the alveoli shows best in young lung. Lung must be made very hard, or thin sections cannot be cut. If the ordinary ninety-five per cent, alcohol does not harden sufficiently, the process may be completed by transferring the tissue for twenty-four hours to absolute alcohol. The celloidin process is well adapted to this structure. Stain the sections with borax -carmine, or hsematoxylin and eosin. Mount in balsam. SECTION OF LUNG OF PIG (Vide Fig. 89) OBSERVE : (L.) 1. The large scalloped openings, A, A, transversely divided infundibula. 2. The divided alveoli, B, B, so sectioned as to cut off both 132 STUDENTS HISTOLOGY bottom and top, and show no epithelial lining excepting at inner edge of periphery. 3. The alveoli, C, C, divided so as to show a cup- shaped bot- tom or top. (The minute granules are the nuclei of the lining cells.) 4. The alveoli, D, D, so cut as to leave most of bottom or top, showing an opening in the center where the sac has been sliced off. FIG. 89. SECTION OF LUNG OF PIG. STAINED WITH H^EMATOXYLIN AND EOSIN (X60). A, A. Infundibula in T. S. B, B, B. Alveoli; so sectioned as to show the outline only. C, C, C. Alveoli; so sectioned as to present cup-shaped cavities. D, D, D. Alveoli; so sectioned as to divide the top (or bottom). E, E. Terminal bronchioles in T. S. 5. Openings, E, E, which are about the same size and bear a general resemblance to those of Obs. 2. (Note that their internal edges are smooth and not ragged. They are terminal bronchi- oles. No larger bronchial tubes have been included in the section.) \ HUMAN LUNG 133 HUMAN LUNG — SECTION SHOWING A SINGLE ALVEOLUS (Fig. 90) OBSERVE : (L.) 1. The outline of alveolus. (The alveoli in human lung will show much distortion, as the tissue cannot be secured in perfect condition.) FIG. 90. TRANSVERSE SECTION OF A SINGLE PULMONARY ALVEOLUS. STAINED WITH H^EMATOXYLIN (X 400). A. A, A. Walls of alveolus. B. Lumen. C. C, C. Capillaries variously sectioned in their tortuous course. D. Pavement epithelial cells intact. E. Detached pavement cell. F. Detached cluster of pavement cells. F'. Granular lining cells. G. Pulmonary artery. (H.) 2. The fibrous wall, A, A. 3. The lumen, B. (The bottom or top has been cut off in making the section.) 4. The tortuous capillaries, C, C, in the fibrous wall. 134 STUDENTS HISTOLOGY 5 The lining epithelial cells, (a) Those remaining attached to the edges of the wall, D. (&) Detached cells. E. (c) Groups partly detached, F, F. 6. The divided pulmonary artery, G. (A medium - sized bron- chial tube existed in the section immediately to the left of the artery.) FCETAL LUNG Harden the lung of a foetus, preferably human, in Miiller's or Orth's fluid. After washing, finish hardening in alcohol ; imbed in celloidin; cut; stain with haematoxylin and eosin; mount in balsam. Observe the polyhedral form of the epithelial cells lining the alveoli. A few such polyhedral cells can be demonstrated in the alveoli of the adult lung with silver staining, and they correspond to the polyhedral cells of the terminal bronchioles. It appears that the plate -like cells lining the alveoli of the adult lung are derived from the polyhedral cells of the foetal lung, which become flattened from the distension which they undergo when the alveoli are inflated. THE TEETH 135 THE TEETH A human tooth is a calcareous structure of extreme hardness, and is divided into an exposed crown, a constricted neck, and one or more concealed roots — the latter being inserted into an alveolus, by means of which the whole is very firmly connected with the maxillary bone. The central portion presents an elongated cavity (pulp -cham- ber) containing vascular, nervous, and connective tissue elements — the pulp. The pulp -cavity is surrounded by the dentine, which consti- tutes the major portion of the tooth The crown portion of the dentine is provided with a covering of enamel, while the root is invested with an osseous cementum, or crusta petrosa. A thin membrane, 1 /* or less in thickness — called the membrane of Nasmyth or the cuticula — covers the enamel in early life, while the cementum receives a periosteal investiture. The vascular and nervous elements of the pulp obtain admission to the pulp -cavity by a perforation or foramen at the apex of the root. The Pulp. — The ground -substance, or stroma of the pulp, is a form of primitive connective tissue, gelatinous rather than mark- edly fibrous. It contains elongated capillary loops, multipolar cells, and medullated and non-medullated terminal nerve -fibrils. Surrounding the pulp mass, and next to the dentinal wall of the chamber, we find a single layer of elongated cells— odontoblasts. These are probably in communication, by means of processes or prolongations, with fibrous elements of the pulp. Dentine. — The dentinal stroma or matrix is made of fibrous tissue containing calcium salts, and is, next to the enamel, the hardest tissue of the body. The matrix is pierced with the denti- nal canals (extremely minute channels, only 5/* in diameter), which radiate from their beginning, next the pulp -chamber, toward the outer portion of the dentine. These canals branch and anastomose, and are lined with an exceedingly thin den- tinal sheath. From the outer extremity of the odontoblasts of the pulp nu- merous prolongations are sent which are probably continued within the dentinal canals as the dentinal fibers. The dentinal canals terminate exteriorly, by very fine lumina, in a system of irregu- 136 STUDENTS HISTOLOGY FIG. 91. DIAGRAM OF THE STRUCTURE AND IMPLANTATION OF A NORMAL- INCISOR TOOTH. (BODECKER.) L. Cuticle of enamel, Nasmyth's membrane. E. Enamel. D. Dentine with uniformly distributed canaliculi. I. Interzonal layer between enamel and dentine. B. Border-line between enamel and cementum of neck. S. Cementum of neck. Ce. Cementum of root. Z. Interzonal layer between dentine and cementum. P. Pericementum. A. Arteriole of pulp, branching into capillaries. V. Vein of pulp taking up capillaries. N. Medullated nerve-fibers of pulp. Eg. Stratified epithelium of gum. Pg. Papillary layer of gum. Pe. Periosteum. Co. Cortical bone of alveolus or socket. Ca. Cancellous bone-tissue of alveolus. M. Medullary spaces of cancellous bone. THE TEETH 137 larly formed openings, interglobular spaces, which are channeled in the outer part of the dentine. The dentinal terminal fibers are in connection with branched cells which occupy the interglobular spaces. The Enamel. — The part of the dentine above the neck of the tooth is protected by a covering of enamel. The enamel consists of prisms, 4 p in diameter, united into bundles by a little cement substance, which pass in a direction nearly at a right angle to the surface of the dentine. They are of extreme density, contain little besides inorganic material, and in a vertical section the whole is traversed by parallel striae, not unlike the markings indicating tree -growth — the lines of Retzius. Cementum.— The fang portion of the dentine is invested with a thin layer of true bone, containing lacunce and canaliculi, but no Haversian canals. The cementum is provided with pericementum (periosteum), which forms the bond of union between the teeth and the process of the maxillary bone. The bone- corpuscles are in connection, through the canaliculi, with the cells in the interglo- bular spaces of the dentine. It will be seen that the connective tissue elements, at least of the pulp, are in eventual histological connection with the bone -corpuscles of the cementum. PRACTICAL DEMONSTRATION The illustrations given in text-books have been drawn from dried teeth, ground down to the requisite thinness by means of corundum or emery wheels. This is a very tedious process, and is impracticable with the student. If such specimens are desired, it will be advisable to purchase them already mounted. They only give the skeleton of the organ, all the soft tissues being destroyed by the drying and grinding. , While dry specimens exhibit the plan of a tooth, the soft tissues must be studied in sections made after the inorganic constituents have been removed. Teeth immediately after extraction are to be treated in the same manner as described for bone. A one-sixth per cent, chromic-acid solution, to which five drops of nitric or hydrochloric acid have been added, may be used. Let the quantity of liquid be liberal, and from time to time, say every three days, add a few drops of the nitric acid. The decalcification should proceed slowly, and may be complete in from two to three or four weeks. The earthy matters will first be dissolved from the surface. Watch the action carefully, ascer- taining the progress of decalcification by pricking a fang with a needle. If the acid be too strong, and the action too rapid, the whole may be destroyed. When the decalcification is complete, a needle may be easily passed through the tooth, and sections may be made with the razor or knife, with or without a microtome. The form will be preserved except as regards the enamel; this 138 STUDENTS HISTOLOGY will be entirely dissolved. The enamel prisms may be demonstrated by treating broken fragments with dilute acid for a short time only. Sections should be stained with carmine and picric acid and mounted in glycerin. For the study of the development of teeth, foetal jaws may be treated as just described; and, when properly decalcified and hardened, should be infiltrated with celloidin, sectioned, and stained. TRANSVERSE SECTION OP FANG OF HUMAN DECIDUOUS CANINE TOOTH— DECALCIFIED (Fig. 92) OBSERVE : (L.) 1. Division into pulp, dentine, cementum, and perice- mentum. 2. Line of junction of pulp and dentine. (If the elements of the pulp are intact, note the layer of deeply stained odontoblasts next the dentine.) 3. External limit of dentine. (Note here the deeply stained granular line of Purkinje. This is the location of the interglobu- lar spaces. The deep color is due to the staining of the contents of their cells.) 4. The striae of the dentine. (The dentinal canals and stained contents.) 5. The laminated cementum. (The yellowish pink dots on the lacunas.) (H.) 6. Elements of the pulp, (a) The layer of odontoblasts. (Note their internal processes connecting with other cells of the pulp; and the external processes passing into the dentinal canals.) (6) The sparsely fibrillated character of the pulp- tissue, (c) Sections of vascular loops. (The nerve-elements may be demonstrated, particularly if the section be made near the apex of the root, where the fibers are medullated. The terminal fibrillae are non- medullated.) 7. Dentinal elements. («) The dentinal canals. (&) The dentinal sheath. (Better demonstrated in transverse sections.) (c) Dentinal fibers. (In transverse sections the canals are well shown lined with a membrane of extraordinary tenuity, with the fiber appearing as a central dot.) (d) Fine dentinal fibers near the outer limit, (e) Interglobular spaces. (An occasional cell may be made out in the larger spaces. They were formerly sup- THE TEETH 139 posed to contain a gelatinous material only. Note the connection between these spaces and the termini of the dentinal fibers.) 8. The cementum. (a) The lacunae. (&) Bone-corpuscles in the last. (The canaliculi are not well demonstrated here, as the tissue is very translucent and feebly stained. These minute canals are better indicated in dried bone.) 9. The pericementum. (Note its dense fibrillar meshwork.) FIG. 92. TRANSVERSE SECTION OF FANG OF A HUMAN DECIDUOUS CANINE TOOTH, DECALCIFIED WITH CHROMIC AND NITRIC ACIDS AND STAINED WITH PICRO- CARMINE (X 400). A, B. Line through the dentine indicating the point at which the edges have been made to join after the omission of an intervening portion. This was necessary in order that the dif- ferent layers might be shown in a single drawing. C, D. Junction line between the pulp and dentine. E, F. Junction line between dentine and cementum. G, G. Odontoblasts of the pulp. H, H. Stellate connective tissue cells of the pulp. I, I. Dentinal processes of odontoblasts. J, J. Dentinal fibers. K, K. Terminal branching dentinal fibers. L, L. Interglobular spaces of dentine. M, M. Lacunae of the cementum. The drawing does not show the periosteal investiture of the crusta. 140 STUDENTS HISTOLOGY GLANDS A gland is an organ — frequently subsidiary to and located within other organs — whose cells manufacture from the blood pro- ducts to be utilized in the performance of some of the functions of the body, or waste products which are to be excreted. Simple glands are tubes or cavities, with connective tissue walls lined with epithelial cells, which are usually placed upon a base- ment membrane. Around, and in close proximity to the lining, is spread a plexus of blood -capillaries. In compound glands, the simple glands (acini or alveoli) are enclosed by connective tissue in groups called lobules, and larger groups called lobes. The same connective tissue is continued to form a capsule over the outside. The essential parts of a gland are, therefore: 1. A duct, or efferent conduit for the secretion. 2. Parenchyma, or cells engaged in secretion. 3. A blood -vascular supply. TUBULAR GLANDS The simplest gland-structure is offered in the form of a tube. Glands are, frequently, little more than tubular depressions in mucous surfaces. Examples are found in the uterus, and small and large intestines. COILED TUBULAR GLANDS Tubular glands are often greatly elongated, with the blind extremity coiled. This variation presents the simplest differentia- tion between the part of the tube which is secretory, and the duct, or drainage part. With this change in function of the different extremities of the tube will occur a change of epithelium. The cells belonging to the duct-end will usually retain the columnar form; while the actively secreting elements will become enlarged, more nearly filling the tube, and assume a polyhedral form from pressure. Examples have already been seen in the sweat-glands of the skin. SIMPLE AND COILED TUBULAR GLANDS 141 FIG. 93. DIAGRAM. SIMPLE TUBULAR GLAND, A. Lining cells— parenchyma. B. Capillary plexus, supplying the parenchyma, 0. Connective tissue supporting capillaries. D. Arterial supply. FIG. 94. DIAGRAM. COILED TUBULAR GLAND. Same references as Fig. 93. 142 STUDENTS HISTOLOGY FIG. 95. DIAGRAM. BRANCHED TUBULAR GLAND. References same as Fig. 93. FIG. 96. DIAGRAM. ILLUSTRATING THE PLAN OF ACINOUS GLANDS, References same as Fig. 93. GLANDS 143 BRANCHED TUBULAR GLANDS With the branching of the duct-portions of gland-tubules, usually occurs a dilatation of the extremities into acini or alveoli, although pure examples of branched tubular glands are afforded in the gastric and uterine glands. The most nearly typical branching of gland -like tubules is afforded by the tubuli uriniferi of the kidney, or the system of tubes in the testicle. The tubules here present other features peculiar to them, which will be referred to under the proper head. ACINOUS GLANDS The dilatation of branching tubules, referred to under the previous heading, results in the formation of acinous glands. They are formed by the subdivision of a main tube or duct, with repeated branching of the secondary tubules. Collections of ter- minal branches often result in globular masses, which are more or less perfectly isolated from one another by connective tissue. In this way compound acini are produced, such as the pancreas, the salivary, mammary, and buccal glands. The large compound acinous glands are also called compound racemose glands. Simple acinous glands do not occur in human tissues. THE PAROTID GLAND The parotid, submaxillary , sublingtial,3ind. bitccal salivary glands are typical glandular structures, with individual peculiarities only in respect to the cell-elements ; these vary according to the nature of the secretion formed in each. The parotid is a compound acinous gland, leading from which is a principal duct — lined with tall columnar cells — which collects the fluid saliva from the different divisions of the organ. As the duct penetrates the gland it branches freely, the lumina becoming smaller and the cells shorter as thQ deeper parts are approached. Each terminal duct is in connection with several acini. The connective tissue adventitia of the duct becomes the thin wall of the acinus, and the lining cells broaden, frequently become poly- 144 STUDENTS HISTOLOGY FIG. 97. SECTION OF A SMALL PORTION OP THE PAROTID GLAND. STAINED WITH H^EMATOXYLIN AND EOSIN (X 250). A. Narrowing of the duct from a small lobule, before entering a larger duct. B. Dilatation of a duct after leaving a small lobule. C. Primary lobules, in nearly L. S. D. Acini in T. S., showing the minute lumen. E. Connective tissue supporting the gland. F. Striated muscular fiber adjacent to the gland. G. Adipose tissue in the loose areolar tissue. hedral, and are bluntly pointed. The cells so nearly fill the acini as to leave a small and not easily recognized lumen. The gland is richly supplied with blood-vessels. THE SUBMAXILLARY GLAND The submaxillary is presented as an example of a typical mixed gland. The general arrangement is not unlike that of the other salivary glands. Its peculiarity appears in the parenchyma, and will be noticed later. Pure mucous glands are found in the submucosa of the mouth, tongue, fauces, trachea, and the larger bronchi. GLANDS 145 FIG. 98. SECTION OF PART OF THE SUBMAXILLARY GLAND (X 250). A. Narrow duet from terminal lobules. B. Small duct in T. S. C. Small duct in oblique section. D. Transversely divided acini, showing large lumen. E. Mucus remaining in the lumina. F. Striated muscular fibers. G. Adipose tissue. THE PANCREAS The histology of the pancreas is, in general, that of a true serous gland, like the parotid. It has been called by physiolo- gists the abdominal salivary gland. The lobules are more tubular and less regular in size and form; and the lumen of the acini is much less easy of demon- stration, in an ordinary hardened section, than the same in the parotid. The branches of the pancreatic duct are provided with a very thick adventitia, are lined with short columnar cells, and seldom present the dilatation which generally occurs in a serous gland on entering the lobule. 146 STUDENTS HISTOLOGY PAROTID AND SUBMAXILLARY GLANDS, AND THE PANCREAS Practical Demonstration The tissue must be fresh, divided in small pieces— not larger than a centi- meter cube — and hardened by placing in ninety-five per cent, alcohol for twelve hours, after which fresh spirit should be substituted. If, after the lapse of another twelve hours, the tissue should not be sufficiently firm, it should be placed in a small quantity of absolute alcohol for three hours. Sections should Fia. 99. SECTION FROM THE PANCREAS. A. Wall of a large duct. B. The somewhat cubical lining cells. C. Arteries. D. Lumen of the acini, T. S. E. Terminal duct leaving a lobule. F. Acini in L. S. be made immediately after hardening — as more prolonged action of the strong spirit will cause the tissue to contract. Sections may be cut with or without a simple microtome — the desideratum being thin rather than large cuts. Stain lightly with haematoxylin and deeply with eosin. After sections of hardened tissue have been examined, the glandular GLANDS 147 parenchyma may be profitably studied in teasings from tissue which has been in Miiller twenty-four hours. Wash the teasings on the slide with a liberal supply of water, removing the same from time to time with blotting-paper. Add a drop of haematoxylin solution ; and, after washing this away, add a drop of glycerin, and cover. This method is very generally useful for teased m* scraped fragments of glandular structures. ! (Figs. 97, 98 and 99) OBSERVE: (L.) 1. The connective tissue. (Most abundant in the parotid gland, and least so in the pancreas.) 2. The ducts. (Note the flattening of the lining columnar cells, as the ducts approach the acini, until mere scales result. Also the thick connective tissue adventitia, especially demonstrable in the pancreas.) 3. The lobules. (These are formed by several acini, and are* most typical in the parotid. It must be remembered that only one plane is visible, and that there is little perspective.) 4. The acini. (Note the lumina — large in the submaxillary, less so in the parotid, and least, and often difficult to make out, in the pancreas. 5. The blood-vessels, muscular and adipose tissue. (The two latter are demonstrable only in the salivary glands, and do not belong properly to the gland itself. The capsule of the pancreas, in common with such structures in general, contains adipose tissue. The abundant interacinous capillary plexuses of the pancreas require the high -power for satisfactory demonstration.) (H.) 6. The parenchyma. («) The small but distinct shortened columnar cells of the acini of the parotid. (Observe that they are frequently so formed that the convexity of one cell fits into the concavity of its neighbor. Where seen in transverse section, the outline is a polygon. Note especially the change in the parenchy- matous elements as the terminal duct merges into an acinus.) (6) The large, swollen cells of the mucous acini — submaxil- Im-y. (Observe the comparative clearness of the cells. They contain a very delicate reticulum, and their nuclei are often obscured and frequently seen to be placed at the junction of the cells.) (c) The rounded, often polyhedral cells of the pancreas. 148 STUDENTS HISTOLOGY (They resemble the parotid elements, although smaller and less granular. The acini are more tubular than in the parotid gland. Even with the low-power one may distinguish small, rounded areas called the bodies of Langerhans. They are probably groups of immature acini.) (e J-h SgRffl^S^ilS &S. • g: §•£"•< S 3 t? ^ S <"D ~3£ B.2 » S't; II ffsr fs- ^2 S- lilM!** l^s'^§-^« &||5' a 2. ^J°2l2.Hg, § s-.Hg'S.m^ "g |?5-3.SS I" sg1?!?! g LlSrl? i 8^8 g •: rrllS"! i 3 < ^a?5 « B a ® SL^S S- W ill}!! ! -*P a S^^ ^ ** 5' tr1 tt* ? 3 «• w °S^! from the organ and pour it into the ascending cava as it lies pos- teriorly in its fissure. The hepatic artery also penetrates the transverse fissure. It accompanie^ the portal vein in its ramifications, giving off nutrient twigs to the connective tissue framework and to the walls of the ves- sels. The terminal branches, very minute, pour any remaining THE LIVER 165 blood into the venous plexus at the margin of the lobules, thus providing arterial blood for the lobular parenchyma. The hepatic duct is also seen emerging from the transverse fis- sure. (For the sake of clearness, we will trace it from without in- ward.) It follows the course of the portal vein with the hepatic artery. Wherever in a section of the organ the portal is divided, the artery and duct will also appear. Bound together with con- nective tissue, the trio reach the walls of the lobules. The ducts now penetrate the lobule and break up into an exceedingly minute plexus — the bile -capillaries. This plexus properly begins in the lobules and drains the bile as formed, passing it into the ducts in a direction opposite to the portal blood -current. THE PORTAL CANALS If it were possible to grasp the vessels as they are found emerg- ing at the transverse fissure, the portal vein, hepatic artery, and hepatic duct, and to forcibly tear them, with their supporting con- nective tissue, out of the liver, a series of channels or canals would thereby be formed. A portal canal, then, is a space in the liver occupied by branches of the portal vein, the hepatic artery, and the hepatic duct, and the contiguous connective tissue. Frequently more than one specimen of each vessel is to be seen in a canal. There may be two or three veins, and as many arteries and ducts, asso- ciated in a single portal canal. Lymphatic chinks are also abun- dant in this connective tissue. From what has been said, it will be understood that a vessel found by itself in this organ must be either an intralobular or a hepatic vein; and these are easily distinguished, as the former are within, while the latter are without the lobules and in the connec- tive tissue framework. On the other hand, a group of vessels will indicate a portal canal, with its large and thin -walled vein, the small thick-walled artery, and, intermediate in size, the duct. THE LOBULAR PARENCHYMA The lobules consist of two capillary plexuses, one containing blood and the other bile. In the meshes of this network, the hepatic cells are located. The blood -capillaries, although extremely tortuous, have a gen- eral direction of convergence toward the central veins. This is 166 STUDENTS HISTOLOGY best seen when the lobules have been divided in a vertical di- rection. The bile -capillaries are among the smallest canals found in vas- cular tissues, having a diameter of only 1 to 2 /*. They pursue a direction in the human liver, as a rule, at right angles to the course of the blood -capillaries, and are not demonstrable, except with considerable amplification, say X 400, and then only in the thinnest portion of the sections. They are, properly speaking, merely minute channels in the parenchyma, and have, it is believed, no wall. The hepatic cells are polyhedral, about twice the size of a white blood-corpuscle, say from 20 to 25 ^ in diameter, usually with a single nucleus and with granular protoplasm, frequently contain- FIG. 108. DIAGRAM ILLUSTRATING THE INTRALOBULAR HISTOLOGY OF THE LIVER. The hepatic cells are connected in columns between the blood-capillaries. The cells are endowed with the power of selecting especially such materials from the blood as are necessary for 'the manufacture of bile. Having accomplished this, the secreted fluid is given up to the bile-capillaries, and by them poured into the ducts, and led out of the liver for subsequent use. The direction of the pressure is indicated by the arrows. This is the histology of gland-struc- tures generally. ing minute fat-droplets and granules of yellow pigment. The ex- istence of a definite limiting membrane has been questioned, as far as the cell of the human liver is concerned, although such a structure can be shown in many of the lower animals. The physiological plan of the intralobular structure is expressed in the diagram, Fig. 108. The blood is brought into relation with the lobular parenchyma — the hepatic cells — by the capillary plexus, THE PARENCHYMA OF THE LIFER 167 and the elements necessary to constitute the bile are selected and carried on, to be drained away by the bile -capillaries and -ducts. PRACTICAL DEMONSTRATION It is best to begin with the liver from a pig. The amount of connective tissue in the normal human liver is very small, and is mainly confined to the support of the interlobular vessels; the boundaries of the lobules are, there- fore, poorly defined, and without the previous observation of some well outlined specimen the student frequently gets but an imperfect notion of the plan of the human organ. Pieces of liver, say a centimeter square by half a centimeter thick, are hardened by twenty-four hours' immersion in strong alcohol. Larger pieces may be prepared with Miiller's fluid. Sections should be cut with a microtome, care being taken to include some of the medium-sized portal canals. The portal vein, with its accompanying vessels, may be easily distinguished from the solitary and Jess frequent branches of the hepatic veins. The elements of these canals, and especially the larger ones, are best kept intact by infiltration of the tissue with celloidin; but very fine sections may, with care, be made from the alcohol -hardened tissue. Even free-hand cuts, after some degree of skill has been obtained by practice, will answer very satisfactorily. Stain with hsematoxylin and eosin. SECTION OF LIVER OF PIG. CUT VERTICALLY TO AND INCLUDING THE CAPSULE OF GLISSON OBSERVE: (Fig. 109) (L.) 1. The capsule of Glisson, C. (Note the prolongations sent into the organ, which divide the entire structure into irregularly polygonal areas — if divided transversely — and elongated, verti- cally-sectioned areas — the hepatic lobules.) 2. The central (intralobular) veins, C. V. (Note that the figure formed by the division of the vein varies according to the direction of the cut, a circle, oval, or elongated slit, as the lobules have been sectioned transversely, obliquely, or vertically.) 3. The hepatic veins, H. V. (Those shown in the section are undoubtedly sublobular. It must be remembered that sub applied to these vessels is misleading, as the lobules are situated on every side, as well as above the sublobular veins.) 4. The portal canals, P. C. (Even the smaller ones, I, I, are reactily differentiated from areas containing hepatic veins, inas- much as a group of vessels can be distinguished — the hepatic veins running alone.) 168 STUDENTS HISTOLOGY 5. The portal veins, V. (Observe that they usually are the largest vessels in the canals. Note their thin walls. They not infrequently contain blood- clots, with deeply stained, scattering, FIG. 109. LIVER OF THE PIG SECTIONED AT A RIGHT ANGLE TO GLISSON' CAPSULE. STAINED WITH H^IMATOXYLIN AND EOSIN (X60). C. Capsule of Glisson. C. V. Central or intralobular veins. O. C. Oblique section of central veins. 1. 1, 1. Interlobular veins. (In small portal canals.) P. C. A large portal canal. A. A. Hepatic arteries. D. Hepatic duct. V. A portal vein. H. V. Hepatic veins— probably sublobular. white corpuscles, appearing with this amplification as dots or granules.) 6. Hepatic arteries, A. (The larger examples may be deter- mined by their thick muscular media and the wavy pink line — the fenestrated membrane. Several may be seen in a single canal.) 7. Hepatic ducts, D. (These are lined with cylindrical epithe- lial cells, hexagonal in transverse section, and the bold, deeply THE PARENCHYMA OF THE LITER 169 stained nuclei give the ducts marked prominence even with the low -power. Indeed, the smaller portal canals are frequently dif- ferentiated by this element alone — this being especially true when the structures have been disturbed, and perhaps torn, in the. pro- cess of mounting.) 8. The lobular parenchyma. (The arrangement of the hepatic cells, forming branching columns, is merely indicated — with the low -power — by their deeply stained nuclei presenting granular areas within the lobular boundaries. Still, by careful attention, the elements will be seen to radiate more or less distinctly from focal points — the central or intralobular veins. (H.) 9. The portal veins. 10. The lymph-spaces in the connective tissue of the portal canals. (Note, in those which are better denned, the nuclei of the endothelium. Do not confound these lymphatics with small veins, as the latter present a tolerably denned wall, while the lymphatic chinks appear like rifts in the connective tissue; it would be diffi- cult to make this distinction without the endothelial cells.) 11. Hepatic arteries. (On account of its solidity, the liver will enable the student to secure sections of blood-vessels present- ing the typical structure more nearly than the specimens obtained from the organs heretofore examined.) Note (a) the elongated nuclei of the muscular elements of the media; (&) the fusing of the adventitia with the connective tissue surrounding the artery ; (c) the sharply defined outer boundary of the intima — the fe- nestrated membrane, which, from the action of the hardening agent, has contracted the elastic fibers and detached (d) the endothelial cells. (Inasmuch as the lining cells of small arteries are very frequently partly detached in alcohol -hardened tissue, they may simulate columnar cells. A like appearance is often presented when an artery has been sectioned obliquely, by the projecting muscle-cells of the media.) 12. Hepatic ducts. Note : (a) The lining cylindrical cells. (&) The nuclei of these cells (as a rule, perfectly spherical ; and, in transections arranged in a circle, affording an appearance per- fectly characteristic). (c) Mucous glands in the wall of the larger ducts, lined with large, nucleated, columnar cells, preeisely like those lining the duct-lumen ; and, hence, liable to be mis- taken for small ducts. (The tube carrying the mucus secreted in 170 STUDENTS HISTOLOGY i these pocket -like glands does not pass directly into the lumen of the duct, but runs along obliquely, much like glands in the bronchi. Not infrequently the glands possess no proper efferent tube, but are mere depressions or diverticula in the thick wall of the bile-duct.) 13. The lobular parenchyma. (Single cells, partly detached, may be found about the edges of the section.) Note: (a) The somewhat polygonal figure; (&) the nucleus; (c) nucleoli ; (d) fibrillated, mesh-like cell-body; and (e) an apparent cell-wall. (The arrangement of the lobular parenchyma will be noted in con- nection with the human liver.) HUMAN LIVER PRACTICAL DEMONSTRATION The sections from which the illustrations have been drawn were made from material hardened in Miiller's fluid. The tissue was then cut, the sections washed by six hours' maceration in water, after which they were treated suc- cessively with weak and stronger alcohol, stained with hsematoxylin and eosin, and mounted in balsam. This treatment aids greatly in the demonstration of the blood-capillaries, as the contained blood-corpuscles, in consequence of some change affected by the chromium salt, take the eosin deeply. The nuclei of cells are also rendered markedly prominent. Pieces of tissue, one centimeter square by half a centimeter thick, may be hardened in alcohol. This method will give very excellent results, providing the sections be cut as soon as the hardening process has become complete. Stain as above. For the demonstration of the isolated hepatic cells, scrape the cut surface of a piece of hardened liver with a scalpel, and throw the scrapings into a watch-glass of hasmatoxylin. After a few moments drain off the stain, and brush the stained tissue elements into a test-tube nearly filled with water. Change the water two or three times ; and when clear, add a few drops of eosin solution. Allow the eosin to stain for a moment only; decant, drain, and fill the tube with alcohol. After ten minutes the spirit may be drained off and the tube partly filled with oil of cloves. A drop of the sediment may then be placed upon the slide, the bulk of the oil removed with paper, and the mounting completed by adding a drop of balsam and the cover-glass. This tissue may be kept in the oil from year to year for class-room purposes. If the oil be pure and the washing thorough, the staining will remain unaffected for two or three years. HUMAN LIVER 171 SECTION OF HUMAN LIVER, CUT AT A RIGHT ANGLE TO THE SUR- FACE, AND STAINED WITH H^MATOXYLIN AND EOSIN (Fig. 110) OBSERVE : (L.) 1. The imperfectly outlined lobules (in consequence of the absence of interlobular connective tissue.) 2. The fusing of the lobules. (At points like B, B, it is impossible to say just where one lobule ceases and the contiguous one begins.) FIG. 110. SECTION OP HUMAN LIVER. STAINED WITH H^EMATOXYLIN AND EOSIN (X60). A. A, A. Central veins sectioned generally at a right angle to the lobule. B. B. Points where adjoining lobules coalesce. Illustrating the difficulty of outlining the lobules in normal human liver. C. Connective tissue of a portal canal. D. Large interlobular vein. E. Hepatic duct belonging to C. F. Hepatic artery of C. may occasionally be seen, which apparently belong to this tissue.) 1). The peculiar lining cells. (They are unlike any other parenchymatous elements found in the body. Note that, while they are evidently of the columnar or cylindrical type, they differ greatly in form and size. The protoplasm is hazy, granular, and frequently striated. They take a dirty brick -red hue from the eosin.) c. The lumen. (Compared with the diameter of the tube- wall, the lumen is very small, and presents a stellate figure. The urine, in passing through the tubule, is, consequently, brought in contact with a very considerable portion of the lining. ) 3. The large proportion of the cortical area occupied by the convoluted tubules, and the exceedingly small amount of inter- tubular connective tissue. 4. The intertubular capillaries. (These are exceedingly small and difficult of demonstration unless filled with blood. The nuclei of the endothelial wall are frequently seen. The cells of the con- voluted tubules are not infrequently detached from the membrana propria, and the space so formed may be mistaken by the careless observer for longitudinal sections of capillaries. These vessels are much better seen in an injected kidney; although if ,an organ be 190 STUDENTS HISTOLOGY selected containing considerable blood, and the corpuscular ele- ments have their color preserved [as in bichromate hardening] , the vessels will be easily demonstrated.) 5. Ascending limb of Henle's loops in the cortical labyrinths. (The general course of these tubules is confined to the pyramids of Malpighi and Ferrein; but occasionally one of them may be seen passing in a tortuous course toward the outer cortex, running between the proximal convoluted elements. They are easily recog- nized by their small size and relatively large lumen. They are lined with short columnar or cuboid cells, which stain deeply blue with the haematoxylin . ) G. The pyramids of Ferrein. a. Collecting tubes. (These will generally be recognized by their large size and the blue color of the staining. They are lined with columnar cells, which are hexagonal in transverse section; and this gives an appearance like pavement epithelium when they are seen from above or below. Endeavor to find a tube split through the center longitudinally, and note the typical columnar cells, as they project inward from the membrana propria toward the now open lumen.) 1). The spiral tubules. (These resemble somewhat the convo- luted tubules, especially as their cells take much the same dirty red color. The cells, however, plainly columnar, are large and hexag- onal in transverse section. The lumen is small.) c. Ascending limbs of Henle's loop. (These are small tubes, and have already been described. ) d. The intertubular capillaries. (Inasmuch as, in the speci- men under consideration, the vessels of the pyramids are mostly in tran verse section, they are not readily made out, especially if the blood-corpuscles have become decolorized. 7. Elements of the medullary portion. Fig. 120. a. Collecting tubes. (These tubes constitute a large propor- tion of the medulla of the organ. They have already been described. As the apex of the Malpighian pyramid is approached, and the straight tubules unite to form the principal collecting tubes, these again uniting to form the papillary ducts, the lining cells will be seen to get shorter and the lumina larger. ) 6 Spiral tubes. (These can, in many instances, be followed down from the pyramids of Ferrein; and examples are frequently seen very near the pelvis of the kidney in the cortical columns.) c. Descending limbs of Henle's loop. (These tubes are the THE KIDNEY 191 most difficult of all the tubuli uriniferi to demonstrate. The section must be very thin, and even then they may be mistaken for blood -capillaries. Their peculiar feature consists in the wavy lumen, which is produced by the alternate disposition of the lining cells.) d. Loops of Henle. (The loops will be recognized by the curving of the tube. They are lined with short columnar cells, FIG. 120. MEDULLARY PORTION OF SPECIMEN SHOWN IN FIG. 118 (X400). A. Collecting tubule in L. S. B. Collecting tubule from above, showing attached bases of lining cells. C. Collecting tubule presenting different appearance of lining cells, according to mode of section. D. Ascending limb of Henle's loop. E. Same as last. The upper end of the tubule not sectioned. F. Descending limb of Henle's loop. Below may be seen the loop and ascending limb. G. Oblique section of large collecting tubule. H. Basal attached extremities of cells lining a large collecting tubule. I. Intertubular capillaries. which are sharply brought out by the haematoxylin. On account of their course, but few complete sections are seen.) e. Ascending limbs. (Conveniently traced from the loops.) /. Intertubular blood-vessels. (Do not mistake tubules con- 192 STUDENTS HISTOLOGY taining blood for capillaries. The human kidney is rarely abso- lutely normal; and blood is frequently found outside the proper channels. The vessels will be differentiated by the histology of their walls. Quite a number of venules will be seen running in groups in the medulla — the venulce rectce. 8. The same elements as in 7 (shown in a transverse section of the middle of a Malpighian pyramid, Fig. 121). FIG. 121. TRANSVERSE SECTION OF PYRAMID OF MALPIGHI. SAME TISSUE AS SHOWN IN FIG. 118. STAINED WITH H^EMATOXYLIN AND EOSIN (X400). A. Group of intertubular blood-vessels. B. Collecting— straight— tubules. C. Descending limb of Henle's loop. D. Ascending limb of Henle's loop. E. Principal— collecting— tubule. F. Principal tubule. Lower portion near the papillary duct. The ring of cells will be seen detached from the membrana propria in some instances. This is due to contraction of the tissue during the hardening. In amphibians the epithelial cells at the neck of the capsule of Bowman are ciliated. In man and the mammals the epithelial cells of the convoluted, spiral, and irregular tubules and the ascending limb of Henle's loop have distinct, vertical striations close to the basement membrane. The cells of the convoluted tubules show PELVIS OF THE KIDNEY AND URETER 193 variations in size and in the number of granules, dependent on the state of secretion. DIAGRAM SHOWING DISTRIBUTION OP TUBULES (AFTER PIERSOL) CORTEX. Labyrinth. Medullary Ray. Malpighian bodies. Spiral tubules. Necks of the tubules. Ascending limbs of loops of Proximal convoluted tubules. Henle. Irregular tubules. Collecting tubules. Distal convoluted tubules. Collecting tubules. Loops of Henle. Descending limbs. V MEDULLA. Ascending limbs. Collecting tubules. PELVIS OF THE KIDNEY AND URETER The coats of the ureter are three in number — mucous, muscu- lar, and fibrous. The muscle is unstriated, and is divided into inner longitudinal and outer circular layers. The pelvis of the kidney, and the calyces and infundibula present a similar structure. The circular muscular fibers are numerous around the papillae, and form a kind of sphincter. Obtain the ureter of a human subject, if possible. Fix the fresh ureter with alcohol or Miiller's fluid. Imbed in celloidin or paraffin. Cut thin sec- tions, stain with hsematoxylin and eosin, and mount as usual. OBSERVE : (L.) 1. The relative thickness of the epithelium. 2. The narrow mucosa. 3. The internal longitudinal muscular layer. 4. The bundles of the external circular muscular layer. 5. The arteries between the muscular bundles. 6. Adipose tissue, more or less abundant in the loose cellular tissue surrounding these canals. (This element will afford a prominent feature of the section of the pelvis of the kidney, while the muscular tissue will be seen to a limited extent only.) (H.) Fig. 122. 7. The epithelium, (a) That it is of the thin, stratified squa- mous type known as transitional. >(&) The broad basal attach- ed 194 STUDENTS HISTOLOGY ment of the deep cells, (c) The elongated form of the cells generally, (d) The more flattened surface cells, (e) The out- line of the last, as seen in the detached specimens. (/) The deeper cells present tapering prolongations, generally at one end only, FIG. 122. SECTION OF THE URETER NEAR THE PELVIS OF THE KIDNEY. STAINED WITH ILEMATOXYLIN AND EOSIN (X400). A. Rich capillary plexus of the mucosa. B. Internal muscular coat. C. External muscular coat. D. Large vessels of the areolar adventitia. E. Deep layer of somewhat cubical cells. F. "Tailed cells" of the middle epithelial layers. G. Surface cells in profile. H. Detached surface cells. and are hence called "tailed cells." They may be confounded with similarly shaped cells from the bladder. THE URINARY BLADDER. The layers of the bladder are mucous, muscular, and fibrous, while part of it has a serous covering derived from the peritoneum. The muscle is unstriated, and is arranged in inner and outer longi- THE URINARY BLADDER 195 tudinal layers, with a circular layer between them. The muscular layers are not well marked. The internal vesical sphincter is formed from an increase in the thickness of the circular layer. Numerous minute ganglia are found along the nerves of the blad- FIG. 123. VERTICAL SECTION OP THE LINING PORTION OF THE BLADDER (MALE) BEHIND THE TRIGONE. STAINED WITH ELEMATOXYLIN AND EOSIN (X400). A. Connective tissue of sub-epithelial region. B. Capillary supply of sub-epithelial region. C. Muscular wall of bladder. D. Deep cells of the epithelial lining. E. Middle region of lining. F. Detached surface cells, showing processes beneath. G. Thin surface cells in profile. H. Squamous surface cells, seen detached, in plan. I. Vacuolated cells. der, which contain both medullated and non-medullated fibers. As in the ureter, the blood supply of the mucous and muscular coats is abundant. Small nodules of lymphoid tissue occur in the mucous membrane. The epithelium is transitional ; but the disposition of the cells varies with the extent to which the bladder is expanded. Prepare sections of human bladder in the same manner as sections were made of the ureter. 196 STUDENTS HISTOLOGY OBSERVE : (L.) 1. The epithelial lining, (a) That it is formed after the transitional type. 2. The mucosa and its capillary supply. 3. The dense muscular portion. (H.) Fig. 123. 4. The epithelium, (a) The size of the cells. (&) The layers of the epithelium, which are deep, middle, and superficial, (c) That the deep cells are polyhedral or columnar, (d) The form of the middle cells ; not unlike those of the corresponding region in the ureter, (e) The large, scaly surface epithelial cells. (Note that while these all appear flat, when seen in plan, it is only those of the extreme surface that are simple scales ; the less superficial examples show, when viewed in profile, prolongations from the under surface, by means of which union is effected with the deeper cells.) THE URETHRA The urethra consists of a mucous coat surrounded by muscular and fibrous tissue. The muscle is unstriated. Numerous papilla cover the surface of the mucous membrane proper. The lining in the female urethra is stratified squamous epithelium. A few small glands are found in the female urethra. The lining of the male urethra varies in the different regions. In the prostatic region it is transitional epithelium , as in the bladder ; in the membranous portion it is stratified columnar ; in the spongy or penile it is simple columnar ; while in the fossa navicularis it becomes strati- fied squamous epithelium, and is continuous with that covering the glans penis. Small, branched, mucous glands (the glands of Littre) occur throughout the male urethra. THE FEMALE GENERATIVE ORGANS 197 THE FEMALE GENERATIVE ORGANS THE VAGINA AND UTERUS The walls of the vagina are lined with a mucous membrane, covered with a thick, stratified squamous epithelium, beneath which are numerous papillae. The submucous, unstriated muscular, and fibrous adventitious coats are not well defined. Glands are not present. The uterus has mucous, muscular, and serous layers, but the muscular layer is much the thickest. The muscle is unstriated. The fibers increase both in size and number during pregnancy. The mucous membrane is covered with a single layer of columnar, ciliated epithelial cells, which create a current in the direction of the cervix. Numerous tubular glands, lined with similar cells, extend down to the muscular layer. During menstruation the mucous membrane, having become thickened, soft, and distended with blood, undergoes disintegration in its outer portion, which is cast off. Accompanying this phenomenon there is an escape of blood from the capillaries. The epithelium is quickly renewed from the deeper portions of the glands of the mucous membrane. PRACTICAL DEMONSTRATION From the body of a (preferably young) human female, as soon as possi-" ble after death, remove centimeter cubes of the organs required, observing that the lining is included. The outer portions are of very little moment comparatively. Secure pieces from the os, cervix, and fundus of the uterus, and the wall of the vagina. The vagina and uterus of a human subject should be secured, because their structure differs considerably from that of the same organs in the lower animals. The differences are greater than is the case with many of the organs hitherto studied. We desire to prepare the tissue so as to keep the original form of cell- elements — to avoid contraction — and Miiller's fluid will accomplish this per- fectly. Allow the pieces to remain for a month in the bichromate solution, with an occasional change. Complete the hardening in alcohol, after washing as usual. Infiltrate with celloidin, and let the sections be vertical to the mucous surface. The tissues should not be handled with the fingers ; other- wise the epithelial lining cells will be detached. Stain with haematoxylin and eosin; mount in balsam. 198 STUDENTS HISTOLOGY VAGINA AND UTERUS OF THE HUMAN FEMALE AT PUBERTY — VER- TICAL DEXTRO-SINISTRAL SECTION OF THE RIGHT LIP OF THE OS, AND INCLUDING PART OF THE VAGINAL CUL-DE-SAC OBSERVE : (L.) 1. The outline of the section. (Commencing at D, Fig. 124, which is placed in the internal os, follow downward, out upon FIG. 124. VERTICAL DEXTRO-SINISTRAL SECTION OF THE RIGHT-HAND SIDE OF THE Os UTERI. SHOWING THE INTERNAL Os, THE EXTERNAL Os, THE VAGINAL CUL-DE-SAC, AND THE UPPER PORTION OF THE VAGINAL WALL (X 60). A. The letter is placed in the internal os. B. Vaginal cul-de-sac. C. Vaginal wall. D. Columnar epithelium of the internal os. In the upper portion the tubular glands are well seen. E. Stratified epithelium of the vaginal lining. F. Change at the external os from stratified flattened to columnar epithelium. the external os, curve upward, reaching, at B, the vaginal cul- de-sac. Descend along the right vaginal wall.) 2. The irregular surface of the internal uterine wall. (Caused by longitudinal section of the glandulee uterinae or glan- VAGINA AND UTERUS 199 dulae utriculares— branched tubular glands. These are increased in depth during pregnancy, and are most prominent in the lower portion of the organ.) 3. The epithelium, (a) The deeply stained layer, lining of the vagina, cul-de-sac, and external os. (b) The wavy course of a as it covers the irregularly formed and often imperfect pa- pillae of the mucosa. (c) The lighter appearance of the lining of the internal os. (d) Projection of the last into the glands. (e) The sharp line of separation between the deeply stained lining common to the vagina and the lighter lining of the uterus at the external os (Fig. 124, F). 4. The mucosa of the uterus. (There are no sharply denned regions in the genito- urinary tract corresponding to the mucosa and submucosa of typical mucous membranes. The arrangement generally is, (1) an epithelial lining; (2) a subepithelial struc- ture, consisting of a more or less prominent or abundant plexus of capillaries supported by delicate connective tissue, and which corresponds to the typical mucosa; (3) the muscular walls proper, consisting of layers in different directions, frequently irregularly disposed and seldom in distinct fasciculi.) 5. The mucosa of the vagina (the surface of which is beset with small papillae, and in which large veins are prominent) . 6. The uterine and vaginal walls (consisting largely of involuntary muscular fibrils, recognized by the elongated and deeply stained nuclei, and containing numerous thick -walled ar- teries and irregular lymph -spaces.) (H.) 7. The uterine epithelium (Fig. 125). (a) That it consists of a single layer of cells. (&) That the cells are columnar or cylindrical, (c) The cells in transverse section are polygonal. (d) They are ciliated. (If the section has been properly prepared from uninjured tissue, the cilia will be seen without difficulty, and especially in the depressions where they are somewhat pro- tected.) (e) The cell-body and nucleus. (Note the elongated, clear, free portion, and the frequent curving of the whole. Near the attached extremities, which often appear pointed, note the small, deeply stained nuclei.) (/) The large mucous crypts which occur in the cervix. Retention of their secretion produces the cysts which are known as the ovula Nabothi. (g) It is dif- ficult to see the basement membrane. 200 STUDENTS HISTOLOGY 8. The abrupt transition from columnar to flattened cells in the epithelium of the external os. (a) The shortening of the columnar cells as the point of change is approached. (Sections must be examined until one is found showing this point well. The illustration [Fig. 125] is not exaggerated, and a properly cut and selected specimen must exhibit clearly the last columnar and the adjoining flattened cell.) 9. The vaginal epithelium (Figs. 125 and 126). (a) That it is FIG. 125. EXTERNAL Os OF FIG. 124. MORE HIGHLY MAGNIFIED (X400). A. Muscular tissue of the os uteri, with numerous blood-vessels. B. Capillary plexuses of subepithelial tissue— mucosa. C. Ciliated columnar cells covering the os. D. Vacuolated cells. E. Shortening of the columnar cells preparatory to F. Change from typical uterine epithelium— ciliated columnar cells— to flattened stratified cells. G. Papillary structure of the niucosa of the external os, after change of epithelium. of the stratified squamous variety, (b) The deepest line of cells following the sinuous line formed by sectioning the papillary mucosa. (c) That the cells are more or less flattened. (d) That their edges, excepting those of the surface, are serrated, (e) The VAGINA AND UTERUS 201 change in form as the surface is approached. (/) The surface cells. (These are very much flattened, and so fused in longitudinal section as to resemble fibers.) (g) Detached surface cells. (At H, Fig. 126, these are shown in plan, having been torn off; those_ intact are, of course, seen in profile.) (h) The nuclei, evenly granular, usually larger than a red blood -corpuscle. 10. The subepithelial vaginal structures. (a) The large and abundant capillaries of the rnucosa. (b) The submucosa, not FIG. 126. VERTICAL SECTION OF THE VAGINAL LINING AT PUBERTY. STAINED WITH H^MATOXYLIN AND EOSIN (X 400). A. Subepithelial capillary plexus. B. Papillary arrangement of the mucosa. C. Large blood-vessels in the submucosa. I). Muscular wall of vagina. E. Deep cells of the lining epithelium. F. Middle strata of lining stellate cells. G. Surface cells in profile. H. Surface cells in plan — detached. clearly separated from the mucosa, but easily recognized by the large vessels and the abundant connective tissue. (c) The muscular portion of the vaginal wall. 202 STUDENTS HISTOLOGY THE FALLOPIAN TUBE OB OVIDUCT The Fallopian tube has a serous coat derived from the peri- toneum; a muscular layer, consisting of unstriated muscular fibers, mostly disposed in a circular manner; and a mucous membrane which lines the tube. The mucous membrane is covered with a FIG. 127. FALLOPIAN TUBE. TRANSVERSE SECTION OF A FOLD OF THE AMPULLA— AFTER HENLE. * * Spaces between the folds. single layer of columnar, ciliated epithelial cells, whose cilia create a current in the direction of the uterus. The mucous mem- brane has numerous longitudinal folds, which are very com- plicated, and which, in tranverse section, appear like glands. Glands, however, are not present. PRACTICAL DEMONSTRATION Harden the Fallopian tube of a healthy young female in alcohol or Miiller's fluid ; imbed in celloidin ; cut transverse sections ; stain in hsernatoxy- lin and eosin; mount in balsam. THE OVARY 203 (L.) Find the three principal layers : the mucous membrane, with its folds ; the muscular layer, consisting of unstriated muscle, divided into an inner circular and an outer longitudinal band ; and the thin serous covering. (H.) The mucous membrane consists of a fibro- elastic basis, covered by a single layer of columnar ciliated epithelial cells, which will be found well preserved at the bottoms of the folds. THE OVARY The ovary consists of a stroma or ground -substance of connec- tive and smooth muscular tissue, in which are scattered various sized spherical bodies, the Graafian follicles . The stroma is divided into three layers or regions, which are not very sharply differentiated. The free surface of the ovary is covered with a single layer of low columnar epithelial cells, called the germinal epithelium. Immediately beneath . the epithelium is a thin layer of fibrous tissue, termed the tunica albuginea. The cortex proper, or second layer, is distinguished by the Graafian follicles, which will be described later. The central portion of the organ, the zona vasculosa, is largely occupied by thick-walled blood-vessels, among which the extremely tortuous arteries are especially evident. Occasionally one may see in this region somewhat ovoid nodules in varying degrees of retro- grade change — the corpora lutea. They present the phenomena resulting from the maturation of the follicle during menstruation. The accompanying illustration, Fig. 128, was drawn from a cor- pus luteum which had formed in the site of a Graafian follicle, the contents of which had escaped at some menstrual epoch, and been followed by impregnation. PRACTICAL DEMONSTRATION The ovary of a young animal is to be preferred. If the organ cannot be obtained from the human subject, the female of almost any domestic animal will provide an excellent demonstration for the histological elements. Let the tissue be hardened with strong alcohol, and sections be cuti vertically to the free surface and stained with hsematoxylin and eosin. The sections should include at least one-half the depth of the organ, so as to exhibit all of the regions. 204 STUDENTS HISTOLOGY SECTION OF THE ADULT HUMAN OVARY OBSERVE : (Fig. 1 28) (L.) 1. The tunica albuginea. (Note that 'the layer is not of uni- form thickness, and is composed largely of spindle-shaped cells, as PIG. 128. SECTION OP AN OVARY FROM A WOMAN 35 YEARS OLD. STAINED WITH H^EMATOXYLIN AND EOSIN (X250). A. Surface of the ovary. B. Stroma. C. Large, tortuous, thick-walled arteries of the central portion of the organ. D. D. Small Graafian follicles of the superficial zone. E. Larger follicles of the deeper portion. F. Membrana propria of a Graafian follicle. G. Membraua granulosa of the follicle. The line leads to the discus proligerus. H. An ovum. I. Germinal vesicle. J. Germinal spot. K. An old corpus luteum. shown by the numerous elongated nuclei. Search particularly for and note the character of the epithelial covering.) 2. The cortical layer, containing numerous Graafian follicles, and possibly a corpus luteum. (Note the aggregation of the smaller follicles in the extreme outer portion of the region.) H UMAN OVARY 205 3. The zona vasculosa. (Note the unusual thickness of the vascular walls and the irregular outlines on section, on account of their tortuous course.) (H.) 4. The Graafian follicles. («•) Their diameter, varying from 25 /*• when young to 5 or 10 mm. when mature, (b) The mem- brana propria. (This is difficult to separate from the stroma of the ovary itself, except in more mature follicles than shown in the section, (c) The membrana granulosa. (This, in general, appears to be the outer layer of the follicle, on account of the difficulty of separating the membrana propria from the stroma proper of the ovary. Note that it is composed, in the smaller and less mature follicles, of pavement cells, and that the cells become thicker with maturation, until columnar cells in a single layer result, (d) The ova. (These are contained within the follicles, excepting that they may have become detached during manipula- tion of the section, and occupy the greater part of the follicles.) (e) The zona pellucida (the thin wall of the ovum). (/) The discus proligerus. (This will be recognized as a mass of polyhe- dral cells, connecting the ovum at one side with the columnar celts of the membrana granulosa. These cells will proliferate later in the development, and completely enclose the ovum.) (g) The .germinal vesicle. (Contained within the ovum. The contents appear granular ; it, as well as the ovum, is fibrillated ; but this demonstration cannot be made, excepting the animal be killed for the purpose, and the tissue elements fixed, before changes, which quickly follow death, occur.) (h) The germinal spot. (Appear- ing as a small dot within the germinal vesicle. The ovum pre- sents the characteristics of what it indeed is — a typical cell, with wall, body, nucleus, and nucleolus.) 5. The corpus luteum. (The example shown in the drawing was developed after the contents of the Graafian follicle, which it represents, had suffered impregnation ; and it has arrived at the later stage of the series of the phenomena connected with its development — the stage of cicatrization. The cicatricial tissue, to which the letter K points, indicates the remains of the membrana granulosa. Outside is seen the thickened membrana propria, while among the contents will be found pigment -granules and fat -glob- ules, imbedded in a structureless, gelatinous stroma. This material results from changes in the clot of blood effused after the escape of the ovum.) 206 STUDENTS HISTOLOGY FORMATION OF THE OVUM As has been previously shown, the ovary is covered with columnar epithelium ; and, singular as it may appear, the fifty thousand Graafian follicles, which it is estimated are devel- oped during the life of the human female, have their origin in these cells. During foetal life this surface epithelium undergoes a very rapid proliferation, and chains of cells become imbedded in the stroma of the ovary. These epithelial prolongations are called ovarial or egg-tubes. A little later in the development, separate portions or links of these chains are cut off by the ingrowth of the stroma. The little groups of cells thus isolated become each a Graafian follicle. Scattered among the columnar cells, larger, more nearly spherical cells are also found, the primordial ova. These are also imbedded in the substance, and one at least will always be found among the minute collections of cells which have been isolated. In the process of development, each group of cells becomes a Graafian follicle with its contained ovum, the columnar cells form- ing the wall proper, and the primordial cell the ovum, with its vesicle and germinal spot. PRACTICAL DEMONSTRATION The ovary from a still-born babe is to be removed with the scissors, exercising the utmost care that the surface be not touched. The ovary of a rabbit or guinea-pig may also be used with advantage. Place immediately in strong alcohol, and in twenty-four hours it will be fit for cutting. Cut extremely thin sections at a right angle to the free surface and including the same; stain with haematoxylin and eosin; mount in balsam. OVARY OF HUMAN INFANT (Fig. 129) OBSERVE : (L.) 1. The free surface. (Note the occasional depressions which mark the involution of the epithelial cells.) 2. The layers. (Note the absence of demonstrable tunica albuginea and the great area occupied by the cortex. The vessels of the central portions are, unlike the ovary of mature life, large, not numerous, and thin-walled.) OVARY OF HUMAN INFANT 207 (H.) 3. The primordial ova of the surface epithelium. 4. The projecting lines or chains of epithelium undivided. (Here the cells seem rather elongated.) 5. Chains which are in process of subdivision. 6. Young Graafian follicles in columns at a right angle to the surface of the ovary. 7. The discus proligerus, in many instances still composed of flattened cells. 8. Follicles showing discus proligerus as columnar cells. 9. Follicles showing great proliferation of discus pro- ligerus. FIG. 129. SECTION OP OVARY OP CHILD. DEATH TEN DAYS AFTER BIRTH (X350). A. Germinal epithelium, covering surface of the ovary. B. Primitive ova. C. C. Projection of surface epithelium within the organ. D. Constriction of the projected chain or cord of epithelium and isolation of portions to form Graafian follicles. E. Chain of Graafian follicles. The stroma is seen filled with previously formed follicles which have now become isolated. F. A large Graafian follicle. It has been cut in half ; the ovum has fallen out, and' the membrana grunulosa is seen lining the cup-shaped cavity. G. Large arteries of the central portion of the ovary. 208 STUDENTS HISTOLOGY 10. Ova in the early stages of development from primordial cells, with granular vesicles. 11. Instances of development of two, possibly three, ova in a single follicle. 12. Large blood-capillary supply of cortex, — vessels generally parallel with the chains of follicles. THE MA MM ART GLAND The mammary gland consists of acini lined by epithelial cells. The acini are grouped into lobules and lobes. There is an abun- FIG. 130. MAMMARY GLAND OP THE DOG. TRANVERSE SECTION OF AN ACINUS IN THE EARLY STAGES OF FAT FORMATION. (HEIDENHAIN.) dant framework of fibrous and adipose tissue. The ducts are lined by columnar epithelium, and open at the nipple. The lobules and acini of the resting gland are small. During activity the acini are lined by low cubical epithelial cells resting on a basement mem- FIG. 131. MAMMARY GLAND OF THE DOG AT THE HEIGHT OF ACTIVITY. (HEIDENHAIN.) brane. Globules of oil form within these cells, and are discharged, making the oil -globules of the milk. The secretion of the gland during the first few days of activity contains numerous cells in which drops of oil are present, the colostrum-corpuscles. Sections of the resting and active mam- mary gland of the dog may be studied with advantage. THE MALE GENERATIVE ORGANS THE MALE GENERATIVE ORGANS THE TESTICLE The testicle is a glandular body (Fig. 132, T), oval in shape and flattened laterally. The epididymis, E, is an elongated and arched structure, which is applied to the upper end. and posterior border of the body of the testicle. It is enlarged at each end. The upper end is termed the globus major; the lower, which is somewhat smaller, the globus minor; while between them is the body of the epididymis. From the lower end of the epididymis a hard convoluted tube, \. 132. DIAGRAM OP THE COURSE OP THE CANALS IN THE TESTICLE AND EPIDIDY- MIS, AND THE PASSAGE OF THE CANAL INTO THE VAS DEFERENS. (LADTH.) T. Testicle. Rt. Rete testis. E Epididymis. PE. Organ of Giraldes. Vd. Vas deferens. * Vasa efferentia. ** Vas aberrans. the vas deferens, Vd, is given off, which soon straightens, and extends upward in the spermatic cord. The testicle is almost completely covered with a serous mem- brane, the tunica vaginalis. Beneath that is a strong fibrous capsule, the tunica albuginea, with partitions or septa converging toward the posterior portion, the mediastinum testis, or corpus 210 STUDENTS HISTOLOGY Highmori. Between the septa are delicate tubular masses — the lobules. The tunica albuginea is the supporting framework of the testicle. Its inner layers convey the blood-vessels, and constitute the tunica vasculosa. The mediastinum receives the blood-vessels and contains also the rete testis, Rt, the termination of the secreting tubules. Two or more seminiferous tubules are contained in each lobule. They are fine thread-like tubes closely packed together — the convo- luted portion. As these tubules approach the mediastinum they PIG. 133. TRANSVERSE SECTION OF THREE SEMINIFEROUS TUBULES OF THE CAT. (SCHAFER.) A. Containing spermatozoa least advanced in development. B. More advanced. C. Containing fully developed spermatozoa. join to form a smaller number of straight tubes, or tubuli recti. The straight tubules enter the mediastinum, making a plexus of tubular spaces, the rete testis, and leave the rete testis at its upper end as fifteen to twenty delicate ducts, the vasa efferentia, Fig. 132. The vasa efferentia, entering the globus major, become coiled into small conical masses, the coni vasculosi, and open into a single convoluted tube, the canal of the epididymis. This canal is remark- ably tortuous; it passes down the back of the body of the testicle into the globus minor, and finally terminates in the vas deferens. (For the character of the lining epithelial cells of the different parts, see page 55. THE MALE GENERATIVE ORGANS 211 PRACTICAL DEMONSTRATION Harden the testicle of some animal, preferably a rat, in Flemming's, Miil- ler's, or Orth's fluid. After washing, complete the hardening in alcohotr Imbed in celloidin, cut transverse sections, stain in heematoxylin and eosin, and mQunt in balsam. (L.) Find the tunica albuginea covering the testicle, consisting of fibrous tissue ; its outer serous layer; the tunica vaginalis, and its looser inner layers, containing many blood-vessels; the tunica FIG. 134. HUMAN SPERMATOZOA, HIGHLY MAGNIFIED. (RETZIUS.) A. Seen from above. B. Head seen from the side. C. Extremity of the tail. vasculosa. Find the thickening of the tunica albuginea behind, called the mediastinum, from which fibrous septa arise, dividing the testicle into compartments. These compartments are seen to contain seminiferous tubules 130 p- to 140 /* in diameter, cut at various angles. (H.) Examine seminiferous tubules in transverse section. Observe a lining of epithelial cells in several layers, and spermatozoa in 212 STUDENTS HISTOLOGY the central part of the tube, in various stages of formation. The tails of the spermatozoa point toward the center of the tube, and the heads are directed toward the epithelial lining. Among the epithelial cells some may be found that are under- going karyokinesis. Careful study of selected specimens has shown that the epithe- lial cells next the basement membrane are of two sorts : (1) The sustentacular cells. (2) The spermatogenic cells. The sperma- tozoa are derived from the spermatogenic cells. Spermatozoa may be obtained from the fresh testicle or epidi- dymis of some one of the domestic animals. The human sperma- tozoon is about 50 /* in length. It consists of a short oval head, a short middle piece or body, and a long tail. The tail possesses active vibratile movements. THE P-ROSTATE GLAND. The prostate gland consists of a number of tubular acini, which are imbedded in a muscular and fibrous framework. The muscle is of the unstriated variety, and is very abundant. The tubular glands are lined by columnar epithelium. They open by a num- ber of ducts into the urethra. Certain small, round, concentrically laminated bodies, the prostatic concretions, often occur in the ducts and acini, especially in old subjects. The secretion of the prostate gland is thin and nearly transparent, containing granules and epithelial cells. ERECTILE TISSUE Erectile tissue is best demonstrated in the penis, preferably of a human infant, cut in transverse section. The two corpora cavernosa and the corpus spongiosum are readily distinguished. The erectile tissue can be studied in the corpora cavernosa. The connective tissue surrounding the cavernous bodies gives off numerous trabeculse, forming a framework, enclosing spaces. These spaces are lined by endothelium, and communicate with one another freely. The arteries open into them, and the veins open from them. During erection they become greatly distended with blood. Similar erectile tissue occurs in the external generative organs of the female. THE SUPRARENAL BODY 213 THE SUPRARENAL BODY This body is attached by areolar tissue to the summit of the kidney, and consists of several folia or leaflets. An examina- tion of one of these leaflets will give us an idea of the organ as a whole. The plan of structure seems to be as follows: In the connective tissue which supports the folia are found arterial branches derived from the phrenic and renal arteries, besides the suprarenal artery itself. These arteries penetrate the organ, break up immediately into capillaries, which finally con- verge toward the center of the leaflet ; the blood is here col- lected in thin -walled veins, by which it is drained into the supra- renal vein, thus leaving the body. The capillary meshes vary in form and size, according to their position. Near the circumference of the leaflets the meshes are small and ovoid; while, as the center is approached, they become elongated. These spaces between the capillaries are filled with compressed, globular, nucleated cells, the smaller containing only perhaps six or eight, while the longer may be occupied by thirty or forty of these cell -elements, which constitute the parenchyma of the organ. This variation in size of the cell -compartments, contributing, as it does, to alter the appearance of the different zones of the tissue, has given rise to a division into cortex and medulla. The cortex is divided from without inward into a zona glomerulosa, zona fasciculata, and zona reticularis. Many nerve- fibers enter the organ with the arteries. They form a plexus, mostly of non-medullated fibers, in the medulla. Ganglion -cells are numerous. The surface is covered with a fibrous capsule con- tinuous with the supporting framework. The suprarenal body is also often known as the suprarenal capsule. This body, the spleen, the thyroid and the thymus glands, with certain other organs of less importance, are some- times called "the ductless glands " PRACTICAL DEMONSTRATION The tissue is best hardened in strong alcohol, and should be cut as soon as the hardening is complete. It will be sufficiently firm to admit of the thinnest sections being made free-hand or with a simple microtome. The sections, stained with hsematoxylin and eosin, give excellent differentiation. 214 STUDENTS HISTOLOGY HUMAN SUPRARENAL BODY. (Figs. 135 and 136) SECTION OF A SINGLE LEAFLET, CUT TRANSVERSELY TO THE CENTRAL VEINS. STAINED WITH H.EMA- OBSERVE: TOXYLIN AND EOSIN. (L.) 1. Section of arterial twigs on the border of the leaflet. 2. The convergence of the parenchyma toward the center. 3. The large and thin- walled central veins. 4. The small size of the parenchymatous areas on the outer borders and their elongation within, making the zona glomeru- losa and the zona fasciculata respectively. 5. Distinguish the zona reticularis next to the medulla. FIG. 135. VERTICAL SECTION OF A SINGLE LEAFLET OF THE SUPRARENAL BODY. STAINED WITH H^EMATOXYLIN AND EOSIN (X 60). A. Fibrous tissues surrounding and connecting the leaflets. B. The outer portion, consisting of small compartments— the so-called zona glomerulosa. C. The central elongated cell-compartments and medulla. D. Large, thin-walled central veins. E. Arteries ramifying in the outer fibrous tissue which supply the parenchyma. HUMAN SUPRARENAL BODY 215 SAME SECTION AS FIG. 135, MORE HIGHLY MAGNIFIED. ZONA FASCICU- LATA (X 400). A. Blood-capillaries, arising from the arteries seen in the proceeding illustration, and ramifying in the connective tissue framework. B. Compartments— lobules— formed by delicate connective tissue prolongations from the fibrous capsule. C. Lobular parenchyma. These large, somewhat rounded cells are generally mononucleated, contain fat-globules, and are frequently pigmented. (H.) Fig. 136. 1. The capillary plexus, forming ovate or elongated meshes. 2. The compressed globular cells of the parenchyma. (Note that the cells are small in the small compartments, as though crowded. This is due, in a measure, to the contraction of the tissue from the rapid hardening. 3. The minute fat-globules in the parenchyma. 4. Yellow pigment- granules in the cells, especially of the medulla. 216 STUDENTS HISTOLOGY THE NERVOUS SYSTEM Structural Elements The elements of the nervous system are : 1. Nerve -Fibers. 2. Nerve -Cells. 3. Connective Tissue and Neuroglia. 4. Peripheral Termini. NERVE-FIBERS Nerve -fibers are of two sorts, medullated or white, and non- medullated or gray. A typical medullated nerve-fiber consists of three portions; viz., a central conducting portion, the axis -cylinder; the medullary sheath, or white substance of Schwann; and the enveloping connec- tive tissue substance, the neurilemma. Such fibers are found largely in the trunks of the cerebro- spinal system, while medul- lated fibers devoid of the neurilemma exist in the optic and acoustic nerves, the spinal cord, and the brain. The axis -cylinder may be seen to be split up longitudinally, and is found to be composed of fine primitive or ultimate fibrillae, which may present minute varicosities or swellings at irregular intervals. The white substance of Schwann, which is largely fatty, pre- sents under the microscope the most prominent feature of medul- lated nerves, affording a nearly complete investment of the nerve- axis. The neurilemma is an elastic envelope, which completely invests the medullary substance. This tubular membrane is nucleated, and at intervals is constricted so as to reach the axis- cylinder. These constrictions are called the nodes of Ranvier. The neurilemma presents a single nucleus, with a small amount of protoplasm between each two of these nodal points. The con- strictions do not, however, affect the even caliber or continuity of the axis -cylinder. The distance between two nodes of Ranvier may be as much as a millimeter, or it may be less than that. Non- medullated nerve -fibers are found chiefly in the sympa- THE NERVE-TRUNKS 217 thetic system. But some non-medullated fibers occur in most of the cerebro- spinal nerves, and they form the chief part of the olfactory nerve. Non-medullated nerve -fibers have an axis -cylin- der and a neurilemma, but no medullary sheath. They branch freely, and aid in the formation of plexuses. There are numerous FIG. 137. SEPARATED NERVE-FIBERS (X 400). A. Neurilemma of a fiber. B. White substance of Schwann, stained with osmic acid, which hides the axis-cylinder. C. Nucleus of the neurilemma. D. One of Ranvier's nodes in an osmic acid - stained fiber, showing the axis-cylinder between the separated portions of Schwann's sheath. E. A medullated fiber, teased in normal salt solution. The medullary substance has become coagulated on exposure and removal. The axis-cylinder is faintly seen. F. Axis-cylinder at torn extremity. CT. Non-medullated fiber. nuclei beneath the neurilemma. The neurilemma is wanting in some situations. The medullated nerve -fibers also lose the medul- lary sheath when they are about to enter upon their peripheral distribution. THE NERVE-TRUNKS The structure of nerve -trunks is most typical in the large nerves composed of medullated nerve -fibers. In transverse sec- tions, medullated nerve -fibers appear like small round cells, in which the axis -cylinders resemble nuclei. The nerve -fibers are collected into bundles called funiculi. The connective tissue, which serves to unite the elements of a nerve -trunk, does not differ materially from the sustentacular tis- sue of other organs. Different terms are applied, according to its use and location, as follows : 218 STUDENTS HISTOLOGY EPINEURIUM. — Forming the sheath of the entire nerve -trunk. PERINEURIUM. — Surrounding the funiculi composing the nerve- trunk. ENDONEURIUM. — Surrounding and uniting the nerve-fibers of the funiculi. NEURILEMMA. — Surrounding the individual nerve -fibers of a bundle. FIG. 138. TRANSVERSE SECTION OF THE ANTERIOR CRURAL NERVE (X 250). A. The epineurium. B. Adipose tissue in the loose areolar tissue of the sheath. C. Lymph-spaces of the epineurium. D. Large blood- vessels of epineurial sheath. E. Perineurium surrounding funiculi. F. Lymph-spaces of last. G. Medullated nerve-fibers in T. S. supported by connective tissue — endotieurium. The formula E. P. E. N., composed of the initials of the nances of the investments from without inward, will aid the memory. The epineurium serves to protect the nerve in its course, and to support the nutrient blood-vessels and the channels of lym- phatics. The fibers run both longitudinally and transversely. The perineurium, arranged in dense bands, forms distinct sheaths NERVE-CELLS 219 for the funiculi, the fibers running, for the most part, circularly. The endoneurium not infrequently divides the nerve -bundles into smaller or primitive bundles. It supports the blood -capillaries, contains small lymph -spaces, and its nuclei are frequently large and prominent. The larger nerve -trunks have their own nerves distributed to the epineurium, — nervi nervorum. The final distribution of the elements of a nerve -trunk is effected by subdivision, first, of the large, and afterward of the primitive bundles or funiculi. The perineurial sheaths are pro- longed, surrounding the dividing bundles, even to their final distri- bution, where, around terminal and single medullated fibers, the sheath remains as a layer of exceedingly delicate flattened cells. The necessity for the endoneurium ceases with the ultimate sub- division of the funiculus. . The medullated nerve -fibers, when they reach the point of their final distribution, branch at a node of Ranvier, and also lose the medullary sheath. PRACTICAL DEMONSTRATION Medullated nerve-fibers can be studied best in preparations that have been teased on a slide. It requires much care and patience to separate tho single nerve-fibers of a bundle. The student should use nerve-tissue that was fixed with osmic acid while fresh (see page 23). The medullary sheaths are black. Also, tease nerve-fibers that were hardened in Muller's fluid and afterwards washed and transferred to alcohol. Stain the fibers on the slide with Van Gieson's picric acid and acid fuchsin mixture (page 32), omitting the heematoxylin. Dehydrate with a few drops of alcohol in succession, which may be removed with blotting-paper ; clear with a drop of oil of cloves ; mount in balsam. The axis-cylinders and nuclei are red ; the med- ullary sheaths are yellow ; the nodes of Ranvier are distinctly visible. A nerve that has been hardened in Miiller's fluid should be imbedded in celloidin, and transverse sections cut ; stain with ha3matoxylin and Van Gieson's picric acid and acid fuchsin. NERVE-CELLS The cells of the nerve -centers are usually called ganglion -cells. They differ greatly in size, some of the largest measuring 100 p* or more in diameter. The nucleus is round, conspicuous, and has a nucleolus. The protoplasm sometimes contains pigment. The cells are surrounded by minute lymph -spaces. Ganglion -cells exhibit various shapes, some being spherical, others pyriform or stellate. The differences in outline are due, in part, to the pro- 220 STUDENTS HISTOLOGY cesses, of which there may be one or many. According to the number of processes, the cells are sometimes named unipolar, bipolar, or multipolar. Every ganglion-cell has a relatively straight process, which is the axis -cylinder process. The other processes, when they are present, divide and subdivide rapidly, to form fine networks or arborizations. They are called protoplasmic processes, FIG. 139. TYPES OF GANGLION-CELLS AS SHOWN BY GOLGI'S METHOD. (BAKER.) A. Type I. * Axis-cylinder process with collaterals. B. Type II. * Axis-cylinder process. or dendrites. The development of protoplasmic processes is seen in an extreme degree on the ganglion -cells of Purkinje in the cere- bellum (Fig. 158). It is probable that the processes of one cell do not unite with those of any other, and that whatever physiologi- cal relations the cell has with other cells are established through proximity, but not by continuity of substance. The researches made according to Golgi's method of impregnating tissues with silver have shown that ganglion-cells are of two principal types : NERVE-CELLS 221 FIG. 140. DIAGRAM OP THE LOWER MOTOR NEURONE. (BARKER.) D. A motor-cell from the anterior horn of the spinal cord, with its protoplasmic processes. Ax. Axis cylinder process. N. of N. Nucleus of neurilemma. S. F. Collaterals. Tel. The ending, in striped muscle-fiber, M'. M. Medullary sheath. N. The nucleus, and N' nucleolus of the N. R. Node of Ranvier. ganglion-cell. 222 STUDENTS HISTOLOGY TYPE I. The cell has, besides the protoplasmic processes, an axis - cylinder process which becomes continuous with the axis- cylinder of a nerve -fiber usually having a considerable length. Minute offshoots are given from the axis - cylinder process, which are called collaterals (Fig. 139, A). TYPE II. In this case the axis -cylinder process divides repeat- edly and soon after leaving the ganglion -cell, forming a network within the nerve -center (Fig. 139, B). A ganglion -cell with its axis -cylinder process is called by many writers a neurone. In the case of some of the medullated nerve- fibers proceeding from the cerebro - spinal centers, the terminal distribution of the axis -cylinder may be as much as a meter distant from the ganglion -cell (Fig. 140). NEUROGLIA The brain and spinal cord have a supporting framework of ordinary connective tissue which enters at the surface from the pia mater. They possess, besides, a special form of supporting tis- FIG. 141. NEUROGLIA FROM BENEATH THE PIA MATER OF THE SPINAL CORD. (X 400.) A. Network of neuroglia-fibrils. B. Spider (Belter's) cells. C. Nerve-fibers in T. S. sue called neuroglia. It ctmsists of a fine reticulum produced by the interlacing of large numbers of delicate processes, which arise from neuroglia cells. These cells have a stellate outline, and, with their TEE PERIPHERAL NERVE-ENDINGS 223 numerous processes, suggest a spider and its legs (Figs. 139 and 155). Some forms are described as "moss -like." Neuroglia- cells are well shown in silver -stained specimens. The neuroglia of the spinal cord is intimately related with the epithelium lining the central canal, from which it originated. Neuroglia, unlike the other supporting tissues, is derived from the ectoderm. THE PERIPHERAL NERVE-ENDINGS The terminal branches of the nerves are so complex in struc- ure and so difficult of demonstration that only a few of the most important can be considered in this work. Sensory nerves sometimes present free endings, as in the strati- fied epithelium of the epidermis and cornea. The medullated nerve- fiber loses its medullary sheath at a node of Ranvier, and divides repeatedly, making a plexus of minute fibrillae in the connective tissue, or just below the epithelium, or between the epithelial cells. Special sensory endings are more complex in structure. Examples are found in the tactile corpuscles, which occur in the papillae of the skin. They are oval bodies having numerous nuclei. TERMINATION OP NERVE -FILAMENTS IN THE EPITHELIUM OF THE CORNEA. (RANVIER.) One or more medullated nerve -fibers enter at the base, losing their medullary sheaths. The axis -cylinders break up into fibrils, which here and there present expansions. The Pacinian bodies or corpuscles of Vater are easily found in the mesentery of the cat, where they are numerous and visible to the unaided eye. They are oval in form and nearly transparent. The most prominent part is the capsule, which consists of lamellas of connective tissue, which are concentric, and resemble the layers 224 STUDENTS HISTOLOGY FIG. 143. TACTILE CORPUSCLE FROM THE SKIN OF THE PALMAR SURFACE OF THE INDEX FINGER OF MAN. (RANVIER.) a. Terminal nerve-fibrils, n. Afferent nerve. Fia. 144. PACINIAN CORPUSCLE FROM THE MESENTERY OF THE CAT. (RANVIER.) c. Capsule. m. Inner bulb. f, n. Afferent nerve. NERVE-ENDINGS IN MUSCLE 225 of an onion. A medullated nerve -fiber enters at the bottom, and soon loses its medullary sheath. The free axis -cylinder terminates in a rounded or bulb -like extremity, surrounded by a homogenous substance, the inner bulb. The special form of nerve -endings, called taste-buds, have already been described (page 149). NERVE -ENDINGS IN MUSCLE The plexuses of non - medullated nerves supplying non- striated muscle have often been referred to (pages 154 and 160). From these plexuses minute fibrillae extend among the muscle -cells. Their exact mode of termination has not been definitely determined. FIG. 145. NERVE-ENDING IN STRIATED MUSCLE-AFTER KUHNE. The medullated nerves supplying striated muscle form an intra- muscular plexus. Bundles of nerve -fibers start from this plexus, one nerve -fiber going to each muscle -fiber. The medullary sheath is lost. The axis -cylinder breaks up into twisted fibrillae, with bulbous ends. These constitute the end -plate, which probably lies below the sarcolemma, and is imbedded in nucleated protoplasm, the sole -plate. 226 STUDENTS HISTOLOGY SPINAL CORD The membranes covering the spinal cord will be discussed later; see page 235. The spinal cord is composed of gray matter (cellular) and white matter (consisting of nerve-fibers), and serves as a medium of communication between the brain and the peripheral nerve- apparatus. The arrangement of its several parts will be best understood by the study of a transverse section, of which Fig. 146 is a diagrammatic representation. The gray substance occupies the central portions of the struc- FIG. 146. DIAGRAM. CERVICAL SPINAL CORD IN TRANSVERSE SECTION. A. Anterior median fissure. B. Posterior median fissure. C. Anterior or ventral horn. D. Posterior or dorsal horn. E. Point of emergence of anterior root of spinal nerve. F. Posterior root of spinal nerve. G. White commissure. H. Anterior gray commissure. I. Posterior gray commissure. J. Substantia gelatinosa Rolandi. The tracts which are named on the diagram have no definite boundaries histologically. They are physiological areas. SPINAL COED 227 ture, and consists of two lateral masses and a connecting link, or commissure. Near the central portion of the figure, a small cir- cular opening occurs — the transversely divided central canal. FIG. 147. HUMAN SPINAL CORD FROM THE DORSAL REGION, STAINED BY THE WEIGERT-PAL METHOD. SLIGHTLY MAGNIFIED. PHOTOMICROGRAPH. is in communication, in the medulla, with the fourth ventri- cle, and will serve as a starting point for our study. The gray matter completely surrounds the central canal, and its outline resembles the capital H, or a pair of crescents with their concavities looking outwards. The anterior (or ventral) horns or cornua are blunt. The posterior (or dorsal) horns are pointed. There are lateral projections from the anterior horns, sometimes called lateral horns. They lie opposite the central canal, and are most marked in the upper thoracic region. The crescents are connected, a portion of the connecting substance passing in front and a portion behind the central canal — the anterior and posterior gray commissural bands. The amount of gray matter is greatest in the cervical and lumbar enlargements of the cord (Fig. 148). The white substance is divided anteriorly by the anterior median fissure, which cuts into the cord nearly to, but not quite as far as the anterior gray commissure. A corresponding division appears posteriorly (the posterior median fissure), which does not divide the cord posteriorly, but the division is indicated by a band of pia mater, which penetrates entirely to the posterior gray com- missure. The two masses of white substance thus indicated are roughly divided into anterior, lateral, and posterior columns by 228 STUDENTS HISTOLOGY the horns of gray matter. They are united just in front of the anterior gray commissure by white matter — the white commissure. The spinal nerves take origin from the gray cornua, the anterior roots from the anterior, and the posterior roots from the posterior cornua. The white substance consists essentially of medullated nerve -fibers which, with the exception of the anterior spinal nerve -roots and the commissural fibers, pass mainly in a longi- tudinal direction. The anterior,/ lateral, and posterior columns into which the white matter isi primarily divided, may be divided secondarily FIG. 148. HUMAN SPINAL CORD FROM THE LUMBAR REGION. STAINED BY THE WEIGERT-PAL METHOD. SLIGHTLY MAGNIFIED. PHOTOMICROGRAPH. into certain other columns or tracts which are indicated in Fig, 146. These tracts are often called by the name of the discoverer. The principal ones are as follows : The direct pyramidal tract: Tiirck. The ascending antero -lateral tract: Gowers. The descending antero -lateral tract. The crossed pyramidal tract. The direct cerebellar tract. The postero- external tract — funiculus cuneatus: Burdach. The postero -internal tract— funiculus gracilis: Goll. The demonstration of the different tracts we owe partly to pathology, since in certain diseases' definite tracts may be involved throughout the length of the cord, while the others may be exempt. The alterations which ensue in the diseased parts make it easy to trace such tracts. SPINAL COED 229 Embryological study has shown also that the medullary sheath appears at different periods of development in the different tracts of nerve-fibers, although the time is constant for each particular tract. The presence of the sheath in certain tracts, and its absence in others, makes it possible to outline the tracts in a series of embryonic cords. Physiological experiments demonstrate the functions belonging to certain tracts. PRACTICAL DEMONSTRATION Nerve-tissue should, under all circumstances, be hardened in Miiller's fluid. The cord should be obtained as nearly fresh and uninjured as possi- ble, cut transversely with a sharp razoj1 into pieces a centimeter long, and placed immediately in the fluid— in the proportion of a liter of the mixture to 100 grams of tissue. The solution should be thrown away after twenty- four hours, and a fresh supply provided. It should be again changed after three days, and again after another week. After four weeks the bichromate should be poured off, and the tissue rinsed once with water, after which the hardening is to be completed with alcohol in the ordinary manner. After hardening, pieces from the different regions should be cut, and this is best effected after imbedding. Transverse sections are the most[ instruc- tive, although the student should afterward study longitudinal sectiobs. The sections may be stained with hsematoxylin and eosin. Sections should also be stained by the Weigert-Pal method. For this purpose the hardening must be done with great care, and must be continued longer. The pieces of tissue are placed in alcohol direct from Miiller's fluid without washing. The details of the method are given on page 30. The Golgi method presents too many difficulties to be attempted by any but advanced students. HUMAN SPINAL CORD — CERVICAL REGION — TRANSVERSE SECTION (Fig. 149) OBSERVE : (L.) 1. General arrangement of gray and white substance, with the latter surrounding the former, which resembles in outline the letter H. 2. Subdivisions of white substance. (a) Anterior median fissure. (Note its passage inward and its cessation before reach- ing the gray substance.) (b) Posterior median fissure. (Note its shallowness as a true fissure, and the extension of the connec- tive tissue from the bottom inward, until the gray substance is met. Compare the two median fissures.) (c) The emergence of the anterior nerve-roots. (This provides the external or lateral 230 STUDENTS HISTOLOGY boundary of the anterior white columns, the internal boundaries being provided by the anterior median fissure.) (d) The lateral columns. (These contain the fibers of the crossed pyramidal tract, and include the white substance between the anterior nerve -roots and the posterior gray cornua. Each lateral column contains nerve-fibers which pass to the cerebellum — direct cerebellar tract ; observe that these tracts have no internal histological boundary. Note the numerous prolongations of the pia mater inward in the lateral columns and blood-vessels in them, (e) The postero-in- PIG. 149. TRANSVERSE SECTION OP THE SPINAL, CORD. MIDDLE CERVICAL REGION (X 60). A. Anterior. B. Posterior. This section was made from the cord of a man who died at the age of 75 years, from senile dementia. The gray substance appeared normal, but of somewhat diminished area. ternal or column of Goll—funiculus gracilis. (These columns occur on either side of the posterior median fissure, and are bounded laterally by a prolongation from the pia mater.) (/) The postero-external column — funiculus cuneatus. (Bounded internally by the postero- internal column, and externally by the posterior gray cornu.) (0r) The white commissure. (Note the SPINAL CORD 231 absence of a white commissure posteriorly, the posterior median septum reaching the gray substance.) 3. Subdivisions of the gray substance, (a) The central canal. (Its size and shape vary a good deal at different levels.) (b) The gray commissures, anterior and posterior, (c) The gray columns, (d) The anterior gray cornua, broad and not reaching the periphery of the cord section, (e) The posterior cornua, narrow and passing completely out, posteriorly, to form the pos- terior root of a spinal nerve. (H.) 4. The white substance. (Select a field, e. g., in the anterior PIG. 150. SAME SPECIMEN AS SHOWN IN FIG. 149. MORE HIGHLY MAGNIFIED. REGION OF ANTERIOR CORNU (X 350). A. Medullated filaments passing out from the gray substance to form the anterior root of a spinal nerve. B. Ganglion-cells. C. Neuroglia-nuclei. I). Blood-vessels. E. One of the transversely divided medullated fibers of the white substance, anterior to the anterior gray cornu. The line leads to the neurilemma. F. White substance of Schwann— of E. G. The axis-cylinder of E. median column, and observe the transversely divided nerves.) (a) The nerves are not collected into funiculi, but each fiber pursues an independent course, (b) The axis-cylinders, which suggest cell-nuclei. (Note the great variation in size.) (c) Most of the axis -cylinders surrounded by more or less concentric rings 232 STUDENTS HISTOLOGY of translucent, unstained white substance of Schwann. (These are medullated fibers. In the spinal cord a neurilemma is wanting for these fibers. With the Weigert-Pal method the medullary sheaths are stained black and their course is easily traced.) (d) The small deeply haematoxylin- stained cells of the neuroglia. (e) The neuroglia-substance, finely granular or fibrillated, be- tween the nerve -fibers. (/) The spider cells (Deiter's) of the neuroglia. (These are not numerous, but easily found near the periphery.) (g) The longitudinal nerve-fibers passing from the anterior gray corau to form the anterior root of a spinal nerve. (h) The different size of the nerve-fibers in different areas of the section. Note the small fibers of the postero- internal column. With certain exceptions, the larger fibers belong to motor tracts and the smaller fibers to sensory tracts, (i) The blood-vessels. (These vessels are largely confined to the fibrous septa, which pass in from the pia.) 5. The gray substance, (a) The central canal. (The canal is lined with columnar ciliated cells in a single layer. The cilia are rarely demonstrable in the human cord, except in children. The central canal in adults is often partly occluded. Observe the clear, homogeneous ground-substance, — substantia gelatinpsa centralis. Compare it with a similar mass covering the posterior horn, — the substantia gelatinosa Rolandi.) (&) The ground - substance. (This consists, first, of exceedingly minute fibers, formed by the repeated subdivision of the axis-cylinders — the primitive fibrillae ; second, of the delicate neuroglia-fibers. It is usually difficult in a section to differentiate between the two. The details can only be made out in preparations stained with silver by Golgi's method.) (c) Large ganglion-cells. (In the anterior horn. The straight, unbranching axis -.cylinder process can frequently be distinguished. Note the large, shining nucleus and the deeply stained nucleolus. These cells are frequently deeply pigmented. They may be divided into two or three separate groups.) (d) Small ganglion-cells. (Best seen in the posterior horn. In the dorsal cord a collection of medium sized cells appears at the point where the gray commis- sure joins the posterior horn, — the column of Lockhart Clarke.) (e) The lateral horn also contains small ganglion -cells. Occa- sional outlying ganglion -cells appear in the white matter of the antero-lateral and posterior columns. (/) Pericellular lymph- spaces. '(Observed as a somewhat clear space around the ganglion- cells.) (g) Blood-vessels. (These are much more numerous here SPINAL CORD 233 than in the white portion ; and arteries of considerable size may frequently be found.) (h) Peri-vascular lymphatics. (Find an artery in transverse section, and observe the clear space around it, which may be mistaken for the result of contraction of the tissue in hardening. Careful study will reveal minute branches of cells, passing between the adventitia of the blood-vessel and the wall of the lymph -space.) The course of the axis-cylinders in the cord, and their relations with the ganglion-cells of the gray matter, are extremely intricate. The Gelgi method, in the hands of Ramon y Cajal and others, has been the means of clearing up many of the obscure parts of this subject. The limits of the present work will only permit of FIG. 151. LARGE GANGLION-CELLS OF THE ANTERIOR HORN OF THE SPINAL CORD. Low POWER. PHOTOMICROGRAPH. allusion to a few of the most important facts. In this connection, it is well to recall the description and diagram of a neurone (page 222). The central nervous system is supposed to consist of many neurones. According to this theory, several neurones may operate together, one superimposed on another. It is to be remembered that the processes of ganglion -cells probably do not anastomose with those of other ganglion -cells. The large ganglion-cells of the anterior horns of gray matter give off axis -cylinder processes to the anterior motor nerve-roots. Some ganglion -cells (column cells) have axis -cylinder processes which run vertically in the white matter of the anterior and lateral 234 STUDENTS HISTOLOGY FIG. 152. DIAGRAM OF THE RELATIONS OF THE CELLS AND FIBERS OF THE SPINAL CORD. (BAKER, AFTER LENHOSSEK.) The right side shows the cells of different classes found in the cord, and their processes. The left side gives the processes of cells whose bodies are either beyond the cord or at other levels, with the distribution of their collaterals. a, a. Motor cells of the anterior horn. c. Commisstiral cells. d. Golgi commissural cell. e. e. Columnar cells of antero-lateral column. f. f. Columnar cells of posterior column. g. Golgi cell of posterior horn. 1. Fibers of posterior root forming the antero-posterior reflex tract. 2. Fibers passing to the column of Clarke. 3. Commissural fibers of posterior root. 4. Fibers that enter the posterior horn, k, k. Collaterals of antero-posterior column. 1, 1. Collaterals from the pyramidal tracts. columns. Commissural cells give off axis-cylinder processes to the opposite side of the cord, by way of the anterior gray commissure. The axis -cylinders of some ganglion -cells terminate in the gray matter itself. The fibers of the posterior nerve-roots, arising from the cells of the spinal ganglia, divide into ascending and descend- ing branches, which enter the posterior columns. The difficulties in the way of tracing the paths of the fibers in the cord are enhanced by the numerous collaterals arising from many axis -cylinders. THE BRAIN AND ITS MEMBRANES 235 THE BRAIN AND ITS MEMBRANES le brain and spinal cord are surrounded by three connective tissue layers — the dura mater, the arachnoid, and the pia mater. The dura mater is the most external and the thickest of the three membranes, and constitutes the periosteal lining of the cranial cavity. It consists largely of elastic tissue, the laminae and blood- vessels of which are supported by connective tissue. The outer surface is in more or less intimate connection with the bone ; the inner surface is covered with a single layer of thin endothelial cells. Beneath is a space — the subdural — containing lymph. The arachnoid, exceedingly thin, presents an outer glistening surface, covered with a layer of endothelial cells. It is devoid of blood-vessels and nerves. It is separated from the dura by the subdural space, from the under (inner) side of which short, fibrous trabeculaa are projected to the pia. Other trabeculaa attach it loosely to the dura mater. Villous projections from the arachnoid enter the subdural space. Near the longitudinal fis- sure they are large and encroach upon the dura mater, forming the Paccliionian bodies. The subaraclinoidal space is thus seen to consist of numerous communicating chambers, and these spaces are everywhere lined with flat cells, and contain lymph, as does the subdural space. The pia mater consists of fibrillated connective tissue, usually in intimate connection with the arachnoid externally, by means of the trabeculae of the latter. The pia mater is exceedingly vas- cular, and everywhere covers the brain and cord; and, unlike the arachnoid, penetrates the sulci of the former and the fissures of the latter, becoming continuous with the connective tissue. The outer surface of the pia mater is also covered with flat eriftothe- lial cells. The subdural and subaraclinoidal spaces are lymph -cavities, and, while not in direct connection one with the other, belong to the general lymphatic system, and are in eventual connection. The arrangement of gray and white nerve -substance in the brain is precisely the reverse of that of the cord. The gray matter forms an external covering or layer of varying thickness, while the white matter occupies the more central regions. Collec- tions of gray matter— the basal ganglia — are also situated in the 236 STUDENTS HISTOLOGY deeper parts of the brain -substance, the study of which does not come within the limits of this work. The brain -substance does not differ essentially from the cord, except in the arrangement of its parts. The nerve -fibers are I iM-i ,j ntL ii , n )• FIG. 153. LAYERS OF THE HUMAN CEREBRAL, CORTEX. (MEYNERT.) The numbers refer to the layers given in the text, page 238. mostly medullated, but have no neurilemma. The gray substance is arranged in five layers, which are in some instances quite sharply defined, and oftener demonstrable only with considerable difficulty. Transversely running bands of medullated nerve -fibers THE CEREBRUM 237 (the stripes of Baillarger) have sometimes been counted as addi- tional layers. PRACTICAL DEMONSTRATION The tissue is to be prepared in the manner usual with nerve -substance —hardened with Miiller, followed by alcohol. Thin sections, stained deeply with haematoxylin and eosin, may be mounted in balsam. Sections stained by Golgi's method should be studied if possible. SECTION OF HUMAN CEREBRUM— CUT PERPENDICULARLY TO THE SURFACE (Fig. 154) OBSERVE : (L.) The membranes. (In the drawing only the arachnoid and pia are shown.) (a) The fine fibrillar mesh of the arachnoid. (&) The nuclei of the flattened cell-covering, (c) The large Fia. 154. VERTICAL, SECTION OF CEREBRAL CORTEX. SUPERIOR FRONTAL CONVOLUTION (X 250). A. Arachnoid. B. Pia mater. 1, 2, 3, 4, 5. First, second, third, fourth, and fifth layers of gray matter. 6. White matter. 238 STUDENTS HISTOLOGY blood-vessels. 00 The pia. (e) Its continuity with the con- nective tissue of the cerebrum. 1. The outer layer — the first — of the gray substance. (This layer is poorly defined, but can usually be made out. It consists of primitive nerve -fibrillae, neuroglia -fibrils, and scattered gan- glion-cells. A few medullated nerve -fibers run horizontally — tangential fibers.) 2. The second layer. (This layer presents about the same thickness as the preceding, and will be recognized by the abun- dance of small triangular nerve -cells. From Golgi preparations it appears that numerous protoplasmic processes pass peripherally, while the axis -cylinders arise from the bases of the small pyramidal ganglion-cells.) 3. The third layer. (This layer— the thickest of all the gray laminae — is called the formation of the cornu Ammonis [Mey- nert] . The large pyramidal cells have numerous protoplasmic processes arising from their sides and prominent ones from the apices, while the axis -cylinders are given off from the blunt bases to enter the white matter [Fig. 155] . Medullated fibers, in more or less distinct bundles, pass between the column -like ganglion- cells.) 4. The fourth layer. (The large cells of the third layer are seen to stop, as we pass inward, and give place to small, irregu- lar nerve -cells, called the granular formation. Between the cells of this layer bundles of nerve -fibers are seen, as they radiate toward the cerebral surface.) 5. The fifth layer. The line of demarcation between this and the fourth layer is feebly shown; but, on close attention, it will be observed that the small cells of the fourth layer rather abruptly give place to spindle-shaped ones, sometimes parallel with the sur- face. The nerve-bundles are here more plainly indicated. 6. The white matter. (The ganglion -cells cease here, and the field is occupied with medullated fibers and neuroglia, the spherical nuclei of the latter becoming prominent from the deep haematoxy- lin staining.) 7. The nutrient blood-vessels. (The capillaries projected from the pia are especialty to be noticed, often of the diameter of a single blood -corpuscle, and appearing as branching lines, composed of these elements — indeed difficult of demonstration when empty. Note the light, perivascular lymph -spaces, well seen around the larger arteries in transverse section.) THE CEREBRUM 239 Certain concentrically striated bodies, corpora amylacea, are often found in the vicinity of the ventricles and along the olfac- tory tract, as well as in other localities. After treatment with iodine solution and sulphuric acid they* take a violet color, there- fore resembling starch in their reactions — hence their name. The arrangement of the layers of the gray matter is subject to considerable modification in different parts of the cerebrum. As in the spinal cord, the difficulties of tracing the course of the nerve- FIG. 155. PYRAMIDAL GANGLION-CELL AND NEUROGLIA-CELL FROM HUMAN CERE- BRAL CORTEX PREPARED BY THE GOLGI METHOD. a. Axis-cylinder process of the ganglion-cell. fibers are increased by the numerous collaterals given off from the axis -cylinder processes of the ganglion -cells. The paths followed by the fibers of the white matter are very complicated. The tracts have been mapped out largely by prepa- rations made by Weigert's method and its modifications. In gen- eral these fibers may be divided into three groups: a. Projection fibers, which constitute the corona radiata, pass- ing from the peduncles of the cerebrum and the basal ganglia to the cortical gray matter. b. Association fibers, which bring parts of the same hemis- phere into relation with one another. c. Commissural fibers, which connect the opposite hemispheres through the corpus callosum and the anterior commissure. 240 STUDENTS HISTOLOGY VERTICAL SECTION OF HUMAN CEREBELLUM Practical Demonstration Cut large sections of cerebellum, hardened with Miiller's fluid and im- bedded as usual ; cut so as to show the ramifications of the arbor vita? ; stain with haematoxylin and eosin. Also stain the cerebellum of a young dog or kitten, prepared according to Golgi's method (page 33), being careful to cut the sections at a right angle to the long axis of the convolutions. OBSERVE : (Fi«s- 156 and 157> (L.) 1. The arrangement in the form of leaflets. 2. The extension of the gray laminae within even the mi- nutest folds of the leaves, so as to completely envelop the central white nerve -substance. (The staining has been so selected by the FIG. 156. LONGITUDINAL SECTION OF ONE OF THE FOLIA OF THE CEREBELLUM (X60). A, A. Line of pia mater. B, B. Sulci. C, C. Outer layer of gray matter. D, D. Inner layer of gray matter, including Purkinje's cells. E, E. White nerve-substance. THE CEREBELLUM 241 FIG. 157. VERTICAL SECTION, CORTEX OF CEREBELLUM. PORTION OF SECTION SHOWN IN FIG. 156, MOKE HIGHLY MAGNIFIED (X 250). A. Outer layer of gray matter. B. Layer of Purkinje's cells. C. Inner gray layer. D. White nerve-substance. tissue as to divide the outer gray matter into two prominent layers. The explanation of this will follow increased amplification.) 3. The central white matter. (The fibrillar character can be made out, and the general plan will be found to consist, as in the cerebrum, of central nerve -fibers radiating toward the cells of the cortical gray substance, the arbor vitae.) (H.) 4. The outer gray layer, or molecular layer. (This is the thickest of the three layers. The prominent elements to be ob- served are : the scattering neuroglia- and ganglion-cells, nerve- fibrils, and blood-vessels, which pass in from the pial invest- ment.) The ganglion -cells of the molecular layer are of two sorts, small and large. The large cells are remarkable for their axis -cylinder processes, which at intervals give off branches whose 242 STUDENTS HISTOLOGY fine subdivisions form a basket-like network about the bodies of the Purkinje cells of the underlying layer. The arborizations of these Purkinje cells spread out in the molecular layer. 5. The middle layer is a thin stratum directly beneath the outer layer. The section becomes deeply stained, from the pres- ence of numerous small cells, among and partly concealed by which are the very large ganglion-cells of Purkinje. These are flask -shaped, and are arranged in a single plane, with their long axes placed vertically. A thread-like prolongation may be seen FIG. 158. CELL OF PURKINJE. HUMAN CEREBELLUM. GOLGI METHOD. penetrating the layer beneath, providing the cell has been cen- trally sectioned. This is the axis -cylinder process, which gives off certain collaterals and becomes the axis -cylinder of a medullated nerve -fiber. Two thick protoplasmic processes project from the outer end of the cell, which ramify in the molecular layer. Their arborizations are luxuriant, and the branchings of the various cells have been likened to a forest. Considering the large size of the cells of Purkinje, their axis -cylinder processes, the richness of the branches of their protoplasmic processes, and the curious basket- work of nerve -fibrils around the cell -bodies, they are among the THE CEREBELLUM 243 most striking of the objects that have been revealed by the Golgi method. (Fig. 158.) The branches of these cells spread out chiefly on planes at right angles to the long axis of the convolution. Sections should there- fore be made across the convolutions. 6. The granular layer. (This is the layer seen so distinctly with the low -power. It consists of innumerable small bodies, deeply stained with haematoxylin, usually spherical, which are ganglion- and neuroglia- cells. The ganglion -cells belong to the second type. The majority of these cells are small. A few are large. The small cells have protoplasmic processes Which ramify in their own granular layer, and axis -cylinders which terminate in the outer molecular layer. On the other hand the large ganglion - cells of the granular layer have protoplasmic processes which extend into the molecular layer, while the axis -cylinder processes ramify in the granular layer. Medullated nerve -fibers from the white matter form a dense plexus in the granular layer. Some of these fibers are continued into the molecular layer. Search care- fully for the axis-cylinder processes of the Purkinje cells, which pierce this layer, and follow them into the white matter below.) 7. The white substance consists of medullated nerve -fibers. INDEX Abbe" condenser, 1, 4. Abdominal cavity. See Peritoneum. salivary gland, 145. Absorption from intestine, 158. of fat, 159. Acid, acetic, 39. alcohol, 29, 39. aniline dyes, 31. chromic, 24. fuchsin, 31, 32. hydrochloric, 25, 29, 39. nitric, 24, 75, 137. osmic, 23. picric, 23, 29, 31, 32. Acidophile granules, 31. Acini of glands, 140. Acinous glands, 143. Acoustic nerve, 216. Adenoid reticulum, 76, 113. tissue, 76, 111. See Lymphoid tissue. Adipose tissue, 65. Adventitia of blood-vessels, 103, 104. Agents, staining, 27. Agminate glands, 160. Ailantus pith, 13. Air-bubbles, 46. -sacs, 128. -vesicles, 128. Alcohol, acid, 29, 39. fixation and hardening, 21. dehydration with, 35, 38. Alimentary canal, 149. Alum-ha3matoxylin, 28. Alveoli of gland, 140. of lung, 128. Amoeboid movements, 83, 104. Amphophile granules, 31. Aniline colors, dyes, etc., 31. dyes, staining with, 31. oil, 35. Anterior commissure, 227. median fissure, 227. Antero-lateral tracts, 228. Aorta, 104. Appendages of skin, 95. Appendix, vermiform, 162. Aponeuroses, 62. Arachnoid, 235. Arbor vitro, 241. ^.reolar tissue, 62. Arrectores pili, 96. Arrowroot-starch, 48. Arteries, 102. large, 103, 104. lymphatics of, 107. small, 103. Arteriolae rectee of kidney, 184. Arterioles, 102. Artery, bronchial, 128. hepatic, 164. pulmonary, 128. renal, 183. typical, 103. Articular cartilage, 68. Artificial gastric juice, 25. Asphaltum varnish, 36. Association fibers, 239. Attraction-spheres, 53. Auerbach's plexus, 154, 160. Axis-cylinder, 216.. processes, 220. collaterals, 222. Bacteria, hardening tissues containing, 22. under the microscope, 47. Baillarger's stripes, 237. Balsam, Canada, 35. Basement membranes, 55. Basic aniline dyes, 31. Basophile granules, 31, 87. Beaker cells. See Goblet cells. Bellini, tubule of, 182. Bergamot oil, 35. Bertini, columns of, 179. Berlin blue, injecting, 34. Bichromate of potassium, 22. Bile-capillaries, 166. -ducts, injection of, 176, 177. Bipolar nerve-cells, 220. Birds, blood of, 82. Bladder, gall-, 177. f (245) 246 INDEX Bladder, urinary, 194. Blastodermic layers, j54. Blood, 81. colorless corpuscles, 83. -corpuscles as a standard of meas- urement, 8. -corpuscles, cover-glass prepara- tions, 84. -corpuscles, crenation of, 82, 90. -corpuscles, double staining, 84. -crystals, 89. effect of reagents upon, 90. . effects of acetic acid, 91. effects of osmic acid, 82. effects of tannic acid, 91. effects of water, 90. [32, 85. Ehrlich-Biondi-Heidenhain stain, embryonal origin of, 91. enumeration of corpuscles, 87. fibrin, 90. fixing and staining, 84. haemin, 90. haemoglobin, 89. haemosiderin, 89. haematoidin, 89. human, 81. leucocytes, 83. of birds, 82. of camelidae, 82. of fishes, 82. of frog. 90. of invertebrates, 82. of lamprey, 91. of reptiles, 82. origin of colored corpuscles, 91. origin of white corpuscles, 87. oxygenation of, 89. -plates, 82. red corpuscles, 81. size of corpuscles, 81. tricolor or triacid stain, 32, 85. -vessels, 102. -vessels, capillary, 103. -vessels, development of, 105. -vessels, injection of, 34. white corpuscles, 83. Bodies, Malpighian of kidney, 180. Malpighian of spleen, 118. Pacchionian, 235. suprarenal, 213. thymus, 121. thyroid, 148. of Langerhans, 148. Bone, 70. circumferential lamellae, 72. Bone, compact, 72. cancellous, 72. -corpuscles, 72. decalcification of, 24, 75. development of, 73. Haversian canals, 71. Haversian lamellae, 72. interstitial lamellae, 72. -lacunae, 70. -marrow, 73. osteoblasts, 73. osteoclasts, 73. perforating fibers of Sharpey, 71. periosteum, 73. spongy, 72. varieties of, 72. Borax-carmine, 29, 39. Boundary region of kidney, 187. Bowman, capsule of, 180. Bowman's muscle-disks, 80. Brain, 235. See Cerebrum. Branched tubular glands, 143. Bronchial artery, 128. tube, 123. Bronchi, 123. Bronchus of pig, 125. Brownian movements, 46. Brunner's glands, 157. Bubbles, air-, 46. Buccal epithelium, 56, 149. glands, 143. cavity, 149. Burdach's column, 228. Calcified cartilage, 74. Calcification, 73. Calyces of kidneys, 178. Canada balsam, 35. Canal, alimentary, 149. central, 227. dentinal, 135. Haversian, 71. of the epididymis, 210. portal, 165. Canaliculi, 70. Cancellous bone, 72. Capillaries, blood-, 103. bile-, 166. lymphatic, 106-110. Capillary bronchial tubes, 123. Capsule of Bowman, 180. of glands, 140. of Glisson, 163. of kidney, 178. of lymph-nodes, 112. INDEX 247 Capsule of spleen, 117. of thymus body, 121. suprarenal, 213. Carbol-turpentine, 35. -xylol, 35. Carbon dioxide freezing, 20. Cardiac glands, 152. muscle-fiber, 80. Care of miscrocope, 43. Carmine and picric acid, 29. Grenadier's borax-, 29. injection, 34. staining, 39. Cartilage, 67. articular, 68. arytenoids, 123. -corpuscles, 67. elastic, 68, 123. fibro-, 68. hyaline, 67. -lacunae, 67. of epiglottis, 123. of larnyx, 123. of Santorini, 123. of trachea, 68, 123. of Wrisberg, 123. perichondrium of, 67. -plates in bronchi, 124. reticular, 68, 123. varieties of, 67. Cavity, abdominal. See Peritoneum, pericardial. See Pericardium, thoracic or pleural. See Pleura. Cedar-wood oil, 4. Cell, 49. -cement, 50. distribution, 50. division, 51. typical, 49. -wall, 49. Celloidin imbedding, 26. Cells, acidophile, 31. amphophile, 31. basophile, 31, 87. bipolar nerve-, 220. blood-, 81. border, 153. central, 153. chief, 153. Deiter's, 222. endothelial. See Endothelium. eosinophile, 31, 86. epithelial. See Epithelium, ganglion-, 219. giant. See Giant cells. Cells, goblet. See Goblet cells. lymphoid. See Lymphoid cells, mucous. See Goblet-cells, multipolar nerve-, 220. nerve-, 219. neutrophile, 31, 86. neuroglia, 222. of Purkinje, 242. outlining of by silver nitrate, 33, 60. parietal, 153. pigment-, 93. prickle, 92. spider, 222. typical, 49. unipolar nerve-, 220. wandering, 83, 104. Cement-substance, 62, 104, 108. of tooth, 137. zinc, 36. Central canal of spinal cord, 227. nervous system, 226-243. Centrosome, 53. Cerebellar tracts, direct, 228. Cerebellum, 240. cells of Purkinje, 242. granular layer, 243. molecular layer, 241. Cerebral cortex, layers of, 238. Cerebrum, 236. association fibers, 239. blood-vessels, 238. commissural fibers, 239. nerve-fibers of, 239. practical demonstration, 237. projection fibers, 239. tangential fibers, 238. pyramidal cells of, 238. Cervix uteri, 198. Chalice cells. See Goblet cells. Chamois leather, 43. Channels, lymph-. See Lymphatics, blood-. See Blood-vessels. Chloroform, 25. Chromatin, 28, 51. Chromosomes, 52. Chromic acid, fixing and hardening, 24. Chromic-acetic solution of Flemming. 23. -osmic solution of Flem- ming, 23. Chyle-receptacle, 106. Ciliary motion, 59. Ciliated cells from oyster, 59. Circulatory system, 102. system, lymphatic, 106. Circumferential lamellae, 72. 248 INDEX Circumvallate papillae, 149. Clark's columns, 232. Classification of tissues, 53. • Cleaning cover-glasses, 41. lenses, 44. Clearing agents, 35, 38. Clove-oil, 35. Coarse adjustment, 1. Cohnheim's fields, 79. Coiled tubular glands, 140. Collaterals of axis cylinders, 222. Collecting tubule, 182. Collodion, 26. Colloid substance, 148. Colorless blood-corpuscles, 83, 104. Colors, aniline, 31. Colostrum-corpuscles, 208. Columns of the spinal cord. See Spinal cord. Columnar epithelium, 57. Columns, cortical, of Bertini, 179. Commissural fibers, 239. Commissure, anterior gray, 227. posterior gray, 227. white, 228. Compact bone, 72. Compound acinous glands, 143. racemose glands, 143. Condenser, Abbe, 1, 4. Coni vasculosi, 210. Connective tissue, 62. tissue, embryonic, 76. tissue, juice-canals of, 106. tissue, mucoid, 76. tissue, special, 76. Conservation of eyesight, 7. Constrictions of Ranvier, 216. Convoluted tubule, 181, 182. Copper sulphate, 24. Cord, spinal. See Spinal cord. umbilical, 76. Cords of lymphoid tissue, 113. Corium of skin, 92. Corn starch, 48. Cornea, nerves of, 223. Cornua of spinal cord, 227. Corpora amylacea of brain, 239. Corpus callosum, 239. Highmori, 210. luteum, 203. Corpuscles, blood-, 81. bone-, 70. cartilage-, 67. colostrum-, 208. connective tissue, 63. Corpuscles, Hassall's, 122. lymph-. See lymphoid cells. of Vater, 223. pus-, 84. Pacinian, 223. salivary, 47, 149. tactile, 94, 223. Cortex of cerebellum, 241. of cerebrum, 237. of ovary, 203. of kidney, 178. of lymph-node, 112. of thymus body, 121. Cortical columns of kidney, 179. Corundum hones, 16. Cotton fibers, 48. Cover-glasses, 41. Cover-glass, placing of, 42. preparations of blood, 84. Cox's Golgi method, 34. Crenation of blood-corpuscles, 82. Creosote, 35. Crossed pyramidal tracts, 228. Crusta petrosa, 135. Crypts of the tonsil, 149. of Lieberkuhn, 156. Crystals of Teichmann, 90. Cul-de-sac of vagina, 198. Currents, thermal, 46. Cuticle of hairs, 95. Cutis anserina, 96. Cuticula of tooth, 135. Cutting sections, 10. Cylinder, axis-, 217. Cyclostomi, blood of, 91. Dammar, 36. Decalcification, 24. of bone, 75. of teeth, 137. Dehydrating, 35, 38. Deiter's cells, 222. Delafield's hsematoxylin, 28. Demilunes of Heidenhain, 148. Demonstrations, practical. See Practical demonstrations. Dendrites, 220. Dentinal canals, 135. fibers, 135. sheath, 135. Dentine, 135. Derivatives of blastodermic layers, 54. Derma, 92. basement membrane, of, 94. Development of blood-vessels, 105. INDEX 249 Development of blood-corpuscles, 91. of bone, 73. Diapedesis, 104. Diaphragm, central tendon of, 108. Diaphragm of the microscope, 1. Direct cell division, 51. Direct cerebellar tract, 228. Direct pyramidal tract, 228. Digestion, method of, 25. Disk, intervertebral, 68. of Bowman, 80. Discus proligerus, 205. Dissociating fluids, 10, 25. Distal convoluted tubule, 182. Distribution, cell, 50. Division of cells, 51. Double staining, 38. Duct of glands, 140. hepatic, 163, 177. of thyroid body, 148. bile-, 176, 177. thoracic, 106, 107. Ductless glands. 117, 213. Dura mater, 235. Dust on lenses, 43. Duodenum, 162. Ear, cartilage of, 68. Ectoderm, derivatives of, 54. Egg-tubes, 206. [32. Ehrlich-Biondi-Heidenhain tricolor stain, Elastic cartilage, 68. fibers, 65. lamina of blood-vessels, 102, 103. tissue, 63. Elder-pith, 13. Eleidin, 92. Embedding. See Imbedding. Embryonic tissue, 76. Enamel of teeth, 137. prisms, 137. Endocardium, 102. Endochondral formation of bone, 73. Endomysium, 78. Endoneurium, 218. Endothelial cells. See Endothelium. Endothelium, 60, 103, 108. of blood-vessels, 103. staining of, 61. stomata of, 62. End-plates, 225. Entoderm, derivatives of, 54. Eosin, 28, 31. and haematoxylin, 38. Eosinophile granules, 31, 86. Epiblast, derivatives of, 54. Epidermis, 92. Epididymis, 209. Epiglottis, 68, 123. Epineurium, 218. Epithelial cells. See Epithelium. Epithelium, definition of, 54. buccal, 56. ciliated, 58. classification of, 55. columnar, 57. distribution of, 55. germinal, 203. glandular, 59. of bladder, 195. of ovary, 203, 206. of kidney, 182, 192. pavement, 56. prickle cells, 92. simple squamous, 56. stratified, 55. striated, 148, 192. squamous, 55. tessellated, 56. transitional, 55, 193, 195. uterine, 199. vaginal, 200. varieties of, 55. Equator of the cell, 52. Erectile tissue, 212. Erlicki's fluid, 24. Erythroblasts, 91. Ether freezing, 20. Eustachian tube, cartilage of, 68. Extraneous substances, 47. Eye-lens, 3. -piece, 3. Fallopian tube, 202. Fat, action of osmic acid on, 23. -cells, 65. -columns, 94. -crystals, 66. -globules in milk, 45. in the liver, 174. staining of, 23. -tissue, 65. Feathers, 48. Female generative organs, 197. Fenestrated membrane, 05, 103. Ferrein, pyramids of, 179. Fibers, association, 239. commissural, 239. cotton, 47. dentinal, 135. 250 INDEX Fibers, linen, 47. nerve-, 216. perforating of Sharpey, 71. projection, 239. silk, 47. tangential, 238. wool, 47. Fibrin, 90. Fibro-cartilage, 68. Fibrous tissue, elastic, 63. white, 62. Field lens, 3. of view, 5. Filiform papillae, 149. Fine adjustment, 1, 6. Fishes, blood of, 82, 91. [227. Fissure, anterior median, of spinal cord, posterior median, of spinal cord, Fixation of tissues, 20. [227. Fixing blood-elements, 84. fluids, etc., 20. Flemming's solutions, 23, 24. Fluid, artificial gastric, 25. dissociating, 10, 25. Erlicki's, 24. Flemming's, 23. Miiller's, 22. Orth's, 22. Stirling's, 25. Toison's, 87. Focal adjustment, 5. Focusing, 5, 45. Foetal blood, 91. blood-vessels, 105. bone, 73. lung, 134. Follicle of hair, 95. Follicles, Graafian, 203. of Lieberkiihn, 156. of lymphoid tissue, 113. solitary, 160. Foramen caecum, 148. Formaldehyde, 22. Form of objects, 45. Free nerve-endings, 223. Freezing microtome, 19. Fresh tissue, 20. Frog, blood of, 90. capillaries of, 105. mesentery of, 60. skin of, 56. Fuchsin, 31. acid, 31, 32. Fungiform papillae, 149. Funiculus cuneatus of spinal cord, 228. Funiculus gracilis of spinal cord, 228. Funiculi of nerve-trunks, 217 Gall-bladder, 177. -duct, 177. Ganglia of heart, 102. of urinary bladder, 195. Ganglion-cells. See Nerve-cells. Gastric fluid, artificial, 25. glands, 152. tubules, 152. Generative organs, female, 197. male, 209. Gentian violet, 31. Germinal epithelium, 203. spot, 205. vesicle, 205. Giant cells, 73, 120. Glands, 140. acinous, 143. agminate, 160. branched tubular, 143. Brunner's, 157. buccal, 143. capsule of, 140. cardiac, 152. coiled tubular, 140. compound racemose, 143. compound tubular, 143. ductless. See Ductless glands. gastric, 152. intestinal, 156, 157. Lieberkuhn, 156. lenticular, 156. Littre', 196. lymphatic. See Lymphatic nodes. mammary, 208. mucous. See Mucous glands. mucous, of bronchi, 127. Nabob's, 199. pancreas, 145. parenchyma of, 140. parotid, 143. peptic, 152. Fever's, 160. pyloric, 152. racemose, 143. salivary, 143, 148. sebaceous, 98. simple tubular, 140. sublirigual, 148. solitary, 160. submaxillary, 144. sudoriferous, 97. sweat-, 97. INDEX 251 Glands, tubular, 140, 143. thyroid, 148. Glandular epithelial cells, 59. Glassy membrane of hair, 95. Glisson's capsule, 163. Globus major, 209. minor, 209. Globules, oil-, 46. Glomerulus of kidney, 184. Glycerine in mounting, 36, 76. Glycogen in the liver, 174. Goblet cells, 58, 126, 127, 157. Gold-staining, 33. Golgi's method, 33. Goll, column of, 228. Gowers, column of, 228. Goose-flesh, 96. Graaflan follicles, 203. Gray commissure, 227. Gray matter of brain, 236. spinal cord, 226. Grenacher's borax-carmine, 29. Griibler's dyes, 31. Gum dammar, 36. Gun-cotton, 26. Haematoxylin, 28. and eosin, 38. Delafleld's, 28. staining process, 36. Weigert's, 30. Hfemin, 90. Haemoglobin, 89. Haemocytometer, 87. Haematoidin, 89. Haemosiderin, 89. Hair, 95, 99, 101. -follicles, structure of, 95. permanent mounting of, 99. Hardening, 20. Hassall's corpuscles, 122. Haversian canals, 71. lamella, 72. system, 71. Heart, 80, 102. blood-vessels of, 102. endocardium, 102. muscular tissue of, 80. nerves of, 102. pericardium, 102. valves of, 102. Heidenhain's demilunes, 148. Henle, loop of, 181. Hepatic duct, 163, 177. veins, 163. Highmori, corpus, 210. Histology, definition of, 53. Hilum of kidney, 178. of lymph-node, 113. of spleen, 117. Hones, 16. Horns of spinal cord, 227. Horny layer of skin, 92. Hyaline cartilage, 67. Hypoblast, derivatives of, 54. Ileum, section of, 161. Illumination, 4. Imbedding with ailantus pith, 13. with celloidin, 26. with paraffin, 13, 25. Immersion lenses, 4. Indirect cell division, 51. Inflammation, 84, 104. Infundibula of kidney, 178. of lung, 129. Injection, methods of, 34. Insects, 48. Intercellular substance, 50. Interglobular spaces, 137. Interlobular veins of liver, 167. vessels of kidney, 184. Intervertebral disks, 68. Interstitial lamellae, 72. Intestines, 151, 156. Brunner's glands of, 157. Lieberkuhn's follicles of, 156. mucosa of large, 162. mucosa of small, 156. Peyer's patches, 160. practical demonstrations, 160- solitary glands of, 160. [162. valvulae conniventes, 151, 162. villi of, 156. Intima of blood-vessels, 103. Intramembranous ossification, 73. Intralobular vein, 163. Involuntary muscle, 76. See Muscle, non-striated. Iodine solution, 29. Iris diaphragm, 1. Irregular tubule, 182. Japanese paper, 43. Juice, gastric, 152. Jelly of Wharton, 76. Juice-canals, 106. Karyokinesis, 23, 51. Keratin, 92. 252 INDEX Kidney, 178. blood-vessels of, 183. boundary region of, 187. Bowman, capsule of, 180. calyces of, 178. capsule of, 178. collecting tubes of, 182. columns of Bertini, 179. connective tissue of, 189. cortex of, 179. labyrinth of, 179. diagram of, 179. epithelium, 182, 192. Ferrein's. pyramids, 179. glomerulus of, 184. Henle's loop, 181. hilum of, 178. infundibula of, 178. labyrinths of, 179. lobules of, 178. Malpighian bodies of, 180. Malpighian pyramids, 178. medulla, 179. medullary rays of, 179. nerves of, 185. papillae of, 178. pelvis of, 193. practical demonstration of, 186. tubes of Bellini, 182. tubules, diagram of, 181, 193. tubules of, 180. Knives, sharpening, 16. Krause's membrane, 79. Labeling of slides, 42. Labyrinth of kidney, 179. Lacteals, 159. Lacunae of cartilage, 67. of bone, 70. Lamellae of bone, 72. Lamina, internal elastic, 103. Lamprey, blood of, 91. Large intestine, 162. Langerhans, bodies of, 148. Larnyx, 123. cartilages of, 68, 123. Lateral columns, 228. Layers of cerebrum, 238. of epidermis, 92. Lens, immersion, 4. microscope, 2, 3, 4. Lenticular glands, 156. Leucocytes, 83, 104. Lieberkuhn, crypts of, 156. Lifting sections, 41. Ligamenta subflava, 65. Ligamentum nuchae, 65. Light transmitted, 9. Limiting membrane, 49. Linen fibers, 47. Lines of Retzius, 137. Littre", glands of, 196. Liver, 163. cells of, 174. fat in, 174. Glisson's capsule, 163. glycogen in, 174. human, 171. of pig, 167. of rabbit, 174. practical demonstrations, 167, 170, scheme of structure, 104. [177. Lobes of glands, 140. Lobules of glands, 140. Logwood, 27. Loop of Henle, 181. Lung, 123. air-sacs, 128. blood-vessels of, 128. connective tissue of, 128. foetal, 134. infundibula of, 129. interlobular septa, 128. pigment within, 128. practical demonstration, 131. terminal bronchiole, 123, 132. vascular supply of, 128. Luschka's tonsil, 149. Lymph, 106. -corpuscles, 85, 106. -node, diagram of, 112. -node, practical demonstration of, -nodes, 111. [113. -spaces, 106, 107. -spaces of nerves, 219. [219. -spaces around ganglion-cells, Lymphatic capillaries, 106, 110. vessels, 107. glands. See Lymph-nodes, system, 106. tissue. See Lymphoid tissue. Lymphatics, 106, 107. practical demonstration, 108. perivascular, 107. valves of, 107, 109. Lymphoid cells, 85, 113. tissue, 76, 111. tissue, diffuse, 113. tissue distribution, 113, 118, 121, 123, 149, 156, 159, 195. Lymphocytes, 85. INDEX 253 Magnification of movements, 46. Magnifying power of objectives, 7. Male generative organs, 209. Malpighian stratum of epidermis, 92. Malpighian bodies of kidney, 180. bodies of spleen, 118. Malpighi, pyramids of, 179. Mammary glands, 208. Marrow, bone-, 73. Measurement of objects, 8. Media of arteries, 103. Mediastinum testis, 209. Medulla of bone, 73. of kidney, 179. of lymph-node, 112. of hair, 95. of thymus body, 121. Medullary cavity of bone, 74. rays of kidney, 179. Medullated nerves, 216. Meissner's plexus, 154, 160. Membrana granulosa, 205. [branes. propria. See Basement mem- %lembrane, basement. See Basement membranes. glassy, 95. limiting, 49. of Nasmyth, 135. of Krause, 79. Membranes of brain, 235. Menstruation, uterus in, 197. Mercuric chlorid method for nervous sys- tem, 34. Mesenteric lymph-node, 113. Mesentery, silvered, 61. Mesoderm or Mesoblast, derivatives of, 54. Methylene blue, 31, 84. Methyl green, 32. Methyl violet, 87. Metric scale, 8. Micro-millimeters, 8. Micron, M, 8. Micrometers, 8. Microscope, 1. adjustment of, 5. care of, 43. illumination, 4. magnifying power of, 7. parts of, 1. sketching from, 9. Microtome, freezing, 19. Minot, 15. Schanze, 15. Stirling, 12. Thoma, 14. Milk, 45, 208. colostrum-corpuscles, 208. fat-globules in, 45. secretion of, 208. Mixed salivary glands, 148. Mirror, 1, 4. Mitosis, 51. Molecular movement, 46. Mounting fluids and methods, 35. of objects, 41. Mounts, rings on, 36, 76. Mouth, 149. Movement, Brownian, 46. amoeboid, 83. vital, 47. Movements, magnification of, 46. Mucoid tissue, 76. Mucosa of bronchial tubes, 123. of stomach and intestine, 151. of mouth and pharynx, 149. of oesophagus, 150. ureter and bladder, 193. uterus, 199. vagina, 199. Mucous glands, 127, 144, 148, 149. cells. See Goblet cells. Miiller's fluid, 22. Multipolar nerve-cells, 220. Muscular tissue, 76. Muscle, blood-vessels of, 80. cardiac, 80. involuntary, 76. See Non-striated muscle, nerves of. 225. non-striated, 76. non-striated, distribution of, 76,96, 102, 107, 112, 118, 123, 150, 151, 193, 194, 196, 197, 202. of hair-follicle, 96. smooth, 76. See Non-striated, striated, 78. voluntary, 78. of oesophagus, 150. Myocardium, 80. Naboth, ovules of, 199. Nails, 98. Nasal mucous membrane, epithelium, 55. Nasmyth's membrane, 135. Needles, 36. Nerve-cells, 219. -cells of suprarenal body, 213. -cells, processes of, 220. -cells pyramidal, 238. -cells, types, 220 254 INDEX Nerve-endings, 223. [225. -endings in non-striated muscle, -endings in striated muscle, 225. -endings in skin, 94, 223, 224. -fibers, 216. -fibers, axis-cylinder of, 216. -fibers in osmic acid, 23. -fibers medullated, 216. -fibers non-medullated, 217. Nerve, acoustic, 216. connective tissue of, 218. funiculi, 217. olfactory, 217. optic, 216. plexuses, 225. practical demonstration, 219. spinal, 228. -staining, Cox, 34. -staining, Golgi, 33. -staining, nigrosin, 32. -staining, Van Gieson, 32. -staining, Weigert's, 30. -trunks, 217. Nervi nervorum, 219. Nervous system, 216. [22, 23. Nervous tissues, fixation or hardening, tissues, development of, 54, 223. tissues, supporting framework of, 222. Neurilemma, 217, 218. Neuroglia, 222. -cells, 223. Neurone, 221, 222. Neutrophile granules, 31, 86. Newt's tail for karyokinesis, 53. Nigrosin, 32. Nitrate of silver, 33, 61, 108. * Nitric acid, 24, 75. Nodes, lymph-. See Lymphatic nodes. of Kanvier, 216. Non-medullated nerves, 217. ? Non-striated muscle, 76. See Muscle, non-striated. Normal salt solution, 36. Nose, epithelium of, 55. Nuclei of cells, 49. Nucleoli of cells, 50. Nuclear stains, 27. spindle, 52. Nucleus, fibrils of, 49. structure of, 49. Objectives, 2, 4. Ocular. See Eye-piece. Odontoblasts, 135. (Esophagus, 150. Oil-immersion objective, 4. -globules, 46. of cedar wood, 4. Oils, essential, 35. Olfactory nerve, 217. Optical axis, 5. Optic nerve, 216. Organisms in urine, 47. Origanum oil, 35. Orth's fluid, 22. Osmic acid, 23. Osmico-bichromate mixture, 23. Ossification, 73. Osteoblasts, 73. Osteoclasts, 73. Os uteri, 198. Ovarial tubes, 206. Ovary, 203. corpus luteum of, 203. germinal epithelium, 203. Graafian follicles of, 203. [206. practical demonstrations of, 203, tunica albuginea, 203. Oviduct, 202. Ovula Nabothi, 199. Ovum, 205. formation of, 206. Oyster, cilated cells from, 59. Pacchionian bodies, 235. Pacinian corpuscles. 223. Pal-Weigert method, 30. Pancreas, 145. practical demonstration of, 146. Paper, Japanese, 41. Papillas, circumvallate, 149. filiform, 149. foliates, 149. fungiform, 149. of skin, 93. of tongue, 149. Papillary eminences of kidney, 178. Paraffin, cementing or soldering, 18. imbedding, 33, 25. Parenchyma of glands, 140. Parotid gland, 143, 148. [146. gland, practical demonstration of, Patch, Peyer's, 160. Pavement epithelium, 56. Pelvis of kidney, 193. Penis, 212. Peptic glands, 152. Perforating fibers of Sharpey, 71. Pericardium, 60, 102, 107. INDEX 255 Perichondrium, 07. Pericementum, 137. Perimysium, 78. Perineurium, 218. Periosteal bone, 74. Periosteum, 73. Peripheral nerve-termini, 223. Peritoneum, 60, 107, 151. Perivascular lymphatics, 107. [107, 238. lymph-spaces of cerebrum, Peyer's patches, 1GO. Pharynx, 149. mucous membrane of, 149. Pia mater, 235. Picric acid, 23, 29, 31, 32. alcohol, 23. Picro-carmine, 29. Pigment-cells of hair, 95. -cells of skin, 93. -cells in suprarenal body, 215. Pineal body, embryonic derivation, 54. Pipettes for haemocytometer, 86. Pituitary body, embryonic derivation, 54. Plates, blood-, 82. cartilage- in bronchi, 124. Pleura, 60, 107, 128. Plexuses, sympathetic, 225. of Auerbach, 154, 160. of Meissner, 154, 160. Penciling of serous surfaces, 108. Pole of the cell, 52. Pollen, 48. Polynuclear or polymorphonuclear leu- cocytes, 86. Portal canal, 165. vein, 163. Posterior commissure of spinal cord, 227t columns, 227. median fissure, spinal cord, 227. Potassium bichromate, 22. Potato starch, 48. Power, magnifying, 7. Practical demonstrations of— blood, 82-91. blood-vessels, 105. bone, 75. bronchial tube, 125. cartilage, 68. cerebellum, 240. cerebrum, 237. development of ovum, 206. elastic tissue, 65. endothelium, 60. epithelium, 55-60. Fallopian tube, 202. Practical demoustrations of — hair, 99. intestine, 160-16'J. karyokinensis, 53. kidney, 186. liver, 167, 170, 177. lung, 131. lymphatics of central tendon of diaphragm, 108. mesenteric lymph-node, 113. mouth, oesophagus, pharynx, etc., 150. muscle, 77-80. nerves, 219. ovary, 203, 206. pancreas, 146. parotid gland, 146. skin, 98. spinal cord, 229. spleen, 118. stomach, 155. submaxillary gland, 146. suprarenal body, 213. teeth, 137. testicle, 211, 212. thymus body, 121. tongue and taste-buds, 150. ureter, 193. urinary bladder, 195. uterus, 197. vagina, 197. white fibrous tissue, 63. Pregnancy, uterus in, 197. Preservation of tissues, 20. Prickle cells of skin, 92. Prismatic color in air-bubbles, 46. Prisms, enamel, 137. Projection fibers, 239. Prostate gland, 212. Prostatic concretions, 212. Protoplasm, 49. reticulation of, 50. [220 Protoplasmic processes of ganglion-cells, Proximal convoluted tubule, 181. Pseudopodia, 83. Pulmonary alveoli, 128. artery, 128. Pulp of teeth, 135. of spleen, 117. Purkinje, cells of, 242. Pus-corpuscles, 84. Pyloric glands, 152. Pyramidal tracts, 228. Pyramid of Ferrein, 179. of Malpighi, 179. Pyroxylin, 26. 256 INDEX Quick hardening with alcohol, 21. hardening with formaldehyde, 22. Racemose glands, 143. Ranvier's nodes, 216. Rapid hardening with alcohol, 21. with formaldehyde, 22. Rays, medullary, of kidney, 179. Razor, form of, 10. stropping, 17. Receptaculum chyli, 106. Red blood-corpuscles, 81. See Blood. marrow, 73. Reproductive organs, 197, 209. Reptiles, blood of, 82. Respiratory organs, 123. Rete Malphighii of skin, 92. mucosum of skin, 92. •testis, 210. Reticular cartilage, 68. connective tissue, 76, 113. [113. Reticulum of adenoid or lymphoid tissue, Retiform tissue. See Reticular connect- ive tissue. Retzius, lines of, 137. Ribbon sections. See Serial sections. Ringing mounts, 36, 43, 76. Root-sheath of hair, 95, 99. Rugae, 151. . Sacs, air-, 128. Safranin, 23, 31. Salamander tail for karyokinesis, 53. Salivary gland, abdominal, 145. corpuscles, 47, 149. glands, 143, 148. Salt solution, normal, 36. Santorini, cartilage of, 123. Sarcolemma, 78. Sarcoplasm, 79. Schanze microtome, 15. Schwann, white substance of, 216. Sebaceous glands, 98. Sebum, 98. Section cutting, 10. free-hand, 10. with microtomes, 12. in series, 15. -lifter, 41. Sections, frozen, 19. serial, 15, 26. * to place on slide, 41. Seminiferous tubules, 211. Sensory nerve-terminations, 223. Serial sections, 15, 26. Serous glands, 145, 148. membranes, 60, 107. membranes, lymphatics of, 108. Sharpening knives, 16. Sharpey's fibers, 71. Sheath, dentinal, 135. of Schwann, 216. Silk fibers, 47. Silver, mesentery stained with, 61. nitrate, 33. staining, 33, 61, 108, 134. staining solution, 33. Skeletal muscle, 78. Sketching from microscope, 9. Skin, arrector pili, 96. blood-vessels of, 94. corium, 92. chamois, 43. eleidin granules, 92. epidermis, 92. hair-follicles, 95. injected, 94. keratin of, 92. negro, 93. nerves of, 94. papillse of, 93. pigment of, 93. practical demonstration, 98. sebaceous glands, 98. stratum corneum, 92. stratum granulosum, 92. stratum lucidum, 92. stratum Malpighii, 92. sudoriferous glands, 97. sweat-glands, 97. tactile corpuscles of, 94, 223. Slides, 41. Small intestine, 156. Smooth muscle. See Muscle, non-striated. Sole-plate, 225. Solitary glands, 160. Solution, Erlicki's, 24. Ehrlich-Biondi-Heidenhain, 32. Flemming's, 23. Miillers, 22. normal salt, 36. Orth's, 22. Stirling's, 25. Toison's, 87. Space, subarachnoid, 235. subdural, 235. Spaces, interglobular, 137. lymph-, 106. venous, 117. Special connective tissues, 76. INDEX 257 Specimens, permanent, 35. Spermatogenic cells, 212. Spermatozoa, 211. Sphincter of renal papillae, 193. of urinary bladder, 195. Spider cells, 223. Spinal cord, 226. anterior column, 227. anterior gray commissure, 227. anterior median fissure, 227. ascending antero-lateral tract, Burdach's column, 228. [228. central canal of, 227, 232. Clarke's column, 232. collateral fibers, 234. columns of, 227, 228. commissures of, 227, 228. crossed pyramidal tract, 228. descending antero - lateral tract, 228. direct cerebellar tract, 228. direct pyramidal tract, 228. funiculus cuneatus, 228. funiculus gracilis, 228. ganglion-cells, 232. GolPs column, 228. Gower's column, 228. gray commissures, 227. gray matter, 226. lateral columns, 227. nerve-fibers of, 231. nerve-roots of, 228. posterior column, 227. posterior median fissure, 227. practical demonstration, 229. staining of, 30, 32, 33, 229. substantia gelatinosa Rol- andi, 232. substantia gelatinosa cen- tralis, 232. Turck's column, 228. white commissure, 228. white matter of, 226. Spiral tubule, 181. Spleen, 117. Malpighian bodies, 118. practical demonstration, 118. Spongy bone, 72. Spot, germinal, 205. Squamous epithelium, 55. Staining agents, 27. aniline dyes, 31. borax-carmine, 29, 39. carmine, 29, 39. Cox-Golgi, 34. Staining, double, 38. Ehrlich-Biondi-Heidenhain,32,85 • eosin, 28, 38. fresh tissue, 20. fuchsin, 31. general or ground, 27. gentian violet, 31. Golgi, 33. haematoxylin, 28, 36. heematoxylin and eosin, 38. in bulk, 29. methylene blue, 31, 84. nuclear, 27. nigrosin, 32. osmic acid in nerve tissue, 23. Pal, 30. picro-carmine, 29. selective, 27. silver, 33, 61, 108, 134. safranin, 23, 31. triacid, tricolor, triple, 32, 85. Van Gieson, 32. Weigert's nerve-, 30. Starch, 48. Stirling microtome, 12. dissociating fluid, 25. Stomach, 151. practical demonstration, 155. lymphatics of, 156. mucous membrane of, 152. nerves of, 154. peptic glands. 153. pyloric glands, 154. Stomata, 62, 109. Stratified epithelium, 55. Stratum corneum of skin, 92. granulosum, 92. lucidum, 92. Malpighii, 92. Striated or striped muscle, 78. See Mus- cle, striated. cardiac muscle, 80. Stripes of Baillarger, 237. Stropping knives, 17. Subarachnoidal space, 235. Subdural space, 235. Sublingual gland, 148. Sublobular vein, 163. Submaxillary glands, 144, 148. glands, practical demonstra- tion, 146. Substance, cement-. See Cement-sub- stance. white, of Schwann, 216. Substances, extraneous, 47. 258 INDEX Substantia, gelatinosa, 232. Succus entericus, 156. Sudoriferous glands, 97. Sulphate of copper, 24. Supernumerary spleens, 118. Suprarenal body, 213. capsule, 213. Sustentacular cells of testicle, 212. Sweat-glands, 97. Sympathetic system, 217. System, cerebro-spinal, 226-243. . circulatory, 102. digestive, 135-177. Haversian, 71. lymphatic, 106. nervous, 216. respiratory, 123. reproductive, 197, 209. sympathetic nervous, 217. Tactile corpuscles, 94, 223. Tangential fibers, 238. Taste-buds, 149. Teasing, 9. Technology, 1. Teeth, 135. cementum, 137. crusta petrosa, 135. decalcification of, 137. dentinal fibers, 135. dentinal tubules, 135. dentine, 135. enamel, 137. interglobular spaces. 137. membrane of Nasmyth, 135. odontoblasts, 135. pericementum, 137. practical demonstration of, 137. pulp, 135. stripes of Eetzius, 137. Teichmann's crystals, 90. Tendon, 62. Tesselated epithelium, 56. Testicle, 209. coni vasculosi, 210. mediastinum of, 209. seminiferous tubules of, 210, 211. spermatozoa, 211. spenuatogenesis, 212. tunica albuginea, 209. tunica vaginalis, 209. vasa efferentia, 210. Thermal currents, 46. Thoracic cavity, 60, 107, 128. duct, 106, 107. Thymus body, 121. Thyme oil, 35. Thoma microtome, 14. Thyro-glossal duct, 148. Thyroid body, 148. body, colloid secretion of, 148. Tissue, adenoid. See Lymphoid tissue. adipose, 65. areolar, 62. connective, 62. definition of, 53. elastic, 63. embryonic, 76. erectile, 212. fat-, 65. fibrous, 62. fixation of, 20. fresh, 20. hardening of, 20. lymphoid. See Lymphoid tissue. mucoid, 76. muscular, 76. nerve., 216. white fibrous, 62. yellow elastic, 63. Tissues, classification of, 53. dehydration of, 35, 38. embryonic derivation of, 54. varieties of, 53. Toison's fluid, 87. Tongue, 149. muscle of, 79. Tonsil of Luschka, 149. Tonsils, 149. Tooth. See Teeth. Touch corpuscles, 94, 223. Trabeculse of lymph-nodes, 112. splenic, 117. Trachea, 123. cartilages of, 68, 123. Tracts of the spinal cord, 228. Transitional epithelium, 55, 193, 195. Tricolor or triacid stain of Ehrlich, etc., True skin, 92. [32, 85. Tube, bronchial, 123. Tubular glands, 140. Tubule, distal convoluted, 182. Fallopian, 202. gastric, 152. Henle's loop, 181. proximal convoluted, 181 spiral, 181. straight, of kidney, 182. straight, of testicle, 210. Tubules, seminiferous, 211. INDEX 259 Tubules, uriniferous, 180, 182. Tunica albuginea, 203, 209. Tiirck, column of, 228. Turpentine carbol-, 35. Turn-table, 36. Tunica vaginalis, 209. vasculosa, 210. Typical cell, 49. Unstriated or unstriped muscle, 76. See Muscle, non-striated. Umbilical cord, 76. Unipolar nerve-cells, 220. Ureter, 193. Urethra, 196. glands of, 196. Urinary bladder, 194. organs, 178-196. Urine, bacteria in, 47. course of, in kidney, 185. Uriniferous tubules of kidney, 180. Uterus, 197. Vagina, 197. Valves of heart, 102. of lymphatics, 107, 109. of veins, 104. Van Gieson's stain, 32. Vasa vasorum, 105. Vascular system, 102, 106. Vas deferens, 209. Vasa efferentia, 210. Vegetable fibers, 47. spores, 48. Veins, 104. hepatic, 163. interlobular, 163. intralobular, 163. portal, 163. sublobular, 163. valves of, 104. Venae stellataa of kidney, 184. Venous spaces of spleen, 117. Venulte recta of kidney, 184. Venules, 102. Vermiform appendix, ILL'. Vesicle, germinal, 205. Vesicles, air , 128. Villi of intestine, 156. Vital movements, 47. Voluntary muscle, 78. See Muscle, stri- ated. Wall of cell, 49. Wandering cells, 83, 104. Weigert-Pal method, 30. Welsbach gas-burner, 4. Wharton's jelly, 76. Wheat starch, 48. White blood-corpuscles, 83, 104. commissure of spinal cord, 228. fibrous tissue, 62. matter of brain, 235. matter of spinal cord, 226. substance of Schwann, 216. Wood fibers, 47. Wool fibers, 47. Wrisberg, cartilage of, 123. Xylol, 35. balsam, 35. Yeast, 48. Yellow elastic tissue, 63. Zinc cement, 36. Zona fasciculata, 213. glomerulosa, 213. pellucida, 205. reticularis, 213. vasculosa, 203. DATE DUE SLIP UNIVERSITY OF CALIFORNIA MEDICAL SCHOOL LIBRARY THIS BOOK IS DUE ON THE LAST DATE STAMPED BELOW flOV 10 1930 FEB 1 1932 MAY !934 x- ..: . i*39 193^ MAY i 8 1937 DEC1 194V 1941 13 194, GCT2V id*} SEP 2 - 5 Aavaan IOOHOS IVOIQBW VINUOJIIVO do AIISHBAIND Hal