i m n- O; I si D D m a A TEXT-BOOK OF HISTOLOGY INCLUDING MICROSCOPIC TECHNIC BY A. A. BOHM, M. D., and M. VON DAVIDOFF, M. D. of the Anatomical Institute in Munich Edited, with Extensive Additions to both Text and Illustrations BY G. CARL HUBER, M.D. Professor of Histology and Embryology and Director of the Histological Laboratory University of Michigan Second Edition Thoroughly Revised and Enlarged WITH 377 ILLUSTRATIONS PHILADELPHIA AND LONDON B. SAUNDERS COMPANY 1910 Copyright, 1900, by W. B. Saunders and Company. Reprinted July, 1901. Revised, reprinted, and recopyrighted August, 1904. Reprinted April, 1905, January, 1906, July, 1908, June, 1909, and October, 1909. Copyright, 1904, by W. B. Saunders & Company. Registered at Stationers* Hall, London, England. Reprinted September, 1910. PRINTED IN AMERICA PRESS OF W. B. SAUNDERS COMPANY PHILADELPHIA TO THEIR TEACHER PROFESSOR C VON KUPFFER THIS BOOK IS DEDICATED BY THE GRATEFUL AUTHORS EDITOR'S PREFACE TO THE SECOND EDITION. The favorable reception accorded to the first American edition of Bohm and Davidoff's Text-book of Histology has justified the as- sumption expressed by the editor in his preface to the former edition, that an English translation of this work would meet with approval from American and English teachers and students* In the preparation of this second American edition the editor has retained in general the same arrangement of the subject-matter as presented in the former edition. The revision of the text has given opportunity to take cognizance of the many contributions to our knowledge of the ultimate structure of tissues and organs and of their histogenesis which have appeared in recent years, and in doing so, many of the chapters, especially those dealing with gen- eral histology, have been subjected to extensive alterations. Re- cognition has also been given to the results obtained by the use of precise methods of plastic reproduction, methods which have been especially useful in giving clearer and more accurate conceptions of the form and relationship of anatomic structures, too small and too delicate to admit of disassociation by means of methods of macera- tion and teasing and too complicated to admit of full interpretation by means of sections. Maziarski's observations on the form and relationship of the ultimate divisions of the tubular systems of many of the more important glands have, been embodied, also the results of numerous reconstructions made in the Histological Laboratory of the University of Michigan. The text of this edition has been extended by some forty pages, and the illustrations have been increased from three hundred and fifty-one to three hundred and seventy-seven. Recognizing the fact that a text-book of Histology is a book which of necessity needs to be constantly used in the laboratory, and its size is, therefore, a matter of some importance, the editor seemed justi- fied, in view of the fact that an increase in the number of text-pages seemed inevitable, to dispense in the present edition with the list of references to the literature (some twenty pages) which appeared in the former edition. G. CARL HUBER. LABORATORY OF HISTOLOGY AND EMBRYOLOGY, UNIVERSITY OF MICHIGAN. EDITOR'S PREFACE TO THE FIRST EDITION. THE " Text -book of Histology " by Bohm and v. Davidoff, as stated Ay the authors in the preface to the first edition, presents as fully as possible, from both the theoretic and technical standpoints, the subject- matter of the lectures and courses in histology given to students in the University of Munich. The authors further state that in the completion of their work they had the constant aid and advice of Professor von Kupffer, and had at their disposal the sections in the collection of the histologic laboratory in Munich, which were freely used in the selection and preparation of the illustrations accompanying the text. The excellence of the text and illustrations of the German edition, attested by all familiar with the work, and the cordial reception which it has received from both students and investigators, justify the belief that an English translation will meet with approval from American and English teachers and students. In the preparation of this American edition the editor has retained substantially all the subject-matter and illustrations of the second German edition, although certain minor changes in the arrangement of the text seemed desirable. Additions to the German text have been freely made. The sections on the Motor and Sensory Nerve-endings and on the Spinal and Sympathetic Ganglia have been greatly expanded, and the Innerva- tion of Glands and Organs has been considered much more fully than in the original. Our knowledge of the normal function of tissues and organs is so dependent on a correct understanding of their innervation that this subject seemed deserving of fuller consideration than is generally given it in text-books of this scope. The glands with internal secretion have also been considered more fully than in the original text, their im- portance necessitating such treatment. More than one hundred illustra- tions, the majority of them from original drawings, have also been added. In making these and other minor additions the editor has striven to stamp his own work with the excellent features of the German text, and trusts that his endeavors may have added to the usefulness of the book. The editor acknowledges with pleasure -his indebtedness to Dr. Herbert H. Gushing for his excellent and accurate translation, and for suggestions received from him. The publishers, Messrs. Saunders & Company, have shown throughout the greatest interest in the work, and deserve the gratitude of the editor for their ready acquiescence in all suggestions made by him, for the excellent reproduction of his drawings, and for the suggestions made to him. The editor is particularly in- debted to his able assistant, Dr. Lydia M. De Witt, for valuable assistance rendered, more especially in the tedious work of proof-correction, for which he expresses his sincere appreciation and gratitude. G. CARL HUBER. UNIVERSITY OF MICHIGAN, ANN ARBOR, MICH. CONTENTS. INTRODUCTION TO MICROSCOPIC TECHNIC PAGE Microscope and Its Accessories 17 Microscopic Preparations . 21 Methods of Maceration 22 Fixing Methods , 23 Infiltration and Imbedding 27 Paraffin 27 Celloidin 30 Celloidin-Paraffin 32 Microtomes and Sectioning 32 Further Treatment of the Section 38 Fixation to the Slide and Removal of Paraffin 38 Staining 41 Section Staining 41 Staining in Bulk 46 Methods of Impregnation 47 Preparation of Permanent Specimens , 52 Methods of Injection . . 53 Reconstruction by Means of Wax Plates 55 GENERAL HISTOLOGY. L THE CELL. Cell-body 59 Nucleus 62 Nuclear and Cell-division 64 Mitosis or Karyokinesis (Indirect Cell-division) 64 Prophases 66 Metaphases 68 Anaphases 69 Telophases ... 70 Heterotypic Form of Mitosis ; . . 70 Amitosis 70 Process of Fertilization 71 Chromatolysis 74 Technic for the Cell 74 II. TISSUES. Epithelial Tissues 80 Simple Epithelium . . . 82 Simple Squamous Epithelium 82 Simple Cubic Epithelium 82 Simple Columnar Epithelium 83 Pseudostratified Columnar Epithelium 83 Stratified Epithelium . . . . 83 Stratified Squamous Epithelium 84 Transitional Epithelium 85 Stratified Columnar Epithelium 85 7 8 CONTENTS. PAGE Glandular Epithelium 87 Unicellular Glands 87 Multicellular Glands 88 General Consideration of the Structure and Classification of Glands ... 88 Tubular Glands 89 Alveolar Glands 91 Remarks on the Process of Secretion 92 Neuro-epithelium 92 Mesothelium and Endothelium 92 Technic for Epithelial Tissues 94 Connective Tissues 96 Mucous Connective Tissue 100 Reticular Connective Tissue loo Fibrous Connective Tissue 101 Adipose Tissue 107 Cartilage 108 Bone 112 Structure of Bone 112 Development of Bone 116 Technic for Connective Tissues 126 Muscular Tissues 134 Nonstriated Muscle-cells 134 Striped Muscle-fibers 136 Development of Voluntary Muscle-fibers 144 Cardiac Muscle '. 145 Technic for Muscular Tissue 147 Nervous Tissues 148 Nerve-cells or Ganglion Cells ; Cell-bodies of Neurones 149 Nerve-fibers 157 Peripheral Nerve Terminations 162 Technic for Nervous Tissues 180 SPECIAL HISTOLOGY. L BLOOD AND BLOOD-FORMING ORGANS, HEART, BLOOD-VESSELS, AND LYMPH- VESSELS. Blood and Lymph 1 86 Formation of Blood 186 Red Blood-corpuscles 187 White Blood-corpuscles 191 Blood Platelets — Thrombocytes ' 194 Behavior of Blood-cells in the Blood Current 196 Lymphoid Tissue, Lymph-nodules, and Lymph-glands 196 Spleen . . 202 Bone-marrow 207 Thymus Gland 2IO II. CIRCULATORY SYSTEM. Vascular System 213 Hearf 213 Blood-vessels 216 Arteries • ... 216 Veins 219 Capillaries 220 Anastomoses, Retia Mirabilia, and Sinuses 222 Lymphatic System 223 Lymph-vessels 223 Lymph-capillaries, Lymph-spaces, and Serous Cavities 224 Carotid Gland (Glandula Carotica, Glomus Caroticum) . . 225 Technic for Blood and Blood-forming Organs 226 Technic for Circulatory System 235 CONTENTS. 9 HI. DIGESTIVE ORGANS. Oral Cavity 235 Teeth 238 Structure of the Adult Tooth 238 Development of the Teeth 243 Tongue 247 Lingual Mucous Membrane and Its Papillae 247 Taste-buds 249 Lymph-follicles of the Tongue (Folliculi Linguales) and the Tonsils ... 251 Pharyngeal Tonsil 251 Glands of the Oral Cavity 253 Salivary Glands 255 Parotid Gland (Serous Gland) 255 Sublingual Gland (Mucous Gland) 255 Submaxillary Gland (Mixed Gland) 4 258 Small Glands of the Mouth 259 Pharynx and Esophagus 262 Stomach and Intestines 264 General Structure of the Intestinal Mucous Membrane 264 Stomach 266 Small Intestine 274 Large Intestine, Rectum, and Anus . . . 281 Blood, Lymph, and Nerve Supply of the Intestine 283 Secretion of the Intestine and the Absorption of Fat 288 Liver .... 289 Pancreas 298 Technic for Digestive Organs 303 IV. ORGANS OF RESPIRATION. Larynx 309 Trachea 310 Bronchi, their Branches, and the Bronchioles 311 Terminal Divisions of Bronchi and Ultimate Air-spaces 313 Thyroid Gland 319 Parathyroid Glands 321 Technic for Organs of Respiration 322 V. GENITO-URINARY ORGANS. Urinary Organs 323 Kidney 323 Pelvis of the Kidney, Ureter, and Bladder 336 Suprarenal Glands 339 Technic for Urinary Organs and Suprarenal Body 342 Female Genital Organs 344 Ovum 344 Ovary 344 Fallopian Tubes, Uterus, and Vagina 354 Male Genital Organs 361 Spermatozoon 361 Testes 362 Excretory Ducts 367 Spermatogenesis 372 Technic for Reproductive Organs • 378 VI. THE SKIN AND ITS APPENDAGES. Skin (Cutis) 379 Hair 389 Nails 394 Glands of the Skin 396 Sweat-glands 396 Sebaceous Glands 398 Mammary Glands . 400 Technic for the Skin and Its Appendages 403 IO CONTENTS. VIL THE CENTRAL NERVOUS SYSTEM. Spinal Cord 406 Cerebellar Cortex 413 Cerebral Cortex . . 416 Olfactory Bulb 421 Epiphysis and Hypophysis 422 Ganglia 424 General Survey of the Relations of the Neurones to One Another in the Central Nervous System 431 Neuroglia 434 Membranes of the Central Nervous System 436 Blood-vessels of the Central Nervous System 439 Technic for Central Nervous System 440 VIII. EYE. General Structure 446 Development of the Eye 446 Tunica Fibrosa Oculi 448 Sclera 448 Cornea ... 449 Vascular Tunic of the Eye . 452 Choroid, Ciliary Body, and Iris 452 Internal or Nervous Tunic of the Eye 457 Pigment Layer 457 Retina .... 457 Region of the Optic Papilla 460 Region of the Macula Lutea 460 Ora Serrata, Pars Ciliaris Retinas, and Pars Iridica Retinae 461 Miiller's Fibers of the Retina 462 Relations of the Elements of the Retina to One Another 462 Optic Nerve 464 Blood-vessels of the Optic Nerve and Retina 465 Vitreous Body 467 Crystalline Lens 467 Fetal Blood-vessels of the Eye 468 Interchange of Fluids in the Eyeball 469 Protective Organs of the Eye 469 Lids and Conjunctiva 469 Lacrimal Apparatus 473 Technic for the Eye 474 IX. ORGAN OF HEARING. External Ear 476 Middle Ear 478 Internal Ear 480 Utriculus and Sacculus 482 Semicircular Canals 483 Cochlea . 484 Organ of Corti . 489 Development of the Labyrinth 496 Technic for Organ of Hearing 497 X. ORGAN OF SMELL. Technic for Nasal Mucous Membrane 5°° XL GENERAL CONSIDERATIONS OF THE SPECIAL SENSE-ORGANS. INDEX 501 ILLUSTRATIONS. FIG. PAGE 1. Microscope . . . . 18 2. Diagram showing the principle of a compound microscope 20 3. Box for imbedding tissues 28 4. Laboratory microtome 33 5. Minot automatic precision microtome 34 6. Minot automatic rotary microtome 34 7. Apparatus for cutting tissues frozen by carbon dioxid 37 8. Movements in honing . .... 38 9. Apparatus for making wax plates, used in reconstruction by Bern's method . . 56 10. Diagram of cell (Huber) 59 1 1 . Cylindric ciliated cells from the primitive kidney of Petroniyzon planeri ... 60 12-26. Processes of mitotic cell- and nuclear division 65, 66 27-29. Mitotic cell-division of fertilized whitefish eggs (Huber) 67 30. Mitotic division of cells in testis of salamander (Benda and Guenther) ... 69 31-36. Processof fertilization (Boveri) . 72, 73 37. Pigment cell from the skin of the head of a pike 77 38. Isolated cells of squamous epithelium (Huber) 82 39. Surface view of squamous epithelium from skin of a frog 82 40. Simple columnar epithelium from the small intestine of man (Huber) .... 83 41. Pseudostratified columnar epithelium 83 42. Stratified pavement epithelium . . 84 43. Cross-section of stratified squamous epithelium from esophagus of man (Huber) 84 44. Isolated transitional epithelial cells from bladder of man (Huber) 85 45. Cross-section of transitional epithelium from the bladder of a young child (Huber) 85 46. Stratified columnar epithelium 86 47. Ciliated cells from bronchus of dog 86 48. Cross-section of stratified ciliated columnar epithelium from trachea of rabbit (Huber) . . . . 86 49. Goblet cells from bronchus of dog . . 87 50. Mucus-secreting cell (goblet cell) (Huber) ... 87 51. Simple tubular glands ... .... 88 52. Excretory ducts and lumina of the secretory portion of a compound tubular gland . 89 53. Lumina of the secreting portion of a reticulated tubular gland 89 54. Glandular classification .... 90 55. Mesothelium from pericardium of rabbit (Huber) 93 56. Mesothelium from mesentary of rabbit (Huber) 93 57. Mesothelium from peritoneum of frog ... 93 58. Mesothelium covering posterior abdominal wall of frog (Huber) 94 59. Endothelial cells from small artery of mesentery of rabbit (Huber) 94 60. Mesenchymatous tissue from the subcutis of a duck embryo 97 61. Development of the different types of connective tissue from the mesenchyma (Huber) 98 62. White fibrils and bundles from teased preparation of a fresh tendon from tail of rat (Huber) 99 62^. Elastic fibers from ligamentum nuchas of ox 99 63. Reticular fibers from a thin section of a lymph-gland (Huber) IOI 64. Reticular connective tissue from lymph-gland of man 102 65. Areolar connective tissue from subcutaneous tissue of rat (Huber) 102 66. Cell-spaces in the ground- substance of areolar connective tissue of young rat (Huber) 103 67. Connective-tissue cells from pia mater of dog (Huber) . . . . 103 68. Pigment cells found on the capsule of sympathetic ganglion of frog (Huber) . 103 69. Leucocyte of frog with pseudopodia 104 70. Fibrous connective tissue from great omentum of rabbit 105 II 12 ILLUSTRATIONS. FIG. PAGE 71. Longitudinal section of tendon 106 72. Cross-section of secondary tendon bundle from tail of rat (Huber) 106 73. Tendon cells from tail of rat (Huber) 107 74. Cross-section of ligamentum nuchse of ox (Huber) 107 75. Fat-cell . . 107 76. Hyaline cartilage 108 77. Section through cranial cartilage of squid 109 78. Insertion of the ligamentum teres into the head of the femur no 79. Elastic cartilage from external ear of man Ill 80. Longitudinal section through a lamellar system (v. Ebner) 113 81. 82. Lamellae seen from the surface (v. Ebner) 113 83. Segment of a transversely ground section from the shaft of a long bone, showing lamellar system . . : .... ... 114 84. Portion of a transversely ground disc from the shaft of a human femur ... 115 85. Longitudinal section through a long bone of a lizard embryo .... 117 86. Longitudinal section of the proximal end of a long bone of a sheep embryo . 118 87. Longitudinal section through area of ossification from long bone of human embryo (Huber) ... 119 88. Longitudinal section through epiphysis of arm bone of sheep embryo .... 122 89. Section through the lower jaw of an embryo sheep 123 90. Cross-section of developing bone from leg of human embryo, showing endo- chondral and intramembranous bone development (Huber) . . ... 124 91. Cross-section of shaft (tibia of sheep) 125 92. Nonstriated muscle from the intestine of a cat 135 93. Cross-section of striated muscle-fibers 136 94. Muscle-fiber from ocular muscles of rabbit 136 95. Striated muscle-fiber of frog, showing sarcolemma (Huber) 137 96. Diagram of structure of fibrils of a striated muscle-fiber (Huber) 138 97. Diagram of transverse striation in the muscle of an anthropoid 138 98. Transverse section through the striated muscle-fiber of a rabbit 139 99. Striated muscle-fiber of man 140 100. Cross-section through the trapezius muscle of man ... 140 101. Branched, striated muscle-fiber from tongue of dog (Huber) 141 102. Cross-section of rectus abdominis of child, under low magnification (Huber) 142 103. Longitudinal section through the line of junction between muscle and tendon 142 104. 105. Longitudinal and cross-section of muscle-fibers from the human myocar- dium 143 106. Longitudinal section of heart-muscle (Huber) 146 107. Bipolar ganglion cell from the ganglion acusticum of a telecost 150 108. Chromatophile granules of a ganglion cell from the Gasserian ganglion of a telecost 151 109. Nerve-cell from the anterior horn of the spinal cord of an ox 151 110. Motor neurones from anterior horn of the spinal cord of new-born cat (Huber) 152 111. A nerve-cell with branched dendrites (Purkinje's cell), from cerebellar cortex of rabbit .... 152 112. Pyramidal cell from cerebral cortex of man 153 113. Nerve-cell from dendrites ending in claw-like telodendria 154 114. Ganglion cell with a T-shaped process 154 115. Ganglion cell from Gasserian ganglion of rabbit (Huber) 155 116. Ganglion cells from spinal ganglion of rabbit embryo 155 117. Neurone from inferior cervical sympathetic ganglion of rabbit (Huber) . . . 156 118. Longitudinal section of nerve-fiber . . 157 119. Transverse section through sciatic nerve of frog 158 1 20. Medullated nerve-fibers from rabbit 159 121. Remak's fibers from pneumogastric nerves of rabbit ... ..... 159 122. Diagram to show composition of a peripheral nerve-trunk (Huber) .... l6l 123. Cross-section through a peripheral nerve 161 124. Peripheral motor neurone (Huber) . 163 125. Motor nerve-ending in voluntary muscle of rabbit (Huber-De Witt) .... 164 126-130. Motor endings in striated voluntary muscles ... 165 131. Motor nerve-ending in striated voluntary muscle of frog (Huber-De Witt) . . 166 132. Motor nerve-ending on heart muscle-cells of cat (Huber-De Witt) .... 166 133. Motor nerve-ending on involuntary nonstriated muscle-cell from intestine of cat (Huber-De Witt) 166 ILLUSTRATIONS. 13 FIG PAGE 134. Peripheral sensory neurone (Huber) 167 135. Termination of sensory nerve-fibers in the mucosa and epithelium of urethra of cat (Huber) 168 136. End-bulb of Krause from conjunctiva of man (Dogiel) 169 137. Meissner's tactile corpuscle (Dogiel) . . 170 138. Genital corpuscle -from glans penis of man (Dogiel) 171 139. Cylindric end-bulb of Krause from intermuscular fibrous tissue septum of cat (Huber) 172 140. Vater-Pacinian corpuscle from the mesentery of a cat 173 141. Pacinian corpuscles from the mesorectum of a kitten (Sala) 174 142. Corpuscle of Herbst from bill of duck 175 143. Intrafusal muscle-fiber from neuromuscular nerve end-organ of rabbit (Huber) 176 144. Cross-section of a neuromuscular nerve end-organ from interosseous muscle of man (Huber) ... ... ... . 176 145. Neuromuscular nerve end-organ from plantar muscles of dog ( Huber-De Witt) 177 146. Neurotendinous nerve end-organ from rabbit (Huber-De Witt) 178 147. Cross-section of neurotendinous nerve end-organ of rabbit (Huber-De Witt) . 179 148. Ranvier's crosses from sciatic nerve of rabbit 181 149. Medullated nerve-fiber from sciatic nerve of frog 181 150. Ganglion cell from anterior horn of spinal cord of calf 182 151. Human red blood-cells 188 152. Rouleau formation of human erythrocytes 1 88 153. Hemin, or Teichmann's crystals, from blood stains on a cloth (Huber) . . . 188 154. Crenated human red blood-cells 188 155. Red blood-corpuscles subjected to the action of water 188 156. Red blood-corpuscles from various vertebrate animals 189 157. White blood-corpuscles from normal blood of man 191 158. Ehrlich's leucocytic granules 192 159. Fibrin from laryngeal vessel of child (Huber) 195 160. Solitary lymph-nodule from human colon 197 161. Transverse section of human cervical lymph-gland, showing the general struc- ture of a lymph-gland 198 162. Section from human lymph-gland 199 163. Section through the human spleen 203 164. Lobule of the spleen (Mall) 205 165. Cells containing pigment, blood-corpuscles, and hemic masses from spleen of dog 206 1 66. Section through human spleen showing reticular fibrils 206 167. Cover-glass preparation from bone-marrow of dog 208 1 68. Section through human red bone-marrow ... 209 169. Small lobule from thymus of child, well-developed cortex 211 170. Hassall's corpuscle and a small portion of medullary substance from thymus of child ten days old (Huber) 211 171. Cross-section of human carotid artery 217 172. Section through human artery, one of the smaller of the medium-sized ... 217 173. Precapillary vessels from mesentery of cat (Huber) 218 174. Cross-section of human internal jugular vein 219 175. Section of small human vein . . . 220 176. Endothelial cells of capillary and precapillary from mesentery of rabbit (Huber) 221 177. Small artery from oral submucosa of cat with nerve-terminations 222 178. Section of a cell-ball from glomus caroticum of man , . 225 1 79. Thoma-Zeiss hemocytometer 232 180. Section through lower lip of man ... .... 237 181. Longitudinal section through a human tooth, showing lines of Retzius . . . 239 182. Portion of ground tooth from man, showing enamel and dentin .... 240 183. Longitudinal section through human molar from the center of the enamel layer 241 184. Cross-section of human tooth, showing cement and dentin 243 185. Nerve termination in pulp of rabbit's molar (Huber) 244 186-189. Four stages in the development of tooth in sheep embryo 245 190. Portion of cross-section through developing tooth 246 191. Fungiform papilla from human tongue (Huber) 247 192. Cross-section of human tongue showing filiform papillae . . 248 193. Longitudinal section of foliate papilla of rabbit, snowing taste-buds (Huber) 249 194. Longitudinal section of a human circumvallate papilla 250 14 ILLUSTRATIONS. FIG. PAGE 195. Schematic representation of a taste-goblet (Hermann) 251 196, 197. Section through pharyngeal tonsil of man . . 252, 253 198. Section through salivary gland of rabbit, with injected blood-vessels ... 255 199. Model of a small portion of a sublingual gland of man 256 200. Section of human submaxillary gland 257 201. Section of parotid gland of man 257 202. Portion of a model of a salivary gland with mucus secretion 258 203. Alveoli from submaxillary gland of dog (Huber) 258 204. Model of a gland of v. Ebner, from a boy 260 205. Section of esophagus of a dog . ... 263 206. Section of human esophagus, showing a cardiac gland with a dilated duct . 264 207. Epithelium of human stomach, covering fold of mucosa between two gastric crypts 266 2c8. Vertical section through fundus of human stomach . . 267 209. Fundus glands from fundus of stomach of young dog (Huber) 267 210. Section through junction of human esophagus and cardia 268 211. Vertical section through human pylorus 270 212. Section of human pylorus 271 213. Section through fundus of human stomach in condition of hunger 272 214. Section through fundus of human stomach during digestion 272 215. Illustrations of models, made after Bern's wax-plate reconstruction method, of glandular structures and duodenal villi of human intestine 273 216. Section through mucous membrane of human small intestine 275 217. Longitudinal section through summit of villus from human small intestine . . 276 218. Section through the junction of the human pylorus and duodenum . . . . 278 219. Section of solitary lymph-nodule from vermiform appendix of guinea-pig . . 279 220. Section through colon of man, showing glands of Lieberkiihn 280 221. Transverse section of human vermiform appendix (Huber) 281 222. Solitary lymph-follicle from human colon 282 223. Section through fundus of injected cat's stomach 283 224. Schematic transverse section of human small intestine (Mall) 285 225. Portion of the plexus of Auerbach from stomach of cat (Huber) 286 226. Section of esophagus of cat showing nerve-terminations (Huber) 287 227. Section through liver of pig, showing chains of liver-cells 289 228. Section through injected liver of rabbit 290 229. 230. Human bile capillaries . 291 231. Diagram of hepatic cord in transverse section 292 232. Section through the human liver, showing the beginning of bile-ducts . . . 292 233. Injected blood-vessels in liver lobule of rabbit 293 235. Reticulum of dog's liver 294 236. Connective tissue from liver of sturgeon, showing reticulum 295 237. Liver of rabbit, showing the so-called stellate cells of Kupffer (Huber) . . 296 238. Section through liver lobule of dog, showing stellate cells 297 239. Transverse section through alveolus of frog's pancreas 299 240. Model of lobule of human pancreas 299 241. 242. Section through human pancreas 300, 301 243. Relation of three adjoining alveoli to excretory duct, illustrating origin of centro-acinal cells . 3°2 244. Section of human pancreas, showing gland alveoli surrounding an area of Langerhans (Huber) ... 302 245. Vertical section through mucous membrane of human larynx 309 246. Longitudinal section of human trachea (Huber) 311 247. Transverse section through human bronchus 312 248. 249. Sections of cat's lungs ... 313 250. Internal surface of human respiratory bronchiole (Kolliker) 314 251. Inner surface of human alveolus, showing respiratory epithelium (Kolliker) . 315 252. Respiratory epithelium in amphibia . 316 253. Scheme of lung lobules after Miller . . . 317 254. Reconstruction in wax of a single atrium and air-sac with the alveoli (Miller) 317 255. Section of human lung, showing elastic fibers (Huber) 318 256. Section through injected rabbit's lung . . 318 257. Cross-section of thyroid gland of man (Huber) 319 258. Section from parathyroid of man (Huber) 321 259. Kidney of new-born infant ... 323 260. Isolated uriniferous tubules 324 ILLUSTRATIONS. 1 5 FIG. PAGE 261. Median longitudinal section of adult human kidney 325 262. Section of cortical substance of human kidney 326 263. Section of proximal convoluted tubules from man 327 264. Epithelium from proximal convoluted tubule of guinea-pig, with surface and lateral views .... 328 265. Cortical portion of longitudinal section of kidney of child (Huber) 328 266. Section of medulla of human kidney ... 329 267 Longitudinal section through papilla of injected kidney 330 268. Section through junction of two lobules of kidney 331 269. Diagrammatic scheme of uriniferous tubules and blood-vessels of kidney . 333 270. Direct anastomosis between an artery and vein in a column of Berlin of child' 335 271. Section of lower part of human ureter . ... 337 272. Transverse section of the wall of the human bladder, giving a general view of its structure 338 273. Section of suprarenal cortex of dog 340 274. Arrangement of intrinsic blood-vessels in cortex and medulla of dog's adrenal (Flint) 341 275. Section from ovary of adult dog (Waldeyer) 345 276. Section from ovary of young girl 340 277-280. Sections from cat's ovary 348 281. Transverse section through the cortex of a human ovary 349 282. Representation of behavior of the chromatin during the maturation of the ovum (Riickert) . . 351 283. Scheme of the development and maturation of an ascaris ovum (Boveri) . . 352 284. Section of oviduct of young woman 7 355 285. Section from uterus of young woman ... 4 357 286. Section of human vagina (Huber) 358 287. Section of human labia minora (Huber) 359 288. Diagram showing the characteristics of spermatozoa of vertebrates 361 289. Human spermatozoa . . 362 290. Longitudinal section through human testis and epididymis 363 291. 292. Sustentacular cells 364 293. Section of human testis (Huber) 365 294. Section through human vasa efferentia 366 295. Cross-section of vas epididymidis of human testis (Huber) 366 296. Section of dog's testis with injected blood-vessels 367 297. Cross- section of vas deferens near epididymis (human) (Huber) 368 298. Cross-section of wall of seminal vesicle (human) (Huber) 369 299. Section of prostrate gland of man (Huber) . . 369 300. Schematic diagram of spermatogenesis as it occurs in ascaris (Boveri) .... 373 301. Schematic diagram of section through convoluted seminiferous tubule of mammal (Hermann ) . . 375 302. Section of convoluted tubule from rat's testicle 376 303. Under surface of the epidermis . ... 380 304. Cross-section of skin of child with injected blood-vessels 381 305- Prickle cells from the stratum Malpighii of man 382 306. Cross-section of human epidermis 383 307. Cross-section of negro's skin 384 308. A reconstruction, showing the arrangement of the blood-vessels in the skin of the sole of the foot (Spaltcholz) 385 309. Nerves of epidermis and papillae from ball of cat's foot 386 310. 311. Meissner's corpuscle from man 387 312. Grandry's corpuscles from duck's bill 388 313. Transverse section of human scalp . . • 390 314. Longitudinal section of human hair and follicle 391 315. Cross-section of human hair with follicle 392 316. Longitudinal section of cat's hair and follicle, showing nerve-termination . . 393 317. Longitudinal section through human nail and its groove 394 318. Transverse section through human nail and its sulcus 395 319. Coiled portion of a sweat-gland from the plantar region of a man, reconstructed by Bern's wax-plate method (Huber — Adamson) 396 320. Cross-section of coiled tubule of sweat-glands from human axilla . . . . 396 321. Tangential section through coiled tubule of sweat-glands from human axilla 397 322. Sebaceous gland with a portion of the hair follicle, reconstructed by Bern's wax-plate method 398 1 6 ILLUSTRATIONS. FIG. PAGE 323. Section of alveoli from sebaceous gland of human scalp (Huber) 399 324. Model of small portion of a secreting mammary gland 400 325. Section of mammary gland of nullipara (Nagel) 401 326. Transverse section through human skin 404 327. Cross-sections of human spinal cord 407 328. Schematic diagram of spinal cord in cross-section (von Lenhossek) 409 329. Schematic cross-section of spinal cord (Ziehen) .. 410 330. Section through human cerebellar cortex vertical to the surface of the convolution 413 331. Schematic diagram of cerebellar cortex 414 332. Cell of Furkinje from human cerebellar cortex 415 333. Granular cell from the granular layer of the human cerebellar cortex .... 415 334. Vertical section of human cerebral cortex 418 335. Large pyramidal cell from human cerebral cortex 419 336. Schematic diagram of cerebral cortex 420 337. Olfactory bulb 422 338. Longitudinal section of spinal ganglion of cat (Huber) 424 339. Ganglion cell from the Gasserian ganglion of a rabbit (Huber) 425 340. Diagram showing the relations of the neurones of a spinal ganglion (Dogiel) 426 341. Neurone from inferior cervical sympathetic ganglion of a rabbit (Huber) . . 427 342. From section of semilunar ganglion of cat (Huber) 428 343. From section of stellate ganglion of dog (Huber) 429 344. From section of sympathetic ganglion of turtle (Huber) 430 345. From section of sympathetic ganglion of frog (Huber) 430 346. Schematic diagram of a sensorimotor reflex arc according to the modern neu- rone theory 431 347. Schematic diagram of a sensorimotor reflex cycle 432 348. Schematic diagram of the reflex tracts between a peripheral organ and the brain cortex . 433 349. Neurogliar cells ( Huber) 434 350. Neurogliar cells from cross-section of the white matter of the human spinal cord (Huber)- ... 436 351. Section through injected cerebral cortex of rabbit 438 352. Schematic diagram of the eye (Leber and Flemming) 447 353. Section through the anterior portion of human cornea 449 354. Corneal spaces of dog > 450 355. Section through the human choroid 452 356. Meridional section of the human ciliary body 454 357. Injected blood-vessels of the human choroid and iris 456 358. Section of the human retina 458 359. Section through point of entrance of human optic nerve 460 360. Section through human macula lutea and fovea centralis 461 361. Schematic diagram of the retina (Ramon y Cajal) , 463 362. Injected blood vessels of the human retina 465 363. Injected blood-vessels of human macula lutea 466 364. Vertical section of upper eyelid of man . . 471 365. Meibomian or tarsal gland, reconstructed after Bern's wax-plate method . . . 472 366. Schematic representation of the complete auditory apparatus (Schwalbe) . . 477 367. Cross-section of the Eustachian tube 479 368. Right bony labyrinth (Quain, after Sommering) 480 369. Membranous labyrinth from five-month human embryo (Schwalbe, after Retzius) 481 370. Transverse section through an osseous and membranous semicircular canal of an adult human being 482 371. Vertical section through the anterior ampulla . . • • 484 372. Longitudinal section of the cochlea of a cat 486 373. Section through a turn of the osseous and membranous cochlear duct of the cochlea of guinea-pig 4^7 374. Organ of Corti (Retzius) 490 375. Surface of organ of Corti, with surrounding structures (Retzius) 493 376. Scheme of distribution of blood-vessels in labyrinth (Eichler) ...... 495 377. Portion of transverse section of the olfactory region of man 499 INTRODUCTION TO MICROSCOPIC TECHNIC. L THE MICROSCOPE AND ITS ACCESSORIES. A detailed description of the microscope and its accessory appa- ratus hardly lies within the scope of this book. If, notwithstanding, a few points be touched upon, it is done only that the beginner may have a working knowledge of the different parts of the instru- ment which he must use. A more intimate knowledge of the theory of the microscope may be acquired by studying such works as those of Dippel, A. Zimmermann, and Carpenter. Histologic specimens are examined with the aid of the micro- scope, an instrument which magnifies the objects by means of its optic apparatus. For this purpose simple microscopes, consisting of one or more converging lenses or lens systems may be used, though they generally do not give sufficient magnification to be of much service in the study of histologic specimens ; they give an erect image of the object observed. When greater magnification is desired, it is necessary to use a compound microscope, consist- ing generally of two or more lens systems, giving an enlarged, inverted, real image of the object observed. The lens system of a compound microscope may be changed according to the needs of the case, and thus a variation in the magnification of the object obtained. The rest of the instrument consists of a framework called the stand, the lower portion of which consists of a foot- plate or base. From the base rises the column or pillar, to which the other parts of the microscope are attached. From below upward come the movable mirror, the stage and substage with diaphragm and condenser, and the tube with pinion and fine adjust- ment. One side of the mirror is concave, and serves to concentrate the rays of light in the direction of a central opening in the stage. The other side is plane. If the objects are to be examined by direct illumination, and not by transmitted light, the mirror is so placed that the rays are reflected away from the opening in the stage. The specimen to be examined is placed on the stage, over the central opening. If the light be too strong, the opening may be diminished in size by means of a diaphragm. In some instru- ments these diaphragms are placed in the opening of the stage, and consist of plates with different sized apertures. A better form is composed of one large disc containing several apertures of different sizes. This is fastened to the under surface of the stage in such a way that by revolving the disc the apertures may be brought one 2 17 1 8 THE MICROSCOPE AND ITS ACCESSORIES. after the other opposite the opening in the stage. A much better diaphragm, constructed on an entirely different principle, is the so- called iris diaphragm. Although its opening is not exactly circu- lar, yet it has the advantage of being easily enlarged or contracted by manipulating a small handle controlling the metal plates sliding over one another. The tube, which is contained in a close-fitting metal sheath, is attached to the upright of the microscope. In the simpler forms Ocular or eyepiece. Draw-tube. Tube. Triple nose-piece. Objectives, Stage. Iris diaphragm and Abbe condenser. Screw for focusing condenser. Mirror. - Rack and pinion for coarse adjustment. Micrometer screw for fine adjustment. Pillar. - Stand. Fig. I. — Microscope. of microscopes the tube is raised, lowered, or twisted by hand. In more complicated instruments the upward and downward move- ments are accomplished by means of a rack and pinion — coarse adjustment. A micrometer screw — fine adjustment — situated at either the upper or the lower end of the upright, controls the fine adjustment. The tube possesses an upper and a lower opening, into which lenses may be laid and screwed. The ocular, into the ends of which lenses are inserted, fits into the upper opening. The LENSES. 19 upper is called the ocular lens, the lower the collective lens. The objective system, which is a combination of several lenses or lens systems, the lowest and smallest of which is known as the front lens, is screwed into the lower opening of the tube. All larger instruments possess several oculars and objec- tives, which together give different magnifications according to the combinations used. For most objects a magnification of 500 diam- eters is all that is required, but to obtain this and still have a clear and bright field the ordinary lenses are hardly sufficient. The greater the magnification, the darker is the field. To avoid this, illuminating mechanisms (condensers, Abbe's apparatus) have been constructed, by means of which the rays of light are concentrated and controlled. This arrangement is absolutely necessary for deli- cate work. Even with the aid of such an apparatus the dry objective sys- tems are not sufficient. With them the rays of light must pass through different media having various indices of refraction. The rays pass from the object through the cover-slip, and then through the air between the latter and the objective system. They are thus deflected in different directions — a defect which would be avoided if the rays were made to pass through a single medium. This latter condition may be practically brought about by placing between the objective and the cover-glass a drop of some fluid having about the same refractive index as the glass. The lens is then lowered into the fluid. As this invention has proved useful, so-called immersion lenses have been made during recent years. There are thus twcr kinds of lens systems — the dry and the immersion lenses. The latter are divided into two groups — lenses with water and those with oil immersion. As oil has a greater index of refraction than water, and one more nearly approaching that of glass, the oil- immersion lenses are at present the best objectives that we possess; Karl Zeiss, of Jena, and other microscope makers, have in late years made lenses from a special sort of glass which reduces to a minimum the chromatic and spheric aberration of the rays of light in their passage through the objective (apochromatic lenses). The rays of light reflected from the mirror and passing through the object are refracted by the objective system in such a way that they are focused in a so-called real image at a point about half-way up the tube. This picture is an inverted one, the right side of the microscopic field being at the left of the real image, and the upper portion below. The picture is, in other words, rotated 1 80 degrees. By means of the ocular the real image is again mag- nified— virtual image — but no longer inverted, although to the eye of the microscopist the field actually appears inverted. To shut out the rays of light, which cause a diffused picture, diaphragms are sometimes introduced into the tube as well as into the ocular. (See Fig. 2.) The objects to be examined are placed upon a glass plate 20 THE MICROSCOPE AND ITS ACCESSORIES. called a slide. ' Microscopic slides are of different sizes, and are usually oblong in shape. Those in most common use are three inches long arid an inch wide. The object is covered by a very much smaller and thinner glass plate — the cover=slip. The whole preparation is then placed upon the stage in such a way that the cover- slip is upward and immediately be- neath the end of the tube. The mirror of the microscope is now so adjusted as to concentrate the rays of light on the preparation, illuminating it as much as is necessary. By means of the rack and pinion, or coarse adjustment, the whole tube is now slowly lowered toward the cover-slip until the bare outlines of the object are dimly seen in the white field. From this point on, the micrometer screw, or fine adjustment, is used in bringing the front lens down to its proper focal distance from the prep- aration. The object is now seen to be clear and well defined. By turn- ing the screw to the right or the left, different parts of the specimen are brought more clearly into view, this result being due to the fact that not all points in the preparation are in the same plane. In studying objects it is always well to draw them, using a sharpened pencil and smooth paper. The be- ginner soon finds that with constant practice he can sketch the different parts .of the field in nearly their proper relationship. This by no means easy work is facilitated by the use of a drawing apparatus called the camera lucida. The best of these is that devised by Abbe. It is fastened to the upper end of the tube, above the ocular. The apparatus is so made that both the preparation and the drawing surface are seen by the same eye. The microscopic field is seen directly, while the drawing sur- face is made visible by means of a mirror. When the apparatus is in place and the drawing commenced, it appears to the one sketching as if his pencil were moving over the preparation itself. Fig. 2. — .Diagram showing the principle of a compound microscope with the course of the rays from the object (a b] through the objective to the real image (bl a1), thence through the ocular and into the eye to the retinal image (a* i>2), and the projection of the retinal image into the field of vision as the virtual image (b3 a3). — (Fig 21, Gage, The Microscope, eighth edition. ) SECTIONS OF FRESH TISSUES. 21 Outlines are reproduced on paper with great exactness both as to form and size ; finer details must of course be sketched in free hand. Every preparation should first be examined with a low power, and only after the student has studied the specimen as a whole and found instructive areas should the higher powers be used. IL THE MICROSCOPIC PREPARATION. In many cases the making of a microscopic preparation is a very simple procedure, especially when fresh objects are to be examined. A drop of blood, for instance, may simply be placed upon a slide, covered with a cover-slip, and examined. Other objects, as the mesentery, thin transparent nerves, detached epithelia, spermatozoa, etc., need no further preparation, but may be examined at once. Portions of larger organs are often studied after having been teased, which may be done by means of two needles fastened in handles. If the objects be composed of fibers running in parallel directions, one needle is thrust into the substance to hold it in place, while the other is used to tear the fibers apart. This method is used in examining muscles, nerves, tendons, etc. Some tissues are so constituted that they can only be investigated by means of sections, which permit a study of their elements and the rela- tionship of the same to each other. In this method an ordinary razor, moistened in some fluid, may be employed. As a rule, it is not the size of the section, but the thinness, which is important. This latter is obtained only by practice. Every microscopist ought to become accus- tomed to making free-hand sections with the razor. It is the simplest of all methods, is very rapid, and is especially useful in the quick identifica- tion of a tissue. In cutting fresh so-called parenchymatous tissues, such as liver and kidney, an ordinary razor is not sufficient. Here a double knife is necessary. This consists of two blades, which are so placed one above the other that their distal ends touch, while their proximal ends are slightly separated. The distance of the blades from each other is regulated by a screw. If this be removed the knives may be separated for cleaning. In making sections, only those portions of the blades are of importance which are very close together but do not actually touch. Sections are cut by drawing the moistened instrument quickly through an organ, as, for instance, a fresh liver. As the organ is cut in two, a very thin section of the tissue remains between the blades. This is removed by taking out the screw and separating the blades in normal salt solution. Organs of a similar consistence can be frozen and then cut with an ordinary razor the blade of which has been cooled. Sometimes good results may be obtained by drying small pieces of tissue, as, for instance, tendon. As sections or small pieces of fresh tissue would soon become dry when placed on the slide, they must be kept moist during examina- tion. They are therefore mounted in so-called indifferent fluids (placed on the slide and immersed in a few drops of the indifferent fluid and covered with a cover-slip). These have the power of preserving living tissues for some time without change. Such fluids, for instance, are 22 THE MICROSCOPIC PREPARATION. the lymph, the aqueous humor, serous fluids, amniotic fluid, etc. Artifi. cial indifferent fluids are much used and should always be kept in stock. Of this class, the following are useful : 1. Physiologic saline solution: A 0.75% solution of sodium chlorid in distilled water. 2. Schultze's iodized serum: A saturated solution of iodin or tincture of iodin in amniotic fluid. 3. Ranvier's solution of iodin and potassium iodid : A satu- rated solution of iodin in a 2% solution of potassium iodid. 4. Kronecker's fluid : Distilled water, 100 c.c. ; sodium chlorid, 5 gm.; sodium carbonate, 0.06 gm. 5. Solution of Ripart and Petit : Copper chlorid, o. 3 gm. ; cop- per acetate, 0.3 gm. ; aqua camphorae, 75 c.c. ; distilled water, 75 c.c. ; and glacial acetic acid, i c.c. After mixing, this solution is yellow, but clears up within a few hours, and should then be filtered. The examination of fresh tissues comes far from revealing all the finer details of their structure. This is partly due to the fact that the indices of refraction of the different elements of the tissues are too nearly alike, in consequence of which the outlines are somewhat dimmed ; and also, that changes occur, even during the most careful manipulation of the tissues, which result in pictures somewhat different from the normal. With many tissues and organs while yet fresh it is also somewhat difficult to obtain a separation of their constituent elements. It is therefore generally necessary to subject tissues or organs to special methods of treatment before they may be studied microscopically with any degree of profit. Certain of these methods, such as have proved by experience to possess reliability, shall receive consideration in the following pages. METHODS OF MACERATION. The reagents employed for the maceration of tissues have in general the property of softening or removing, partly or completely, certain con- stituents of the tissues, while they at the same time harden or fix other tissue elements. Generally the ground-substance or intercellular sub- stance is softened or removed while the cellular or other constituents undergo fixation. Tissues thus treated when subjected to teasing, crushing, shaking, or brushing with a camel' s-hair brush, are readily broken up into their constituent elements, giving useful and instructive preparations. 1. Alcohol, 30% (Ranvier). Dilute one volume of alcohol with two volumes of distilled water. Small pieces of tissue are macerated in this solution in twenty-four hours to forty- eight hours. It is often advantageous to fix the pieces thus macerated for about an hour in -|% to i% osmic acid. Useful for macerating epithelia. 2. Dilute solutions of chromic acid, i% to -^% • Small pieces of tissue remain in this solution one to several days. Use- ful for macerating epithelia. 3. Concentrated aqueous solution of caustic potash. Small pieces of tissue are macerated in fifteen minutes to an hour. They are then transferred to a saturated aqueous solution of acetate FIXING METHODS. 23 of potassium, which interrupts the action of the macerating fluid. Useful for macerating epithelia and involuntary and heart muscle. 4. Hydrochloric acid, 20% to 30% aqueous solution. Mace- rates small pieces of tissue in twelve to twenty-four hours. The pieces are then thoroughly washed in water. Useful for isolating the uriniferous tubules and macerating glands. 5. Nitric acid, 10% to 20% aqueous solution or made up with normal salt solution. Macerates small pieces of tissue in twenty- four to forty-eight hours. Wash thoroughly in water. Useful for macerating involuntary and voluntary muscle. 6. J. B. MacCallum ("Contributions to Medical Science," Baltimore, 1900) recommends the following nitric acid mixture for isolating heart -muscle fibers of embryos and adults : Nitric acid, i part; glycerin, 2 parts ; water, 2 parts. The hearts remain in this fluid from eight hours to three days, according to their size, and are then transferred to a 5 % aqueous solution of glycerin. This method is especially useful for obtaining prepara- tions showing the arrangement of the heart -muscle fibers. 7. Nitric acid and chlorate of potassium (Schulze). Powder the chlorate of potassium and add sufficient nitric acid to make a thin paste. Embed the tissue to be macerated in this paste, in which they remain from one to several hours. They are then washed in water. Useful for isolating the branched, voluntary muscle -fibers of the tongue of a frog. 8. Concentrated sulphuric acid. Useful for isolating the corni- fied cells of the epidermis, nails, and hair. FIXING METHODS. The fixing fluids most used for general purposes are the following : Alcohol. — Alcohol is frequently used as a fixing fluid. It is at the same time a hardening fluid, as the water of the tissues is withdrawn and their albumin coagulated. Small or thin pieces are put immediately into absolute alcohol, in which they remain for from twelve to twenty- four hours. The period required for fixation may be greatly shortened by changing the absolute alcohol at the end of one or two hours. In the case of larger pieces, a successive immersion in gradually increasing, strengths of alcohol (50%, 70%, 90%) is the method chosen. Pieces i c.c. in size remain for twenty -four hours in each grade of alcohol, larger pieces for a proportionately longer time. Alcohol used in this way is a hardening fluid rather than a fixing fluid. Carney's Acetioalcohol Mixture. — Glacial acetic acid I part. Absolute alcohol 3 parts. Fixes very rapidly. Pieces of i centimeter in thickness are fixed in one-half hour to one hour. The after-treatment is with absolute alcohol, which should be renewed at the end of twenty-four hours. Carney's Acetic Acid-alcohol=chloroform Mixture. — Glacial acetic acid I part. Chloroform 3 parts. Absolute alcohol 6 " Fixes very rapidly, even larger pieces in from one-half to -one hour. The after-treatment is with absolute alcohol. 24 THE MICROSCOPIC PREPARATION. Osmic acid is a reagent that kills quickly, fixes protoplasm exceed- ingly well, but nuclei not so well, and colors certain tissues. Only small pieces can be fixed in this fluid, as it does not easily penetrate the tissues. It is ordinarily used in a \c/c to \cfo aqueous solution, the objects remaining immersed twenty-four hours. They are then washed in running water for the same length of time, after which they are trans- ferred to 90% alcohol. Very small objects may be treated with osmic acid in the form of vapor (vaporization). This is done as follows: A very small quantity of osmic acid solution is put in a small dish. The object is then suspended by a thread in such a way that it does not come in contact with the fluid. The dish should be covered with a well-fitting lid. Flemming's Solution. — A solution with a similar action, but fixing nuclear structures better than osmic acid, is the chromic-osmic-acetic acid solution of Flemming : Osmic acid, I c/c aqueous solution . . 10 parts. Chromic acid, I c/o aqueous solution ... 25 " Glacial acetic acid, \°/o aqueous solution .10 " Distilled water 55 " Small pieces are fixed in a small quantity of the fluid for at least twenty- four hours, sometimes for a longer period, extending even to weeks. They are then washed for twenty-four hours in running water and passed through 50%, 70%, and 80%, each twenty-four hours, into 90% alcohol. Flemming also recommends a stronger solution, which is made as follows : Osmic acid, 2^ aqueous solution .... 4 parts. Chromic acid, I f/o aqueous solution ... 1 5 " Glacial acetic acid ." I part. Fol's Solution. — Fol has recommended the following modification of Flemming's solution : Osmic acid, I % aqueous solution .... 2 parts. Chromic acid, I c/c aqueous solution ... 25 " Glacial acetic acid, 2cfo aqueous solution .5 " Distilled water 68 " The after-treatment is the same as for Flemming's solution. Hermann's Solution. — Very good results sometimes follow the use of the platinum-acetic-osmic acid solution of Hermann (89, i). It is employed as is Flemming's solution : Osmic acid, 2 ^ aqueous solution .... 4 parts. Platinum chlorid, \c/0 aqueous solution . .15 " Glacial acetic acid i part. After fixing with this solution, Flemming's solution, or any other osmic mixture, the subsequent treatment with alcohol may be followed by crude pyroligneous acid. The objects are placed for from twelve to twenty-four hours in the latter and then again immersed in alcohol. The result is a peculiar coloring of the specimen which often makes subsequent staining (see below) unnecessary (Hermann). Corrosive Sublimate. — An excellent fixing fluid is made by saturating distilled water or a physiologic saline solution (see p. 22) with FIXING METHODS. 25 corrosive sublimate. Small pieces, about 0.5 cm. in diameter, are im- mersed in this fluid for from three to twenty-four hours, are then washed in running water for twenty -four hours, and then transferred into 70% alcohol. After twenty-four hours the tissues are placed in 8oc/c for the same length of time, and then preserved in gof/c alcohol. It often occurs that after changes in temperature crystals of sublimate are formed on the surface or in the interior of the object. For their removal a few drops of a solution of iodin and potassium iodid are added to the alcohol (P. Mayer). It is a matter of indifference whether the joc/f , 8o(/c or 90% alcohol is thus iodized. In the further treatment of the object, as well as in sectioning, any such crystals of sublimate will not be found to be a hindrance. Indeed, in the case of very delicate objects it is often more advantageous to undertake their removal after sectioning by adding iodin to the absolute alcohol then used. Acetic Sublimate Solution. — This is an excellent fluid, and at present much used for embryonic tissues and for organs containing only a small quantity of connective tissue. To a saturated aqueous solution of sublimate, 5^ to 10% of glacial acetic acid is added. After remaining two or three hours or more in this solution, the objects are transferred to 35% alcohol, after which they are passed through the higher grades of alcohol. Picric Acid. — Small and medium-sized objects (up to i c.c.) are fixed in twenty-four hours in a saturated aqueous solution of picric acid (about 0.75%), although an immersion lasting for weeks is not detrimental, especially if the objects be of considerable size. The tissues are transferred to 70% or 80% alcohol, in which they remain until the alcohol is not colored by the picric acid. They are then preserved in 90% alcohol. Instead of a pure solution of picric acid, the picrosulphuric acid of Kleinenberg or the picric=nitric acid of P. Mayer may be used. The first is made thus : i c.c. of concentrated sulphuric acid is added to 100 c.c. of a saturated aqueous picric acid solution. This is allowed to stand for twenty-four hours, then filtered, and diluted with double its volume of distilled water. The picric-nitric acid solution is made by adding 2 c.c. of pure nitric acid to 100 c.c. of a saturated picric acid solution. Filter after standing for twenty-four hours. Rabl's Solutions. — C. Rabl (94) recommends the following mixtures, especially for embryos : ( i ) Concentrated aqueous solution of corrosive sublimate, i vol. ; concentrated aqueous solution of picric acid, i vol. ; distilled water, 2 vols. (2) i per cent, aqueous solution of platinum chlorid, i vol. ; concentrated aqueous solution of corrosive sublimate, i vol. ; distilled water, 2 vols. In both cases, after being washed twelve hours in water (in the first preferably in alcohol) the specimens are transferred to gradually increased strengths of alcohol. Vom Rath's Solutions. — O. vom Rath (95) recommends, among others, the following two solutions: (i) Picric=osmic=acetic acid solution. Add to 1000 c.c. of a cold saturated picric acid solution i gm. of osmic acid, and after several hours 4 c.c. of glacial acetic acid. Objects are fixed, according to their size, in four, fourteen, and forty- eight hours, and then transferred to T$c/c alcohol. (2) Picric=sub= limate=osmic acid solution. A mixture of 100 c.c. of a cold saturated aqueous picric acid solution with 100 c.c. of saturated sublimate solution is made, into which is poured 20 c.c. of a 2 c/( osmic acid solu- 26 THE MICROSCOPIC PREPARATION. tion. 2 c.c. of glacial acetic acid may also be added. Tissues fixed by either of these fluids may be treated with pyroligneous acid or tannin. The crystals of sublimate must be removed by iodized alcohol. Nitric Acid. — Small objects may be fixed in about six hours in 3% to 5% nitric acid (sp. gr. 1.4). A longer immersion is injurious, as certain nuclear structures are affected. After washing thoroughly in running water, the tissues are treated as usual with alcohols of increasing concentration. Chromic acid is used in a Y3% to i% aqueous solution. Small pieces are fixed for twenty-four hours, larger ones for a longer time, even weeks. The quantity of the fixing fluid should be at least more than fifty times the- volume of the tissues to be fixed. The objects are subsequently washed in running water and run through the ascending alcohols. This last should be done in the dark. Two or 3 drops of formic acid may be advantageously added to each 100 c.c. of chromic acid solution (C. Rabl). Miiller's Fluid.— Potassium bichromate 2 to 2.5 gm. Sodium sulphate I " Water loo c.c. With this solution it requires several weeks for proper fixation, and the process must be conducted in the dark. During the first few weeks the solution should be changed every few days, and later once a week. According to the results desired, the pieces are either washed out in run- ning water and subsequently treated in the usual manner with alcohol, or they are placed directly in 70%, which is later replaced by 80 % and goj/o alcohol. It is important that all these procedures should take place in the dark. The use of Erlicki's fluid (potassium bichromate, 2^ gm.; cupric sulphate, 0.5 gm., and water, 100 c.c.) is quite similar to that of Miiller's, except that it acts much more quickly. A temperature of 30° C. to 40° C. shortens the process in both cases considerably, Miiller's fluid fixing in eight and Erlicki's in three days. Tellyesnicky's Fluid. — This solution gives better nuclear fixation than Miiller's fluid. Potassium bichromate 3 gm. Glacial acetic acid 5 c-c- Water 100 " Small pieces of tissue remain in this fluid for one or two days. Larger pieces may also be used, but require a longer period of fixation. Wash thoroughly in flowing water. Dehydrate in graded alcohol, beginning with 15%. Zenker's Fluid. — Potassium bichromate 2.5 gm. Sodium sulphate I " Corrosive sublimate 5 " Glacial acetic acid 5 c-c- Water 100 " It is advisable to add the glacial acetic acid in proper proportion to the quantity of the solution to be used, and not to add it to the stock solution. The tissues are allowed to remain for from six to twenty-four hours in this INFILTRATION AND IMBEDDING. 2/ mixture, in which they float for a short time. They are then washed in running water for from twelve to twenty-four hours, and transferred to gradually concentrated alcohols. Crystals of sublimate which may be present are removed with iodized alcohol. Zenker's fluid penetrates easily, and fixes nuclear and protoplasmic structures equally well without decreasing the staining qualities of the elements. Formalin (Formol). — Of recent years formalin, which is a 40 % solution of the gas formaldehyd in water, has been much used as a fixing fluid. It is best employed in the form of a solution made by add- ing 10 parts of formalin to 90 parts of water or normal saline solution. Small pieces of tissue remain in this solution for from twelve to twenty- four hours, larger pieces or organs a number of days or weeks, and are then transferred to 90% alcohol. Potassium Bichromate and Formalin. — Potassium bichromate, 2% to 3% aqueous solution 90 parts. Formalin 10 " Tissues remain in this fluid from several days to several weeks, de- pending on their size. Wash thoroughly in water and dehydrate in alcohol. Useful for fixation of central nervous system. We have attempted to give only the fixing and hardening fluids com- monly employed for general purposes. There are numerous other fluids used for special purposes ; these will be noticed under the headings of the corresponding tissues and organs. INFILTRATION AND IMBEDDING. Few tissues have a consistency, even after fixation, which enables them to be cut into sections thin enough to be studied under high magni- fication, without being especially prepared for this purpose. To admit of sectioning, it is generally necessary to imbed them in media which offer no resistance to the knife, while giving them firmness, and do not obscure the structure of the sections when cut, or which may be removed from the sections by methods which are not harmful to them. The media used for imbedding may be classed under two heads : ( i ) Such as are fluid when warm, and may in this state be caused to penetrate the tissue, and are solid when cold ; ( 2 ) such as are fluid when in solution, and in this state will penetrate tissues, but which become solid on the evaporation of the solvent. The best example of the former class of substances is paraffin ; and of the latter, celloidin (collodion or photoxylin). i. PARAFFIN IMBEDDING. In describing the method of paraffin infiltration and imbedding it is assumed that the tissues have been previously fixed and hardened and are in alcohol ready for further manipulation. From the hardened tissues small flat pieces are cut with a sharp knife or razor. If possible, they should be square, rectangular, or triangular in shape, their surfaces not exceeding i^ square inch, and their thickness from ^ to ^ of an inch. Pieces of larger size may be imbedded, if desired, provided the requisite care be exercised. The pieces selected are placed in absolute alcohol, in which they remain until thoroughly dehydrated. From the latter they 28 THE MICROSCOPIC PREPARATION. can not be passed directly into paraffin, as alcohol dissolves only a small percentage of paraffin, and, consequently, the preparation would not be infiltrated with the imbedding mass. The pieces of tissue are therefore first placed in some fluid which mixes with absolute alcohol and at the same time dissolves the paraffin. There are many reagents which have this property, such as xylol, toluol, chloroform, and a number of oils (oil of turpentine, oil of cedar, oil of origanum, etc.). Of these xylol may be recommended for general use. In the xylol the tissues remain for from two to twelve hours, the time depending somewhat on the size of the pieces and on the density of the tissue. When thoroughly permeated by the xylol, they are transparent. From the xylol (toluol, chloroform, or oils) the tissues are placed in melted paraffin. Two kinds of paraffin are generally used, one having a melting point of 38° to 40° C. — soft paraffin — and another with a melting point of 50° to 58° C. — so-called hard paraffin. The paraffin should always be filtered before using. This is best done by using a hot-water filter. It is essential that melted paraffin have a constant temperature while the tissues are being infiltrated. This is attained by placing the receptacle containing the paraffin in a paraffin oven regulated by means of a thermostat to a temperature about two degrees above the melting point of the hard paraffin. Filtered hard and soft paraffin may be kept in suitable glass beakers in respective compartments in the paraffin oven. After the tissues are thoroughly permeated with the xylol, this is poured off and melted soft paraffin added, and the dish replaced in the paraffin oven. In the soft paraffin the tissues remain from one to four hours, at the end of which time the soft paraffin is poured off and hard paraffin added, and the dish again placed in the oven. In the hard paraffin the tissues remain from Fig. 3,-Box for imbedding tissues. two to twelve hours, depending on the size of the pieces. They are now ready to be imbedded. Two metallic L's are placed together on a glass or metal plate in such a way as to make a rectangular box. (Fig. 3.) This is filled with melted hard paraffin taken from the oven. Before the paraffin cools, the piece of tissue to be imbedded is taken from the hard paraffin in the oven and placed with one of its flat surfaces against one end of the box. If several pieces of tissue are to be imbedded, a piece may thus be placed in each end of the box. While transferring the tissues from the hard paraffin to the imbedding box they should be handled with forceps, the blades of which have been warmed in a flame. As soon as the paraffin in which the tissues are imbedded has cooled sufficiently to allow the formation of a film over the melted paraffin, the imbedding box is placed in a dish of cold water. This cools the paraffin quickly and prevents its becoming brittle. A stay of from five to ten minutes in the cold water hardens the paraffin so that the L's may be removed, and the paraffin block containing the imbedded tissue may be taken from the plate. It is well to place the paraffin block thus obtained back into the cold water for a short time, so that it may become hard all the way through. As the paraffin often adheres closely to the glass or metal plate and the L's, it is advisable to cover these parts with a very thin layer of glycerin before imbedding. There is then no difficulty in separating them from the paraffin block. INFILTRATION AND IMBEDDING. 29 If a large number of small pieces of tissue are to be imbedded, it is often more convenient to imbed them in a small flat dish of suitable size. The dish to be used is covered on its inner surface with a thin layer of glycerin and partly filled with hard paraffin and the several pieces of tissue to be imbedded transferred to it and arranged on the bottom of the dish. As soon as a film forms over the paraffin the dish is placed care- fully in cold water and the paraffin allowed to harden. The large piece of paraffin thus obtained may then be cut into several smaller pieces, each containing a piece of the imbedded tissue. On transferring an object from one fluid into another, so-called currents of diffusion occur, which produce, especially in such tissues as contain cavities, shrinkage and tearing. This often results in totally changing~"the finer structure of the tissues. It is therefore necessary to proceed with greater caution than in the method above indicated. Mix- tures containing different percentages of alcohol and the intermediate fluid (xylol, toluol, chloroform) may be prepared, and the object, ac- cording to its delicacy, passed through a greater or smaller number of such solutions. In ordinary cases a single mixture of alcohol and the intermediate fluid is sufficient, the object remaining in the solution for a length of time varying with its size before being passed into the pure in- termediate fluid. This part of the treatment may of course be slowed or hastened according to the number of such mixtures, each succeeding one containing more and more of the intermediate fluid. After the object has been passed into the pure intermediate fluid it should be just as care- fully passed into the infiltrating fluid. If paraffin is to be used and the object be delicate, the following method is advisable : The object is placed in a glass vessel half filled with the intermediate fluid, into which a few pieces of soft paraffin are dropped. The vessel is then covered and allowed to remain at the temperature of the room. When the paraffin is dissolved the cover is removed and the vessel placed in a par- affin oven kept at a temperature corresponding to the melting point of the paraffin. The volatile intermediate fluid evaporates gradually, and in a few hours the object is infiltrated with an almost pure soft paraffin. It may now be transferred into pure melted hard paraffin. In this the tissue remains for a longer or shorter time, according to its size. It is often of advantage to infiltrate the tissues in a partial vacuum. In this way there is obtained a better infiltration of the tissues with the paraffin, and this seems to obtain a better consistency. Especially is this method to be recommended in imbedding larger embryos or tissue with cavities. A simple and convenient method is as follows : a glass bottle of suitable size is warmed and partly filled with melted hard paraffin and placed at one end of a copper plate, the other end of which is heated by a flame, care being taken to heat the copper plate only sufficiently to keep melted the paraffin in the bottle. The bottle is fitted with a rubber cork with two holes, into which have been inserted two L~ shaped glass tubes, provided, the one with a short rubber tube, which is clamped, the other with a tube of sufficient length to reach to a Chapman water-pump. The tissues are placed in the paraffin, the bottle tightly corked, and the water-pump allowed to play for about half an hour, after which the tissues are imbedded in the paraffin used during this procedure. High temperatures are, as a rule, injurious to tissues. This should always be borne in mind, and the student should aim to keep his specimens at the lowest possible temperature conducive to proper infiltra- tion. If for any reason higher temperatures become necessary, the ex- THE MICROSCOPIC PREPARATION. posure of the tissues to their action should be as brief as possible. The paraffins most used have a melting point of 40° to 60° C. The kind of paraffin used should depend upon the temperature of the room in which the sectioning is to be done. It is even well to have different mixtures of hard and soft paraffins at hand, so that, if the temperature of the room be low, tissues may be imbedded in a softer mixture, and vice versa. The process of infiltrating and imbedding in paraffin is repre- sented by the following diagram (instead of xylol, other intermediate fluids may be used) : Alcohol, 90% t Abs. alcohol Alcohol-xylol mixture t —Xylol -<— t Xylol-paraffin (cold) • t »• Xylol-paraffin (in paraffin oven) t Soft paraffin -^ - f Hard paraffin t Imbedding The size and density of the tissues must necessarily regulate the length of time necessary for their proper infiltration. It is therefore hardly possible to give any definite figures. In presenting the following table we have taken as a standard any tissue that has the general consistency of liver fixed in alcohol. The time is given in hours, and should in each case be regarded as a minimum. A longer stay in any one fluid will, under favorable circumstances, do no harm. SMALL OB- JECTS UNDER MIDDLE-SIZED OBJECTS UP LARGE OB- JECTS UP TO 10 VERY LARGE OB- JECTS, ALTHOUGH NOT MORE THAN A I MM. IN DIAMETER. TO 5 MM. IN DIAMETER. MM. IN DIAMETER. FEW CM. IN Di- Absolute alcohol . . . 2 6 24 For a longer or Xylol 1A •\ 6 shorter time in the fluids, ac- cording to the From now on in par- size of the object. affin oven : Soft paraffin l/> •3 6 Hard paraffin .... i 3 6 2. CELLO1DIN. The best and most convenient celloidin to use in microscopic work is Schering's granular celloidin, put up in i -ounce bottles. Of this a stock or thick solution is prepared by dissolving 6 gm. of the celloidin in 100 c.c. of equal parts of absolute alcohol and ether. Of this, when required, a thin solution is prepared by diluting a quantity of the stock solution with an equal quantity of the ether and alcohol solution. INFILTRATION AND IMBEDDING. $1 The hardened tissues are cut into small pieces, which should not be much more than J^ of an inch in thickness and not have a surface area of more than ^ of a square inch. Much larger pieces of tissue may be imbedded in celloidin. This is not advised, however, unless it is necessary to show the whole of the structure to be studied. The pieces to be imbedded are placed for twenty-four hours in absolute alcohol, and are then transferred for twenty-four hours to a mixture of equal parts of abso- ute alcohol and ether. Then they go into the thin celloidin solution, where they remain for from twenty-four hours to several days, depending on the size and density of the pieces to be imbedded. The pieces of tissue are then transferred to the thick celloidin solution, where they again remain for from twenty-four hours to several days. If it is desired to imbed large pieces, especially if these be of the medulla or brain, the stay in the cel- loidin solutions should be lengthened to several weeks. The hardening of the celloidin may now be obtained by one of several methods. A sufficient quantity of the stock or thick celloidin solution to cover well the tissues to be imbedded is poured into a flat dish large enough to allow the pieces to be imbedded to be arranged on its bottom and leave a space of about ^ of an inch between adjacent pieces. The dish is then covered, not too tightly, and set aside to allow the ether and alcohol to evaporate. In one or two days the celloidin is usually hard enough to cut into small blocks, each block containing a piece of the imbedded tissue. The blocks of celloidin are now further hardened by placing them in 80% alcohol. A stay of several hours in this alcohol is usually sufficient to give them the hardness required for section cutting. After the celloidin pieces have obtained the right degree of hardness they are to be stuck to small pieces of pine wood or vulcanized fiber so that they may be clamped into the microtome. This is done in the following way : A piece of celloidin containing a piece of tissue is trimmed with a sharp knife so that only a rim of celloidin about ^ of an inch in thickness surrounds the piece of tissue. It is now placed for a few moments in the ether and alcohol solution. This is to soften the surfaces of the celloidin. One end of a small pine -wood or vulcanized-fiber block about one inch long, the cut end of which has a surface area slightly larger than the celloidin block, is dipped for a few moments into the ether and alcohol solution and then into the thick celloidin. The celloidin block is now taken from the ether and alcohol solution, dipped into the celloidin, and pressed against the end of the wooden or vulcanized-fiber block, which has been coated with the celloidin. The whole is now set aside for a little while to allow the celloidin to harden slightly, and is then placed in 80% alcohol. In the alcohol it may remain indefinitely ; it may, however, be used for cutting as soon as it again becomes hard. The piece of tissue to be imbedded may be mounted at once on pine-wood or vulcanized-fiber blocks from the thick celloidin solution by pouring a small amount of thick celloidin over one end of the block and placing the piece of tissue from the thick celloidin solution onto the layer of celloidin on the block. In three to four minutes a layer of the thick celloidin solution is poured over the piece of tissue and the end of the block. It may be necessary to do this several times if the piece of tissue is large or of irregular shape. The block is now set aside for about five minutes, and is then placed in 8b% alcohol, where it remains until the celloidin is hard, or until it is desired to cut sections. 32 THE MICROSCOPIC PREPARATION. The tissues may be imbedded by pouring the thick celloidin, to- gether with the objects, into a small box made of paper. The surface of the celloidin hardens in about an hour (preliminary hardening), after which the whole is transferred to 80% alcohol, in which the final hardening takes place. The paper is then removed, the block of celloidin trimmed to a convenient size and fastened on a block. While being cut, celloidin preparations are kept moistened with 80% alcohol. Organs consisting of tissues of varying consistency, as well as very dense objects, can be cut with better results in celloidin than in paraffin. On the other hand, celloidin sections can never be cut as thin as paraffin sections, and the after-treatment (see below), fixation on the slide, etc., are much more complicated than in the case of paraffin sec- tions. The following is a diagram showing the process of infiltration and imbedding in celloidin. 90^ alcohol t Abs. alcohol t Abs. alcohol and ether (in equal parts) t Thin celloidin solution f Thick celloidin solution t Imbedding t Sof0 alcohol 3. CELLOIDIN-PARAFFIN. To combine the advantages which infiltration in celloidin and in paraffin offer, a method of celloidin -paraffin infiltration is recommended. Preparations that have been imbedded in celloidin or photoxylin and hardened in 80% alcohol are placed for about twelve hours in 90% alco- hol, from which they are transferred to a mixture of equal parts of oil of origanum and 90% alcohol. They are then immersed for a short time in pure origanum oil, then in a mixture of equal parts of origanum oil and xylol, and finally in pure xylol. From this point the regular method of infiltrating with paraffin is followed, care being taken that the pieces re- main for as short a time as possible in the different fluids, in order that the celloidin may not become brittle. Very thin sections may be obtained by painting the cut surface with a thin layer of a very dilute celloidin solution. This hardens and gives the tissue a greater consistency. This treatment is useful in the combined celloidin-paraffin method, as well as when paraffin alone is used. THE MICROTOME AND SECTIONING. Instruments known as microtomes have been devised in order that section cutting may be rendered as independent as possible of the skill of the individual, but more especially t<3 obtain series of sections of uni- form thickness. Their construction varies greatly. Some of these in- THE MICROTOME AND SECTIONING. 33 struments, as the so-called rocking microtomes, are so specialized that they only cut paraffin objects when the knife' is transversely placed. Others have a more general function, celloidin as well as paraffin objects being sectioned with the knife in any position. To the latter class belong the sliding microtomes. In figure 4 is shown an instrument which may be recommended for general laboratory work. This instrument consists of a horizontal base which rests on the table, and a vertical plate (#), and a slide (^) which supports a block (V), to which is fastened a knife by means of a thumb- screw (V). On the other side of the vertical plate is a metal frame ( every time the milled head is moved i notch (toward the manipulator) the slide carrying the clamp holding the tissue is elevated 5 microns ; 2 notches would elevate the tissue 10 microns (ToW °f an incn) ') 4 notches, 20 microns (j^Vo" °f an incn)> etc. It is not essential to have a lever attached to the instrument as above described, although this is very convenient ; if not present, the milled head is moved the desired number of notches with the hand. Minot has recently devised two kinds of microtomes which deserve spe- cial mention, and are especially to be recommended for accurate work. One of these (see Fig. 5) is known as the "Precision Microtome." It consists of a square frame made of cast-iron, to which the knife is fastened. .3 Fig. 5. — Minot automatic precision microtome. Fig. 6. — Minot automatic rotary microtome. 34 THE MICROTOME AND SECTIONING. 35 Beneath the frame which supports the knife are two horizontal ways, upon which runs the sliding carriage supporting an adjustable object-carrier. The object is raised by a micrometer screw, fed automatically by a large- toothed wheel attached to the bottom of the screw. Both paraffin and celloidin sections may be cut with this instrument. The other type of mi- crotome is known as the "New Rotary Microtome." In this instrument (see Fig. 6) the knife is carried by two upright standards which can be adjusted as to their distance from the object. The object, which needs to be imbedded in paraffin, is fixed to an object -carrier, which may be adjusted to any plane, and which is fixed to a vertical carriage, held by adjustable gibs against the vertical ways, and which is raised or lowered by a crank, working in a slide, and attached to an axle turned by the wheel. The vertical carriage also carries the micrometer screw, to which is attached a toothed wheel ; this is turned by a pawl which acts upon it. This instrument may be most highly recommended for the cutting of serial sections. In cutting paraffin sections with the sliding microtome the knife is placed at an angle of about 35° to 40° to the horizontal plate of the microtome. Sections are cut more easily with the knife in this posi- tion than when the knife is placed at right angles to the microtome, as is often recommended, and it does not seem that the tissues suffer materially from distortion when they are cut with the knife at an angle, as is some- times claimed. Before fastening the paraffin blocks into the clamp on the microtome, preparatory to cutting sections, the paraffin is trimmed with a sharp knife from the end of the paraffin block until the tissue is nearly exposed, care being taken, however, to leave a flat surface. The top of the paraffin block is then beveled off on three sides to within a very short distance of the tissue. The fourth side, that which faces the knife when the block is clamped in the microtome, should be trimmed only to within about y& of an inch of the tissue. This edge of paraffin is made use of, as will be seen in a moment, for preventing the sections from curling while they are being cut. The paraffin block is now ready to be clamped in the microtome. This is done in such a way that the paraffin block just escapes the knife when drawn over it. A number of rather thick sections (20 to 40 microns) are cut by moving the micrometer screw from right to left 4 to 8 notches every time the knife has been drawn over the paraffin block and has been brought back again, until it is noticed that the knife touches all parts of the top of the paraffin block, or until the tissue is fairly exposed. (In this description reference is made to the simple labora- tory microtome shown in Fig. 4. ) The succeeding sections may now be kept. It may perhaps be well to state that it is better not to try to cut very thin sections at the beginning; sections 15 to 20 microns in thickness will answer very well. To begin with, then, the milled head of the mi- crometer screw is turned 4 notches from left to right, and the knife is drawn over the block with a steady, even pull, and without using undue pressure. Usually the sections will curl up as they are being severed from the paraffin block. This may very readily be prevented by holding the tip of a camel's-hair brush, which has been pointed by drawing it between the lips, against the edge of the section as soon as it begins to curl. A little practice will enable one to do this almost automatically. The sections are transferred to paper by means of the camel's-hair brush, which process is facilitated if the brush has been slightly moistened with saliva, as the section will then adhere lightly to the brush. 36 THE MICROSCOPIC PREPARATION. If the tissues are well imbedded and not too hard, and if the knife is sharp and properly adjusted, paraffin sections may be cut in such a way that each succeeding section adheres to the preceding one, so that actual ribbons of paraffin sections may be made. In order to do this, the knife should be at right angles to the microtome. The paraffin block should be trimmed in such a way that when clamped in the microtome ready for cutting sections, the surface of the paraffin block facing the knife should be exactly parallel to its edge, also to the opposite side of the block. In other words, 2 sides of the paraffin block should be parallel to each other and to the knife ; then if the paraffin is of the right consistency, which' must be ascertained by trying, the sections as they are cut will ad- here to each other and form a ribbon. If the sections do not adhere to each other it is quite probable that the paraffin is a little too hard. This may often be remedied by holding an old knife or other metallic instru- ment which has been heated in a flame near the two parallel surfaces for a few moments. Care should be taken not to allow this instrument to touch the paraffin. This is a very convenient and rapid way of cutting par- affin sections. To facilitate the cutting of a paraffin possessing a rela- tively low melting point in a room with a high temperature, the cooled knife of Stoss may be used. This is so made that a stream of ice-water may be passed through a tube running through the entire length of the back of the blade. Paraffin sections may be cut in ribbons — serial sec- tions— on an ordinary sliding microtome; for this purpose, however, the "automatic rotary microtome" of Minot is especially recommended. Celloidin Sections. — Before fastening the block of wood or vul- canized fiber to which the celloidin blocks have been fixed in the clamp on the microtome, the celloidin should be trimmed with a sharp knife from the top of the block until the tissue is nearly exposed, care being taken to leave a flat surface. The sides of the celloidin block are then trimmed down, if necessary, to within about Jg- of an inch of the tissue. The block is now clamped in the microtome at such a level that it just escapes the knife when drawn over it. The knife is placed at an angle of about 45°, or at even a greater angle. During the process of cutting, the knife, as also the tissue, must be kept constantly moistened with 80 % alcohol. This is perhaps most easily accomplished by taking up the 80% alcohol with a rather large camel' s-hair brush and dipping this on the celloidin block and on the knife. A number of rather thick sections are cut until the knife touches the entire surface of the block or until the tis- sue is well exposed. The sections may now be kept. The block is raised 20 to 15 microns, and the knife, which should be well moistened with 80% alcohol, is drawn over the block with a steady pull, not with a jerk. The sections are transferred from the knife to distilled water. This is perhaps most conveniently done by placing the ball of one of the fingers of the left hand under the edge of the knife, in front of the sec- tion, and drawing the section down onto the finger with the camel's-hair brush. The finger is then dipped into the distilled water when the sec- tion floats off. If the sections can not be stained within a few hours after they are cut, they are best transferred to a dish containing 8oc/c alcohol, in which they may be left until it is desired to stain them. The sliding microtomes may be provided with an arrangement for freez- ing tissues — a so-called freezing apparatus. This consists of a metal plate on which the tissue is laid; an ether or rigolene atomizer plays upon its lower surface, cooling and finally freezing the object, which is then cut. THE MICROTOME AND SECTIONING. 37 A drop of fluid (physiologic saline solution, water, etc.) is placed upon the knife, in which the section thaws out and spreads. A better and more rapid method of freezing tissues consists in the use of compressed carbon dioxid, as recommended by Mixter. Cylinders containing about twenty pounds of the liquid gas may be obtained from Bausch & Lomb, who also make a small microtome designed for this purpose. In figure 7 is shown the lower third of a cylinder for compressed carbon dioxid firmly fastened to a thick board, and connected by means of a short piece of strong rubber tubing with the freezing box of the microtome. The handle of the escape valve is from 8 to 10 inches long, so that the quantity of escaping gas may be readily controlled. The pieces of tis- sue are placed on the freezing box of the microtome and the escape valve slowly opened until a small quantity of the gas escapes. Small pieces of tissue are frozen in about thirty seconds to a minute ; tissues taken from alcohol should be washed for a short time in running water before freez- ing. A strong razor may be used for cutting sections ; or better, a well- sharpened blade of a carpenter's plane, as suggested by Mallory and Wright. Sections are transferred to distilled water or normal salt solu- tion, and if fixed may be stained at once. Sections of fresh tissue should be taken from the normal salt solution and transferred to a fixing fluid. Bardeen has devised a microtome to be used with compressed carbon dioxid, which presents many advantages. It admits of better control of the temperature of the freezing stage and there is less carbon dioxid wasted than with other instruments of this type. It freezes almost instantane- ously, since the expanding carbon dioxid is caused to pass through a spiral passage contained in the freezing chamber. In this apparatus the microtome is attached to the steel cylinder containing the carbon dioxid. It is impossible to cut thin sections with a knife that is not sharp, or with one that is nicked. A few directions as to sharpening a micro= tome knife may therefore not be out of place. For this purpose a good Fig. 7- — Apparatus for cutting tissues frozen by carbon dioxid. Belgian hone is used, which should be moistened or lubricated with filtered kerosene oil or with soap as necessity demands. While sharpening the knife it is grasped with both hands — with one by the handle, with the 38 THE MICROSCOPIC PREPARATION. other by the end. The hone is placed on a table with one end directed toward the person sharpening. If the knife is very dull, it is ground for some time on the concave side only (all microtome knives are practically plane on one side and concave on the other), with the knife at right angles to the stone. It is carried from one end of the stone to the other, edge foremost, giving it at the same time a diagonal movement, so that with each sweep the entire edge is touched (see Fig. 8). In drawing back the knife, the edge is slightly raised. The knife is ground on the concave side until a fine thread (feather edge) appears along the entire edge. It is then ground on both sides, care being taken to keep the knife at right angles to the stone, to keep it flat, and to use practically no pres- sure. It is a good plan to turn the knife on its back when the end of the stone is reached. On the return stroke, the knife is again held at right angles to the stone, the same diagonal sweep is used (see Fig. 8), so that the whole edge of the knife is touched with each sweep. The grinding on both sides is continued until the thread above mentioned has disappeared. The knife should now be carefully cleaned and stropped, with the back of the knife drawn foremost. The strop should be flat and rest on a firm surface. Fig. 8. — Diagram showing direction of the movements in honing. THE FURTHER TREATMENT OF THE SECTION. 1. FIXATION TO THE SLIDE AND REMOVAL OF PARAFFIN. Sections obtained by means of the microtome undergo further treat- ment either loose or, better, fixed to a slide or cover -glass, thus making further manipulation much easier. The simplest, surest, and most convenient method of fixing par- affin sections to the slide is by means of the glycerin-albumen of P. Mayer (83.2). Egg-albumen is filtered and an equal volume of glycerin THE FURTHER TREATMENT OF THE SECTION. 39 added. To prevent decomposition of the fluid a little camphor or sodium salicylate is placed in the mixture. A drop of this fluid is smeared on the slide or cover-slip as evenly and thinly as possible. A section or a series of sections arranged in their proper sequence is then placed upon the slide so prepared. Any folds in the section are smoothed out with a brush, and the section or the whole series gently pressed down upon the glass. When the desired number of sections are on the slide or cover-slip, they are warmed over a small spirit or gas flame until the paraffin is melted. At the same time the albumen coagulates. The sections are now fixed, and are loosened from the glass only when agents are used which dissolve albumen, as, for instance, strong acids, alkalies, and certain staining fluids. If it is desired that a given space, say the size of a cover-slip, be filled up with sections as far as possible, an outline of the cover-slip to be used may be drawn upon a piece of paper and placed under the slide in the required position. A second and in many respects better method is the fixation of the section with distilled water (Gaule). The paraffin sections are spread in proper sequence on a thin layer of water placed on the slide. There should be sufficient water to float the sections. The slide is then dried in a warm oven kept at 30° to 35° C., or gently heated by holding it at some distance from a spirit or gas flame (the paraffin should not melt). By this treatment the sections are entirely flattened out. The superfluous water is either drained off by tilting or drawn off with blot- ting-paper, the sections are definitely arranged with a brush, and the whole is placed for several hours in a warm oven at 30° to 35° C. The sections thus dried are exposed, over a flame, to a temperature higher than the melting point of the paraffin, and from now on can be subjected to almost any after-treatment. The slide or cover-slip should be thor- oughly cleaned (preferably with alcohol and ether), as otherwise the water does not remain in a layer, but gathers in drops. The advantage of this method lies in the fact that the evaporated water can have no possible influence on the subsequent staining of the sections, while albumen, especially if it be in a thick layer, is sometimes stained, thus diminishing the transparency of the preparation. This method, although trustworthy for alcohol and sublimate prepara- tions, often fails with objects' that have been treated with osmic acid, chromic acid and its mixtures, nitric acid, and picrosulphuric acid. In such cases advantage may be taken of the so-called Japanese method, which is a combination of the above fixation methods. A little Mayer's albumen is placed on the slide and so spread about that hardly a trace of the substance can be seen. The slide is then put in a warm oven heated to 70° C. This temperature soon coagulates the albumen, after which the sections are fixed to the slide by the water method (Rainke, 95). The procedure can be varied by adding to the distilled water one drop of glycerin-albumen or gum arabic to every 30 c.c. of water (vid. also Nussbaum). When a large number of paraffin sections are to be fixed to cover-slips, the following method may be recommended : A small porcelain evapo- rating dish is nearly filled with distilled water and placed on a stand which elevates it 6 to 8 inches from the table. A number of sec- tions are placed on the water, which is then heated by means of a gas flame until the sections become perfectly flat, care being taken not to raise the temperature of the water sufficiently to melt the paraffin. Each 4O THE MICROSCOPIC PREPARATION. section is then taken up on a cover-slip coated with a very thin layer of Mayer's albumen fixative. During this procedure the cover-slips are held by forceps, and the sections are guided by means of a small camel's-hair brush. When all the sections have thus been placed on cover-slips they are placed for four to six hours in a warm oven maintained at 30° to 35° C. Removal of Paraffin. — Before paraffin sections, either fixed or loose, are subjected to further manipulation, the paraffin surrounding the tissues must be removed. This may be done by means of several agents having a solvent action on paraffin, such as xylol, toluol, oil of turpen- tine, etc. After the paraffin has been dissolved, the sections are trans- ferred to absolute alcohol and by this means prepared for further treatment with aqueous or weak alcoholic solutions. Dextrin Method of fixing Paraffin Sections. — This method is to be recommended for class-room purposes where 30 to 50, or even more sections need to be stained at one time. The following solutions are kept on hand : Solution i : A solution of equal parts of white sugar and boiling distilled water 300 c.c. A solution of equal parts of distilled water and dextrin 100 " Absolute alcohol 200 " Mix the sugar and dextrin solutions in a mortar, and add very slowly, while constantly stirring, the absolute alcohol ; filter through fine muslin. Keep in a wide-mouthed bottle through the cork of which there has been placed a broad camel's-hair brush. Solution 2 : Photoxylin IO gm. Absolute alcohol looc.c. Ether 500 " The sections to be stained are cut and arranged on a clean piece of paper. A clean glass plate is coated with a thin layer of solution No. i. The sections are arranged on this and pressed against the plate with the finger. The plate is now placed in a warm oven (temperature 40° C.), where it remains for several hours. The plate is then warmed over a flame until the paraffin of the sections begins to melt and is then placed in a tray containing xylol, where it remains until the paraffin is dissolved. It is then transferred to a tray containing 95^ alcohol and the xylol re- moved. The plate is next taken from the tray and the alcohol drained off. The plate is now covered with a thin layer of solution No. 2, and set aside, at an angle, until the photoxylin dries. The plate is now placed in the staining fluid, in which, or in the water used in washing off the staining fluid, the thin layer of photoxylin, to which the sections ad- here, separates from the plate. This thin film may now be treated as one section and carried on in this form through the several stages of staining and clearing until the process is completed. The individual sections are cut from the film with scissors. Celloidin preparations can not be fixed to the slide with the same degree of certainty, although many sections may be treated at one time. The celloidin sections can be collected in their sequence on strips of paper by gently pressing such a strip, on the blade of a knife, onto the STAINING. 41 section floating in the alcohol. The sections adhere to the paper, and in this way the entire surface of the strip may be covered by series of sections. To prevent the drying of the sections, a number of such strips are laid in rows on a layer of blotting-paper moistened with 70% alcohol. A glass plate of corresponding size is painted with very fluid celloidin. After the layer of celloidin is dry, the strips of paper are laid, one by one, on the glass plate, with sections downward, and the fingers gently passed over the reverse side. This process is continued until the entire surface of the glass is covered. On carefully raising the strips it is seen that the sections will adhere to the layer of celloidin. (To prevent drying, sections must be kept moistened with 70% alcohol.) After first drying the sections with blotting-paper, a second layer of very thin celloidin is painted on the surface of the glass plate. When this layer is also dry, the plate with its adherent sections is placed in water. Here the double layer of celloidin containing the sections is separated from the glass, and is ready for further manipulation. Before mounting, the sheet of celloidin is cut with scissors into convenient portions. In the case of celloidin sections, if it be desirable to preserve the surrounding celloidin, care should be taken that the preparations should not come in contact with any agents dissolving celloidin. These latter are alcohols from 95^ upward, ether, several ethereal oils, especially oil of cloves, but not the oils of origanum, cedar wood, lavender, etc. 2. STAINING. It is in most cases necessary to stain tissues to bring clearly to view the tissue elements and their relation to each other. The purpose of staining is therefore to differentiate the tissue elements. The differential staining is due to the fact that certain parts of the tissue take up more stain than others. Staining of sections may be looked upon as a microchemic color reaction, and has therefore a value beyond the mere coloring of sections so that they may be seen more clearly. Broadly speaking, stains used in microscopic work may be divided into basic stains, which show special affinity for the nuclei of cells and are therefore known as nuclear stains, and acid stains, which color more readily the protoplasm — protoplasmic stains. Certain stains, which we may know as selective stains (they maybe either basic or acid), color one tissue element more vividly than others, or to the exclusion of others. Since the various tissue elements show affinity for different stains, prepa- rations may be colored with more than one stain. Accordingly we have simple, double, triple, and multiple staining. Certain stains are also especially adapted for staining in bulk or mass — that is, staining a piece of tissue before it is sectioned. SECTION STAINING. Carmin. — Aqueous Borax=carmin Solution. — 8 gm. of borax and 2 gm. of carmin are ground together and added to 150 c.c. of water. After twenty-four hours the fluid is poured off and filtered. The sections, previously freed from paraffin and treated with alcohol, are placed in this fluid for several hours (as long as twelve), and then washed out in a solution of 0.5 to ic/c hydrochloric acid in 70% alcohol. They are then transferred to 70% alcohol. Alcoholic Borax=carmin Solution. — 3 gm. of carmin and 42 THE MICROSCOPIC PREPARATION. 4. gm. of borax are placed in 93 c.c, of water, after which 100 c.c. of 70% alcohol is added. The mixture is stirred, then allowed to settle, and later filtered. Sections are treated as in the aqueous borax-carmin solution. Paracarmin is the carmin stain containing the most alcohol, and is therefore of great value. Carminic acid I gm. Aluminium chlorid 0.5 " Calcium chlorid 4 " Alcohol, 70% ioo c.c. Paracarmin stains quickly, is not liable to overstain, and is there- fore peculiarly adapted to the staining of large objects. Specimens are washed in 70% alcohol, with the addition of 0.5% aluminium chlorid or 2.5% glacial acetic acid in case of overstating (P. Mayer, 92). Czocor's Cochineal Solution. — 7 gm. of powdered cochineal and 7 gm. of roasted alum are kept suspended in ioo c.c. of water by stirring while the mixture is boiled down to half its volume. After cooling it is filtered and a little carbolic acid added. This fluid stains quite rapidly and does not overstain. Before the sections are placed in alcohol they should be washed with distilled water, as otherwise the alum is precipitated on the section by the alcohol. Partsch recommends the following solution of cochineal : Finely pow- dered cochineal is boiled for some time in a 5% aqueous solution of alum, and filtered on cooling, after which a trace of hydrochloric acid is added. It stains sections in two to five minutes. Alum-carmin (Grenadier). — ioo c.c. of a 3% to 5% solution of ordinary alum, or preferably ammonia-alum, are mixed with o. 5 gm. to i gm. of carmin, boiled for one-fourth of an hour, and after cooling filtered and enough distilled water added to replace that lost by evaporation. This fluid stains quickly but does not overstain. Wash the sections in water. Hematoxy lin. — Bohmer's Hematoxylin : Hematoxylin crystals I gm. Absolute alcohol 10 c.c. Potassium alum 10 gm. Distilled water 200 c.c. Dissolve the hematoxylin crystals in the alcohol, and the alum in the distilled water. While constantly stirring, add the first solution to the second. The whole is then left for about fourteen days in an open jar or dish pro- tected from the dust, during which time the color changes from violet to blue. After filtering, the stain is ready for use. Sections, either loose or fixed to the slide or cover-slip, are placed in this solution, and after about half an hour are washed with water. If the nuclei are well stained the further treatment with alcohol may be commenced. Should the sections be over- stained, a condition showing itself in the staining of the cell -protoplasm as well as the nuclei, the sections are then washed in an acid alcohol wash (six to ten drops of hydrochloric acid to ioo c.c. of 70% alcohol) until the blue color has changed to a reddish-brown and very little stain comes from the section — usually about one to two minutes. They are then washed in tap -water, and passed into distilled water before placing in alcohol. STAINING. 43 Delafield's Hematoxylin : Hematoxylin crystals 4 gm. Absolute alcohol 25 c.c. Ammonia alum, saturated aqueous solution 400 " Alcohol, 95$, ioo " Glycerin ioo " Dissolve hematoxylin crystals in absolute alcohol and add to the alum solution, after which place in an open vessel for four days, filter, and add the 95% alcohol and glycerin. After a few days it is again filtered. This fluid is either used pure or diluted with distilled water. Staining is the same as with Bohmer's hema- toxylin. Friedlander's Glycerin-hematoxylin : Hematoxylin crystals 2 gm. Potassium alum 2 " Absolute alcohol ioo c.c. Distilled water ioo " Glycerin ioo " Dissolve the hematoxylin crystals in the absolute alcohol and the alum in the water ; mix the two solutions and add the glycerin. The mixture is filtered and exposed for several weeks to the air and light, until the odor of alcohol has disappeared, and then again filtered. It stains very quickly. Sections are afterward washed in water and are placed for a short time in acid alcohol if the nuclei are to be especially brought out. Ehrlich's Hematoxylin : Hematoxylin crystals 2 gm. Absolute alcohol 60 c.c. Glycerin "1 saturated with .... 60 " Distilled water J ammonia alum .... 60 " Glacial acetic acid 3 " The solution is to be exposed to light for a long time. It is ready for use when it acquires a deep-red color. Stain as above. Hemalum (P. Mayer, 91). — i gm. of hematein is dissolved by heating in 50 c.c. of absolute alcohol. This is poured into a solu- tion of 50 gm. of alum in i liter of distilled water and the whole well stirred. A thymol crystal is added to prevent the growth of fungus. The advantages of hemalum are as follows : The stain may be used im- mediately after its preparation, it stains quickly, never overstains, especially when diluted with water, and penetrates deeply, making it useful for staining in bulk. After staining, sections or tissues are washed in distilled water. Acid Hemalum. — To the above hemalum solution is added 2% of glacial acetic acid. Stains even more rapidly than hemalum, and gives excellent nuclear differentiation. Wash sections in tap -water. Heidenhain's Iron Hematoxylin. — Good results, particu- larly in emphasizing certain structures of the cell (centrosome), are ob- tained by the use of M. Heidenhain's iron hematoxylin (92. 2). Tissues are fixed in saline sublimate solutions, alcohol, or Carney's fluid. Very thin sections (in case of amniota not over 4, a) are fixed to the slide with water and put into a 2 . 5 ffc aqueous solution of ammonium sulphate of iron for four to eight hours (not longer). After careful rinsing in water, the sections are brought into a solution of hematoxylin prepared as fol- lows : Hematoxylin crystals i gm., absolute alcohol 10 c.c., and dis- 44 THE MICROSCOPIC PREPARATION. tilled water 90 c.c. This solution should remain in an open vessel for about four weeks, and, before using, should be diluted with an equal volume of distilled water. Staining takes place in twelve to twenty-four hours, after which the sections are rinsed in tap-water and again placed in a like solution of ammonium sulphate of iron, until black clouds cease to be given off from the sections. They are rinsed in distilled water, passed through alcohol into xylol, and mounted in balsam. Should a protoplasmic stain be desired, rubin in weak acid solution may be employed. Coal-tar or anilin stains. — Ehrlich classifies all anilin stains as salts having basic or acid properties. The basic anilin stains, such as safra- nin, methylene-blue, methyl-green, gentian violet, methyl-violet, Bis- marck brown, thionin, and toluidin-blue are nuclear stains, while the acid anilin stains, such as eosin, erythrosin, benzopurpurin, acid fuchsin, lichtgriin, aurantia, orange G, and nigrosin stain diffusely and are used as protoplasmic stains. Safranin : Safranin , . . I gm. Absolute alcohol 10 c.c. Anilin water . . • 90 " Anilin water is prepared by shaking up 5 c.c. to 8 c.c. of anilin oil in 100 c.c. of distilled water and filtering through a wet filter. Dissolve the safranin in the anilin water and add the alcohol. Filter before using. Stain sections of tissues fixed in Flemming's or Hermann's solutions for twenty-four hours, and decolorize with a weak solution of hydrochloric acid in absolute alcohol (i : 1000). After a varying period of time (usu- ally only a few minutes) all the tissue elements will be found to have become bleached, only the chromatin of the nucleus retaining the color. Bismarck Brown. — A very convenient color to handle is Bismarck brown. Of this, i gm. is boiled in 100 c.c. of water, filtered, and yi of its volume of absolute alcohol added. Bismarck brown stains quickly without overstaining, and is also a purely nuclear stain. Wash in absolute alcohol. Methyl-green stains very quickly (minutes), i gm. is dis- solved in 100 c.c. of distilled water to which 25 c.c. of absolute alcohol is added. Rinse sections in water, then place for a few minutes in 70 f/0 alcohol, transfer to absolute alcohol for a minute, etc. Other so-called basic anilin stains can be used in a similar manner. Thionin or toluidin-blue in dilute aqueous solutions are espe- cially useful. Nuclei appear blue and mucus red. Double Staining. — When certain stains are used in mixtures or in succession, all portions of the section are not stained alike, but certain elements take up one stain, others another. This elective affin- ity of tissues is taken advantage of in plural staining. If two stains are employed, one speaks of double staining. Picrocarmin of Ranvier. — Two solutions are prepared, a satu- rated aqueous solution of picric acid and a solution of carmin in ammonia. The second is added to the first to the point of saturation. The whole is evaporated to one-fifth of its volume and filtered after cooling. The solution thus obtained is again evaporated until the picrocarmin remains in the form of a powder. A i % solution of the latter in distilled water is the fluid used for staining. STAINING. 45 To stain with this solution, one or two drops are placed on the slide over the object and the whole put in a moist chamber for twenty-four hours. A cover- slip is then placed over the preparation, the picrocarmin drained off with a piece of blotting-paper, and a drop of formic-glycerin (i : 100) brought under the cover-slip by irrigation. Proper differentia- tion takes place only after a few days, and the acid-glycerin may then be replaced by the pure glycerin. In objects fixed with osmic acid, the nuclei appear red, connective tissue pink, elastic fibers canary yellow, muscle tissue straw color, keratohyalin red, etc. Weigert's Picrocarmin. — The preparation of Weigert's picro- carmin is somewhat simpler. 2 gm. of carmin are stirred in 4 c.c. of ammonia and allowed to remain standing in a well-corked bottle for twenty-four hours. This is mixed with 200 c.c. of a concentrated aqueous solution of picric acid to which a few drops of acetic acid are added after another twenty-four hours. The result is a slight precipitate that does not dissolve on stirring. Filter after twenty-four hours. Should the precipitate also pass through the filter, a little ammonia is added to dis- solve it. Both picrocarmin solutions dissolve off sections fixed to the slide with albumen. Carmin=bleu de Lyon (of Rose). — Sections or pieces of tis- sue are first stained with carmin (alum- or borax-carmin). Bleu de Lyon is dissolved in absolute alcohol and diluted with the latter until the solu- tion is of a light bluish color. In this the sections or pieces of tissue are after-stained for twenty-four hours (developing bone stains, for instance, blue). Picric acid is often used as a secondary stain, either in aque- ous (saturated solution diluted i to 3 times in water) or in alco- holic solution (weak solutions in 70^, 80^, and absolute alcohol). Sections previously treated with carmin or hematoxylin are stained for two to five minutes, washed in water or alcohol, and transferred to abso- lute alcohol, etc. Sections stained in safranin can be exposed to the ac- tion of an alcoholic picric acid solution. A solution of picric acid in 70% alcohol may be used to wash sections stained in borax-carmin. This often gives a good double stain. Sections can also be first treated with picric acid and afterward stained with alum -carmin. Hematoxylin. — Van Gieson's Acid fuchsin-picric acid Solu- tion.— Stain in any one of the hematoxylin solutions and after rinsing sections in water counter-stain in the following : Acid fuchsin, \% aqueous solution . ... 5 c.c. Picric acid, saturated aqueous solution . . loo " Dilute with an equal quantity of distilled water before using. The hematoxylin stained sections remain in the solution for from one to two minutes, are then rinsed in water, dehydrated and cleared. Hematoxylin=eosin. — Sections already stained in hematoxylin are placed for two to five minutes in a i °/c to 2 % aqueous solution of eosin or in a \CJC solution of eosin in 60% alcohol. They are then washed in water until no more stain comes away, after which they remain for only a short time in absolute alcohol. In place of the eosin solution a i c/c aqueous solution of benzopurpurin may be used or the following solution of erythrosin (Held) : Erythrosin I gm. Distilled water 150 c.c. Glacial acetic acid 3 drops. 46 THE MICROSCOPIC PREPARATION. Hematoxylin-safranin of Rabl (85). — Sections of preparations fixed with chromic-formic acid or with a solution of platinum chlorid are stained for a short time with Delafield's hematoxylin, then counterstained for twelve to twenty-four hours with safranin and washed with absolute alcohol until no more color is given off. Bioridi-Heidenhain Triple Stain. — Of the many triple stains in use we mention only the most important, the rubin S — orange G — methyl-green mixture of Ehrlich and Biondi, employed according to the modification of M. Heidenhain. The best results are obtained with ob- jects fixed in saline sublimate solution. The three stains just mentioned are prepared in concentrated aqueous solutions. (In 100 c.c. of distilled water there are dissolved respectively about 20 gm. of rubin S, and 8 gm. of orange G and methyl-green.) These concentrated solutions are combined in the following proportions: rubin S 4, orange G 7, methyl- green 8. The stock solution thus obtained is diluted with 50 to 100 times its volume of distilled water before using. The sections should be as thin as possible and fixed to the slide by the water method. They remain for twenty-four hours in the stain, and are then rinsed in distilled water or in 90% alcohol or in such with the addition of a little acetic acid (i to 2 drops to 50 c.c.). Before staining it is occasionally of advantage to treat the sections with acetic acid (2 : 1000) for one to two hours. STAINING IN BULK. Instead of staining in sections, entire objects can be stained before cutting. This method is in general much slower, and demands, there- fore, special staining solutions, as, for instance : Alcoholic Borax-carmin Solution. — Pieces ^ cm. in diameter remain in the stain at least twenty -four hours, are then decolorized for the same length of time in acid alcohol (0.5% to i °/o hydrochloric acid in 70% alcohol), and after washing in 70% alcohol are transferred to 90% alco- hol. Larger objects require a correspondingly longer time. Paracarmin. — Treatment as in section staining; length of time according to size of object. Alum-carmin of Grenacher. — This never overstains. Time of stain- ing according to size of object. Wash in water, then transfer to 70% and 90% alcohol. Hemalum, when diluted with water, is very useful for staining in bulk. After staining, objects should be washed with distilled water. Bohmer's hematoxylin stains small pieces very sharply. Use the same as hemalum. Hematoxylin staining according to R. Heidenhain 's method is especially recommended for staining in bulk. Stain objects fixed in alcohol or picric acid twenty-four hours in a °-33% aqueous solution of hematoxylin ; transfer for an equal length of time to a o. 5 cfc aqueous solution of potassium chromate, changing often until the color ceases to run. Wash with water and pass into strong alcohol. This stain also colors the protoplasm, and is so powerful that very thin sections are an absolute condition to the clearness of the prepa- ration. If the objects have been fixed with picric acid and the latter has not been entirely washed out, staining in bulk by the above methods pro- duces very striking differentiation. METHODS OF IMPREGNATION. 47 Pieces of tissue stained in bulk may be infiltrated, imbedded, and cut according to the ordinary methods. Under these circumstances, section staining is not necessary unless a still further differentiation be desired. In general, then, the treatment of the object is somewhat as fol- lows : First, it is fixed in some one of the fixing fluids already described, then carefully washed, and in certain cases stained in bulk before infiltrat- ing with paraffin or celloidin ; or the staining may be postponed until the tissue has been cut. In the latter case, the sections are subjected to the stain either loose or fastened to the slide or cover-slip. In all cases it is absolutely essential that the paraffin be entirely removed. After the sections have been stained and washed, they are transferred to absolute alcohol in case it be desired to mount them in some resinous medium. They may also be mounted in glycerin or acetate of potash, into which they may be passed directly from distilled water. The method of staining tissues in sections or in bulk is shown in the following diagrams : In Bulk, gofc alcohol In Sections. Celloidin sections Paraffin sections Water in 90 tfo alcohol Distilled water Wash in water \ "jo% alcohol Wash in acid alcohol I Remove paraffin 4- Absolute alcohol I gofc alcohol Distilled water 70$ alcohol Absolute alcohol Alcohol Alcohol Absolute alcohol METHODS OF IMPREGNATION. The impregnation methods differ from the staining methods in that in the latter the coloration is obtained by reagents in solution, while in the former the tissues are filled with fine particles which enter into com- bination with certain constituents of the tissue elements and are reduced in them. Silver Nitrate Method. — This method was suggested by Krause ; it was, however, brought to prominence by v. Recklinghausen. It is especially useful for staining the intercellular substances of epithelium, endothelium, and mesothelium and the ground-substance of connective 48 THE MICROSCOPIC PREPARATION. tissues. The method may be used on fresh tissues or on fixed tissues ; the employment of fresh tissue is, however, more satisfactory. The tis- sues to be impregnated are spread in thin layers, and immersed in a o.$'/c to i'/( solution of silver nitrate for from ten to fifteen minutes; they are then rinsed in distilled water and placed in fresh distilled water or 70% alcohol or a 4% solution of formalin and exposed to direct sun- light, where they remain until they assume a brown color. The sunlight reduces the silver, in the form of fine particles which appear black on being examined with transmitted light. The preparations thus obtained may be examined in glycerin or dehydrated and mounted in balsam. (See methods of injection for staining the endothelial cells of blood and lymph vessels. ) Gold Chlorid Method. — In gold chlorid impregnation the cells and fibers of certain tissues are stained while the intercellular substances remain uncolored. The coloration is obtained by a reduction of the gold (either by sunlight or certain reagents — formic acid, acetic acid, citric acid, oxalic acid), in the form of very fine particles which impart to the tissues a pur- plish-red color. This method is especially useful for bringing to view the terminations of nerve-fibers, both motor and sensory ; however, it may also be employed for staining other tissue elements. The method of gold impregnation was introduced by Cohnheim and was used by him in staining the nerve terminations in the cornea. It has received numerous modifications since its introduction. The following may be mentioned : Cohnheim' s Method. — Small pieces of muscle are placed in a i % solution of gold chlorid acidulated by a trace of acetic acid. In this they become yellow (in from a few minutes to half an hour). They are then rinsed in distilled water, placed in water slightly acidulated with acetic acid, and kept in the dark. As a rule, the pieces will change in color, becoming yellowish-gray, grayish- violet, and finally red, from one to three days generally being required for this process. The parts best adapted to examination are those in the transitional stage of violet to red. This procedure has been subjected to innumerable modifications ; of these, the most used are : ( i ) The method of Lowit : Small pieces are placed in a solution of i vol. formic acid and 2 vols. distilled water until they have become transparent (ten minutes). They are then placed in a i % solution of gold chlorid, in which they become yellow (one-quarter hour). They are now again placed in formic acid, in which they pass through the same color changes as above. Finally, they are washed and teased, or subsequently treated with alcohol and cut. (2) Kiihne (86) acidifies with 0.5% solution of acetic acid (especially in the case of muscle), then treats the specimens with a i% solution of gold chlorid, and reduces the gold with 20 to 25% formic acid dissolved in equal parts of water and glycerin. (3) Ranvier (89) acidifies with fresh lemon juice filtered through flannel, then treats with a i % solution of gold chlorid (quarter of an hour or longer), and finally either places the specimen in water acidulated with acetic acid (i drop to 30 c.c. water) and subjects it to light for one or two days, or reduces it in the dark, as in L6 wit's method, in a solution of i vol. formic acid and 2 vols. water. (4) Gerlach uses the double chlorid of gold and potassium, but in weaker concentrations than a i$> solution, otherwise he continues as in the method of Cohnheim. (5) Golgi (94) also uses the same double chlorid, but acidifies with 0.5% arsenious acid, and then reduces in i% arseni- ous acid in the sunlight. METHODS OF IMPREGNATION. 49 Golgi'sChromsilver or Chromsublimate Method. — This method depends on the formation of a very fine precipitate, which forms in cer- tain tissue elements or in preexisting spaces, when treated first with a solution of bichromate of potassium and secondarily with a solution of silver nitrate or bichlorid of mercury. The nature and precise location of this precipitate is not well understood. It is very probable, however, as Kallius suggests, that an albumin-chromsilver compound, of an unknown constitution, is formed in the cells and processes or in spaces filled with the precipitate. This method is especially useful in bringing to view the cellular elements of the nervous system, both central and peripheral ; further, the end-ramifications of gland ducts, and now and then cell boundaries. Usually only a small percentage of the tissue elements or the spaces of any given tissue are colored. This may, how- ever, be regarded as one of the advantages of the method, since it enables a clearer view of the parts colored. The precipitate appears black in transmitted light. It is necessary to state, however, that this method is very unreliable, and that failures are often met. with, also that an amorphous precipitate is generally formed, both in and about the tissues, which in part at least destroys the usefulness of the preparations obtained. Golgi's methods will perhaps be better understood if we first give a short historic sketch of their development. In the year 1875 Golgi applied his method as follows : He fixed (olfactory bulb) in Miiller's fluid, and increased the percentage of bichromate on changing the fluid (up to 4 f0). Fixation lasted five or six weeks in summer and three or four months or more in winter. He then took out pieces of the tissue every four or five days and treated them experimentally with a 0.5^ to ifi> silver nitrate solution. In summer this process took about twenty-four hours, and in winter forty-eight hours, although a longer treatment was not found to be detrimental. This method must be regarded as very uncertain, since the length of time during which the specimens remain in Miiller's fluid must be very closely calculated, as it depends largely upon the temperature of the medium. As soon as the silver reaction was established, the pieces were preserved either in the silver solu- tion itself or in alcohol. The sections were finally washed in absolute alcohol, cleared with creosote, and mounted in Canada balsam. The impregnation disappeared in a short time. In the year 1885 Golgi made a further announcement regarding his method, recommending for fixation the pure bichromate of potassium, as well as Miiller's fluid. Pieces of the brain and spinal cord (from I to 1.5 c.c. in size) from a freshly killed ani- mal were used, and the reaction sometimes took place in from twenty-four to forty-eight hours after death. For fixing, potassium bichromate solution in gradually ascending strengths (i^ to 5^,) was employed, large amounts of the fluid being used and placed in well-sealed receptacles. The fluid was repeatedly changed, and camphor or salicylic acid was added in order to prevent the growth of fungi. Since it is difficult to determine exactly when fixation in potassium bichromate reaches the precise point favorable to sub- sequent treatment with nitrate of silver, because the process depends entirely upon the temperature and quantity of the fluid, it becomes necessary, after about six weeks' treat- ment with the bichromate, to experiment every eight days or so to see whether the silver nitrate gives good results. The strength of the latter should be about 0.66^0 and the quantity about 2OO c.c. to a I c.c. object. At first a plentiful precipitate is thrown down, in which case the solution should be changed, and this probably repeated once more after a few hours. After twenty-four hours, at the most forty-eight hours, this process is usually completed, and the tissues may be sectioned. The sections must then be care- fully dehydrated with absolute alcohol, cleared in creosote and mounted without a cover- glass in Canada balsam (the section is mounted on a cover-glass with Canada balsam, and the cover-slip then fastened over the opening of a perforated slide with the specimen downward). In order to obtain a uniform penetration of the objects by the potassium bichromate, the latter may be first injected into the vessels. Golgi uses potassium bichromate-gelatin (2.5^ of the salt, based on the amount of the softened gelatin ; com- pare Golgi, 93). After the injection and cooling of the specimen the latter is cut in 4 5*^"'v. Fig. 66. — Cell - spaces in the ground- substance of areolar connective tissue (sub- cutaneous) of a young rat. Stained in silver nitrate. Fig. 67. — Three connective-tissue cells from the pia mater of a dog. Stained in methylene-blue (intra vitani). nective-tissue cells or wandering or migratory cells. The former are again divided, according to their shape and structure, into true connective-tissue cells or corpuscles, plasma cells, mast-cells, and pigment-cells. The connective-tissue cells or corpuscles are flattened, variously shaped cells of irregular form, usually having many branches. The protoplasm is free from granules ; the nucleus, situated in the thicker portion of the cell-body and of oval shape, shows a nuclear net- work and one or several nucleoli. The cells assume the shape of - - -- Fig. 68. — Two pigment cells found on the capsule of a sympathetic ganglion of a frog. the space that they occupy and nearly fill. The branches of neigh- boring cells often anastomose through the fine channels uniting the cell-spaces. IO4 THE TISSUES. Plasma cells (Unna) vary in size and shape according to the space which they occupy. They may be round, oval, or /spindle- shaped, and measure from 6 M to 10 /A The nucleus is round or oval. They are characterized by the fact that their protoplasm stains intensely in basic aniline dyes, often of a color differing from that of the solution used. Accord- ing to some observers, the plasma cells are thought to be developed / from the connective-tissue cells, v, ^^bn while others regard tnem as de- , rived from the white blood-cells • /^Tl^'A (lymphocytes). They are found u: Nucleus. t-'-i -I -I " • \J^fJj °'--\ / %. various mucous membranes and m. lymphoid tissues generally. Mast-cells (Ehrlich) are rela- tively large cells of round, oval, or irregular shape, the protoplasm of which contains relatively large granules which stain chiefly in basic aniline dyes, which granules Fig. 69.— Leucocyte of a frog with are often found in such numbers pseudopodia. The cell has included a that they COVCr Up the nucleus, bacterium which is in process of diges- —, o-^nnlps arf> Drained bv a tion. (After Metschnikoff, from O. Hertwig, 93, II.) number of basic aniline dyes, often of a color differing from that of the stain used. They are found generally in mucous membranes, generally near the vessels, in the skin, in involuntary muscle, and in the bone-marrow. Pigment cells are branched connective-tissue cells, in the proto- plasm of which are found brown or nearly black granules. In man they occur in the choroid and iris and in the dermis. In the lower animals they have, however, a much wider distribution, and in the frog and other amphibia they are very large and irregular. These cells have the power of withdrawing their processes and, to a limited degree, of changing their location (dermis). The wandering or migratory cells are described in this connec- tion not because they form one of the structural elements of areolar connective tissue, but because they are always associated with it. They are lymph- or white blood-cells, which have left the lymph- or blood-vessels and have migrated into the lymph canalicular system. They possess ameboid movement, and wander from place to place, and are the phagocytes of Metschnikoff. They seem to be intrusted with the removal of substances either superfluous or detrimental to the body (as bacteria). These are either digested or rendered harm- less. The wandering cells even transport substances thus taken up to some other region of the body, where they are deposited. In the peritoneum and other serous membranes the network formed by the fibrous tissue lies in one plane, and does not branch THE CONNECTIVE TISSUES. IO5 and intercross in all directions, as where areolar tissue is found in larger quantity. (Fig. 70.) (<£) Tendons, aponeuroses, and ligaments represent the densest variety of fibrous connective tissue, and are composed almost wholly of white fibrous tissue. This is found in the form of rela- tively large bundles of white fibrils, having a parallel or nearly parallel course. In tendons these bundles are known as primary tendon bundles or tendon fasciculi. The fibrils of white fibrous con- Fibrils. —--&?- Nucleus. ~m~~ Fig. 70. — Fibrous connective tissue (areolar) from the great omentum of the rabbit ; X400. nective tissue forming the fasciculi are cemented together by an in- terfibrillar cement substance. Here and there the fasciculi branch at very acute angles and anastomose with other fasciculi. The fa's- ciculi are grouped into larger or smaller bundles, the secondary tendon bundles, which are surrounded by a thin layer of areolar con- nective tissue, and in part covered by endothelial cells. Between the tendon fasciculi there is found a ground-substance, interfasdcu- lar ground-substance, identical with the ground-substance in areolar connective tissue. In this there are cell-spaces occupied by the tendon cells, morphologically similar to the branched cells of areolar connective tissue. The tendon cells are arranged in rows between the tendon fasciculi. They have an irregular, oblong body, containing a nearly round or oval nucleus. Two, three, or even more wing- like processes (lamellae) come from the cell-body and pass between the tendon fasciculi. In cross-section the tendon cells have a stellate shape. The secondary tendon bundles are grouped to form the tendon, and the whole is surrounded and held together by a layer of areolar connective tissue, called the peritendineum. From this, septa pass in between the secondary tendon bundles, forming the internal peri- tendineum. The blood- and lymph-vessels and the nerve-fibers reach the interior of the tendon through the external and internal peritendineum. io6 THE TISSUES. The structure of an aponeurosis and a ligament is like that of a tendon. The structure of a fascia, the dura mater, and the more fully Tendon cell. Tendon fibers. Tendon fasciculus. Fig. 71. — Longitudinal section of tendon ; Fig. 72 — Cross-section of secondary X 270. tendon bundle from tail of a rat. developed gland capsules, differs from that of the formed connective tissues above described, in that the fasciculi are not so regularly arranged, but branch and anastomose and intercross in several planes. (c) Elastic Fibrous Tissue. — In certain connective tissues the elastic fibers predominate greatly over the fibers of white fibrous connective tissue. These are spoken of as elastic fibrous tissues and their structural peculiarities warrant the making of a special sub- group. The ligamentum nuchae of the ox consists almost exclu- sively of elastic fibers, many of which attain a size of about io//. The elastic fibers branch and anastomose, retaining, however, a generally parallel course. They are separated by a small amount of areolar connective tissue, in which a connective-tissue cell is here and there found, and are grouped into bundles surrounded by thin layers of areolar connective tissue ; the whole ligament receives an investment of this tissue. In cross-sections of the ligamentum nuchae, the larger elastic fibers have an angular outline ; the smaller ones are more regularly round or oval. (Fig. 74.) In man the ligamenta subflava, between the laminae of adjacent vertebrae, are elastic ligaments. In certain structures (arteries and veins), the elastic tissue is arranged in the form of membranes. It is generally stated that THE CONNECTIVE TISSUES. 107 such membranes are composed of flat, ribbon-like fibers or bands of elastic tissue arranged in the form of a network, with larger or smaller openings ; thus the term fenestrated membranes. F. P. Mall has reached the conclusion that such membranes are composed of three layers — an upper and a lower thin transparent layer in which no openings are found and which are identical with the sheaths of elastic fibers described by this observer, and a central layer, contain- ing openings, and staining deeply in magenta. This substance is identical with the central substance of elastic fibers. Areolar con- nective tis- sue. Nucleus of con- nective-tissue cell. Fig. 73. — Tendon cells from the tail of a rat. Stained in methylene- blue (intra vitam}. Fig. 74. — Cross-section of ligamentum nucha; of ox. 4. ADIPOSE TISSUE. In certain well-defined regions of the body occur typical groups of fixed connective-tissue cells which always change into fat-cells (fat organs,Toldt). Connective-tissue cells in various other portions of the body may also change into fat-cells, but in this case the fat, as such, sometimes disappears, allowing the cells to resume their original con- nective-tissue type, only again to appear and a second time change the character of the tissue. The formation of fat is very gradual. Very fine fat globules are deposited in the cell ; these coalesce to form larger ones, until finally the cell is almost entirely filled with a large globule (vid. also H. Rabl, 96). As the fat globule grows larger and larger, the protoplasm of the cell, to- gether with its nucleus, is crowded to the periphery. The protoplasm then appears as a thin layer just within the clear cellular membrane. The nucleus becomes flattened by pressure, until in profile view it has the appearance of a long, flat body. In regions in which large masses of fat- cells are developed, they are seen to be gathered into rounded groups of various sizes (fat lobules) separated by strands of con- nective tissue. Such lobules have, as was first pointed out by Toldt, a typical and very rich blood-supply from the time that they are recognized as fat organs in the embryo. A small artery ^rr.vj. Nucleus. Protoplasm. Fat drop. Cell-membrane. 75- — Scheme of a fat-cell. IO8 THE TISSUES. courses through the center of the fat lobule, breaking up into capillaries which form a network around the fat cells. The capil- laries unite to form several veins which are situated at the periphery of the lobule. Where fat cells develop from connective-tissue cells, even though these are present in considerable number this typic arrangement of the blood-vessels is wanting. Microscopically, fat is easily recognized by its peculiar glistening appearance (by direct light). It has a specific reaction to certain reagents. It becomes black on treatment with osmic acid, and is stained red by Sudan III and blue in cyanin. 5. CARTILAGE. Cartilage is readily distinguished from other connective tissues by its ground-substance or matrix, — intercellular substance, — which yields chondrin on boiling. Three varieties are found in higher vertebrates: (i) hyaline cartilage; (2) elastic cartilage; (3) white fibro-cartilage or connective-tissue cartilage. The simplest type is hyaline cartilage, so named because of its Cartilage cell. — :''Jj Fig. 76. — Hyaline cartilage (costal cartilage of the ox). Alcohol preparation ; X 300. The cells are seen inclosed in their capsules. In the figure a are represented frequent but by no means characteristic radiate structures. homogeneous and transparent ground-substance, which, however, in reality consists of fibrils and an interfibrillar substance, the two having essentially the same refractive index. In this ground- substance are found the cartilage cells, occupying spaces known as lacunae. The spaces or lacuna:; are surrounded by a narrow zone of ground-substance, which does not stain as does the ground- THE CONNECTIVE TISSUES. IOQ substance and which refracts the light more strongly. This zone is generally known as the capsule of the cartilage cells. As pre- viously stated, the matrix or ground-substance, develops in the exoplasm of the protoplasmic syncytium from which cartilage has its origin, while the endoplasm and nuclei form the cartilage cells. Cartilage cells, as such, are of various shapes, and have no typical appearance. They are usually scattered irregularly throughout the matrix, but are often arranged in groups of two, three, four, or even more cells. At the periphery of cartilage, either where it borders upon a cavity (articular cavity) or where it joins the perichondrium, the cells are arranged in several rows parallel to the surface of the tissue. Cartilage cells often contain glycogen, either in the form of drops or diffused throughout their protoplasm. Cartilage grows by intussusception, and an appositional growth, although in a lesser degree, also takes place. It occurs where the cartilage borders upon its connective -tissue sheath or perichondrium, Fig. 77. — From a section through the cranial cartilage of a squid (after M. Fiirbringer, from Bergh). a vascular, fibrous-tissue membrane composed of white and elastic fibers, which covers the cartilage except where it forms a joint sur- face. The relations of the cartilage and perichondrium are extremely intimate. Fibers are seen passing from the perichondrium into the cartilaginous matrix, and the connective-tissue cells appear to change directly into cartilage-cells. Certain observers (Wolters, Spronk, and others) have described a system ofcanaliculi in the ground substance, which are said to unite the lacunae and are thought to serve as channels for the passage of lymph. Such structures are, however, not generally recognized. It is an interesting fact, however, that the cartilage of certain inverte- brate animals, the cephalopoda, shows cells with anastomosing pro- cesses. (Fig. 77.) In this case the cartilage-cell is similar to a bone-cell, thus theoretically allowing of the possibility of the meta- morphosis of the elements of cartilage into those of bone (M. Fiir- bringer). no THE TISSUES. Hyaline cartilage occurs as articular cartilage, covering joint surfaces, as costal cartilage and in the nose, larynx, trachea, and White fibrous connec- tive tissue. White fibrocartilage. Insertion of liga- mentum teres. Hyaline cartilage Fig. 78. — Insertion of the ligamentum teres into the head of the femur. section ; X 650. Longitudinal bronchi. All bones except those of the vault of the skull and the majority of the bones of the face are preformed in hyaline cartilage. In white fibrocartilage (Fig. 78) there are from the beginning, even in precartilage, fibrous strands in the ground-substance. They preponderate over the matrix and, as a rule, have a parallel direc- tion. White fibrocartilage is found in the intervertebral and inter- articular disks, the symphysis pubis, and in the insertion of the ligamentum teres ; it deepens the cavity of ball-and-socket joints, and lines the tendon grooves. In some places elastic fibers are found imbedded in hyaline car- tilage — -fibro-elastic cartilage. The elastic fibers send off at acute angles finer or coarser threads which interlace to form a delicate or THE CONNECTIVE TISSUES. Ill dense network which permeates the hyaline matrix (Fig. 79), pass- ing over into the corresponding elements of the perichondrium. Elastic cartilage is found in the external ear, the cartilage of the Eustachian tube, the epiglottis, a portion of the arytenoid cartilages, and the cartilages of Wrisberg and Santorini. -Cartilage-cell. — Elastic fibers. Fig. 79- — Elastic cartilage from the external ear of man; X 7°o- a> Fine elastic network in the immediate neighborhood of a capsule. The ground-substance of cartilage undergoes changes as age advances. In certain cartilages there is observed a fibrillar forma- tion, in the ground-substance between the cells. The fibers are coarse and differ from white fibrous or yellow elastic fibers. This change is observed in laryngeal cartilages as early as the twentieth year, and is sometimes designated as an asbestos-like alteration of cartilage. Calcification occurs in many cartilages — laryngeal, tracheal, costal — and consists of the deposition in the ground-sub- stance of fine granules of carbonate of lime, first in the immediate vicinity of the cartilage cells. Calcification is observed as early as the twentieth year in the laryngeal cartilages. Ossification may be regarded as a normal occurrence in many cartilages. It begins with an ingrowth of blood-vessels from the perichondrium into the matrix. These vessels are surrounded by connective tissue. Around such locations ossification occurs. Chievitz has shown that the laryngeal cartilages begin to ossify in men at about the twentieth year, and in women at about the thirtieth year; and the tracheal I I 2 THE TISSUES. cartilage in men about the fortieth year, and in women about the sixtieth year. To obtain chondrin, a piece of cartilage matrix is placed in a tube containing water. This is hermetically closed and heated to 120° C, after which it is opened and the fluid filtered and treated with alcohol. A precipitate of chondrin is the result. This sub- stance is insoluble in cold water, alcohol, and ether, but soluble in hot water, although, on cooling, it gelatinizes. In contrast to gel- atin, chondrin is precipitated by acetic acid. This precipitate does not redissolve in an excess of this acid but disappears in an excess of certain mineral acids. 6. BONE. (a) Structure of Bone. — Bone nearly always develops from a connective-tissue foundation, even where it occurs in places formerly occupied by cartilage. The inorganic substance of bone is deposited in or between the fibers of connective tissue, while the cells of the latter are trans- formed into bone-cells. As in connective tissue, so also in bone, the ground-substance is fibrous. Between the fibers remain uncalcified cells, bone-cells. each of which rests in a cavity of the matrix — lacuna. Primarily, bone consists of a single thin lamella, its later com- plicated structure being produced by the formation of new lamellae in apposition to the first. During its development the bone becomes vascularized, and the vessels are inclosed in especially formed canals known as vascular or Haversian canals. The bone-cells have processes that probably anastomose, and that lie in special canals known as bone canaliculi. Whether, in man, all the processes of bone-cells anastomose is still an open question. The appearance presented by a transverse section of the shaft of a long bone is as follows : In the center is a large marrow cavity, and at the periphery the bone is covered by a dense connective- tissue membrane, the periosteum. In the new-born and in young in- dividuals the periosteum is composed of three layers — an outer layer, consisting mainly of rather coarse, white fibrous-tissue bundles that blend with the surrounding connective tissue ; a middle fibro-elastic layer, in which the elastic tissue greatly predominates ; and an inner layer, the osteogenetic layer, vascular and rich in cellular elements, containing only a few smaller bundles of white fibrous tissue. In the adult the osteogenetic layer has practically disappeared, leav- ing only here and there a few of the cells of the layer, while the fibro-elastic layer is correspondingly thicker (Schulz, 96). A large number of Haversian canals containing blood-vessels, seen mostly in transverse section, are found in compact bone-substance. THE CONNECTIVE TISSUES. I 1 3 Lamellae of bone are plainly visible throughout the ground-sub- stance, and are arranged in the following general systems : First, there is a set of bone lamellae running parallel to the ex- ternal surface of the bone, while another set is similarly arranged around the marrow cavity. These are the so-called fundamental, or outer and inner circumferential lamella (known also as periosteal and marrow lamella}. Around the Haversian canals are the con- centrically arranged lamellae, forming systems of Haversian or con- centric lamella. Besides the systems already mentioned, there are found interstitial or ground lamella wedged in between the Haversian Fig. 80. — Longitudinal section through a lamellar system. a d Figs. 8 1 and 82. — Lamellae seen from the surface; X 460 (after v. Ebner 75). a, Primitive fibrils and fibril-bundles ; c, bone-corpuscles with bone-cells ; d, bone canaliculi. or concentric systems of lamellae. Some authors group the inter- stitial lamellae with the systems of fundamental lamellae. Lying scattered between the lamellae are found spaces known as bone corpuscles (Virchow) or lacuna. These are present in all the lamellar systems. It is very probable that all the lacunae are in more or less direct communication with each other by means of fine canals called canaliculi ( I . I fi. to 1 . 8 // in diameter). It can be demon- strated without difficulty that the lacunae of a single lamellar sys- tem communicate not only with each other, but also with those of 8 THE TISSUES. adjacent systems. In the lamellae adjoining the periosteum and mat- row cavity the canaliculi end respectively in the subperiosteal tissue and in the marrow cavity. The canaliculi of the Haversian lamellae empty into the Haversian canals. , ' - " - _ - - ' -'-'' ' Outer circum- ferential lamellae. /- - ----- Haversian or concentric lamellae. '- — Haversian ^-'-— Interstitial lamellse< Inner circum- ferential lamellae. Fig. 83. — Segment of a transversely ground section from the shaft of a long bone, show- ing all the lamellar systems. Metacarpus of man ; X 5°- The lamellae of bone are compose'd of fine white fibrous-tissue fibrils, embedded in a ground-substance, in which they are arranged in layers, superimposed in such a way that the fibrils in the several layers cross at about a right angle, forming an angle of 45° with THE CONNECTIVE TISSUES. 115 the long axis of the Haversian canal. It is as yet undecided whether the mineral salts (phosphate and carbonate of lime, sodium chlorid, magnesium salts, etc.) are deposited in the ground-substance (v. Ebner) or in the fibrillae (Kolliker). The lacunae (13;* to 3 1 // long, 6 // to 15 //-wide, and 4 // to 9 // thick) have, in common with the canaliculi, walls which present a greater resistance to the action of strong acids than the rest of the solid bone-substance. In each lacuna there is found a bone-cell, the nucleated body of which practically fills the lacuna, while its processes extend out into the canaliculi. The Haversian canals contain blood-vessels, either an artery or a vein or both. Between the vessels and the walls of the canals are perivascular spaces bounded by endothelial cells, resting on the adventitious coats of the vessels and the sides of the canals. Into these spaces empty the canaliculi of the Haversian system. Lymph- spaces beneath the periosteum and at the periphery of the marrow Haversian canal. Fig. 84. — Portion of a transversely ground disc from the shaft of a human femur; X40Q. cavity communicate directly with the canaliculi of the circumferen- tial systems. All the lacunae and canaliculi should be thought of as filled by lymph plasma which circulates throughout, bathing the bone-cells and their processes. The formed elements of the lymph are prob- ably too large to force their way through the very small canaliculi. The plasma current probably flows from the periosteal and marrow regions toward the Haversian canals. Between the lamellae are bundles of fibers (some of which are calcified), which can be demonstrated by heating the bone, or in de- calcified preparations on staining by certain methods. These are the so-called fibers of Sharpey ; in the adult they contain elastic fibers. In the circumferential lamellae are found canals, not surrounded by concentric lamellae, which convey blood-vessels from the perios- teum to the Haversian canals. These are called Volkmanrf s* canals. The structure of bone-marrow will be discussed with the blood- forming organs. I 1 6 THE TISSUES. (£) Development of Bone. — Nearly all the bones of the adult body are, in the earlier stages of embryonic life, preformed in embry- onic cartilage. As development proceeds, this embryonic cartilage assumes the character of hyaline cartilage, its cells becoming vesic- ular, and probably disappearing. In the* matrix,1 however, there are formed spaces that are soon occupied by cells and vessels which grow in from a fibrous-tissue membrane (the future periosteum) sur- rounding the cartilage fundaments of the bones. These cells deposit a bone matrix in the cartilage spaces. Bone developed in this man- ner is known as endochondral or intracartilaginous bone. In certain bones — namely, those of the vault of the skull and nearly all the bones of the face — there is no preformation in cartilage, these bones being developed from a connective -tissue foundation. They are known as intramcmbranous bones. As will become evident upon further discussion of the subject, the formation of fibrous-tissue bone (intramembranous) is not confined to bones not preformed in cartilage. In bones preformed in cartilage, fibrous-tissue bone de- velops from the connective-tissue membrane surrounding the carti- lage fundaments, the two types of bone-development going on simul- taneously in such bones. Attention may further be drawn to the fact that nearly all endochondral bone is absorbed, so that the greater portion of all adult bone, even that preformed in cartilage, is developed from a foundation of fibrous tissue. The two modes of ossification — endochondral or intracartilaginous and intramem- branous— even though appearing simultaneously in the majority of bones, will, for the sake of clearness, be discussed separately. I. Endochondral Bone=developrnent. — The cartilage that forms the fundaments of the bones preformed in cartilage has at first the appearance of embryonic cartilage, consisting largely of cells with a small amount of intercellular matrix. These fundaments are sur- rounded by a fibrocellular membrane — the perichondrium. Ossifi- cation is initiated by certain structural changes in the embryonic cartilage, in one or several circumscribed areas, known as centers of ossification. In the long bones a center of ossification appears in the middle of the future diaphysis. In this region the intercellular matrix increases in amount and the cells in size ; thus the embry- onic cartilage assumes the character of hyaline cartilage. This is followed by a further increase in the size of the cartilage-cells, at the expense of the thinner partitions of matrix separating neighbor- ing cells, while at the same time lime granules are deposited in the matrix remaining. During this stage the cells appear first vesicu- lar, distending their capsules, then shrunken, only partly filling the enlarged lacunae. They stain less deeply, and their nuclei show degenerative changes. The center of ossification, in the middle of which these changes are most pronounced, is surrounded by a zone in which these structural changes are not so far advanced and which has the appearance at its periphery of hyaline cartilage. Simultaneously with these changes in the cartilage, a thin layef THE CONNECTIVE TISSUES. 117 of bone is deposited by the perichondrium (in a manner to be described under the head of intramembranous bone-development) and the perichondrium becomes the periosteum. This in the mean- time has differentiated in£o two layers — an outer, consisting largely of fibrous tissue with few cellular elements, and an inner, the osteogenetic layer, vascular and rich in cellular elements and con- taining few fibrous-tissue. fibers. Ossification in the cartilage begins after the above-described Vesicular cartilage- cells. -Primary periosteal bone lamella. •Periosteal bud. Fig. 85. — Longitudinal section through a long bone (phalanx) of a lizard embryo. The primary bone lamella originating from the periosteum is broken through by the peri- osteal bud. Connected with the bud is a periosteal blood-vessel containing red blood- corpuscles. structural changes have taken place at the center of ossifica- tion. Its commencement is marked by a growing into the cartilage of one or several buds or tufts of tissue derived principally from the osteogenetic layer of the periosteum. As the periosteal buds grow into the cartilage, some of the septa of matrix separating the altered cartilage-cells disappear, and the cells become free and probably degenerate. In this way the cartilage at the center of ossi- I 10 THE TISSUES. fication becomes hollowed out, and there are formed irregular anas- tomosing spaces, primary marrow spaces, separated by partitions or trabeculae of calcified cartilage matrix. Into these primary mar- row spaces grow the periosteal buds, consisting of small blood- vessels, cells, and some few connective-tissue fibers, forming embry- onic marrow, tissue. Some of the cells which have thus grown into teal bone ry marrow Fig. 86. — Longitudinal section of the proximal end of a long bone (sheep embryo) ; X3Q. the primary marrow spaces arrange themselves in layers on the trabeculae of calcified matrix, which they envelop with a layer of osseous matrix formed by them. The cells thus engaged in the formation of osseous tissue are known as osteoblasts. Ossification proceeds from the center of ossification toward the THE CONNECTIVE TISSUES. extremities of the diaphysis (in a long bone), and is always preceded as at the center of ossification, by the characteristic structural changes above described. Beginning at the center of ossification and proceeding toward either extremity of the diaphysis, the enlarged and .vesicular cartilage-cells will be observed to be arranged in quite reg- ular columns, separated by septa or tra- beculse of calcified cartilage matrix. The cells thus arranged in columns show the degenerative changes above described. They are shrunken and flattened, and their nuclei, when seen, stain less deeply than the nuclei of normal cartilage-cells. Beyond this zone of columns of altered cartilage-cells are found smaller or larger groups of less changed cartilage-cells, and beyond this zone, hyaline cartilage. The arrangement of the cartilage- cells in the columns above mentioned is, according to Schiefferdecker, mainly due to two factors — the current of lymph plasma which flows from the center of ossification toward the two extremities of the cartilage fundament, and the mutual pressure exerted by the groups of carti- lage-cells in their growth and prolifera- tion. Ossification proceeds from the cen- ter of the diaphysis toward its two ex- tremities by a growth of osteoblasts and small vessels into the columns of carti- lage-cells. Here, also, these degenerate, leaving in their stead irregular, oblong, anastomosing spaces, separated by septa and trabeculae of calcified cartilage ma- trix on which the osteoblasts arrange themselves in layers, and which they envelop in osseous tissue. In a longi- tudinal section of a long bone, preformed in cartilage, the various steps of endo- chondral t>one-development may, there- fore, be observed by viewing the prepa- ration from either end to the center of the diaphysis, as may be seen in figures 86, 87. The former represents the appear- ance as seen under low magnification, the latter a small portion of such a section from the area of ossification, more highly magnified. Adjoining the primary marrow spaces is vesicular cartilage and columns and groups of cartilage-cells and finally hyaline car- tilage. Fig. 87. — Longitudinal sec- tion through area of ossification from long bone of human em- bryo. C2O THE TISSUES. In the upper portion of figure 87 is observed a zone composed of groups of cartilage-cells, adjoining this a zone composed of columns of vesicular and shrunken cartilage-cells, the nuclei of which are indistinctly seen. These columns are separated by septa and trabeculae of calcified matrix. This zone is followed by one in which the cartilage-cells have disappeared, leaving spaces into which the osteoblasts and small blood-vessels have grown. In cer- tain parts of the figure, the osteoblasts are arranged in a layer on the trabeculae of calcified cartilage, some of which are enveloped in a layer of osseous matrix, less deeply shaded than the darker car- tilage remnants. As the development of endochondral bone proceeds from the center of ossification toward the extremities of the diaphysis in the manner described, the primary marrow spaces at the center of ossi- fication are enlarged, a result of an absorption of many of the smaller osseous trabeculae and the remnants of calcified cartilage matrix enclosed by them. In this process are concerned certain large and, for the most part, polynuclear cells, which are differentiated from the embryonic marrow. These are the osteoclasts (bone break- ers) of Kolliker (73). They are 43 JJL to 91 // long and 30 ju. to 40 // broad, and have the function of absorbing the bone. The spaces which they hollow out during the beginning of the process appear as small cavities or indentations, containing osteoclasts either single or in groups, and are known as Htnvship's lacunce. All bone absorption goes hand in hand with their appearance. At the same time, the osseous trabeculae not absorbed become thickened by a deposition of new layers of osseous tissue (by osteoblasts), during which process some of the osteoblasts are enclosed in the newly formed bone and are thus converted into bone-cells. In this way there is formed at the center of ossification a primary or embryonic spongy or cancellous bone, surrounding secondary marrow spaces or Havcrsian spaces, filled with embryonic marrow. This process of the formation of embryonic cancellous bone follows the primary ossification from the center of ossification toward the extremities of the diaphysis. It should be further stated, that long before the developing bone has attained its full size — indeed, before the end of embryonic life — the embryonic cancellous bone is also absorbed through the agency of osteoclasts. The Haversian spaces are thus converted into one large cavity, which forms a portion of the future marrow cavity of the shaft of the fully developed bone. The absorption of the embryonic cancellous bone begins at the center of ossification and extends toward the ends of the diaphysis. Some time after the beginning of the process of bone develop- ment at the center of ossification of the diaphysis, centers of ossification appear in the epiphyses, the manner of the develop- ment of bone being here the same as in the diaphysis. Several periosteal buds grow into each center of ossification, filling the irregular spaces formed by the breaking down of the degener- THE CONNECTIVE TISSUES. 121 ated cartilage-cells. Osteoblasts are arranged in rows on the trabeculae of cartilage thus formed, which they envelop in osseous tissue. As development proceeds, the primary osseous tissue is converted into embryonic cancellous bone as above described. In the development of the epiphyses, as in the development of the smaller irregular bones, the formation of bone proceeds from the center or centers of ossification in all directions, and not only in a direction parallel to the long axis of the bone as described for the diaphysis. The epiphyses grow, therefore, in thickness as well as in length, by endochondral bone-development. There remains between the osseous tissue developed in the dia- physis and that in the epiphyses, at each end of the diaphysis, a zone of hyaline cartilage in which ossification is for a long time delayed ; this is to permit the longitudinal growth of the bone. These layers of cartilage constitute the epiphyseal cartilages. Here the periosteum (perichondrium) is thickened and forms a raised ring around the cartilage. As it penetrates some distance into the substance of the cartilage, the latter is correspondingly indented. (Fig. 86.) The im- pression thus formed appears in a longitudinal section of the bone as an indentation, — the ossification groove (encoche d 'ossification , Ranvier, 89). That portion of the perichondrium filling the latter is called the ossification ridge. The relation of the elements of the perichondrium to the cartilage in the region of the groove just described is an extremely intimate one, both tissues, perichondrium and cartilage, merging into each other almost imperceptibly. It is a generally accepted theory that so long as the longitudinal growth of the bone persists, new cartilage is constantly formed at these points by the perichondrium. In the further production of bone this newly developed cartilage passes through the preliminary changes necessary before the actual commencement of ossification — i. e., it goes through the stages of vesicular cartilage and the f}rmation of columns of cartilage-cells, in place of which, later, the osteoblasts and primary marrow cavities develop. By the development of new cartilage elements from the encoche tre longitudinal growth of the bone is made possible ; at the same tine, those portions of the cartilage thus used up in the process of ossification are immediately replaced. (Fig. 88.) The following brief summary of the several stages of endochon- dral bone-development may be of service to the student : 1. The embryonic cartilage develops into hyaline cartilage, beginning at the centers of ossification. 2. The cartilage-cells enlarge and become vesicular. In the diaphysis of long bones such cells are arranged in quite regular colunns, while in the epiphyses and irregular bones this arrange- ment is not so apparent. 3 Calcification of the matrix ensues ; the cartilage-cells disap- pear (degenerate) ; primary marrow spaces develop. 4-\ Ingrowth of periosteal buds. The osteoblasts are arranged 122 THE TISSUES. in layers on the trabeculae of calcified cartilage, which they envelop with osseous tissue. 5. Osteoclasts cause the absorption of many of the smaller osseous trabeculae ; others become thickened by a deposition of new layers of osseous tissue. Osteoblasts are enclosed in bone- tissue and become bone-cells. In this way there is formed embry- onic cancellous bone, bounding Haversian spaces inclosing embry- onic marrow. 6. In the diaphysis, the greater portion of the embryonic can- cellous bone is also absorbed (by osteoclasts) ; the Haversian spaces unite to form a part of the marrow space of the shaft of the bone. 2. Intramembranous Bone. — This, the simpler type of ossifi- cation, occurs in bone developed from a connective-tissue founda- tion, and is exemplified in the formation of the bones of the Blood- vessel. Ossification ridge. Epiphyseal cartilage. Fig. 88. — Longitudinal section through epiphysis of arm bone of sheep embryo ; X I;* a, b, Primary marrow spaces and bone lamellae of the diaphysis. cranial vault and the greater number of the bones of the face, aid also in bone developed from the periosteum (perichondrium) sir- rounding the cartilage fundaments of endochondral bone. All fibrous-tissue bone is developed in the same way. The intramembranous bone-development begins by an apprcxi- mation and more regular arrangement of the osteoblasts of the osteogenetic layer of the periosteum about small fibrous-thsue bundles. The osteoblasts then become engaged in the formation of the osseous tissue which envelops the fibrous-tissue bundles. In this way a spongy bone with large meshes is formed, consisting of irregular osseous trabeculae, surrounding primary marrow spaces. These latter are filled by embryonic marrow and blood-vesses de- veloped from the tissue elements of the periosteum not engaged in the formation of bone. THE CONNECTIVE TISSUES. 123 Intramembranous bone first appears in the form of a thin lamella of bone, which increases in size and thickness by the formation of trabeculae about the edges and surfaces of that previously formed and in the manner above described. A layer of intramembranous bone thus surrounds the endochondral bone in bones preformed in hyaline cartilage. The two modes of ossification may, therefore, be observed in either a cross or a longitudinal section of a develop- ing bone preformed in hyaline cartilage. In such preparations the endochondral bone can be readily distinguished from the intra- •/r V KSffi c "•* '&*%'•&*% •te- ~j*i • " . - • £'fe» —Primary marrow space. j ' 2* '< )/ ft, » • •^®>-€» 1 i a^'-a •« Fig. 89. — Section through the lower jaw of an embryo sheep (decalcified with picric acid) ; X 3°°- At a and immediately below are seen the fibers of a primitive marrow cavity lying close together and engaged in the formation of the ground- substance of the bone, while the cells of the marrow cavity, with their processes, arrange themselves on either side of the newly formed lamella and functionate as osteoblasts. membranous bone by reason of the fact that remnants of calcified cartilage matrix may be observed in the osseous trabeculae of the former. It will be remembered that these osseous trabeculse de- velop about the calcified cartilage matrix remaining after the dis- appearance of the cartilage-cells. In figure 90, which shows a cross-section of a bone from the leg of a human embryo, these facts are clearly shown. A study of this figure shows the endochondral bone, with the remnants of the cartilage matrix (shaded more 124 THE TISSUES. deeply) inclosed in osseous tissue, making up the greater portion of the section and surrounded by the intramembranous bone. In figure 91, more highly magnified, the relations of endochon- dral to intramembranous bone and the details of their mode of development are shown ; also the structure of the periosteum. As was stated in the previous section, soon after the formation of the endochondral bone, this is again absorbed ; the process of endochondral bone-formation and absorption extending from the center of ossification toward the ends of the diaphysis. Before the absorption of the endochondral bone, the intramembranous bone has attained an appreciable thickness and surrounds the marrow cavity formed on the absorption of the endochondral bone. Before, Fig. 9°- — Cross-section of developing bone from leg of human embryo, showing endo- chondral and intramembranous bone-development. however, the marrow cavity can attain its full dimensions, much of the intramembranous bone must also undergo absorption. While intramembranous bone is being developed from the periosteum and thus added to the outer surface of that already formed, osteoclasts are constantly engaged in its removal from the inner surface of the intramembranous bone. The marrow cavity is thus enlarged, the process continuing until the shaft attains its full size. The compact bone of the shaft is developed from the primary spongy intramembranous bone after the following manner : The primary marrow spaces are enlarged by an absorption, through the agency of osteoclasts, of many of the smaller trabeculae of osse- THE CONNECTIVE TISSUES. 125 ous tissue and by a partial absorption of the larger ones, the primary marrow spaces thus becoming secondary marrow spaces, or Haversian spaces. The osteoblasts now arrange themselves in layers Connective _— j~ tissue. Outer fibrous - layer of periosteum. Osteogenetic liL: layer of ^> periosteum. " , ;; : Osteoblasts.. __ Marrow-^ space. Blood-ves-_ ' Osteoblasts.--'-" Remnants of cartilage matrix. Bone-cells.— Osteoblasts. Fig. 91. — From a cross-section of a shaft (tibia of a sheep) ; X 55°- In l^e lower part of the figure is endochondral bone- formation (the black cords are the remains of the cartilaginous matrix) ; in the upper portion is bone developed from the periosteum. about the walls of the Haversian spaces and deposit lamella after lamella of bone matrix, concentrically arranged, until the large Haversian spaces have been reduced to Haversian canals. During 126 THE TISSUES. this process many of the osteoblasts become inclosed in bone matrix, forming bone-cells and the blood-vessels of the Haversian spaces remain as the vessels found in the Haversian canals. The spongy intramembranous bone not absorbed at the commencement of the formation of the system of concentric lamellae, remains between the concentric systems as interstitial lamellae. The circum- ferential lamellae are those last formed by the periosteum. Calcifica- ation of the osseous matrix takes place after its formation by the osteoblasts. From what has been stated it may be seen that the shafts of the long bones and bones not preformed in cartilage develop by the process of intramembranous bone-formation, while the cancellous bone in the ends of the diaphysis and in the epiphyses is endochon- dral bone. Further, that long bones grow in length by endo- chondral bone-development, and in thickness by the formation of intramembranous bone. In the development of the smaller irreg- ular bones, both processes may be engaged ; the resulting bone can not, however, be so clearly defined. TECHNIC Ranvier's Method. — One of the methods for examining connective- tissue cells and fibers is that recommended by Ranvier (89) ; it is as follows : The skin of a recently killed dog or rabbit is carefully raised, and a o. i °/0 aqueous solution of nitrate of silver injected subcutaneously by means of a glass syringe. The result is an edematous swelling in which the connective- tissue cells and fibers (the latter somewhat stretched) come into imme- diate contact with the fixing fluid and are consequently preserved in their original condition. In about three-quarters of an hour the whole eleva- tion should be cut out (it will not now collapse) and small fragments placed upon a slide and carefully teased. Isolated connective-tissue cells with processes of different shapes, having the most varied relations to those from adjacent cells, are seen. The fibers themselves either consist of several fibrils, or, if thicker, are often surrounded by a spirally encir- cling fibril. By this method numerous elastic fibers and fat-cells are also brought out. If a drop of picrocarmin be added to such a teased prepa- ration and the whole allowed to remain for twelve hours in a moist chamber, and formic glycerin (a solution of i part formic acid in 100 parts glycerin) be then substituted for twenty-four hours, the following in- structive picture is obtained : All nuclei are colored red, the white fibrous connective-tissue fibers pink, the fibrils encircling the latter brownish- red, and the elastic fibers canary yellow. The peripheral protoplasm of the fat-cells is particularly well preserved, a condition hardly obtain- able by any other method. Connective tissue with a parallel arrangement of its fibers is best studied in tendon, those in the tails of rats and mice being particularly well adapted to this purpose. If one of the distal vertebrae of the tail be loosened and pulled away from its neighbor, the attached tendons will become separated from the muscles at the root of the tail and appear as thin glistening threads. These are easily teased on a slide into fibers and THE CONNECTIVE TISSUES. fibrils. Such preparations are also useful in studying the action of reagents (see below) . The substance resembling mucin which cements the fibrillae together is soluble in lime-water and baryta-water — a circumstance made use of and recommended by Rollet (72, II) as a method for the isolation of connective-tissue fibrils. In necrotic tissue the fibers show a degenera- tion into fibrils (Ranvier, 89). If connective tissue be heated in water or dilute acids to 120° C., and the fluid then filtered, a solution is obtained from which collagen can be precipitated by means of alcohol. This is insoluble in cold water, alcohol, and ether, but is soluble in hot water and when dissolved in the latter and cooled, becomes transformed into a gelatinous substance. Unlike mucin and chondrin this substance does not precipitate on the addition of acetic and mineral acids. Tannic acid and corrosive sublimate will cause pre- cipitation, as also in the case of chondrin, but not with mucin (vid. also Hoppe-Seyler). Elastic tissue may be obtained by treating connective tissue with potassium hydrate solution, and if the alveoli of the lungs be treated for some time with this reagent, very small elastic fibers can be obtained. By this means the connective-tissue fibers are dissolved, but not the elastic fibers. Particularly coarse fibers are found in the ligamenta subflava. According to Kiihne, connective and elastic tissues are differ- ently affected by trypsin digestion — /. >., alkaline glycerin -pancreas extract at 35° C. — white fibrous connective tissue being resolved into fibrils, while elastic tissue is entirely dissolved. To F. P. Mall also belongs the credit for a few data, which we insert, as to the different reactions which various connective-tissue sub- stances show when treated by the same reagents. When a tendon is boiled it becomes shorter, but if it be fixed before boiling, there is no change. Adenoid reticulum shrinks when boiled, but after a short time swells, and finally dissolves. Both tendon and adenoid reticulum shrink at 70° C. If, however, they be first treated with a 0.5% solution of osmic acid, the shrinkage will not take place until 95° C. is reached. If the reticulum or the tendon has become shrunken through heat, they are easily digested with pancreatin, and putrefy very readily. Tendon fibers do not become swollen in glacial acetic acid, either concentrated or m strengths of 0.05% or less, but in strengths of 0.5% to 25% they swell, and if placed in a 25% solution they will dissolve in twenty-four nours. They also swell in hydrochloric acid in strengths of 0.1% to 6%. In strengths of 6% to 25% the fibers remain unchanged for some time, and only dissolve in a concentrated solution of this acid. Reticulated tissue, on the other hand, swells in a 3% hydrochloric acid solution, but remains unchanged in strengths of 3% to 10%. It dissolves in twenty-four hours in solutions of 25% and over. After treatment with a dilute solution of acid, tendon dissolves more rapidly on boiling than does reticular tissue. Tendon exposed to the action of the gastric juice of a dog does not dissolve more rapidly than elastic tissue ; but if placed in an artificial solu- tion of gastric juice, tendon dissolves first, then reticular tissue, and finally elastic fibers. Pancreatin affects neither tendon nor reticulated tissue, but if boiled, both tissues are easily digested by its action. If taken out of the body, neither tendon nor reticulum will become affected by putre- faction. In the body, however, and especially at high temperatures (37° C.), both tissues are decomposed within a few days. 128 THE TISSUES. Elastic fibers remain unchanged in acetic acid, and even when boiled in a 20% solution they only become slightly brittle. They are, however, rapidly destroyed by concentrated hydrochloric acid, although in a 10% solution at ordinary temperature no change is seen. In a 50% solution the fiber is dissolved in seven days, and in a concentrated solu- tion in two days. The inner substance of the fiber is first attacked, then the membrane. To demonstrate this membrane, the fibers are boiled several times in concentrated hydrochloric acid and the whole then poured into cold water. Occasionally, a longitudinal striation of the membrane is seen, indicating a fibrillar structure. Concentrated solutions of potassium hydrate disintegrate the fibers in a few days ; weak solutions, more slowly. A i c/o solution of potassium hydrate requires months to produce the effect ; a 2 % solution, one month ; a 5 % , three days ; a 10%, one day ; and 20% to 40%, only a few hours. A weak solution of potassium hydrate, even when brought to the boiling-point, does not dissolve elastic fibers, nor does it cause them to become brittle. If, how- ever, they be boiled in a 5% or 10% solution of potassium hydrate, the membranes of the fibers will be isolated. A cold 20% solution has the same effect in one or two days. Pepsin induces a disintegration of the contents of the fiber, leaving the membranes intact. To demonstrate the inner substance of elastic fibers and their membranes, magenta red has been recommended (a small granule is added to 50 c.c. glycerin and 50 c.c. water). By this method the internal substance is colored red while the sheath remains colorless. Orcein, Unna's Method. — Make a solution consisting of Grubler's orcein i part, hydrochloric acid i part, absolute alcohol 100 parts. The sections are stained in a porcelain dish. The stain is heated over a flame or in an oven until the stain becomes quite thick. Rinse thor- oughly in alcohol, clear in xylol, and mount. Elastic fibers stain a dark brown, white fibrous tissue a light brown. Fuchsin-resorcin Elastic Fibers Stain (Weigert). — A solution containing i % of basic fuchsin and 2 % of resorcin is made and brought to boiling. To 200 c.c. of this solution there is added 25 c.c. of liquor ferri sesquichlorati (Germ. Pharm.). Boil for about five minutes, stir- ring the meanwhile. Filter on cooling, and place the filter paper and the precipitate collected in a porcelain dish and add 200 c.c. of 95% alcohol and bring to boiling. Filter on cooling and add to the nitrate 4 c.c. of hydrochloric acid and enough alcohol to bring it up to 200 c.c. Stain sections for about one hour. Sections are then washed in alcohol or acidulated alcohol, or, better still, in alcohol to which a few crystals of picric acid have been added. Clear in xylol and mount. Elastic fibers are stained dark blue or bluish-black if washed in picric alcohol. Differential Stain for Connective-tissue Fibrilla? and Reticu- lum (Mallory). — Fix tissues in corrosive sublimate or in Zenker's so- lution. (Tissues fixed by other methods may also be used, although the results are not quite so satisfactory, if the sections are immersed for fifteen to thirty minutes in a saturated corrosive sublimate solution just before staining. ) The sections, which may be cut in celloidin or paraffin, are stained for one to three minutes in a ^% aqueous solution of acid fuch- sin, rinsed in water, and placed in a i % aqueous solution of phosphomo- lybdic acid for five to ten minutes, and then washed in two changes of water. They are now stained in the following solution for two to twenty THE CONNECTIVE TISSUES. 1 29 minutes: Griibler's aniline blue soluble in water, 0.5 gm. ; Griibler's orange G, 2 gm. ; oxalic acid, 2 gm. ; distilled water, 100 c.c. After staining, the sections are washed in water and dehydrated in 95% alcohol, blotted on the slide, and cleared in xylol and mounted in xylol balsam. The connective-tissue fibers and reticulum stain blue. Dr. Sabin's modification of this method deserves mention. Fix in Zenker's fluid, cut in paraffin, and fix sections to the slide with the water method. After removing the paraffin, stain sections in y1^^ acid fuchsin until red, and without washing fix in a saturated aqueous solution of phosphomolybdic acid diluted ten times for about ten minutes. Wash in 95% alcohol and stain for a very short time in the following solution : Griibler's aniline blue soluble in water, i gm. ; orange G, 2 gm. ; oxalic acid, 2 gm. ; boiling water, 100 c.c. Wash in alcohol, blot on the slide, clear in xylol and mount in xylol balsam. Digestion Method for Demonstrating the Connective-tissue Framework of Organs and Tissues (Mall, Spalteholz, Hoehl, Flint). — For bringing out the framework of white fibrous and reticular fibers of organs and tissues digestion by means of trypsin may be recom- mended. For the account here given we follow Flint. The tissues are fixed in graded alcohol, corrosive acetic, or Van Gehuchten's chloroform- acetic-alcohol mixture. After complete dehydration, small pieces of tissue, not to exceed 3 mm. in thickness, are placed in paper cups and dropped into a Soxhlet apparatus and extracted with ether for a period of six to eight days in order to free the tissue of the fat. After the fat has been removed, the tissues are brought into water, through graded alcohol, and then digested in pancreatin. (Griibler's pancreatin is rec- ommended ; that of Park, Davis & Co. may be used.) The -pancreatin solution to be used is made by adding as much pancreatin as can be taken up on the end of an ordinary scalpel handle to 100 c.c. of a 0.5% solution of bicarbonate of soda. This solution is changed every forty- eight hours. To prevent putrefaction enough chloroform is added to cover the bottom of the dish. The digestion is continued until the cell- ular element has been removed — five to ten days. It is often necessary to repeat the fat extraction and digestion several times. After the cellu- lar elements have been removed the tissue is thoroughly washed in flowing water, and may then be mounted in glycerine and studied with a stereo- scopic microscope, or it may be dehydrated and imbedded in celloidin and sectioned. Such sections may then be stained in fuchsin and thor- oughly washed in alcohol ; this removes the stain from the celloidin, leaving only the connective tissue stained. Slide Digestion. — The method may also be applied for digesting tissues on the slide. Fix as above described, imbed in paraffin, and cut very thin sections which are fixed to the slide by the water method. Remove the paraffin and place the sections from alcohol into the Soxhlet apparatus, where they are extracted with ether for a number of hours. Bring the sections through graded alcohol into water, in which they re- main several hours. The sections are now digested in the above-men- tioned pancreatin solution for several hours to several days, or until the cellular elements have been removed. Wash carefully in water. The remaining connective tissue may now be stained in iron-lac hematoxylin or in an aqueous solution of toluidin blue or in an aqueous solution of fuchsin. Dehydrate, clear, and mount. I3O THE TISSUES. Fresh adipose tissues can be obtained in lobules and in small groups of cells from the mesenteries of small animals. As a rule, the highly refractive fat globule hides from view the nucleus and protoplasm of the cell. The latter structures can be brought out by the subcutaneous injection of silver nitrate solution, this forming the edematous elevation previously described. Fresh fat is soluble in ether and chloroform, especially if the latter be heated. Strong sulphuric acid does not dis- solve fat. The stains made from the root of the henna plant color fat red (the color disappearing in ethereal oils). Quinolin-blue, dissolved in dilute alcohol, stains fat a dark blue. If a 40% potassium hydrate solution be then added, everything will become decolorized except the fat. The most important reagent for demonstrating adipose tissue is osmic acid (and its mixtures). Small pieces of adipose tissue are treated for twenty-four hours with a 0.5% to i% osmic acid solution ; if mixtures containing osmic acid be used, the specimens are generally im- mersed for a somewhat longer period. The pieces are then washed with water, and should not be placed directly into alcohol of full strength, as all the structures would then become intensely black (Flemming, 89), but carried into alcohols of ascending strength. When treated in this way the globules of fat take a more intense stain than the other tissues, which, nevertheless, are blackened to some extent. Fat that has been subjected to osmic acid treatment dissolves readily in turpentine, xylol, toluol, ether, and creosote, with difficulty in oil of cloves, and not at all in chloroform. Such preparations are best carried from chloroform into paraffin. Fat that has been stained with osmic acid can be decolorized by nascent chlorin. The specimens are placed in a jar of alcohol in which crystals of potassium chlorid have been previously placed. Hydro- chloric acid is then added (to i%) and the vessel tightly sealed (P. Mayer, 81). L. Daddi has recently recommended Sudan III as a stain for fat. This reagent can be applied in two ways : ( i ) Either the animals are fed with the coloring matter for some days, in which case all the fat will be colored red, or (2) either fresh or fixed pieces of tissue or sections are stained. Fixation before staining must be done in media that do not dis- solve fat, as, for instance, Miiller's fluid. A saturated alcoholic solution of the stain is used and allowed to act from five to ten minutes. The specimen is then washed with alcohol and mounted in glycerin. The author's experiments with Sudan have been very satisfactory. Thin lamellae of fresh cartilage are examined after separating them from the soft parts and placing them in indifferent fluids. Cartilage removed from the hyposternum or episternum or scapula of a frog is especially adapted for examination. Larger pieces of uncalcified carti- lage may be used if cut into sufficiently thin sections with a razor moist- ened with an indifferent fluid. Under the microscope such sections show a finely punctated background with capsules containing cartilage -cells, provided the latter have not fallen out in the process of cutting, in which case lacunas will be observed. Osmic acid and corrosive sublimate are by far the best fix- ing agents for cartilage. If the cartilage be calcified, it is fixed for some time in picric acid, which at the same time acts as a decalcifying agent. Although alcohol fixes cartilage fairly well, it causes shrinkage of the THE CONNECTIVE TISSUES. 13! cells. The ground substance may be specifically colored by certain reagents, safranin producing an orange and hematoxylin a blue stain. On treating cartilage by certain methods, systems of lines appear in its ground substance, possibly indicating a canalicular sys- tem in the cartilage. In order to make these structures visible, Wolters recommends staining thin sections for twenty -four hours in a dilute solu- tion of Delafield's hematoxylin (violet blue). They are then treated with a concentrated alcoholic solution of picric acid. The capsules are seen to best advantage if small pieces of car- tilage are treated with a 1% solution of gold chlorid. Connective-tissue and elastic fibers in cartilage are easily demonstrated by staining the specimens with picrocarmin. The con- nective-tissue fibers are colored a pale pink, the elastic fibers yellow. The latter may also be stained with a i °/0 aqueous solution of acid fuchsin. If a section of fresh cartilage be placed in a weak solution of iodo-iodid of potassium (Lugol's solution), glycogen can sometimes be seen in the cartilage-cells, stained a peculiar mahogany brown. If elastic fibers be present, they also are stained brown, but of a different shade. Thin bone lamellae, such as occur in the walls of the ethmoidal cells, can be cleaned of all the soft parts and examined without further manipulation. If larger bones are scraped with a sharp knife, pieces suitable for microscopic examination are sometimes obtained. Microscopic Preparation of Undecalcified Bone. — A long bone is thoroughly freed from fat and other soft parts by allowing it to macerate, after which it is thoroughly washed and dried, thus freeing it from its organic material. Then, by means of two parallel cuts with a saw, as thin a disc as possible is cut out. The section is now ground still thin- ner, either between two hones or upon a piece of glass covered with emery. One surface of the bone is then polished and fastened by means of heated Canada balsam to a thick square plate of glass with the polished side toward the glass. Care should be taken that no air-bubbles are inclosed between the section and the glass. As soon as the specimen is firmly adherent, the other side is ground upon the emery plate or hone, during which manipulation the glass to which the bone has been fastened is held between the fingers. As soon as the section is sufficiently thin and transparent, it is polished. In order to remove the Canada balsam and powdered bone from the section, the glass and bone are dried and placed in some solvent of Canada balsam, such as xylol. This loosens the specimen from the glass, after which it is immersed in absolute alcohol, thoroughly washed, and dried in the air. On examining the bone through the microscope, its lacunae will appear black on a colorless back- ground. The reason is, that the air has taken the place of the evapo- rated alcohol and the spaces appear black by direct light. Sections thus prepared may be permanently mounted as follows : Small pieces of dry Canada balsam are placed both upon a slide and a cover- glass and warmed until they have become fluid, then allowed to cool until a thin film forms over the balsam ; the bone disc is then placed upon the balsam on the slide and quickly covered with the cover-glass. A firm pressure will evenly distribute the balsam, and if 132 THE TISSUES. the whole has been done with sufficient rapidity the air will have been caught in the open spaces of the bone before the Canada balsam has had a chance to enter these spaces. Other substances may be used to demonstrate the spaces in bone. Ranvier (75) recommends the following method: A few c.c. of a concentrated alcoholic solution of anilin blue (which is soluble in alcohol and not soluble in water and sodium chlorid solution) are placed in an evaporating dish containing the dry bone. The solution is very carefully evaporated, as the alcohol may otherwise ignite. The specimen, which will soon be covered on both surfaces by a blue powder, is taken out and ground upon a rough glass plate until thoroughly clean. While being polished the bone should be kept moist by a solution of sodium chlorid. On heating in the evaporating dish, the air is driven from the spaces and replaced by the anilin blue. As already stated, anilin blue is insoluble in sodium chlorid solution, and it therefore remains unaffected by the latter during the process of grinding and cleaning. Hence it remains in the lacunse and canaliculi of the bone, which then appear blue. The specimen may either be mounted in glycerin-sodium chlorid and the edge of the cover-glass sealed with varnish, or the section may be washed for a short time in water (in order to remove the sodium chlorid), dried, and finally mounted in Canada balsam as directed. A method adapted to the study of the hard and soft parts together is that first used by von Koch in studying corals. The specimen is first fixed, and if it be a long bone, the marrow cavity should first be opened to permit the fixing agent to come in contact with all parts of the tissue. After fixing, the bone is stained and then placed in absolute alcohol, and when completely dehydrated the pieces are placed in chloro- form, then in a thin solution of Canada balsam in chloroform, and finally put into an oven kept at a temperature of about 50° C. for from three to four months. By this means the pieces are completely penetrated by the Canada balsam, and as the latter becomes very hard on cooling, the sections may be afterward ground without difficulty. Long as this pro- cedure may seem, it is still the one which enables us to see the soft and hard parts of bone in a relationship the least changed by manipu- lation. In bone, as also in cartilage, there sometimes occur amorphous as well as crystalline deposits of lime-salts. Upon the addition of acetic acid the carbonate of calcium gives off bubbles ; upon the addition of sulphuric acid, short, thin needles will be formed — crystals of gypsum. Hematoxylin stains the lime-salts blue, with the exception of the oxalate of lime. Alkaline solution of purpurin stains calcium carbonate red. Caustic potash does not affect lime. In order to study the organic constituents of bone, it must first be decalcified and thus rendered suitable for sectioning — /. e. , the lime-salts must first be removed, and that without destroying the cellular elements of the bone. The process of decalcification consists in substi- tuting the acids of the decalcifying fluids for those of the bone salts. As a consequence^ new combinations are formed, soluble in water or in an excess of the decalcifying acids themselves. The decalcifying fluids most frequently used are : (a) Hydrochloric acid (i% aqueous solution), used in quantities amounting to about fifty times the volume of the specimen. The solution THE CONNECTIVE TISSUES. 133 is changed daily, and the bone remains immersed until it is soft enough to be cut. This stage is reached when a needle can be introduced with no resistance. (<£) An aqueous solution of nitric acid in strengths of 3% to 10%, ac- cording to the delicacy of the specimen, and of a specific gravity of 1.4. Instead of water, 70% alcohol may be used as a solvent for the acid. Thoma has recommended for this purpose a solution consisting of i vol. nitric acid of a specific gravity of 1.3, and 5 vols. alcohol. This fluid is changed daily and decalcifies small objects in a few days. The specimens are then washed several times in 70$ alcohol to remove as much as possible of the acid. 95% alcohol, with the addition of a little precipitated calcium carbonate, has been recommended for washing sections that have been treated by Thoma' s method. After from eight to fourteen days the specimens are again washed with clear 95% alcohol. (c) The process of decalcification recommended by v. Ebner (75) is of considerable value, as it also reveals the fibrillar structure of the bone lamellae. A cold saturated solution of sodium chlorid is diluted with 2 vols. of water, and 2^> of hydrochloric acid added. This fluid decalcifies very slowly, and must either be changed daily or a small quantity of hydrochloric acid occasionally added. As soon as the speci- men is thoroughly decalcified, it is washed with a half-saturated solution of sodium chlorid. A little ammonia is now added from time to time until the reaction of the fluid and bone is neutral. (*/) Very small pieces that contain very little lime-salts, as, for in- stance, bones in an e.mbryonal condition where calcification has only just begun, can be deprived of their lime-salts by means of acid fixing solu- tions like Flemming's fluid, chromic acid, picric acid, etc. (e) Bone should be first fixed in some one of the fixing fluids and then decalcified. Schmorl's Method for Demonstrating the Bone Corpuscles and their Processes in Decalcified Preparations. — The tissues are fixed in Miiller's fluid or in Miiller's fluid with formalin, decalcified in V. Ebner's fluid, and imbedded in celloidin. The sections are stained in either of the following thionin solutions: concentrated 50% alcoholic thionin solution, 10 c.c.; i per cent, carbolic acid water, 90 c.c.; or concentrated 50% alcoholic thionin solution, 10 c.c.; distilled water, 100 c.c.; liquor ammonite, 10 drops. Bring sections from water into trie stain, in which they remain from five to ten minutes or longer. Rinse sections in water, and place them in a saturated aqueous solution of pic- ric acid for one to two minutes or longer. Rinse in water and wash in 70% alcohol until no more stain is given off. Dehydrate in alcohol, clear in xylol, and mount in balsam. The bone corpuscles and processes are stained brownish-black, the ground substance yellow, the cells red- violet. Schmorl' s method for staining the boundary -sheaths of the bone cor- puscle: Harden, decalcify, imbed, and stain as in the preceding method. After staining wash in water for two minutes or longer ; rinse in alcohol for one-half minute, and again rinse in water and place the sections in a saturated aqueous solution of phosphomolybdic os phospho- tungstic acid for three minutes or longer ; wash in water which needs to be changed frequently for ten minutes. The sections are now placed for three to five minutes in a 10% aqueous solution of liquor ammoniae, after 134 THE TISSUES. which they are washed in 90% alcohol, dehydrated, cleared in xylol, and mounted. The boundary-sheaths are stained bluish-black, the bone cells dark blue, and the bone substance light blue. Fibers of Sharpey. — Sections treated by Ranvier's method show the perforating fibers of Sharpey as bright, sharply defined ribbons, appearing as streaks or circles, according to the section made (longitudinal or trans- verse). If decalcified specimens be first rendered transparent by glacial acetic acid, and then immersed for a minute in a concentrated aqueous solution of indigocarmin, washed with water, and then mounted in gly- cerin or Canada balsam, the fibers of Sharpey will appear red and the remaining structures blue. Thin sections of bone can be deprived of their organic elements by bringing them for from one-half a minute to a minute into a platinum crucible at a red heat. In such preparations cal- cified Sharpey's fibers may be seen (Kolliker, 86). Virchow's bone corpuscles may be isolated in the following manner : Very thin fragments or discs of bone are immersed for some hours in concentrated nitric acid. They are then placed on a slide and covered with a cover-glass ; pressure with a needle upon the latter will isolate the lacunae, and occasionally also their numerous processes, the canaliculi. C MUSCULAR TISSUE. Almost all the muscles of vertebrates have their origin from the middle germinal layer. In the simplest type the protoplasm of the formative cell changes into contractile muscle substance, the cell in the meantime undergoing a change in shape (unstriped muscle-cell). In other cases contractile fibrils are formed which are separated by the remains of the undifferentiated protoplasm (striped muscle-cells). In this case the cells either increase very little in length and possess only a single nucleus (heart muscle), or they grow considerably longer and develop many nuclei (voluntary skeletal and skin muscles). A peculiarity of muscle-substance is that it contracts in only one direction, while undifferentiated protoplasm contracts in all directions. J. NONSTRIATED MUSCLE-CELLS. The smooth, unstriped, or nonstriated muscle-cells belong to involuntary muscle, and are found in the walls of the intestine, trachea, and bronchi, genito- urinary apparatus, blood-vessels, in certain glands, and also in connection with the hair follicles of the skin. The involuntary muscle-cells are spindle-shaped cells, which are 40-200 // long and 3-8 // broad. The longest are found in the pregnant uterus, where they attain a length of 500 p. At the thick- ened middle portion of the cell is a long rod-like nucleus, typic of this class of cells. Nonstriated muscle-cells are doubly refractive — anisotropic. The cell substance is longitudinally striated, the striation being due to relatively coarse fibrils situated in the outer MUSCULAR TISSUE. 135 portion of the cell substance (M. Heidenhain, Schaper, Benda). These fibrils have a longitudinal course, and probably run the en- tire length of the cell ; whether they branch and anastomose must be regarded as an open question. In the interior of the cell sub- stance there are found much finer fibrils, which branch and anasto- mose. Between the fibrils there is found a homogeneous substance, which we may know as the sarco- plasm, in which granules are often seen, situated at the poles of the nuclei. It is generally stated that nonstriated muscle-cells are united into membranes and bundles by a small amount of intercellular cement substance which may be darkened by silver nitrate. Recent investigations have, however, revealed the fact that nonstriated muscle-cells are encased in delicate connective membranes, which membranes unite to form compart- ment-like spaces, of fusiform shape, in which the muscle-cells are found. These membranes are not to be re- garded as cell-membranes — sarco- lemma — since one membrane serves the sheath for two contiguous Nucleus. Protoplasm. -- as muscle-cells (Schaffer, v. Lenhossek, Henneberg). The existence of such membranes is clearly shown in invol- untary muscle tissue subjected to trypsin digestion. In such preparation stained in iron- lac -hematoxylin it may be observed that the membranes are not complete, but are fenestrated, showing a varying number of round or oval openings (Henneberg). The membranes are also clearly shown in tissue fixed in corrosive sublimate and stained in Mallory's differential con- nective-tissue stain, the membranes showing as delicate blue lines while the muscle-cells are stained of a red or orange color. (See Fig. 92.) Ac- cording to certain observers (Kultschitzky, Barfurth), nonstriated muscle-cells are thought to be joined by intercellular, protoplasmic bridges. It may, however, be clearly shown that such intercellular bridges are artifacts, due to peripheral vacuolization and to shrinkage of the muscle-cells (Schaffer, v. Lenhossek, Henneberg). What Fig. 92. — Nonstriated muscle from the intestine of a cat. X 3°°- a, Isolated muscle-cell ; b, from cross-section of nonstriated muscle, stained after Mallory's differential connective- tissue stain. Observe the apparent difference in size of the cross-cut cells; four of the cells show( nuclei ; the black lines separ- ating the cells represent the connec- tive-tissue membranes. c, Cross- sections ;of the connective-tissue membranes separating involuntary muscle-cells ; d, an area showing so-called intercellular bridges; they are attached to the connective tissue membranes surrounding the cells (Mallory's differential connective- tissue stain). 136 THE TISSUES. has been described as intercellular bridges may readily be seen in corrosive sublimate preparation, stained in Mallory's differential connective-tissue stain, especially in portions of the preparation not well fixed. In such preparation the so-called intercellular bridges end at the connective membranes separating cells, to which they are attached but which they do not penetrate. Nonstriated muscle- cells develop from the mesenchyme. (Exceptions to this statement appear to be found in the nonstriated muscular tissue of the iris [Szili] and in the sweat-glands, where the muscular tissue appears to be developed from ectodermal cells.) The nuclei of the mesen- chymal cells elongate and become rod-shaped, with oval ends, while the cells become spindle-shaped, the protoplasm staining somewhat more deeply than that of the surrounding mesenchymal cells. Further details as to the development of nonstriated muscular tissue are lacking. 2. STRIPED MUSCLE-FIBERS. Soon after the segmentation of the mesoderm begins, certain cells of the mesoblastic somites or myotomes commence the forma- tion of muscle-substance in their interior, a process which is accom- panied by increase in the number of nuclei, the formation of a mem- brane, a lengthening of the cells, and the appearance of fibrils in the peripheral protoplasm of the cells. ^K — Free ending. Nucleus. Fig- 93- — Cross-section of striated muscle-fibers : I, Of man ; 2, of the frog. The relations of the nuclei to the muscle-substance and sarcolemma are clearly visible ; X 670. Fig. 94. — Muscle-fiber from one of the ocular muscles of a rabbit, showing its free end ; X.I75- Voluntary or striated muscle-cells are large, highly differen- tiated, polynuclear cells, which may attain a length of 12 cm., with a width of 10— 100 fj.. They are consequently known as muscle-fibers. Their free ends are usually pointed ; the ends attached to tendon rounded (Fig. 94). MUSCULAR TISSUE. 137 Each striated muscle-fiber consists of a delicate membrane, the sarcolemma, a muscle protoplasm, in which are recognized very fine fibrils and a semifluid interfibrillar substance (the sarcoplasm) and the muscle nuclei. The sarcolemma is a very delicate, transparent, and apparently structureless membrane, which resists strong acetic acid, even after boiling for a long time. If we examine in an indif- ferent fluid fresh muscle-fibers, the contents of which have been broken without rupturing the sarcolemma, we may see this sheath as a fine glistening line. (Fig. 95.) The fibrils of the muscle-protoplasm constitute the contractile part of the muscle-fiber. They are exceedingly fine and extend the entire length of the muscle-fiber. These fibrils are, however, not of the same composition throughout, but are made up of segments which show different physical properties and stain differently. The structure of the fibrils may be expressed in the form of a diagram (Fig. 96) giving the more recently expressed views of the structure of these fibrils. The fibrils present alternating darker and lighter segments, which taken together give the striation which is so char- Fig. 95. — Striated muscle-fiber of frog, showing sarcolemma. acteristic of striated muscle. The darker segments are slightly longer, are doubly refracting, anisotropic, and in general stain more deeply than do the lighter segments, which are slightly shorter and are singly refracting, isotropic. The darker segments, known as the transverse discs, or Briicker's lines, are indicated in the diagram by the letter Q ; the lighter segments, known as the intermediate discs of Krause, are indicated by the letter j. In the intermediate discs of Krause there is found a dark line, which is doubly refractive, which is known as Krause's membrane (z) (Grundmembran), and which, according to certain observers (M. Heidenhain, J. B. Mac- Callum), is continuous through the fibril bundles, as will be stated more fully later. This membrane divides disc j into two equal parts. The transverse disc (Q) is likewise divided into equal parts by a narrow, isotropic band, known as the median disc of Hensen, and designated by the letter H. In the median discs of Hensen — H — there is found a thin membrane, known as the median mem- brane of M. Heidenhain, and designated as M, which, like the mem- brane of Krause, is continuous through the fibril bundles, uniting 138 THE TISSUES. the fibrils (M. Heidenhain). By grouping the unequally refracting substances (or unequally staining substances) a fibril may be divided into successive portions or protoplasmic metameres which may be termed sarcomeres (Schafer) and which are bounded by the mem- brane of Krause (z). In such a sarcomere or muscle-casket we may recognize, beginning with Krause's membrane, z, an isotropic intermediary disc, j ; an anisotropic, transverse disc, Q, divided by a less refracting Hensen's disc, H, into two equal parts, Hensen's disc showing the median membrane of Heidenhain, M ; again an iso- Fig. 96. — Diagram of the structure of the fibrils of a stri- ated muscle-fiber. The light spaces between the fibrils may represent the sarcoplasm. Fig- 97- — Diagrams of the transverse stria- tion in the muscle of an arthropod ; to the right with the objective above, to the left with the ob- jective below its normal focal distance (after Rol- lett, 85): Q, Transverse disc; h, median disc (Hensen) ; E, terminal disc (Merkel); A7, acces- sory disc (Engelmann) ; J, isotropic substance. tropic intermedian disc, j, and Krause's membrane, z. Krause's membrane, as above stated, is continuous across the small bundles into which the fibrils are grouped, and is also attached to the sar- colemma (M. Heidenhain, J. B. MacCallum). This is shown to the left in Fig. 96, where the sarcolemma appears festooned, with Krause's membrane attached, thus indicating clearly the sarcomeres. One of the best objects for the study of transverse striation is the muscle of some of the arthropods (beetles). In the striated MUSCULAR TISSUE. 139 muscle of beetles and other arthropods there is, however, a further division into isotropic and anisotropic substance. Here it will be noticed that the disc j is separated by an anisotropic disc, known as the accessory disc of Engelman, and designated by the letter N, into an isotropic disc j, next to the anisotropic transverse disc Q, and an isotropic disc, known as Merkel's terminal disc, and designated by the letter E, situated next to Krause's membrane (z). (See lower portion of Fig. 96.) The muscle fibrils present a different appear- ance when focused high than they do when focused low, as may be seen from the diagram given in Fig. 97; those parts which appear light on high focusing appear dark on deep focusing. Sarcoplasm. Cohnheim's area. Sarcolemma. Sarcoplasm. Cohnheim's area. Sarcolemma. Fig. 98. — Transverse section through striated muscle-fibers of a rabbit. I and 3, from a muscle of the lower extremity ; 2, from a lingual muscle ; X 9°°- In 2» Cohn- heim's fields are distinct; in I, less clearly shown ; in 3, the muscle-fibrils are more evenly distributed. It has recently been suggested by J. B. MacCallum that Krause's membrane with the primitive fibrils form a continuous network in the muscle-fiber, the meshes of which would be fairly regular, the fibrils of such a network which run parallel to the long axis of the muscle-fiber being larger than the cross fibrils. Such a network is not to be confused with a network which may be brought out on staining striated muscle-fibers with gold chlorid, which network is due, in part at least, to a staining of the sarcoplasm. The ultimate fibrils are grouped into small bundles (0.3-0. 5 // in 140 THE TISSUES. diameter), forming tint fibril bundles or muscle-cohimns of Kolliker. In the muscle-columns the fibrils are so placed that the larger segments fall respectively in the same plane. (See Fig. 96.) The same disposi- tion of the fibrils prevails in all the numerous muscle-columns form- ing a muscle-fiber, and all the muscle-columns bear such a relation to each other that the larger segments of the fibrils fall in the same plane. The semifluid, interfibrillar substance, the sarcoplasm, pene- trates between the fibrils of the muscle-columns and separates these from each other and from the sarcolemma. In fresh preparations the substance forming the fibrils appears somewhat darker and dimmer, while the sarcoplasm appears clearer. The sarcoplasm is found in greater abundance between the muscle-columns than between the fibrils in the columns. The sarcoplasm between the muscle-columns appears in the form of narrower or broader lines, parallel to the long axis of the muscle-fibers, giving the cross -striated muscle-fiber also a longitudinal striation. The sarcoplasm between the muscle- columns is seen to best advantage in cross-sections of the muscle- fiber. Here it appears in the form of a network inclosing the mus- cle-columns. Thus, we have in a cross-section slightly darker areas, the cross-sections of the muscle-columns, known as Cohn- heim's fields or areas, separated by the network of sarcoplasm. (Fig. 98.) Fig. 99. — From a striated muscle of man ; obtained by teasing ; X ' 2O°- ^> -A- median disc lying in the transverse disc Q; z, the membrane of Krause borders above and below on the light isotropic discs. Fig. 100. — From a cross-section through the trapezius muscle of man, showing dark fibers rich in protoplasm, and light fibers containing very little pro- toplasm (after Schaffer, 93, II) : d, Dark fibers ; a, light fibers ; b and c, transitional fibers from light to dark. In figure 99 is shown a portion of a striated muscle-fiber of man very highly magnified. The larger and darker transverse disc (0 formed by the larger segments of fibrils is divided by a light line (H), Hensen's median disc; the clearer band, largely isotropic substance, is divided by a dark line, the membrane of Krause, z. After a prolonged treatment with 98 % alcohol the muscle-fibers MUSCULAR TISSUE. 141 of the water-beetle (Hydrophilns piceus] can be made to separate into transverse discs (Rollet, 85). One of these discs would cor- respond to the segment Q, and it is very probable that this is the portion which has long been known under the name of Bow- man's disc. Other reagents, as weak chromic acid, cause a separation of the muscle-substance into longitudinal fibrils. In this case the discs Q are split up longitudinally into a number of very small columns which were at one time regarded as the primary elements of the fiber and termed by Bowman sarcoiis elements. In adult skeletal and skin muscle-fibers of mammalia the posi- tions of the nuclei vary. There are muscles in which the nuclei are imbedded in the sarcoplasm between the muscle-columns (so-called red muscles, as the semitendinosus of the rabbit) ; in other muscles they lie immediately beneath the sarcolemma (white muscles, as the semimembranosus of the rabbit ; Ranvier, 89). In the striated mus- cle-fibers of the lower vertebrates and of mammalian embryos the nuclei lie between the fibrillae, or muscle-columns. The red muscle- fibers are rich in sarcoplasm, and the fibrils are grouped in well- marked and large muscle-columns surrounded by sarcoplasm which often contains granules of various sizes, the interstitial granules of Kolliker, often especially abundant at the poles of the nuclei. The white muscle-fibers have a relatively small quantity of sarcoplasm. In cross-sections of the light fibers the fibrils show as fine points, not distinctly grouped, and surrounded by the homogeneous sarcoplasm. Both varieties occur in almost every human muscle, and the relative number of each varies greatly in the different muscles (Schaffer, 93, II, Fig. 100). Muscles with transversely striated fibers are, with the exception of those of the heart, subject to the will of the individual, and are characterized by a rapid contraction in which the anisotropic substance increases in size at the cost of the isotropic discs ; the former appears to play the chief role. Besides morphologic dif- ferences, the red and white muscle-fibers appear to possess differ- ences of a physiologic character, in that the contraction in the red Fig. 101. — Branched, striated muscle-fiber from the tongue ot a frog. variety is slower than that in the white (Ranvier, 80). Only the stri- ated muscles of the esophagus, the external cremaster, and a few others, as well as the somewhat differently constructed muscles of the heart, are involuntary. 142 THE TISSUES. •:- p&. c- Fig. 102. — Cross-section of rectus abdominis of child, as seen under low magnification. Muscle. it > »- ••//./ i'i j PUflrw fHHiii , ti&Jr!'*. r<''), • Stil'ii'i^/f^^i Fig. 103, — Part of a longitudinal section through the line of junction between muscle and tendon; X 15°- At the line where the tendon-fibrils join the sarcolemma (a), the nuclei of the muscle are very numerous. Sublimate preparation. MUSCULAR TISSUE. 143 Transversely striated muscle-fibers are usually unbranched. The muscle-fibers of the tongue and of the ocular muscles do, how- ever, show occasionally communicating branches ; the same are but very rarely seen in other muscles. In regions where striated muscle-fibers terminate under the epithelium, as in the tongue and in the skin of the face, the end of the fiber terminating under the epithelium is often very much branched ; the cross-striation and nuclei may be observed in the finest branches. (Fig. 101.) Each muscle-fiber is surrounded by a thin connective-tissue en- velope, the endomysium, which binds them into primary and second- ary bundles, the muscle-fasciculi. These are surrounded by a denser sheath of similar character, the perimysiuui. The muscle is made up of numerous fasciculi, all bound together by a thicker con- nective-tissue covering, the epimysium. (Fig 102.) Blood-vessels are very numerous in transversely striated mus- Contractile substance. — Nucleus. Fig. 104. Fig. 105. Longitudinal and cross-section of muscle-fibers from the human myocardium, hard- ened in alcohol ; X 640. The muscle-cells in the longitudinal section are not sharply defined from each other, and appear as polynuclear fibers blending with each other. Between them lie, here and there, connective-tissue nuclei. cular tissue. One or several arteries enter each muscle and form superficial and deeper plexuses by, anastomosis. In these plexuses the arteries are accompanied by veins. On reaching the perimysium the arteries give off terminal branches which run transversely over the muscle fasciculi, at quite regular intervals. From these branches precapillaries and capillaries are given off which have a course which is in general parallel to the muscle-fibers ; these capillaries anastomose frequently and collect to form small veins, which are situated between the terminal arterial branches, the terminal arterial and venous branches thus alternating in such a way that one venous branch is situated between two arterial branches or vice versa. The veins, even the smallest, are provided with valves (Spalteholz). 144 THE TISSUES. At its junction with tendon the muscle-fiber with its sarcolemma is rounded off into a blunt point, the fibrils of the tendon being cemented to the sarcolemma. The longitudinal growth of muscle-fibers takes place principally at the distal ends of the fibers, at which point their nuclei are numer- ous. (Fig. 103, at «.) Schaffer (93, II) has recently suggested that there is a formative tissue between the tendon and muscle-substance, from which, on the one hand, muscle-fibers are developed, and, OR the other hand, connective-tissue fibrils and cells are formed. As recent investigations have shown, the development of muscle continues throughout the life of the individual. Muscular tissue is consequently to be regarded as in a perpetual stage of transition, the destruction and compensatory reproduction of its elements going on hand in hand. Its destruction is ushered in by a process which can be compared to a physiologic contraction. Nodes or thickened rings are formed, and at these points the muscle-substance separates into fragments with or without nuclei (sarcolytes), which are then absorbed, in most cases without phagocytic aid. This loss of sub- stance is replaced by new elements developed from the free sarco- plasm, which is characterized by rapid growth and increase in the number of its nuclei. The result is that new elements are formed which have been called myoblasts. The process by which myo- blasts are changed into the finished muscle-fibers is exemplified in the embryonal type of development of the tissue. Development of Voluntary Muscle-fibers. — The striated, voluntary muscular tissue, as above stated, develops from the myo- tomes, segmentally arranged differentiated portions of the meso- derm. In the myotomes are developed round or oval cells known as myoblasts, which proliferate by mitotic cell division. According to the observations of certain observers, the myoblasts elongate and become spindle-shaped, while the nuclei proliferate, without an ac- companying division of the cell body, to form the muscle-fibers, which may thus be regarded as polynuclear cells developing from a single cell. Other observers, notably Godlewsky, state that only relatively few of the muscle-fibers develop in this way, the majority being formed by a fusion of myoblasts, forming a syncytium, a muscle-fiber being thus a syncytial structure developed from a vary- ing number of myoblasts. The contractile fibrils are differentiated from the protoplasm of the differentiating myoblasts. When first seen, they present a uni- form structure, and only later can a differentiation into isotropic and anisotropic substance be recognized. The discs Q and j appear first ; the other parts of the sarcomeres somewhat later. The first formed fibrils divide longitudinally to give rise to new fibrils. Em- bryonic striated muscle tissue, even after striation of the fibers may be observed, forms a very compact tissue with only narrow inter- spaces between the cells. In the further development of this tissue certain of the embryonic muscle-fibers undergo degeneration MUSCULAR TISSUE. 145 (Bardeen, Godlewski), and mesenchymal tissue, blood-vessels, and nerve-fibers make their appearance between the developing muscle- fibers. CARDIAC MUSCLE. Cardiac muscle or heart muscle is striated muscle, but differs physiologically and structurally from voluntary striated muscle. It resembles involuntary muscle in that it is not subject to the will. Heart muscle after fixation with many reagents used in the labora- tories, and when treated with macerating fluids, or subjected to the action of silver nitrate, appears to consist of irregularly shaped ob- long cells, cemented end-to-end to form heart muscle-fibers; such fibers appear to anastomose by means of side processes possessed by the cells. A number of recent investigators, notably v. Ebner and M. Heidenhain, have, however, shown that what has been re- garded as cement lines uniting cells are to be otherwise interpreted, since they are known to bound nonnucleated areas of heart muscle, and since the contractile fibrils possessed by heart muscle pass through such lines without interruption. It would appear, there- fore, that heart muscle must be regarded as a syncytium in which no distinct and separate cells occur, but rather of a complex plexus of branching and anastomosing fibers which differ in size and shape. Heart muscle-fibers consist, as was shown for voluntary striated muscle-fibers, of contractile, primitive fibrils, which are grouped into fibril bundles or muscle columns, between which there is found un- differentiated protoplasm, the sarcoplasm. They are surrounded by a sarcolemma, which differs, however, from the sarcolemma of volun- tary muscle-fibers in not being so well developed. The primitive fibrils present the same structure as described for similar fibrils of voluntary muscle, each sarcomere consisting of Krause's membrane, z; two intermediary discs, j; the transverse disc, Q, bisected by Hensen's median disc, H, which in turn contains the median mem- brane of Heidenhain, M. (See Fig. 96.) Krause's membranes (z) and the median membranes (M) extend across the fibril bundles ; the former are attached to the sarcolemma (M. Heidenhain). The primitive fibrils are grouped into fibril bundles or muscle columns, which in cross-sections are often band-shaped and are placed radially with reference to the center of the heart muscle-fibers. The sarcoplasm is present in relatively larger quantity than in voluntary striated muscle, especially between the fibril bundles, giving the fibers a distinct longitudinal striation. The primitive fibrils pass uninterruptedly through the anastomoses between the fibers. The nuclei, which are round or oval and possess a distinct chromatin network, are situated near the center of the fibers, occurring at ir- regular intervals, and are surrounded by an axial core of undifferen- tiated protoplasm, in which are found granules which stain in basic stains, also fat droplets, and, especially in older individuals, pigment granules. The structures which have been regarded as intercellular 146 THE TISSUES. cement lines may be especially stained in certain anilin stains. In such preparations it may be seen that they often do not extend through an entire fiber, are frequently irregular, often presenting the appearance of steps, and now and then involve only one or two fibril 'bundles. They are frequently seen to bound portions of a muscle-fiber which are nonnucleated. They are looked upon by M. Heidenhain as representing growth areas. See Fig. 106, in which such intercalated growth areas (cement lines ?) are represented darker than the remaining structures. Heart muscle-fibers are surrounded by delicate connective-tissue sheaths, very much as described for nonstriated muscle tissue. These are well shown in tissue fixed in corrosive sublimate and stained after Mallory's differential connective-tissue stain. The » f& l^- s —b Fig. 106. — Longitudinal section of heart-muscle of a grown individual, fixed in cor- rosive sublimate and stained in hematein : a, Intercalated disc (so-called cement line); b, nucleus of heart muscle-fiber ; c, red blood-corpuscles ; d, nucleus of blood capillary. fibers are grouped into bundles or fasciculi which are surrounded by internal perimysium. Development of Heart Muscle-tissue. — Heart muscle-tissue de- velops from the mesenchyme, and shows from the beginning a syncytial structure, in that the cells are united by protoplasmic branches (von Ebner, M. Heidenhain, Godlewsky). As development proceeds, the interspaces between the cells become smaller and the protoplasmic bridges larger and more prominent, forming a distinct syncytium, through which the nuclei are scattered. In this syncytial protoplasm are developed the contractile fibrils, which may be traced uninterruptedly for long distances. These fibrils show at first a uniform structure, and later differentiate into isotropic and aniso- tropic discs, Q and j discs appearing first as in voluntary striated MUSCULAR TISSUE. 147 muscle, and later the other parts of the sarcomeres (Godlewsky). It may be stated that, according to J. B. MacCallum (and other observers), the heart-muscle develops from spindle-shaped cells lying close together in the protoplasm of which there is found a fairly regular network. As development proceeds, fibrils or fibril bundles which run parallel to the long axis of the cells make their appearance at the nodal points of this network. The muscle-cells of the so-called fibers of Purkinje lie immediately beneath the endocardium, and are remarkable in that their proto- plasm is only partially formed of transversely striated substance, and that only at their periphery. Such cells are found in great numbers in some animals (sheep), but rarely in man. Heart muscle has a rich blood supply, which will be considered more fully when the heart is discussed as an organ. For the nerve-endings in smooth and striated muscle-fibers see the chapter on Nervous Tissues. TECHNIC. Fresh, striated muscle-fibers may be isolated by teasing them in an indifferent fluid. After a short time the sarcolemma may separate as a very fine membrane. If a freshly teased muscle be placed in a cold saturated solution of ammonium carbonate, the sarcolemma will become detached in places within five minutes (Solger, 89, III). Striated muscle-fibers may be examined in an extended condi- tion by placing an extremity in such a position as to stretch certain groups of muscles. A subcutaneous injection of 0.25-0.5 c.c. of a i$> osmic acid solution is then made. The acid penetrates between the fibers and fixes them. Pieces of muscle are then cut out and washed in dis- tilled water. Teased fibers, even if not stained, will show the stria- tion plainly if mounted in glycerin. Muscles thrown into a state of tetanic contraction by electric stimulation may also be fixed in this state and later examined. Cross-sections of muscles, extended and fixed in osmic acid, also show the relation of the fibrils to the sarcoplasm (Cohnheim's fields). A remarkable quantity of sarcoplasm in proportion to the number of fibrils is seen, for instance, in the muscles which move the dorsal fin of hippocampus ; among the mammalia a similar condition is found in the pectoral muscles of the bat (Rollett, 89). In the muscles of all adult vertebrates (except the mammalia) the nuclei lie between the fibrils. In young mammalia they also have this position, but in the adult animals only the nuclei of red muscles are found between the fibrillse ; in all other muscles the nuclei are under the sarcolemma. The fibrillar structure of muscle-fibers can be seen by teasing old alcoholic preparations, or tissue treated with weak chromic acid (0.1%) or one of its salts. In alcoholic preparations of mammalian muscle, the cross- striation is clearly seen, and is intensified by staining with hematoxylin. This stain colors everything anisotropic in the muscle, but does not affect the remaining structures. Similar results may be obtained with other 148 THE TISSUES. stains, such as basic anilin dyes, but not with the same precision as with hematoxylin. A certain species of beetle (Hydrophilus'} is admirably adapted for the study of the finer details of striation. The beetle is first wiped dry and then immersed alive in 93% alcohol. On examining in dilute glycerin after from twenty-four to forty-eight hours, the substance of its muscles will show disintegration into Bowman's discs (vid. p. 141). The latter swell up in acids and are finally dissolved, as may be seen, by adding a drop of formic acid to a specimen prepared as above (Rollet, 85). In order to study the relation of muscle to tendon, small mus- cles with their tendons are put into a 35% potassium hydrate solution for a quarter of an hour, after which the specimen is placed upon a slide and teased at the line of junction of the two tissues. This will separate the muscle-fibers from their respective tendon-fibrils (Weismann). Similar results may be obtained by immersing a frog in water at a temperature of 55° C., in which the animal soon dies with muscles perfectly rigid. As soon as the water begins to cool (one-quarter hour) the frog is removed and a small piece of its muscle cut out and teased in water on a slide (Ranvier). Cardiac muscle-cells are isolated by maceration for twenty-four hours in a 20% solution of fuming nitric acid (potassium hydrate with a specific gravity of 1.3 will do the same in one-half or one hour). The margins of the cells may be brought more clearly into view by placing pieces of heart muscle for twenty-four hours in a 0.5% aqueous solution of silver nitrate and then cutting into sections. Isolated fibers of Purkinje are obtained by immersing pieces of endocardium (0.5 mm. in size) in 33% alcohol and then teasing them on a slide. The sheep's heart is especially well adapted for this purpose. Nonstriated muscle-fibers are isolated in the same way as heart muscle. In thin cross -sections (under 5 p. in thickness) of intestinal muscle, preferably of a cat, fixed in osmic acid, the intercellular bridges may be seen here and there between the fibers. D. THE NERVOUS TISSUES. • The entire nervous system, peripheral as well as central, is com- posed of cells possessing one or many processes. These cells develop early in embryonic life from certain ectodermal cells (neuro- blasts] of the neural canal, which is formed by a dorsal invagination of the ectoderm. The neuroblasts soon develop processes, — many of them in loco, others only after wandering from the neural canal. The processes of the nerve-cells are of two kinds : (i) un- branched processes having a nearly uniform diameter throughout, with lateral offshoots known as collateral branches ; these, as we shall see, generally form the central part of a nerve-fiber, and are known as neuraxes (Deiters1 processes, axis-cylinder processes, neurites, neuraxones or axones) ; and (2) processes which branch soon after leaving the cell-body and break up into many smaller THE NERVOUS TISSUES. 149 branches ; these are the dcndrites, dendrons, or protoplasmic branches. In the spinal ganglia and the homologous cranial ganglia these mor- phologic differences in the processes are not observed, the neuraxis and the dendrites of each presenting essentially the same structure. To the entire nerve -cell, cell-body and processes the term neurone (Waldeyer, 91) has been applied; ncnra (Rauber), or neu- rodendron (Kolliker, 93). The neuraxes of many neurones attain great length. Those of some of the neurones, the cell-bodies of which are situated in the lower part of the spinal cord, extend to the foot. In other regions neuraxes nearly as long are to be found, and in the majority of neu- rones the neuraxes terminate some distance from the cell-body. It is therefore manifestly impossible in the majority of cases to see a neu- rone in its entirety. Usually, only a portion of one can be studied in any one preparation. Consequently, the more detailed descrip- tion which follows will deal with the neurone in this fragmentary manner. The cell-bodies of the neurones, to which the term "nerve-cells" or "ganglion cells" is usually restricted, the den- drites and neuraxes, often forming parts of nerve-fibers, and their mode of terminating, will receive separate consideration. NERVE-CELLS, OR GANGLION CELLS ; THE CELL-BODIES OF NEURONES. The cell-bodies of neurones are usually large. The bodies of the motor neurones of the human spinal cord measure 75 to 150 //, their nuclei 45 //, and their nucleoli 1 5 /u. The smallest nerve-cells, the neurones of the granular layer of the cerebellum, are 4 to 9 fi in diameter. The protoplasm of nerve-cells shows a distinct fibrillar structure and the fibrils may be followed into the processes. (Fig. 107.) Their nuclei are also large, with very little chromatin, but as a rule are supplied with a large nucleolus. After treatment by certain special methods, the protoplasm of the ganglion cells shows granules or groups of granules which show special affinity to certain stains, consequently known as chromato- phile granules ; these are densely grouped around the nucleus, so that the cell-body shows an inner darker and an outer lighter por- tion. These chromatophile granules, also spoken of as tigroid granules or as the tigroid substance (v. Lenhossek), as a rule are not arranged in concentric layers, but lie mostly in groups, giving to the protoplasm a mottled or reticular appearance. In the cells of the an- terior horns (man, ox, rabbit) the granules join to form flakes, which are also more numerous in the region of the nucleus. In all cases the granules or flakes are continued into the dendrites of the cell. Here they change their shape into long pointed rods, with here and there nodules, which are probably the chief causes of the varicosities so often seen in dendrites (Golgi's method). The cell usually has a clear, nongranular peripheral border (not a membrane), and in the THE TISSUES. case of large cells there is a similar area around the nucleus, the inner border of which belongs to the nuclear membrane. H. Held has found that the chromatophile granules are brought out by treat- ment with alcohol and acid fixing fluids, but not in alkaline or neu- tral. They appear, according to the treatment, as fine or coarse granules. They can not be seen in fresh nerve-cells. He conse- quently regards them as artefacts — precipitations of the protoplasm due to reagents (vid. A. Fischer). At its junction with the cell the neuraxis spreads out into a cone which is entirely free from granules, and apparently fitted into a depression in the granular substance of the cell (implantation cone or axone hillock). The shape, number, and size of the tigroid granules vary with the physiologic activity of neurones. They practically disappear from the neurones in certain diseased conditions or after the administration of poisons which affect more particularly nerve-cells ; also after extreme fatigue. The cellular substance between the chromatophile granules con- sists also of very fine, highly refractive granules, which appear to be arranged in a reticulum surrounding the chromatophile granules Nucleus. Nucleolus. Fibrillar structure. Medullary sheath. Fig. 107. — Bipolar ganglion cell from the ganglion acusticum of a teleost (longitu- dinal section). The medullary sheath of the neuraxis and dendrite is continued over the ganglion cell ; X 800. (vid. Nissl, 94, and v. Lenhossek, 95), and the recent observations of Apathy and Bethe make it very probable that in the intergranular substance of the protoplasm of the nerve-cell there exist veiy fine fibrils which may be traced into the processes of the cell, and from the branches of one neurone to and into the branches of other neu- rones without interruption. It requires, however, further observation before more positive statements may be made concerning them. Besides the granules above mentioned, and which are revealed by special methods, there are found in the protoplasm of many of the larger nerve-cells pigment granules of a yellow or brown color which stain black with osmic acid. The dendrites are usually relatively thick at their origin, but gradually, as a result of repeated divisions, taper until their widely distributed arborescent endings appear as minute threads of widely different shapes. When treated by certain methods, they present uneven surfaces studded with varicosities and nodules, in contradis- tinction to the neuraxes, which are smooth and straight. Their ter- minal branches end either in points or in small terminal thickenings. The groups of terminal end-branches of a dendrite (also of a neur- axis) are known as telodendria (Rauber), or end-branches. The THE -NERVOUS TISSUES. branches of the dendrites form a dense feltwork, which, together with the cell-bodies of the neurones and with other elements to be described later, constitute the gray substance (gray matter) of the brain and spinal cord. All neurones, with possibly a few exceptions, possess only a single neuraxis. Neurones without a neuraxis have never been found in vertebrates. The neuraxis usually arises from a cone- shaped extension of the cell-body free from chromatophile granules, the implantation cone or axone hillock, more rarely from the base of one of its dendrites, or from a dendrite at some distance from the cell- body. Its most important characteristics are its smooth and regular contour and its uniform diameter. At some distance from the cell- body, usually near its termination, now and then in its course, a neuraxis may divide into two equal parts. Golgi (94) called attention to the fact that the neu- raxes of certain neurones (Pur- kinje's cells in the cerebellum, pyramidal cells of the cerebral cortex, and certain cells of the spinal cord) give off lateral pro- cesses, the collateral branches. Fig. 108. — Chromatophile granules of a ganglion cell from the Gasserian ganglion of a teleost : a, Nucleus ; b, implantation Fig. 109. — Nerve-cell from the ante- rior horn of the spinal cord of an ox, showing coarse chromatophile flakes. Two types of cell are recognized according to the disposition of their neuraxes : In the first the neuraxis is continued as a nerve- fiber ; in the second and rarer type it does not long preserve its independence, nor is it continued as a nerve-fiber, but soon breaks up into a complicated arborization, the neuropodia of Kolliker (93). The latter type of cell occurs in the cortex of the cerebrum and cerebellum and in the gray matter of the spinal cord. The cells of the two types can be simply described as having long (type I) or short, branched neuraxes (type II). The neuraxes of the cells of type I possess the collateral branches which end in small branching tufts. In its simplest form, a neurone consists of a cell-body and a neu- raxis with its telodendrion. In more complicated types one or several 152 THE TISSUES. dendrites may be present, as also collaterals from the neuraxis, and in rare cases even several neuraxes. According to the number of its processes, a ganglion cell is known as unipolar, bipolar, or multipolar. — Dendrite. Neurjixis. Neuraxis. Dendrite. Fig. HO. — Motor neurones from the anterior horn of the spinal cord of a new-born cat. Chrome-silver method. Although neurones present a great variety of morphologic dif- ferences,— large and variously shaped cell-bodies or small ones scarcely larger than the nucleus ; large and numerous dendrites or -J75- Telodendrion. Dendrite. Cell-body. Neuraxis. Fig. in. — A nerve-cell with branched dendrites (Purkinje's cell), from the cerebellar cortex of a rabbit ; chrome-silver method ; X I25- few and less conspicuous ones, — and although these various forms are widely distributed and intermingled in the different parts of the nervous system, yet in many regions there are found nerve-cells of fixed and characteristic morphologic appearance, which would THE NERVOUS TISSUES. 153 enable a determination of their source. A few of the most charac- teristic types are here figured and may receive brief consideration. In the anterior horn of the spinal cord are found large multipolar neurones (motor neurones), with numerous dendrites, which termi- nate after repeated branching in the neighborhood of the cell-body, while the neuraxis with its collateral branches proceeds from the cell-body and becomes a part of a nerve-fiber. (Fig. 1 10.) In the cerebellum are found large neurones, discovered by Pur- kinje, and known as Purkinje's cells, with flask-shaped cell-body, from the lower portion of which arises a neuraxis with collateral branches, b -- Branching of a dendrite. Neuraxis and collaterals. Fig. 112. — Pyramidal cell from the cerebral cortex of man ; chrome-silver method : a, b, c, Branches of a dendrite. from the upper portion one or two very large and typic dendrites the smaller branches of which are beset with irregular granules. (Fig. in.) In the cortex of the cerebrum occur large neurones, each with a cell-body the shape of a pyramid (pyramidal cell of the cerebral cortex), from the apex of which arises one large dendrite, and from angles at the base, or from the sides of the cell-body, several smaller dendrites. The neuraxis arises from the base directly or from one of the basal dendrites. (Fig. 112.) 154 THE TISSUES. In figure 113 is shown a neurone with relatively small cell-body and short dendrites, from the granular layer of the human cere- bellum. The function of the dendrites has given rise to considerable dis- cussion. Golgi and his school regard them as the nutrient roots of the cell, a theory which is opposed by Ramon y Cajal (93, 1 ), van Gehuchten (93, I), and Retzius (92, II). According to the latter, all the processes of the nerve-cell are analogous structures ; they pass out from a sensitive element, and probably have a correspond- ingly uniform function. In the spinal ganglia and the homologous cranial ganglia, are grouped the cell-bodies of neurones (peripheral sensory neurones, peripheral centripetal neurones) which differ in many respects from those above described. In the peripheral sensory neurones the Neuraxis. — Telodendrion. — Nucleus. Fig. 113. — Nerve-cell with dendrites ending in claw-like telodendria ; from the granular layer of the human cerebellum ; chrome-silver method ; X l Io- Fig. 114. — Ganglion cell with a pro- cess dividing at a (T-shaped process); from a spinal ganglion of the frog ; X 23°- neuraxes and dendrites have essentially the same structure, both forming part of a nerve-fiber. From a relatively large, nearly round, oval, or pear-shaped cell-body there arises a single process, which, at a variable distance from the cell-body, divides into two branches forming a right or obtuse angle with the single process (T-shaped or Y-shaped division of Ranvier, 78). Both of these branches form the central axis of a nerve-fiber ; one of the branches passing as a nerve-fiber to the spinal cord or brain, as the case may be ; the other forming a nerve-fiber which passes to the periphery. (Figs. 1 14 and 115.) The ganglion cells of the spinal ganglia and homodynamic structures of the brain are therefore apparently unipolar cells, but, as Ranvier has shown, their processes are subject to a T-shaped or Y-shaped division. The branches going to the periphery are re- THE NERVOUS TISSUES. 155 garded as dendrites, the others as neuraxes. As to the significance to be attached to the single process, the theory of v. Lenhossek Fig. 115. — Ganglion cell from the Gasserian ganglion of a rabbit ; stained in methylene- blue (intra vitam). (94, I) that it represents an elongated portion of the cell, and that therefore the origin of the dendrite and that of the neuraxis are in this case close together, is very plausible. In the embryo these ganglion cells are at first bipolar, a process arising from each end, of a spindle-shaped cell ; as de- velopment proceeds, the two pro- cesses approach each other and ultimately arise from a drawn-out portion of the cell - body, the single process. (Fig. 116.) The sympathetic ganglia are composed mainly of the cell- bodies and dendrites (also some structures to be mentioned later) of neurones of the sympathetic nervous system. In nearly all vertebrates, and with but few ex- ceptions in any one ganglion, these neurones are multipolar and Fig. 116 — Three ganglion cells from a spinal ganglion of a rabbit embryo. The cells are still bipolar. Their processes come together in later stages, and finally form the T-shaped structure seen in the adult animal ; chrome - silver method ; resemble morphologically the multipolar ganglion cells of the anterior horn of the spinal cord, though they are somewhat smaller. In the cell-body there may be ob- 156 THE TISSUES. served fine chromatophile granules and a large nucleus and nucleolus. From the cell-body there proceed a varying number of dendrites which branch and rebranch and terminate, as a rule, near the cell- body, forming plexuses in the ganglia. The neuraxis arises either directly from the cell-body from an implantation cone, or from one of the dendrites at a variable distance from the cell-body. (Fig. 1 17.) In nearly all ganglia a few unipolar or bipolar cells are to be found. In the sympathetic nervous system of amphibia the sympathetic neurones are unipolar ; the single process present is the neuraxis. A most important result of the more recent investigations on the nervous system is the theory of the independence of the neurone. Each neurone develops from a single cell (neuroblast), and func- tionates as an independent cell under physiologic and pathologic conditions. Only very rarely has any direct connection between two neighboring neurones been demonstrated, so rarely that the Fig. 117. — Neurone from inferior cervical sympathetic ganglion of a rabbit ; methylene- blue stain. scattered observations at hand do not vitiate the above statement. Recent investigations have, however, shown that, while a neurone is a distinct anatomic unit, it is always found associated with other neurones. Nowhere in the body of a vertebrate does one find a neurone completely disconnected from other neurones. This asso- ciation of one neurone with one or several other neurones is always effected by a close contiguity existing between the telodendria (end-branches) of the neuraxis of one neurone with the cell-body or dendrites of one or several other neurones. The telodendrion of the neuraxis of one neurone may form a feltwork inclosing the cell- body of one or several neurones, forming structures known as terminal baskets or end-baskets, or the end ramifications of the neuraxis of a neurone may come in very close proximity to the end-branches of the dendrites of one or several neurones. By this contiguity of the telodendria of the neuraxis of one neurone with THE NERVOUS TISSUES. 157 the cell-bodies or the dendrites of other neurones, they are, without losing their identity, linked into chains, so that a physiologic conti- nuity exists between them. In such neurone chains the dendrites are regarded as cellulipetal, transmitting the stimulus to the cell ; the neuraxes as cellulifugal, transmitting the impulse imparted by the cell to the motor nerve-endings or central organs (Kolliker, 93). The entire nervous system may therefore be said to be made up of such neurone chains, the complexity of which varies greatly according to the number of neurones which enter into their construction. This subject will be considered more fully in a chapter on the nervous system. .„. Fibrils of axial cord. •»• Neurilemma. — Segment of Lantermann. THE NERVE-FIBERS. The neuraxes of the cells of type I, and the dendrites of the peripheral sensory neurones (spinal ganglia and homologous cranial ganglia), form the chief elements in all the nerve-fibers. In the nerve-fibers they pos- sess a distinctly fibrillar structure. The fibrils composing them, the axis-fibrils, are imbedded in a semifluid substance, the neuroplasm (Kupffer, 83, II) the whole being surrounded by a very delicate membrane, the axolemma. In the nerve- fibers, the axis-fibrils and the neuroplasm form axial cords which are surrounded by a special membrane or membranes, the presence or absence of which serves as a basis for a classification of nerve- fibers. Two kinds are distinguished, medullated and nonmedullated nerve - fibers. In medullated nerve-fibers, the axial cords (neuraxes of cells of type I, and dendrites of spinal ganglion cells) are sur- rounded by a highly refractive substance very similar to fat, which is blackened in osmic acid, the so-called medullary or my din sheath. In a fresh condition this sheath is homogeneous, but soon changes and presents segments separated from each other by clear fissures. These seg- ments vary in size and are known as " Schmidt- Lantermann-Kuhnt's segments." On boiling in ether or alcohol the entire medullary sheath of a nerve-fiber does not dissolve, but a portion is left in the shape of a fine network which is not affected by exposure to the action of trypsin. From the latter circumstance it has been thought that this network consists of a substance very similar to horn, and is therefore known as neurokeratin (horn-sheath, Ewald and Kiihne). On burning isolated neurokeratin, an odor exactly like that of burn- Fig. 118. — Longitudinal section through a nerve-fiber from the sciatic nerve of a frog; X830. 158 THE TISSUES. ing horn is given off. It is thought that the meshes of this neuro- keratin network contain the highly refractive substance similar to fat, composing the greater portion of the medullary sheath. The medullary sheath is interrupted at intervals of from 80 to 900 //, the constrictions thus formed being known as the nodes of Ranvier. The smaller the fiber, the less the distance between the nodes. In a fiber with a diameter of 2 p. the internodal segments are usually about 90 p in length. In peripheral nerves the medullary sheath is in its turn sur- rounded by a clear, structureless membrane, the neurilemma or sheath of Schwann. Nerve-fibers contain here and there relatively long, oval nuclei (neurilemma-nuclei) which are surrounded by a small quantity of protoplasm, and are situated in small excavations between the neurilemma and the medullary sheath. In the higher vertebrates a single nucleus is found midway between each two Connective _„ tissue. Fibrils of axial cord. Medullary sheath. ?— Fibrils. Fig. 119. — Transverse section through the sciatic nerve of a frog ; X 820. At a and b is a diagonal fissure between two Lantermann's segments ; as a result, the medul- lary sheath here appears double. (Compare Fig. 118.) nodes ; in the lower vertebrates (fishes) several scattered nuclei (5-16) may be found in each internodal segment. At the nodes, where the medullary sheath is interrupted, the neurilemma is thickened and contracted down 'to the axial cord (contraction-ring). Just beneath the contraction-ring, Ranvier found that the axis- cylinder presents a slight, biconic swelling (retirement bicbnique). Thus the sheath of Schwann represents a continuous tube through- out the length of the fiber in contrast to the medullary sheath. In the nerve-fibers of the spinal cord and brain there is no neurilemma, although the medullary sheath is present. In the fresh nerve-fiber the axial cord fills the space (axial space) within the medullary sheath, and appears transparent. After treatment with many fixing fluids the neuroplasm coagulates and shrinks, no longer filling the entire axial space, but appears in the latter as a wavy cord composed of an apparently homogeneous THE NERVOUS TISSUES. 159 mass, the fibrillae of which are no longer recognizable. Such pic- tures, which formerly were supposed to represent the normal condi- tion of the nerve-fibers, gave rise to the conception of an axis-cyl- inder (vid. Technic). That which is known as an axis -cylinder is therefore, in reality, the changed contents of the axial space. It may be stated, however, that the term axis-cylinder is still much used, since the methods commonly employed in the investigation of the nervous system do not preserve the axial cord in its integrity, but nearly always result in the for- mation of an axis-cylin- der. Consequently, al- though we shall make use of the term, its limit- ations are to be kept in mind. Medullated nerve - fibers vary greatly in di- Ranvier's / node. _ Axial cord, — Medullary sheath. Nucleus. Ranvier's node. — Nucleus. Fig. I2O. — Medullated nerve-fibers from a rabbit, varying in thickness and showing internodal segments of different lengths. In the fiber at the left the neuri- lemma has become slightly separated from the under- lying structures in the region of the nucleus ; X I4°- Fig. 121. — Remak's fibers (nonmedullated fibers) from the pneumogastric nerve of a rabbit ; ameter, but whether this points to a corresponding variation in function has not been fully decided. Fine fibers possess a diameter of 2— 4//, those of medium size 4—9 //, and large fibers 9—20 fj. (Kolliker, 93). A division of medullated fibers during their course through a nerve is relatively rare. The greater number of fibers pass unbranched from their central origin to the periphery, and only when in the neighborhood of their terminal arborization do they begin to divide. A point of division is always marked by a node of Ranvier. l6o THE TISSUES. The segmental structure of nerve-fibers would seem to give the impression that they are formed by a number of cells fused end to end. After what has been said with regard to ganglion cells and their processes, this can be the case only so far as the nerve-sheaths are concerned. According to this theory, the formative cells of the latter gather in chains along the neuraxes or dendrites, forming a mantle around them, and in the adult nerve-fibers taking the shape of the segments or internodes just described (His, 87 ; Boveri, 85). The points at which the sheath-cells are joined would then corre- spond to the nodes of Ranvier. Other investigators have concluded that the whole nerve-fiber is developed from a terminal apposition of ectodermal cells. In this case not only the sheaths of the fibers but also the corresponding portions of the nerve processes are formed by them (Kupffer, 90). In both theories the neurilemma corresponds to the cell-membrane ; in the former the neurilemma nucleus corresponds to that of the sheath-forming cell, in the latter to that of the formative cell of the whole nerve segment. It should be noticed that, according to the second theory, a fiber segment is the product of a single cell, while according to the first it is evolved from at least two cells (ganglion cell (process) and sheath-forming cell). The former theory is now very generally accepted. The nonmedullated nerve-fibers, Remak 's fibers, possess no medullary sheath ; the axial cord shows nuclei which can be re- garded as belonging to a thin neurilemma. The majority of the neuraxes of the neurones of the sympathetic nervous system are of this structure, although small medullated nerve-fibers (the neuraxes of sympathetic neurones) are found in certain regions. All nerve-fibers, medullated as well as nonmedullated, in the central and peripheral nervous systems lose the sheaths here de- scribed before terminating ; the axis-cylinders (axial cords) ending without special covering (naked axis-cylinders). These terminal branches are, in fixed and stained preparations, beset with small thickenings — varicosities — which vary greatly in size and shape. Nerve-fibers presenting such appearances are spoken of as varicosed fibers. The varicose enlargements may be regarded as small masses of neuroplasm ; the fine uniting threads, as representing the axial fibrils. In the peripheral nervous system the nerve-fibers are grouped to form nerve-trunks. The nerve-fibers, as has been stated and as will be seen from the diagram (Fig. 122) on the next page, are the neuraxes of neurones, the cell-bodies of which are situated in the spinal cord or brain and in the sympathetic ganglia, and the den- drites of peripheral sensory neurones, the cell-bodies of which are found in the spinal and homologous cranial ganglia. In the nerve-trunks the nerve-fibers are gathered into bundles termed funiculi. The nerve-fibers constituting such a bundle are separated by a small amount of fibro-elastic tissue, containing here and there connective-tissue cells, the endoneurium. This is continu- THE NERVOUS TISSUES. 161 ous with a dense, lamellated fibrous sheath surrounding each funicu- lus, the perineurium. Between the lamellae of this sheath are lymph- spaces, communicating with the lymph-clefts found between the Neuraxis of peripheral sensory neurone. Dendrite of per- ipheral s e n - sory neurone. Nerve-trunk. Spinal ganglion. -£- Anterior horn of gray matter of spinal cord. Neuraxis of peripheral motor neurone. Sympathetic ganglion. Neuraxis of sympathetic neurone. Fig. 122. — Diagram to show the composition of a peripheral nerve-trunk. ?"- Axis-cylinder. 8 Xeurilemma. #*-—-'•-—- Endoneurium Perineurium. Fig. 123. — Part of a cross-section through a peripheral nerve treated with alcohol. The small circles represent the cross-sections of medullated nerve-fibers ; the axis-cylin- ders show as points in their centers. The nerve is separated by connective tissue into large and small bundles — funiculi ; X 75- II 1 62 THE TISSUES. nerve-fibers of the funiculi ; consequently, the lamellae are covered by a layer of endothelial cells. In the larger funiculi, septa of fibrous connective tissue pass from the perineurial sheath into the funiculi, dividing them into compartments varying in shape and size ; these are spoken of as compound funiculi. The funiculi of a nerve- trunk are bound together by an investing sheath of loose fibro-elastic tissue, continuous with the perineurial sheaths, which penetrates between the funiculi, and which contains fat-cells, blood-vessels, and lymph-vessels ; the latter are in communication with the lymph- spaces of the perineurial sheaths. When a nerve-trunk divides, the connective-tissue sheaths above mentioned are continued on to the branches, and this even to the smallest offshoots. Thus, single fibers even possess a connective- tissue sheath, — Henle's sheath, — which consists of a few connective- tissue fibers and of flattened cells. PERIPHERAL NERVE TERMINATIONS. According to the character of the peripheral organs in which the telodendria of nerve-fibers (neuraxes of type I cells and dendrites of spinal ganglion cells) occur, the nerve-fibers are known as motor and sensory nerve-fibers, the terminations as motor and sensory nerve-endings. Motor Nerve-endings (the Telodendria of Nerve-fibers Ending in Muscle Tissue). — The motor nerve -endings in striated, voluntary muscle tissue will first be considered. The motor nerve-endings in voluntary muscle tissue are the endings of neurones (peripheral motor neurones), the cell-bodies of which are situated in the ventral horns of the spinal cord and in the medulla. The neuraxes of these cells leave the cerebrospinal axis as medullated nerve-fibers (motor fibers) which, after branching, end in the muscle-fibers in the so-called motor endings. In figure 124 is represented, by way of diagram, a complete peripheral motor neurone. Each motor nerve - fiber branches repeatedly before terminating, although this branching does not often take place until near the termination of the nerve- fiber. Kolliker estimates that in the sternoradialis of the frog, each motor fiber innervates about twenty muscle-fibers ; but whether this number may be regarded as the average number of muscle-fibers receiving their motor nerve-supply from one motor neurone can not be stated with any degree of certainty at the present time. Each motor ending represents the termination of one of the ter- minal medullated branches of a motor nerve-fiber. The neuraxis of this fiber passes under the sarcolemma and terminates in a teloden- drion (end-brush) in an accumulation of sarcoplasm, in which are found numerous muscle nuclei, forming a more or less distinct ele- vation on the side of the muscle-fiber, Doyere's elevation. The medullary sheath accompanies the nerve-fiber until it passes under the sarcolemma, when it stops abruptly. The neurilemma of the MOTOR NERVE-ENDINGS. 163 nerve-fiber becomes continuous with the sarcolemma of the muscle- fiber at the place where the neuraxis passes under the sarcolemma. Henle's sheath continues over the motor ending as a thin sheath, containing here and there flattened nuclei, the telolemma nuclei. With the majority of the reagents used to bring to view the motor endings, notably chlorid of gold, the sarcoplasm, in which Dendrite. Neuraxis. - — Medullary sheath. Nucleus of neurilemma. Internodal segment. Motor ending. Collateral branch. Neurilemma. Node of Ranvier. Axis-cylinder of medullated nerve-fiber. Muscle-fibers. Fig. 124. — Diagram of peripheral motor neurone. the telodendrion of the nerve-fiber is found, has a granular appear- ance, and is consequently differentiated from the remaining sarco- plasm of the muscle-fiber. To this the term granular sole plate has been applied, the nuclei contained therein being known as sole nuclei, the whole ending as the motor end-plate. If the above interprets,- 164 THE TISSUES. tion of the structure of the motor nerve-ending is correct, there would seem to be no reason why the sarcoplasm in which the telo- dendria occur should be considered other than the sarcoplasm of the muscle-fiber, the nuclei as muscle-nuclei ; the terms motor end- plate, granular sole plate, and sole nuclei would therefore seem un- necessary and misleading. It may be stated in this connection that Bardeen has recently shown that in teased muscle-tissue subjected to trypsin digestion the muscle substance may be removed from the fiber leaving the sarcolemma and on its inner surface a por- tion of the nerve -ending, with the neurolemma continuous with the sarcolemma. He has also shown that the motor ending is in part differentiated in connection with developing muscle-fibers before a sarcolemma can be shown on such fibers. In figures 126 to 130 are shown motor nerve-endings from several vertebrates as seen when stained with gold chlorid. The mass of sarcoplasm in which the neu raxes terminate as above described is about 40 to 60 p long, 40 /abroad, and 6 to 10 /* thick ; these dimensions vary greatly, however ; they may be greater or less than the averages here given. In amphibia the motor nerve-endings are not so localized as in the majority of vertebrates, as above described, but are spread over a relatively greater surface of the muscle-fiber, and there is no distinct accumulation of the sarcoplasm, and the muscle-nuclei are Fig. 125. — Motor nerve-ending in voluntary muscle of rabbit, stained in methylene- blue (intra vitam) (Huber, DeWitt, "Jour. Comp. Neurol.," vol. vn) : A, Surface view ; B, longitudinal section through motor ending ; C, cross-section : a, a, a, neuraxes of nerve-fibers ; s,s,s, sarcolemma ; «/,«/, neurilemma ; d, Doyere's elevation; mn, muscle nuclei ; tn, telolemma nucleus. relatively less numerous. The telodendrion of the nerve-fiber is, however, under the sarcolemma, between it and the contractile sub- stance of the muscle-fiber. (Fig. 131.) Usually only one motor ending is found on each striated muscle- fiber. This may be situated near the center of the muscle-fiber or at a variable distance from the center, nearer one or the other of its extremities. Now and then two nerve-endings are found on one muscle-fiber, in which case the nerve-endings are found in close proximity. MOTOR NERVE-ENDINGS. i65 — |=-r=^=-. __ Nerve. Fig. 126. Fig. 127. Figs. 128 and 129. So-called granular sole. End-brush. Sarco- lemma. Fig. 130. Figs. 126-130. — Motor endings in striated voluntary muscles. Fig. 1 26, from Pseudopus Pallasii; X l6°- Fig. 127, from Lncerta viridis ; X 160. Figs. 128 and 129, from a guinea-pig; X 7°°- Fig. 130, from a hedge-hog; X 1200. As a consequence of the treatment the arborescence is shrunken and interrupted in its continuity. In Figs. 126 and 127 the end plate is considerably larger than in 128 and 129. In Fig. 126 it is in connection with two nerve-branches. Fig. 130 shows a section through an end-plate. The latter is bounded externally by a sharply denned line, which can be traced along the surface of the muscle-fiber. This is to be regarded as the sarcolemma. THE TISSUES. Heart muscle and nominated muscle receive their motor nerve- supply from neurones of the sympathetic nervous system. The cell-bodies of these neurones are situated in sympathetic ganglia ; the neuraxes, the majority of which form nonmedullated nerve- fibers, branch repeatedly, forming primary and secondary plexuses which surround the larger or smaller bundles of heart muscle-fibers or involuntary muscle-cells. From these plexuses, naked, vari- cosed axis-cylinders, or small bundles of such, penetrate between the heart muscle-fibers or involuntary muscle-cells, also forming Fig. 131. — Motor nerve-ending in striated voluntary muscle of a frog ; methylene- blue stain (intra vitani) (Huber, DeWitt) : A, Surface view ; £, cross- section ; s, s, sarcolemma ; »/, neurilemma. Fig. 132. — Motor nerve-ending on heart muscle-cells of cat ; methylene-blue stain (Huber, De Witt). Fig. 133. — Motor nerve-ending on involuntary nonstriated muscle-cell from intestine of cat ; methylene-blue stain (Huber, De Witt). plexuses. The fine fibers of this terminal plexus give off from place to place small, lateral twigs, which end on the muscle-fiber and muscle-cells. In heart muscle these lateral twigs may end in one or two small granules, or in a small cluster of such granules (Fig. 132); in involuntary, nonstriated muscle the ending is very simple, the small lateral twigs terminating in one or two small granules. (Fig. 133.) Sensory Nerve-endings. — The sensory nerve-endings are, in their essentials, the peripheral telodendria of dendrites of peripheral SENSORY NERVE-ENDINGS. 167 sensory neurones. The cell-bodies of such neurones, as has been stated, are found in the spinal and homologous cranial ganglia. Of the two branches arising from the single process possessed by each peripheral sensory neurone, the one going to the periphery is regarded as the dendrite and forms the axis-cylinder of a medullated nerve-fiber, such nerve-fibers constituting the sensory nerves of the Nucleus and nucleolus. Cell-body. Process of cell. Neuraxis, endb in spinal cord or brain. T-shaped division of Ranvier. Dendrite, a sensory nerve- fiber in nerve-trunk. Telodendrion of terminal branch of dendrite. Fig. 134. — Diagram of a peripheral sensory neurone. peripheral nerve-trunks. A peripheral sensory neurone may there- fore be diagramed as in figure 134. The statement was made above that the essential portion of a sensory nerve-ending is a telo- dendrion (end-brush) or several telodendria of the dendrite of a peripheral sensory neurone. The character of a sensory nerve- i68 THE TISSUES. ending depends, therefore, on the complexity of this end-brush and on its relation to the other tissue elements which take part in the formation of the sensory nerve-endings. Bearing this in mind, the Fig- 135- — Termination of sensory nerve-fibers in the mucosa and epithelium of the ure- thra of cat; methylene-blue preparation (Huber, "Jour. Comp. Neurol.," vol. x). following classification of such nerve-endings can be made : i. Free Sensory Nerve-endings. — In these the telodendrion is not SENSORY NERVE-ENDINGS. 169 inclosed in an investing capsule which forms a structural part of the ending. 2. Encapsulated Endings. — In which the telodendrion or several telodendria are surrounded by an investing capsule which separates them more or less completely from the surrounding tissue. 1 . Free sensory nerve=endings are found in all epithelial tis- sues and in fibrous connective tissue of certain regions. A sensory nerve-fiber terminating in such an ending usually proceeds without branching to near its place of termination, where, while yet a medullated fiber, it branches and rebranches a number of times, always at the nodes of Ranvier, the resultant branches diverging at various angles. If the free sensory endings are in epithelial tissue, these larger medullated branches are situated in tne connective- tissue mucosa under the epithelium. From these larger medullated branches, are given off smaller ones, also medullated, which may divide further, and which pass up toward the epithelium, and near its under surface divide into nonmedullated branches. Nonmedullated branches are also given off from the medullated ones as they approach the epithelium, leaving the parent fibers at the nodes of Ranvier. Many of the nonmedullated branches thus formed, after coursing a variable distance under the epithelium, enter it and break up into numerous very small branches, which, after repeated divi- sion, terminate between the epithelial cells in small nodules or discs of variable size and configuration. The small branches result- ing from a division of one of the larger nonmedullated Branches constitute one of the terminal telodendria or end-branches of the dendrites of peripheral sensory neurones terminating in free sensory nerve-endings. In fibrous connective tissue the same genera] arrangement of the branches prevails. In figure 135 is shown the peripheral distribution of the dendrite of a peripheral sensory neurone terminating in a free sensory nerve-ending. 2. Encapsulated Sensory Nerve-endings. — These nerve-end- ings may be divided into two quite distinct groups, — such as have a relatively thin fibrous-tissue capsule, containing mainly telodendria of the nerve or nerves terminating therein, and such as have a distinctly lamel- lated, fibrous tissue capsule, usually investing, besides the nerve-termi- nation, other tissue elements. To the former group belong three types of sensory nerve - endings, which, owing to their similarity of struc- ture, may be described together. ~, ,, , , „ r T? Fig. 136. — End-bulb of Krause These are the end-bulbs of Krause, from conjunctiva of man ; methylene- Meissner's tactile corpuscles, and blue stain (Dogiel, "Arch. f. mik. the genital corpuscles. They have Anat">" voh XXXVI1)' all been investigated recently by I/O THE TISSUES. Dogiel, and the account here given follows closely his descrip- tion. End-bulbs of Krause. — Under this designation there are described a variety of endings which vary slightly in size and shape. They are found in the conjunctiva and edge of the cornea, in the lips and lining of the oral cavity, in the glans penis and clitoris, and prob- ably also in other parts of the dermis. In form they are round, oval, or pear-shaped. Their size varies from 0.02 to 0.03 mm. long and from 0.015 to 0.025 mm. broad for the smaller ones, and from 0.045 to o. 10 mm. long and from 0.02 to 0.08 mm. broad for the larger ones. They have a relatively thin capsule in which nuclei are quite numerous. One, two, or three medul- lated nerves go to each end-bulb. These may lose their medul- lary sheath at the capsule or at a variable distance from it. The naked axis-cylinders, soon after entering the capsule, divide into two, three, or four branches, which form several circular or spiral turns in the same or in opposite directions. These fibers then divide into varicose branches, which undergo further division, the resulting branches interlacing to form a bundle of variously tangled fibers which may be loosely or tightly woven. Between the nerve-fibers and their branches, within the capsule, there is found a semifluid sub- stance, which is granular in fixed preparations. Meissner 's Corpuscles. — These corpuscles are found in man in the subepidermal connective tissue of the hand and foot and outer surface of the forearm, in the nip- ple, border of the eyelids, lips, glans penis and clitoris. They are most numerous in the palmar sur- face of the distal phalanx of the fingers. They are oval in shape, sometimes somewhat irregular, and vary in size, being from 45 fj. to 50 fj. broad and from no // to 1 80 fj. long. They possess a thin connective-tissue capsule, in which are found round or oval nuclei, some t of which have an oblique position to the axis of the corpus- cle. One medullated nerve ends in the smaller corpuscles, two or three or even more in the larger ones. After piercing the capsule, the medullated nerves lose their medullary sheaths, the naked axis-cylinders making a variable Fig. 137. — Meissner"s tactile corpus- cle ; methylene-blue stain (Dogiel, "In- ternal. Monatsschr. f. Anat. u. Fhys.," vol. IX). SENSORY NERVE-ENDINGS. 17! number of circular or spiral turns, some of which are parallel, others crossing at various angles. These larger branches are all beset with large, spindle-shaped, round, or pear-shaped varicosities. The larger branches, after making the windings mentioned, break up into many varicose branches, which interlace and form a most com- plex network. One usually finds one or several larger naked axis- cylinders, which pass up through the axis of the spiral of fibers thus formed ; these give off branches which contribute to the spiral formation. Genital Corpuscles. — These corpuscles are found in the deeper part of the mucosa of the glans penis and the prepuce of the male and the clitoris and neighboring structures of the female. Their shape varies ; they may be round, oval, egg- or pear- Fig. 138. — Genital corpuscle from the glans penis of man ; methylene-blue stain corpuscle from the glans penis of man ; (Dogiel, "Arch. f. mik. Anat.," vol. XLl). shaped, or even slightly lobulated. Their size varies from 0.04 to o. 10 mm. in breadth and from 0.06 to 0.40 mm. in length. They are surrounded by a relatively thick fibrous capsule, consisting of from three to eight quite distinct lamellae, between which irregu- lar flattened cells with round or oval nuclei are found. Within this capsule, there is found a core, which seems to consist of a semi- fluid substance, slightly granular in fixed preparations, the nature of which is not fully known. The number of sensory nerves going to each corpuscle varies from one to two for the smaller ones, and from eight to ten for the larger corpuscles. The medullated nerves, after entering the corpuscle, divide dichotomously, the resultant branches assuming a circular or spiral course, and interlacing in various ways, within the capsule. After a few turns, the medullated THE TISSUES. branches lose their medullary sheaths and undergo further di- vision, often dividing repeatedly. The nonmedullated nerves re- sulting from these divisions, the majority of which are varicose, form a most complicated network, the whole nerve network pre- senting a structure which resembles a tangle of fine threads. In the meshes of this network is found the semifluid substance of the core. Now and then some of the larger fibers of the network leave the corpuscle and terminate in neighboring cor- puscles, or pass to the epithelium, where they end between the cells. These three sensory nerve-endings — end-bulbs of Krause, Meissner's tactile corpuscles, genital corpuscles — are, as Dogiel has stated, very similar in structure. Each has a thin connective -tissue capsule, surrounding a core, consisting of a semifluid substance, concerning which our knowledge is as yet imperfect. One or sev- eral medullated nerves go to each corpuscle, which, after losing their medullary sheaths, divide and subdivide into numerous fine varicose branches, which are variously interwoven, forming a more or less dense plexus of interlacing and, according to Dogiel, anas- tomosing fibers. The chief differences are those of form and size, and of position with reference to the epithelium. Of the three forms of endings, the genital corpuscle is the largest, and occupies the deep- est position in the subepithelial connective tissue ; Meissner's cor- puscle is intermediate in size, and is found immediately under the epithelium ; while the end-bulbs of Krause are the smallest of these Fig. 139. — Cylindric end-bulb of Krause from intermuscular fibrous tissue septum of cat; methylene-blue stain. three forms of sensory endings and may be found in the papillae or in the deeper connective tissue. A somewhat smaller nerve-ending of long, oval, or cylindric form, known as the cylindric end-bulb of Krause, is found in various parts of the skin and oral mucous membrane, in striated muscle and in tendinous tissue. These corpuscles consist of a thin nucle- ated capsule, investing a semifluid core. The nerve-fiber, after losing its medullary sheath and fibrous sheath (the latter becomes continuous with the capsule), passes through the core, generally without branching, as a naked axis-cylinder, terminating at its end, usually in a small bulb. (Fig. 139.) The majority of the sensory nerve-endings with well-developed SENSORY NERVE-ENDINGS. 173 lamellated capsules are relatively large structures. We shall con- sider especially the Vater-Pacinian corpuscles, the neuromuscular end-organs, and the neurotendinous end-organs. Vater-Pacinian Corpuscles. — These corpuscles are of oval shape and vary much in size, the largest being about o. 10 of an inch long and 0.04 of an inch broad. The greater portion of the corpuscle is made up of a series of concentric lamellae, varying in number from twenty to sixty. These lamellae are made up of Fig. 140. — Vater-Pacinian corpuscle from the mesentery of a cat; X 45- The figure shows a general view of the corpuscle, a, Axis-cylinder in the core ; ik, core ; tnn, medullated nerve-fibers entering the core (" Atlas and Epitome of Human His- tology," Sobotta). white fibrous tissue fibers, rather loosely woven, between which is found a small amount of lymph, containing usually a few leucocytes. The lamellae are covered on both surfaces by a layer of endothelial cells (Schwalbe). Between two consecutive lamellae there is found an interlamellar space, also containing lymph. The axis of the cor- puscle is occupied by a core, consisting of a semifluid, granular substance, in the periphery of which oval nuclei are said to be found. Usually one large medullated nerve-fiber goes to each cor- puscle. The fibrous tissue sheath of this nerve-fiber becomes con- tinuous with the outer lamellae of the capsule. The medullary sheath accompanies the axis-cylinder through the concentric lamel- lae until the core is reached, where it disappears. The naked axis- cylinder usually passes through the core to its distal end, where it divides into three, four, or five branches which terminate in large, irregular end-discs. The axis-cylinder may, however, divide soon after it enters the core into two or three or even four branches, these passing to the distal end of the core before terminating in the end-discs above mentioned. Both Retzius and Sala state that the naked axis-cylinders, after entering the core, give off numerous short side branches, terminating in small knobs, which remind these ob- servers of the fine side branches or thorns seen on the dendrites of Purkinje's cells and of the pyramidal cells of the cortex, when stained 174 THE TISSUES. after the Golgi method. In company with the large nerve-fibers here mentioned, Sala has described other nerve-fibers, quite independent of them and much finer, which after entering the corpuscle divide repeatedly, the resulting fibers forming a plexus around the central fiber. A small arteriole enters the corpuscle with the nerve-fiber, dividing into capillary branches found between the lamellae of the capsule. The Vater-Pacinian corpuscles have a wide distribution. They are numerous in the deeper parts of the dermis of the hand and foot, and also near the joints, especially on the flexor side. They have been found in the periosteum of certain bones and in tendons and intermuscular septa, and even in muscles. They are further found in the epineurial sheaths of certain nerve-trunks and near Fig. 141. — Pacinian corpuscles from mesorectum of kitten : A, Showing the fine I/ranches on central nerve-fiber ; B, the network of fine nerve-fibers about the central fiber; methylene-blue preparation (Sala, "Anat. Anzeiger," vol xvi). large vessels. They are numerous in the peritoneum and mesentery, pleura and pericardium. In the mesentery of the cat, where these nerve-endings are large and numerous, they are readily seen with the unaided eye as small, pearly bodies. In the bill and tongue of water birds, especially of the duck, are found nerve-endings, known as the corpuscles of Herbst, which re- semble the Vater-Pacinian corpuscles ; they differ from the latter in having cubic cells in the core. (Fig. 142.) Neuromuscular Nerve End-organs. — These nerve end-organs consist of a small bundle of muscle-fibers, surrounded by an invest- SENSORY NERVE-ENDINGS. 175 ing capsule, within which one or several sensory nerves terminate. They are spindle-shaped structures varying in length from 0.75 to 4 mm., and in breadth, where widest, from 80 to 200 ju (Sherrington, 94). In them there is recognized a proximal polar region, an equatorial region, and a distal polar region. The muscle-fibers of this nerve-ending, known as the intrafusal fiber st which may vary in number from 3 or 4 to 20 or even more, are much smaller than the ordinary voluntary muscle-fibers and differ from them structur- ally, and result from a division of one or several muscle-fibers of the red variety. In the proximal polar region the intrafusal fibers present an appearance which is similar to that of voluntary muscle- fibers of the red variety ; in the equatorial region they possess rela- — Nucleus of lamellae. End-cell of core. Lamellae. Axis-cylinder in core. Cubic cells of core. Termination of medul- lary sheath. Axis-cylinder of nerve-fiber. Medullary sheath of nerve-fiber. Neurilemma and sheath of Henle. Fig. 142. — Corpuscle of Herbst from bill of duck; X °°o. tively few muscle-fibrils and are rich in sarcoplasm and the muscle- nuclei are numerous ; the striation is here indistinct. In the distal polar region the intrafusal fibers are again more distinctly striated and, a short distance beyond the end-organ, become greatly reduced in size, and terminate as very small fibers, still showing, however, a cross-striation. In figure 143 is shown a single intrafusal muscle- fiber. Owing to the length of such a fiber it was necessary to rep- resent it in several segments. The intrafusal muscle-fibers are surrounded by a capsule con- sisting of from four to eight concentric layers of white fibrous tissue. At the proximal end this capsule is continuous with the connective 176 THE TISSUES. tissue found between the muscle-fibers — endo- and perimysium. It attains its greatest diameter in the equatorial region of the nerve end-organ, and becomes narrower again at its distal end, where it may end in tendon or become continuous with the connective tissue Fig. 143. — Intrafusal muscle-fiber from neuromuscular nerve end-organ of rabbit : A, From proximal polar region ; B} equatorial region ; C, distal polar region. of the muscle. Immediately surrounding the intrafusal fibers is found another connective -tissue sheath known as the axial sheath, and between this and the capsule there is found a lymph-space bridged over by trabeculse of fibrous tissue, to which the name periaxial lymph-space has been given. (Fig. 144.) By degenerating the motor nerves going to a muscle, Sherrington lr^->'"v'Sv "--"••' life^^ft' '- .-• Fig. 144. — Cross-section of a neuromuscular nerve end-organ from interosseous (foot) muscle of man ; fixed in formalin and stained in hematoxylin and eosin. determined that the nerve-fibers ending in the neuromuscular nerve end-organs were sensory in character. The manner of termination in these end-organs of the nerve-fibers ending therein has been studied by Kerschner, Kolliker, Ruffini, Huber and DeWitt, Dogiel, and SENSORY NERVE-ENDINGS. 177 others. One or several (three or four) large medullated nerves, sur- rounded by a sheath of Henle, terminate in each neuromuscular end- Fig. 145. — Neuromuscular nerve end-organ from the intrinsic plantar muscles of dog ; from teased preparation of tissue stained in methylene-blue. The figure shows the intrafusal muscle-fibers, the nerve-fibers and their terminations ; the capsule and the sheath of Henle are not shown (Huber and DeWitt, "Jour. Comp. Neurol.," vol. vn). organ. As these nerves enter the capsule, the sheath of Henle blends with the capsule. The medullated nerve-fibers now and THE TISSUES. then divide before reaching the nerve end-organs, and divide several times as they pass through the capsule, periaxial space, and axial sheath. Within the axial sheath, the medullary sheath is lost, and the naked axis-cylinders terminate in one or several ribbon - like branches which are wound circularly or spirally about the intrafusal fibers (annulospiral ending) or they may terminate in a number of larger branches which again divide, these ending in irregular, round, oval, or pear-shaped discs {flower-like endings), which are also on the intra- fusal fibers. These flower-like endings are usually at the ends of the annulo- spiral fibers. In the smaller end- organs only one area of nerve-termi- nation has been observed ; in the larger, two, three, or even four such areas may be found. Neuromuscular nerve end-organs are found in nearly all skeletal muscles (not in the extrinsic eye muscles nor in the intrinsic muscles of the tongue), but they are especially numerous in the small muscles of the hand and foot. They are found in amphibia, reptilia, birds, and mammalia, presenting the same general structure, although the ultimate termination of the nerve-fibers varies somewhat in the different classes of vertebrates. Neurotendinous Nerve End -organ (Golgi Tendon Spindle). — In 1880 Golgi drew attention to a new nerve end-organ found in tendon, describing quite fully its general structure and less fully the nerve termination found therein. These, nerve end-organs are spindle-shaped structures, which in man vary in length from 1.28 mm. to 1.42 mm., and in breadth from 0.17 mm. to 0.25 mm. (Kolliker). Ciaccio mentions a neurotendinous nerve end- organ found in a woman, which was 2 Fig. 146. — Neurotendinous or 3 mm. long. A capsule consisting of nerve end-organ from rabbit; teased from 2 to 6 fibrous tissue lamella^, and preparation of tissue stained in broadest at the equatorial part of the methylene-blue (Huber and DeWitt, "Jour. Comp. Neural.," vol. x). end-organ, surrounds a number of in- SENSORY NERVE-ENDINGS. 179 trafusal tendon fasciculi. The capsule is continuous at the prox- imal and distal ends of the end-organ with the internal periten- dineum of the tendon in which it is found. The number of the intrafusal tendon fasciculi varies from eight to fifteen or even more. They are smaller than the ordinary tendon fasciculi, from which they originate by division, and structurally resemble embryonic tendon, in that they stain more deeply and present many more nuclei than fully developed tendon. The intrafusal tendon fasciculi are surrounded by an axial sheath of fibrous tissue, between which and the capsule there is found a periaxial lymph-space. r J Fig. 147. — Cross-section of neurotendinous nerve end-organ of rabbit ; from tissue stained in methylene-blue : m, Muscle-fibers ; t, tendon ; t, capsule of neurotendinous end-organ ; m n, medullated nerve-fiber (Huber and DeWitt, " Jour, of Comp. Neurol.," vol. X). The termination of the nerve-fibers ending in these end-organs has been studied by Golgi, Cattaneo, Kerschner, Kolliker, Pansini, Ciaccio, Huber and DeWitt. One, two, or three large medullated nerve-fibers, surrounded by a sheath of Henle, end in each end- organ ; as they pass through the capsule, the sheath of Henle blends with the capsule. The medullated nerve-fibers before enter- ing the capsule usually branch several times, branching further within the capsule and axial sheath. Before the resultant branches terminate on the intrafusal tendon fasciculi, the medullary sheath is ISO THE TISSUES. lost, the naked axis-cylinder further dividing into two, three, or four branches, each of which runs along on the intrafusal fasciculi, giving off numerous short, irregular side branches, which partly enclasp the tendon fasciculi and end in irregular end-discs. Some of the ter- minal branches pass between the smaller fibrous tissue bundles of the fasciculi, ending between them. In these end-organs, the larger nerve-branches are found near the center of the bundle of intrafusal tendon fasciculi, the terminal branches and the end-discs nearer their periphery. The neuroten- dinous nerve end-organs are widely distributed, being found in all tendons although not equally numerous in all. Like the neuromus- cular nerve end-organs, they are especially numerous in the small tendons of the hand and foot. Sensory nerve end-organs, which resemble in structure the neurotendinous end-organs here described, though somewhat smaller than these, have been found in the tendons of the extrinsic eye-muscles. In this brief account of the mode of ending of the telodendria of the dendrites of peripheral sensory neurones (sensoiy nerve-fibers) it has not been possible to discuss any but the more typical varie- ties of sensory nerve -endings. Other nerve -endings will be consid- ered in connection with the several organs to be treated later. For a fuller discussion of this subject, the reader is referred to special works and monographs. TECHNIC. Fresh medullated nerve-fibers, when teased in an indifferent fluid, show the peculiar luster of the medullary sheath, and also the nodes of Ranvier, the neurilemma with its nuclei, and the segments of Lantermann. At the cut ends of the fibers, the typical coagula- tion of their medullary portions is seen in the form of drops of myelin. All these structures can also be seen after using i oJ0 osmic acid. A nerve (not too thick) is placed in a i % aqueous osmic acid solution, then washed for a few hours in distilled water, and finally carried over into absolute alcohol. After dehydration, small pieces are cleared with oil of cloves and the fibers teased apart upon a slide. The medullary sheath is stained black and hides the axial space, the nodes are clear, the neu- rilemma is sometimes seen as a light membrane, and the nuclei of the fibers are of a lenticular shape, and stained brown. The nodes of Ranvier may also be demonstrated by means of silver nitrate solution. Fresh nerve-fibers are either teased in distilled water to which a trace of i% silver nitrate solution has been added (the nodes of Ranvier appear after a short time as small crosses), or whole nerves are placed for twenty-four hours in a 0.5% aqueous solution of silver nitrate, washed for a short time with water, hardened in alcohol, after which they are imbedded in paraffin and cut longitudinally. Exposure to light will soon bring out the ' ' crosses of Ranvier ' ' at the nodes. The appearance of these crosses is due to the fact that the silver nitrate solution first penetrates at the nodes of Ranvier, and then passes by capillary attraction along the axial cord for some distance. After the reduction of the silver, the cruciform figures appear colored THE NERVOUS TISSUES. 181 black. Occasionally, a peculiar transverse striation is seen in the longi- tudinal portions of the crosses. These are known as Frommann's lines. Their origin and significance have not as yet been satisfactorily ex- plained. To demonstrate the fibrils of the axial cord a piece of a small nerve is stretched on a match or toothpick and fixed for four hours in a 0.5% osmic acid solution, after which it is washed in water for the same length of time and immersed in 90% alcohol for twenty-four hours. The preparation is now stained for another twenty-four hours in a saturated aqueous solution of fuchsin S and then placed for three days in abso- lute alcohol. Finally, the nerve is passed as rapidly as possible through toluol, toluol- paraffin, and then im- bedded in paraffin. The proper orientation of the specimen is of the greatest importance, as is also the cutting of thin sections. In a lon- gitudinal section red fibrils of almost uniform thickness and evenly dis- tributed throughout the axial space Axis-cylin- d are seen lying in the colorless neuro- Medullary sheath. Fig. 148. — Ranvier's crosses from sci- atic nerve of rabbit treated with silver ni- trate solution ; X * 2O- Frommann' s lines can be seen in a few fibers. Fig. 149. — Medullated nerve-fiber from sciatic nerve of frog. In two places the medullary sheath has been pulled away by teasing, showing the "naked axis-cylinder" ; X 2I2- plasm, and parallel to the long axis of the nerve-fiber. In cross-section the axial fibrils appear as evenly distributed dots. Attention must be called to the fact that the fibrils are not equally well stained in all cases (Kupffer, 83, II ; compare also Jacobi and Joseph). When the fiber is less carefully treated, the fibrils fuse with the neuroplasm to form the ' ' axis-cylinder ' ' of authors. As the appearance of the latter is due to a shrinkage of the contents of the axial space, it is easy to understand that one reagent may have a greater effect in this re- spect than another. The thinnest axis-cylinders are produced by chromic acid and its salts, while thicker ones are seen in nerve-fibers fixed in alcohol. These variations are best seen in cross-sections, in which the 182 THE TISSUES. Dendrite. axis-cylinders sometimes appear as round dots and again as stellate figures. The latter are due to pressure on the shrinking axial cord by the unevenly coagulated medullary sheath. As the medullary sheath in such prepara- tions crumbles away in many places, large areas of the axis-cylinder may often be isolated by teasing (Fig. 149). Sensory and motor nerve-endings may be stained after gold chlorid and chrome-silver methods (see methods of impregnation, page 47), or after the infra vitam methylene-blue method suggested by Ehrlich and variously modified by other investigators. If freshly teased fibers be treated with glacial acetic acid, the axis-cylinders swell up and issue from the ends of the fibers in irregular masses showing fine longitudinal striation (Kolliker, 93). The structures of the axial space dissolve in i% hydrochloric acid, as well as in a 10% solution of sodium chlorid (Halliburton). For the isolation of ganglion cells, 33% alcohol, o.i to 0.5% chromic acid, or \CJ0 solution of potassium bichromate may be used. Small pieces of the spinal cord and brain containing ganglion cells are treated with a small quantity of one of the above solutions for one or two weeks. After this interval the prepara- tions may be teased and the isolated ganglion cells stained on a slide and mounted in glycerin. They may even be fixed in situ by injecting a i % solution of osmic acid or 33% al- cohol into the areas of the brain or spinal cord containing ganglion cells. The region thus treated is then cut out and teased. The nonmedullated or "Remak's fibers" are ob- tained by teasing a sym- pathetic nerve, or, better, a piece of the vagus pre- viously treated with osmic acid. Between the black- ened medullated fibers of the pneumogastric are seen numerous unstained fibers of Remak. The fibers of the olfactory nerve are stained brown by osmic acid. Ehrlich' s methylene-blue method consists in an infra vitam staining of ganglion cells, nerve-fibers, and nerve-endings. The method is much more applicable to the staining of peripheral ganglia (spinal and sympathetic ganglia), peripheral nerves, and nerve-endings than to stain- ing the elements of the central nervous system, although the latter may also be stained by means of this method. Two methods for bringing the stain in contact with the nerve-tissues are now in use : ( i ) injecting the methylene-blue solution into the living — Neuraxis. Fig. 150. — A ganglion cell from anterior horn of the spinal cord of calf ; teased preparation ; X 140. By this method only the coarsest ramifica- tions of the dendrites are preserved ; the rest are torn off. THE NERVOUS TISSUES. 183 tissues through the blood-vessels ; (2) adding a few drops of the stain to small pieces of perfectly fresh tissues removed from the body. The solu- tion used for injecting tne tissues is prepared as follows : i gm. of methyl - ene-blue1 is mixed in a small flask with 100 c.c. of normal salt solution and heated over a flame until the solution becomes hot. It is then allowed to cool ; when filtered, it is ready for use. A cannula is tied into the main artery of the part in which it is desired to stain the nerve elements, and sufficient of the foregoing methylene-blue solution injected to give the part a decidedly blue color. After the injection the part to be studied remains undisturbed for about one-half hour, after which time small, or at least thin, pieces of the tissue to be studied are removed to a slide moistened in normal salt solution, and exposed to the air. The tissues remain on the slide until the nerve-cells, nerve-fibers, or nerve- endings seem satisfactorily stained. After placing the tissues on the slide, they are examined under the microscope (without covering with a cover- glass) every two or three minutes, until such examination shows blue color in the neuraxes of the nerve-fibers and their terminations, or in the nerve-cells, if there be any in the tissues examined. Care should be tak^n not to miss the time when the staining has reached its full develop- ment, as the blue color usually fades again and only inferior preparations are obtained. Tissues thus stained may be fixed by one of two methods (or modifi- cations of these methods), the selection of the method depending some- what on the results desired. If it is desired to gain preparations giving the general course of nerves, the formation of nerve-plexuses, the relations of afferent and efferent nerves to the nerve-cells in ganglia, or the gen- eral arrangement of the terminal branches of nerve-fibers in nerve end- organs, the tissues are placed in a saturated aqueous solution of ammo- nium picrate (Dogiel) in which the blue color of the tissues is in a short time changed to a purplish color. In this solution the tissues remain for from twelve to twenty-four hours, and are then transferred to a mixture consisting of equal parts of a saturated aqueous solution of ammonium picrate and glycerin, in which they remain another twenty-four hours ; they may, however, without detriment remain in the mixture several days. The tissues are then mounted in this ammonium picrate -glycerin mixture. If, on the other hand, it is desired to section tissues stained infra •vitam in methylene-blue, the following method, slightly modified from that given by Bethe, is suggested. The following fixative is prepared : Ammonium molybdate, i gm.; distilled water, 10 c.c.; hydrochloric acid, i drop. The solution is prepared by grinding the ammonium molybdate to a fine powder, removing it to a flask, and adding the required quantity of water. The flask is now heated until the ammonium molybdate is entirely dissolved, when the hydrochloric acid is added. Before using this fixative it is necessary to cool it to 2°-5° C. It is, there- fore, well to prepare it before" the injection is made, and surround it with an ice mixture. In this fixative the tissues remain for from twelve to twenty-four hours. After the first six to eight hours it is not necessary to keep the fixative below ordinary room-temperature. After fixation the tissues are washed for an hour in distilled water. They are then hard- ened and dehydrated in absolute alcohol. It is advisable to hasten this step as much as possible, though not at the risk of imperfect dehydration. 1 Methylenblau, rectificiert nach Ehrlich, Griibler, Leipzig. 184 THE TISSUES. The tissues are then transferred to xylol and imbedded in paraffin, sec- tioned, fixed to the slide or cover-glass with albumin fixative, and may be double stained in alum-carmin or alum-cochineal. After staining in either of these stains, the sections are thoroughly dehydrated and cleared in oil ofbergamot. The oil is washed off with xylol and the sections are mounted in Canada balsam. In staining nerve-fibers with methylene-blue by local application of the stain to the tissues, the tissues to be studied are removed from an animal which has just been killed, divided in small pieces, and placed on a slide moistened with normal salt solution. A few drops of a -%-$% to -Y5% solution of methylene-blue in normal salt solution are added from time to time — sufficient to keep the tissues moistened by the solution, but not enough to cover them. The preparations are examined from time to time, under the microscope, to see whether the nerve elements are stained. The length of time required for staining by this method varies. Some- times the nerve elements are stained in half an hour ; again, it may re- quire two and one-half hours ; on an average, about one hour. As soon as the tissues seem well stained they are fixed as previously directed. Dogiel has found that sympathetic ganglia and sensory nerve-fibers of the heart removed from the human body several hours after death may be stained by means of the foregoing method. In order to obviate the necessity for the low temperature of the pre- vious method, Bethe (96) has recommended the following procedure : According to the method of Smirnow and Dogiel, he first employs as a preliminary fixing agent a concentrated aqueous solution of ammonium picrate. In this he places the tissue, previously treated with methylene- blue, for from ten to fifteen minutes. Without further washing the larger objects are immersed in a mixture composed of ammonium molybdate (or sodium phosphomolybdate) i gm., distilled water 20 c.c., and pure hydrochloric acid i drop. The following mixtures may also be employed for the same purpose : ammonium molybdate (or sodium phosphomo- lybdate) i gm. , distilled water 10 c.c., 2% solution of chromic acid 10 c.c., and hydrochloric acid i drop ; or, for very thin gross specimens or sections, ammonium molybdate (or sodium phosphomolybdate) i gm., distilled water, 10 c.c., 0.5% osmic acid 10 c.c., and hydrochloric acid i drop. Small objects are permitted to remain no longer than from three quarters of an hour to one hour in either of the first two mixtures, and not more than from four to twelve hours in the third. After fixing, the specimens are washed with water, carried over into alcohol, then into xylol, and finally imbedded in paraffin. Subsequent staining with alum-carmin, alum -cochineal, or one of the neutral anilin dyes gives good results. A very promising method recommended by Meyer (95) consists in injecting subcutaneously about 20 c.c. of normal salt solution contain- ing from i% to 4% of methylene-blue into a young rabbit, and repeating the operation in one to two hours. Within the next two hours the animal usually dies and the central nervous organs are then removed and small pieces fixed according to Bethe' s method. The method of Chr. Sihler may be recommended for demonstrating the nerve-endings in striated muscle : Muscle bundles of the thickness of a goose quill are first placed for eighteen hours in a solution composed of acetic acid i vol., glycerin i vol., and i% solution of chloral hydrate 6 vols., and then teased in pure glycerin. Afterward they are placed in a THE NERVOUS TISSUES. 185 mixture of Ehrlich's hematoxylin i vol. , glycerin i vol. , and i c/c chloral hy- drate solution 6 vols. , in which the specimens are allowed to remain for from three to ten days. The pieces are now placed in glycerin acidified with acetic acid (solution No. i), in which the color becomes differentiated, the nerves and nerve-endings in the muscles and vessels being deeply stained, while the remaining portion of the specimen becomes decolor- ized. After having stained with No. 2, the pieces may be preserved in pure glycerin, to be treated later with acetic acid (solution No. i). These methods are most successful in reptilia and mammalia, more difficult in the other classes of vertebrate animals. SPECIAL HISTOLOGY. I. BLOOD AND BLOOD-FORMING ORGANS, HEART, BLOOD-VESSELS, AND LYMPH-VESSELS. A. BLOOD AND LYMPH. J. FORMATION OF BLOOD. EARLY in the development of the embryo there appear in a por- tion of the extra-embryonic area of the blastoderm, known as the area vasculosa, definite masses of cells, derived from the mesen- chyme, and spoken of as blood islands, which are intimately connected with the formation of the blood. If these blood islands be examined at a certain stage, free cells are seen lying in their center, appar- ently derived from the central cells of the islands ; the cells sur- rounding them represent the elements which later go to form the primitive vascular walls. The free elements are the first blood-cells of the embryo. The blood-cells thus developed enter the circula- tion by means of blood channels formed by the confluence of the blood islands. These grow toward the embryo and later join the large central vessels. The origin of these blood islands is still an open question. Some authors contend that they arise from the mesoblast (P. Mayer, 87, 93 ; K. Ziegler ; van der Stricht, 92), others that they are of entodermic origin (Kupffer, 78 ; Gensch ; Riickert, 88 ; C. K. Hoffmann, 93, I ; 93, II ; Mehnert, 96). At a certain period the embryonic blood consists principally of nucleated red cells, which proliferate in the circulation by indirect division. The colorless blood-cells, the development of which is not yet fully understood, appear later. It is possible that they also are elements of the blood islands, which do not contain any hemoglobin. In a later period of embryonic life the liver becomes a blood-forming organ. Recent investigations have, however, shown that it does not take a direct part in the formation of the blood, but only serves as an area in which the blood-corpuscles proliferate during their slow passage through its vessels. The blind sac-like endings of the venous capillaries seem to be particularly adapted for this purpose, as in them the blood current stagnates, and it is here that the greater number of blood-cells reveal mitotic figures. The newly formed elements are finally swept away by the blood stream and enter the general circulation (van der Stricht, 92 ; v. Kostan- ecki, 92, III). Many investigators believe that the red blood-cells 1 86 BLOOD AND LYMPH. l8/ have an entirely different origin in the liver — namely, from the large polynuclear, giant cells, which are thought to arise either from the cells of the capillaries or from the liver-cells (Kuborn, M. Schmidt). Late in fetal life and in the adult, the red bone-marrow and the spleen are the organs which form the red blood-cells. The lym- phatic glands and the spleen produce the white blood-cells. In ad- dition to the nucleated red corpuscles which are present up to a cer- tain stage of development, nonnucleated red blood-cells also appear. The number of the latter increases, until finally they are found almost exclusively in the blood of the new-born infant. The blood of the adult consists of a fluid, coagulable substance, the blood plasma, and of formed elements suspended in this inter- cellular substance. The fluid medium of the blood is of a clear yellowish color and of alkaline reaction, having a specific gravity of about 1030. It is made up of water, of which it contains about 90 % , and various organic and inorganic substances. The formed elements are : (a) Red blood-corpuscles (erythrocytes) ; (£) white blood-corpuscles (leucocytes); and (r) the blood platelets of Biz- zozero (82), hematoblasts of Hayem, or the thrombocytes of Dekhuysen. Besides these, there are present particles of fat, and, as H. F. Miiller (96) has recently shown, also hemokonia. 2. RED BLOOD-CORPUSCLES. In man and nearly all mammalia the great majority of the red blood-corpuscles are nonnucleated, biconcave circular discs with rounded edges. They have smooth surfaces, are transparent, pale yellowish-red in color, and very elastic. No method has as yet been devised to demonstrate a nucleus in these cells, and there is no doubt that the red blood-discs of the human adult and of mammalia are devoid, in the histologic sense, of a nucleus capable of differen- tiation (compare Lavdowsky; Arnold, 96). They are therefore peculiarly modified cells. They possess a somewhat more resistant external zone of exoplasm, which has been interpreted as a cell membrane by certain observers (Lavdowsky), but which does not present the characteristics of a true cell membrane. If fresh blood be left for some time undisturbed, the blood-discs adhere to each other by their flattened surfaces, grouping them- selves in rouleaux, By certain reagents the clear and transparent contents of the blood-corpuscles can be separated into two substances — a staining and a nonstaining. The first consists of the blood pigment, or hemoglobin, which can be dissolved ; the second of a colorless sub- stance, the strorna, which presents itself in various forms (protoplasm of the cell). The stroma probably contains the hemoglobin in solution. Hemoglobin is a very complex proteid which may be decom- posed into a globulin and a pigment hematin. The hemoglobin of the majority of animals crystallizes in the form of rhombic prisms ; 1 88 BLOOD AND BLOOD-FORMING ORGANS. in the squirrel, however, in hexagonal plates, and in the guinea-pig in tetrahedra. Hematin combines with hydrochloric acid to form hemin, or Teichinanris crystals, of brownish color, rhombic shape, and microscopic size. They are of much value in lego-medical work, since they may be obtained from blood, no matter how old, and are characteristic of hemoglobin. They may be obtained from very small quantities of blood pigment. If a small drop of blood pressed from a small puncture is placed on a slide and covered with a cover-glass, the red blood- cells soon become changed. This is due to the evaporation of water in the blood plasma, causing an increased concentration of the sodium chloride contained, which in turn draws water from the blood-cells The shrinkage which follows produces a characteristic Fig. 151. — Hu- Fig. 152. — So-called Fig. 153. — Hemin, or man red blood-cells; "rouleau" formation of Teichmann's crystals, from X 1500: a, As seen human erythrocytes ; X blood stains on a cloth, from the surface ; 6, 1500. as seen from the edge. Fig. 154. — " Crenated" human red blood- Fig. 155. — Red blood-corpuscles sub- cells; X 1500. jected to the action of water; X 15°°: a, Spheric blood -cell ; b, "blood shadow." change in the form of the cells, which assume a crenated or stellate shape. The red blood-cells of blood mounted in normal salt become crenated in a short time for the same reason. Red blood- cells are variously affected by different fluids. In water they become spheric and lose their hemoglobin by solution. Their remains then appear as clear, spheric, indistinct blood shadows, which may, how- ever, be again rendered distinct by staining with iodin. Dilute acetic acid has a similar but more rapid action, with this peculiarity, that before becoming paler the blood-cells momentarily assume a darker hue. Bile, even when taken from the animal furnishing the blood, exerts a peculiar influence upon the red blood-cells ; they first become distended, and then suddenly appear to explode into BLOOD AND LYMPH. 189 small fragments. Dilute solutions of tannic acid cause the hemo- globin to leave the blood-cells, and coagulate in the form of a small globule at the edge of the blood-cell. In alkalies of moderate strength the red blood-cells break down in a few moments. Besides the disc-shaped red blood-cells, every well-made prep- aration shows a few small, spheric, nonnucleated cells containing hemoglobin. These, however, have received as yet but little attention. M. Bethe makes the statement that human blood and the blood of mammalia contain corpuscles of different sizes, bearing a definite numerical relationship to each other. " If they be classified according to their size, and the percentage of each class be calculated, the result will show a nearly constant proportional graphic curve varying but slightly, according o Fig. 156. — Red blood-corpuscles from various vertebrate animals; XIOO° (Walker's model) : a, From proteus (Olm) ; b, from frog ; c, from lizard ; d, from sparrow ; e, from camel ; /and^-, from man ; h, from myoxus glis ; i, from goat ; k, from musk-deer. to the animal species." According to M. Bethe, dry preparations of human and animal blood may be distinguished from each other, with the exception of the blood of the guinea-pig which presents a curve identical with that of human blood. The red blood-cells of mammalia, excepting those of the llama and camel species, are in shape and structure similar to those of man. The red blood-cells of the llama and camel have the shape of an ellipsoid, flattened at its short axis, but also nonnucleated. We have already made mention of the fact that the embryonal erythrocytes are nucleated ; the question now arises as to how, in the course of their development, they lose their nuclei. Three pos- sibilities confront us : First, either the embryonal blood-cells are destroyed and gradually replaced by previously existing nonnucle- 190 BLOOD AND BLOOD-FORMING ORGANS. ated elements ; or, second, the nonnucleated red cells are formed from the nucleated by an absorption of the nucleus (or what appears to be such' to the eye of the observer, Arnold, 96) ; or, finally, the nucleus is extruded from the original nucleated cell. According to recent investigations (Howell) the third possibility represents the change as it actually takes place. In all vertebrate animals except mammalia, the red blood- corpuscles are nucleated. They are elliptic discs with a biconvex center corresponding to the position of the nucleus. The blood- cells of the amphibia (frog) are well adapted for study on account of their size. They are long and, as a rule, contain an elongated nucleus with a coarse, dense chromatin framework, which gives them an almost homogeneous appearance. The cell-body may be divided, as in mammalia, into stroma and hemoglobin. When sub- jected to certain reagents, the contour of the cells appears double and sharply defined. This condition is, however, no proof of the existence of a membrane. The blood-cells of birds, reptiles and fishes are similarly constructed. The diameter of the erythrocytes varies greatly in different ver- tebrate animals, but is constant in each species. The red blood-cells of man measure on the average 7.5 // (7.2 fj. to 7.8 //), in their long diameter, and I.6// to 1.9^ in their short diameter. We append a table of their number in a cubic millimeter and size in man and certain animals as compiled by Rollett (71, II) and M. Bethe : , No. IN SPECIES. SIZE. CUBIC MILLI- METER. Man . . . (Homo] . 7.2-7.8^ . . . 5,000,000 Monkey . . . (Cercopith. ruber) •70 • . . . 6,355,000 Hare . . . (Lepus cuniculus) . 7.16 . . . . 6,410,000 Guinea-pig .... . . . (Cavia cob.) .... . 7-48 - . . . 5,859,5oo Dog . . . (Cam's fam.) .... . 7-2 . . . . 6,650,000 Cat . . . (Felis dom.) .... . 6.2 . . . . 9,900,000 Horse . . . (Equus cab.) .... •5-58 • • • • 7,403,50° Musk-deer .... . . . (Moschus jav.) . . . •2.5 Spanish goat . . . . . . (Capra /its.) ... .4.25 . . . . 19,000,000 Domestic chaffinch . . . . (Fringilla dom. ) . . . Length, 11.9 Breadth, 6.8 Dove . . (Columba) L. 14.7 . . . . 2,010000 B. 6-5 Chicken . . . ( Callus dom. ) . . . . L. 12. 1 B. 7.2 Duck . . . (Anas bosch.) . . . . L. 12.9 B. 8.0 Tortoise . L. 21.2 . . . . 629,000 B. 12.45 Lizard . . . (Lacerta agil.) . . . . L. 15-75 • . . . 1,292,000 B. 9-1 Snake . . . (Coluber natr.) . . . . L. 22. 0 . . . . 829,400 B. 13.0 Frog . . . (Hana temp.) . . . . L. 22.3 . . . . 393,200 B. 15-7 Toad . . . (Bufo vulg.) .... . L. 21.8 . . . . 389,000 B. 15-9 Triton . . . (Triton crist.) . . . . L. 29-3 • . . . 103,000 B. 19-5 BLOOD AND LYMPH. Salamander No. IN SIZE. CUBIC MILLI- METER. Length, 37.8 80,000 Breadth, 23.8 L. 58 35,ooo B. 35 L. 13.4 B. 10.4 Carp (Cyprinus Gobio) . . . L. 17.7 B. 10. i SPECIES. (Salamandra mac.} (Proteus angu.) Sturgeon (Acipenser St.) . . 3. WHITE BLOOD-CORPUSCLES. The white blood-cells contain no hemoglobin and are nucleated elements which, under certain conditions, possess ameboid move- ment. Their size varies from 5 fj. to 12 //, and they are less numer- ous than the red blood-corpuscles, one white blood-cell to from three hundred to five hundred red cells being a normal proportion. Fig. 157. — From the normal blood of man; X I2O° (from dry preparation of H. F. Miiller) : a, Red blood-cell ; b, lymphocyte ; c and d, mononuclear leucocytes ; et transitional leucocyte ; f and g, leucocytes with polymorphous nuclei. Flemming ascribes a fibrillar structure to the protoplasm of white blood-cells, and was the first to observe a centrosome situated near the nucleus. M. Heidenhain made the observation that the white blood-cells possessed several centrosomes grouped to constitute a microcenter (microcentrum) about which the fibrillar structure of the protoplasm was arranged radially." The meshes of the fibrillar net- work are filled with a more fluid interfibrillar substance, in which are found the specific granules to be mentioned later. In the normal blood the white blood-cells vary in size and structure, and the fol- lowing varieties are distinguished : (i) Small and large lymphocytes ; (2) mononuclear leucocytes ; (3) transitional leucocytes ; (4) leuco- cytes, either polymorphonuclear or polynuclear. The lymphocytes form about 20% of the white blood-cells. 1 92 BLOOD AND BLOOD-FORMING ORGANS. They vary in size from 5 // to 7. 5 fj. and possess a relatively large nucleus, the chromatin of which is in the form of relatively large granules, which stain rather deeply. The nucleus is surrounded by a narrow zone of protoplasm, often seen clearly only to one side of the cell in the form of a crescent. It does not stain readily in acid dyes. The leucocytes vary in size from 7 /j. to 10 fi. The mononuclear leucocytes, constituting about 2^ to 4^ of the white blood-cells, have a nearly round or oval nucleus, which usually does not stain very deeply, and which is relatively smaller than that of the lympho- cytes. The transitional leucocytes, forming also about 2 ^ to 4^ of the white blood-cells, are developed from the mononuclear variety and represent transitional stages in the development of mononuclear leucocytes to those with polymorphous nuclei. The nucleus in the transitional form is similar in size and structure to that of the mononuclear variety, but Of a more or less pronounced horse- shoe-shape. The leucocytes with polymorphous miclei, developed from the transitional forms, are very numerous in the blood, form- ing about 70^ of the entire number of white blood-cells. They are also the cells which show the most active ameboid movement when examined on the warm stage. They possess variously lobu- lated nuclei, the several nuclear masses often being united by del- icate threads of nuclear substance. A leucocyte with a poly- morphous nucleus becomes a polynu clear cell in case the bridges of nuclear substance uniting the several lobules of the nucleus break through. In the protoplasm of the transitional leucocytes, the polymorphonuclear, and the polynuclear forms are found fine and coarse granules. Our knowledge of these granules has, however, at /3yd Fig. 158. — Ehrlich's leucocytic granules; X '800 (from preparations of H. F. Miiller) : a, Acidophile or eosinophile granules, relatively large and regularly distributed ; e, neutrophile granules ; /3, amphophile granules, few in number and irregularly dis- tributed ; y, mast cells with granules of various sizes ; t!, basophile granules, (a, 6, and e, From the normal blood ; y, from human leukemic blood ; /3, from the blood of guinea-pig.) been greatly extended since Ehrlich has shown that the granules of leucocytes show specific reactions toward certain anilin stains, or combinations of such stains. He divides the granules of the leuco- cytes into five classes which he terms respectively a-, (3-, 3-, ?-, and e- granules. In human blood are found the a-granules, which show an affinity for acid-anilin stains, are therefore known as acidophile gran- BLOOD AND LYMPH. 1 93 ules, and, since they are most readily stained in eosin, are generally spoken of as eosinophile granules. In normal blood from I ^ to 4^ of the polymorphonuclear leucocytes and now and then a transi- tional cell have eosinophile granules. The granules are coarse and stain bright red in eosin. Nearly all the leucocytes with granules (from 65 ^ to 68 % of all white blood-cells) have e-granules or, since they are stained in color mixtures formed by a combination of acid and basic anilin stains, neutropliile granules. The neutrophile granules are much finer than the eosinophile and are not stained in acid stains. The y- and ^-granules are stained in basic anilin stains, and are known as basophile granules. They are coarse and irregular, and the leucocytes containing them form from o. 5 % to I Blood-vessels ; ff, fibrous capsule ; h, hilum ; kz, germ-center ; nl, lymph-nodule ; sc, cortical substance ; gm, medullary substance ; tr, trabeculse ; via, afferent lymph-vessels ; vie, efferent lymph-vessels ( " Atlas and Epitome of Human Histology," Sobotta). poorly developed, as in the human lymph-glands, where they are often almost wanting. The lymphoid tissue of the gland is so distributed that at its periphery a large number of more or less clearly defined lymph-nodules are found, which are in part separated from each other by the trabeculae just described, the cortical nodules. The nodules are structural units and have a typical blood supply, and are in structure like the lymph-nodules of simple and ag- LYMPHOID TISSUE, LYMPH-NODULES, AND LYMPH-GLANDS. 199 minated follicles above mentioned. They form a peripheral layer which is, however, not clearly defined in the neighborhood of the hilum. This layer is known as the cortex of the lymph-gland. (Fig. 161.) The lymphoid tissue of the interior of the gland, the medullary substance, is in the shape of cords — medullary cords — - which are continuous with the lymphoid nodules of the cortical portion. These connect with each other and form a network of lymphoid tissue, in the open spaces of which lie the trabeculae. At their periphery the nodules and medullary cords are bordered by a wide-meshed lymphatic tissue, the lymph-sinus of the gland, parts of which lie (i) between the capsule and the cortical substance, (2) Mitosis. Germ center. — Lymph-sinus. ^SSS^sk"*"*?^ * t §*JW*V» -• *-*^e * Medullary cord. Fig. 162. — From a human lymph-gland ; X 24°- At a are seen the concentrically arranged cells of the lymph-nodules. (Fixation with Flemming's fluid.) between the nodules of the latter and the trabeculae, (3) between the medullary cords and the trabeculae, and (4) between the medul- lary substance and the capsule at the hilum. At the hilum the loose lymphoid tissue represents a terminal sinus (Toldt). These sinuses are lined throughout by endothelial cells, which are continu- ous with those of the afferent and efferent lymph-vessels. The lymph flows into the gland through the afferent vessels, and passes along into the interior through the spaces offering the least resist- ance (sinuses). The latter represent those peripheral portions of the nodules and of the medullary cords in which the lymphoid tissue is present in Joose arrangement. The lymph consequently envelops 2OO THE BLOOD AND BLOOD-FORMING ORGANS. not only the lymph-nodules of the cortical substance, but also the medullary cords, and finally streams into the terminal sinus and then into the efferent channels. As a result the lymph takes with it the newly formed cells of the lymph-nodules and the medullary cords, and passes out richer in cellular elements than on its entrance. The lymph-glands receive their blood supply mainly through the hilum ; relatively small arterial branches may penetrate the capsule. Generally, a number of arterial branches enter at the hilum, from whence they may pass directly into the medullary substance, or pass for a distance in trabeculae. In their course branches are given off which pass to the medullary cords, in which they break up into capillary vessels situated in the periphery of the cords. These unite to form small veins which anastomose freely, and unite to form larger veins. The cortical nodules receive their blood supply from arterial branches which enter their proximal sides (side toward the hilum) and course through the center of the nodules, giving off capillary vessels which pass, without much anastomosis, to the periphery of the nodules, where they unite to form plexuses ; the capillaries of these plexuses join to form the veins of the nodules, which are thus situated at their periphery. These veins unite to form larger veins, which leave the glands at the hilum (Calvert). Medullated and nonmedullated nerves penetrate the lymph- glands accompanying the blood-vessels on which they terminate. Hemolymph Glands. — A typical lymph-gland possesses afferent and efferent lymph-vessels and a closed blood-vascular system completely separated from the lymph -vascular system, as may have been seen from the foregoing description. Attention has, however, been called in recent years to certain lymph -glands in which the complete separation of the vascular and lymphatic systems does not obtain, — glands in which the formed elements of blood and lymph are intermingled in the meshes of the adenoid reticulum, and which contain blood-sinuses in place of the lymph-sinuses observed in the typical lymph-glands. These have been designated as hemolymph glands (Blutiymphdrusen, hemal glands, hemal lymphatic glands). In the typical hemolymph glands there are no afferent and efferent lymphatic vessels; the. glands are inter- calated in the vascular system. Certain less clearly defined hemo- lymph glands possess afferent and efferent lymphatics and blood- sinuses, the two systems being not completely separated. These may be considered transitional forms. Lymph-glands with blood-sinuses were first described by Gibbes, who found such glands in the region of the renal artery. They were further considered and more fully described by Robertson, to whom the term hemolymph glands is to be credited, and by Clark- son, Vincent and Harrison, Drummond, Warthin, Weidenreich and Lewis. It appears from their description that they are widely distributed among vertebrates, although not equally well developed LYMPHOID TISSUE, LYMPH-NODULES, AND LYMPH-GLANDS. 2OI in the different types studied. Warthin has discussed more fully than other observers the hemolymph glands of man, and his account will here be followed in the main. It may be parenthetically stated that the hemolymph glands are numerous and well developed in the sheep (Warthin, Weidenreich) ; not so well differentiated in the dog and cat ; on the other hand, well developed in the rat (Lewis). We learn from the account of Warthin that the hemolymph glands are numerous in man, in the prevertebral retroperitoneal region, in the cervical region, and less numerous in the thorax. They vary in size from that of several millimeters to that of several centimeters. They present a variety of structure, depending mainly upon the arrangement of the lymphoid tissue and blood-sinuses. The great majority of these glands show a resemblance in structure to splenic tissue (splenolymph glands) ; others resemble more closely marrow-tissue (marrow lymph-glands). Between the two varieties of lymph-glands there are found transition forms, as also between these and lymph-glands (Warthin). The hemolymph glands (splenolymph glands) are surrounded by a capsule varying in thickness and composed of white fibrous and elastic tissue and nonstriated muscle-cells. From it trabeculae of the same structure pass into the gland, which after division are lost in the substance of the gland. Beneath the capsule there is found a continuous or discontinuous blood-sinus, bridged over by reticular fibers, from which anastomosing sinuses pass to the interior of the gland. These blood-sinuses are, in part at least, lined by endothelial cells. The sinuses in the gland substance are also bridged by trabeculse and reticular fibers. The sinuses divide the lymphoid tissue into anastomosing masses and cords. This tissue consists of an adenoid reticulum, in the meshes of which are found white and red blood-cells. The small lymphocytes are numerous; next in frequency are found the rnononuclear leucocytes ; transi- tional and polymorphonuclear cells. Basophile and eosinophile cells are also found. According to Weidenreich, the eosinophile cells are numerous ; he is also of the opinion that the eosinophile granules are derived from disintegrating red blood-cells. In the reticulum and in the blood-sinuses are found mononuclear phago- cytes, the origin of which has not been fully determined. Certain observers (Schumacher, Weidenreich) trace their origin to the cells of the reticulum ; Thoma regards them as developed from endo- thelial cells, while Drummond and others regard them as altered leucocytes. They contain disintegrating red blood-cells and pig- ment (according to Weidenreich, eosinophile cells). The majority of the hemolymph glands present a hilum through which the blood-vessels enter. The arteries, soon after entering the gland, divide into smaller branches, certain of which communicate directly through blood-capillaries with the blood-sinuses (Lewis) ; others pass to the adenoid tissue. The larger veins are in the trabeculae 2O2 BLOOD AND BLOOD-FORMING ORGANS. (at the hilum). On leaving the trabeculae their walls are formed of endothelium and adenoid reticulum, which separates them from the blood-sinuses. They end (or begin) in lacunae with thin walls which are perforated and communicate with the blood-sinuses (Weidenreich). Nerves have been traced to the hemolymph glands by Lewis (dog, rat). They probably end in the involuntary muscle of the capsule and trabeculae. Typical hemolymph glands have no lymph-vessels. In certain glands both blood- and lymph-sinuses are found. In such glands there is apparently an intermingling of blood and lymph, so that red blood-cells may pass into the lymphatics. The marrow lymph-glands are not so numerous. They have a thin capsule consisting of fibrous tissue but containing little elastic and muscular tissue. The blood-sinuses are not so well developed. In the lymphoid tissue the basophile and eosinophile cells are more numerous than in the splenolymph glands, and large cells similar to the large bone-marrow cells are now and then met with. As appears from the accounts of the majority of observers who have studied hemolymph glands, they have a hemolytic function, in that the red blood-cells are destroyed in them. Robertson and Clarkson ascribe to them a blood-forming function. This has also been observed by Warthin in the case of marrow lymph-glands, under certain conditions. The hemolymph glands are seats of origin for the white blood-cells which appear also to be destroyed here (eosinophile cells, Weidenreich). C THE SPLEEN. The spleen is a blood-forming organ, in which white blood-cells and, in embryonic life and under certain conditions in adult life also, red blood-cells are formed — the former in the adenoid tissue (Mal- pighian corpuscles) and spleen pulp, the latter only in the spleen pulp. The spleen is covered by peritoneum, and possesses a capsule consisting of connective tissue, elastic fibers, and nonstriated muscle- cells. This capsule sends numerous processes or trabeculae into the interior of the organ, which branch and form a framework in which the vessels, especially the veins, are imbedded. This con- nective-tissue framework breaks up to form the reticular tissue which constitutes the ground substance of the spleen. On examining a section of the spleen with the low-power mag- nifying glass, sections of the trabeculae, and round or oval masses of cells, having a diameter of about 0.5 mm., and in structure and appearance similar to the lymph-nodules (Malpighian corpuscles), are clearly defined ; between and around these structures is a tissue rich in cells, blood-vessels and blood-corpuscles, known as the spleen pulp. THE SPLEEN. 203 The organ has a very typical blood supply. Its arteries enter at the hilum, or indented surface, and its veins pass out at the same place. On the penetration of the vessels through the capsule, the latter forms sheaths around them (trabeculse), but as soon as the arteries and veins separate, the trabeculae envelop the veins alone. The arteries break up into smaller branches, which in turn divide into a large number of tuft-like groups of arterioles. Soon after their separation from the veins, the adventitia (outer fibrous tissue coat) of SOl Fig. 163. — Portion of section of human spleen ; X I5- The figure gives a general view of the structure of the spleen : a, Arteries with lymphoid sheaths ; cf, fibrous capsule ; Mk, Malpighian corpuscle ; //, spleen pulp ; tr, trabeculae ; v, vein in trabecula ("Atlas and Epitome of Human Histology," Sobotta). the arteries begins to assume a lymphoid character. This lymphoid tissue increases here and there to form true lymphoid nodules, pos- sessing all the characteristics already mentioned — reticular tissue, germ centers, etc. These are the Malpighian bodies, or corpuscles ; they are not very plentifully represented in man. The Malpighian bodies with their germ centers are formative centers for the lympho- cytes. The newly formed cells pass into the pulp and mix with its elements, which are then bathed by the blood emptying from the 2O4 BLOOD AND BLOOD-FORMING ORGANS. arterial capillaries into the channels of the pulp. The lymphoid sheaths and nodules derive their blood supply from arteries which arise from the lateral branches of the splenic vessels, and which divide into capillaries inside of the lymph sheaths or nodules, and only assume a venous character outside of the lymphoid substance. These vessels constitute the nutritive vascular system of the spleen. The small arterial branches above mentioned break up into very fine arterioles which gradually lose their lymphoid sheath, so that branches with a diameter of 0.02 mm. no longer possess a lymphoid sheath, but again assume an adventitia of the usual type. The smallest arterioles now pass over into capillaries which are for a time accompanied by the adventitia (capillary sheath), while the terminal branches have the usual structure of the capillary wall and are gradually lost in the meshes of the pulp. (See below.) On the other hand, the beginnings of the venous capillaries may be dis- tinctly seen in the pulp spaces. Groups of these capillaries com- bine to form larger vessels, which, however, still retain a capillary structure, and these again form small veins which unite to form the larger veins. F. P. Mall, whose recent contributions on the structure of the spleen have greatly extended our knowledge of the microscopic anatomy of this organ, states that the trabecular and vascular systems together outline masses of spleen pulp about i mm. in diameter, which he has named spleen lobules. Each lobule is bounded by three main in- terlobular trabeculae, each of which sends three intralobular trabe- culae into the lobule which communicate with each other in such a manner as to divide the lobule into about ten smaller compartments. An artery enters at one end of the lobule and, passing up through its center, gives off a branch to the spleen pulp found in each of the ten compartments formed by the intralobular trabeculae. . The spleen pulp in these compartments is arranged in the form of anastomosing columns, or cords, to which Mall has given the name of pulp cords. The branches of the main intralobular artery, going to each compartment, divide repeatedly ; the terminal branches course in the spleen-pulp cords, and in their path give off numerous small side branches which end in small expansions known as the ampulla of Thoma. An ampulla of Thoma may be divided into three parts. The first part, which is the ampulla proper, is lined by spindle-shaped cells, directly continuous with the endothelial cells of the artery. The second third, which often communicates with neigh- boring ampullae, contains large side -openings. The remaining third, which is the intermediary segment of Thoma (Thoma 's Ziviscken- stuck], is difficult to demonstrate. It is bridged over by fibrils of reticulum, and its communication with the vein is not wide. The circulation through the spleen is therefore not a closed one, through a system of capillaries completely closed, but rather through spaces in the spleen -pulp, certain of which are more direct, leading from the terminal arteries to the veins. According to this view, then, THE SPLEEN. 2O5 "the blood passes from the ampullae into the pulp spaces, then through the pores into the walls of the veins to form columns of blood discs which are pushed from the smaller to the larger veins of the spleen." The pulp spaces usually contain very few blood- corpuscles, in preparations fixed and prepared in the usual way, since on removal from the animal the muscular tissue of the capsule and trabeculae contracts and presses the blood from pulp spaces into the veins. If, however, the muscular tissue of the spleen is paralyzed before the tissue is fixed, numerous blood-corpuscles are found in the pulp spaces. In the above account of the ultimate distribution of the splenic vessels we have followed very closely the recent observations of F. P. Mall. The accompanying diagram (Fig. 164), slightly, though immaterially, modified from one given Capsule. Intralobular trabecula. - - I i : Artery to one of the ten - compartments. Intralobular artery. : — Interlobular trabecula. - - Intralobular trabecula , Malpighian corpuscle. -- Intralobular venous spaces. I Intralobular vein. - Ampulla of Thoma. — Spleen pulp cord. — Interlobular vein. — Intralobular vein. l-'ig. 164. — Diagram of lobule of the spleen (Mall, "Johns Hopkins Hospital Bulletin," Sept., Oct., 1898).' by F. P. Mall, shows clearly the trabecular and vascular systems of a spleen lobule. In larger spleens there may be some two hun- dred thousand of these lobules. In a dog weighing 10 kg. there are on an average some eighty thousand (F. P. Mall). The splenic pulp consists of a reticulum, in the meshes of which are found (i) fully developed red blood-cells; (2) now and then nucleated red blood-cells; (3) in many animals giant cells ; (4) cells containing red blood-corpuscles and the remains of such, with or without pigment ; (5) the different varieties of white blood-cells, especially a relatively large proportion of mononuclear leucocytes. Pigment granules, either extra- or intracellular, also occur in the splenic pulp. The pigment probably originates from disintegrating erythrocytes. Besides these are found, especially in 2O6 BLOOD AND BLOOD-FORMING ORGANS. teased preparations, long, spindle-shaped and flat cells, which are probably derivatives of the connective-tissue cells of the pulp and of the endothelium and muscular fibers of the vessels. Fig. 165. — Cells containing pigment, blood-corpuscles, and hemic masses from the spleen of dog ; X 1800 (from cover-glass of H. F. Miiller). Fig. 1 66. — From the human spleen ; X 8° (chrome-silver method) : a, Larger fibers of a Malpighian body ; b, reticular fibrils (Gitterfasern). In embryonic life and under certain conditions in postembryonic life (after severe hemorrhage and in certain diseases) red blood-cells are developed in the spleen pulp. The nucleated red blood-cells THE BONE-MARROW. 2O/ from which they develop may lose their nuclei in the spleen pulp or only after entering the circulation (compare Bone-marrow). Lymphatic vessels have been observed in the capsule and tra- beculae, but not in the spleen pulp nor Malpighian corpuscles. The spleen receives medullated and nonmedullated nerve-fibers ; the latter are much more numerous. The medullated nerve- fibers are no doubt the dendrites of sensory neurones. Their mode of ending has, however, not been determined. It is probable that they will be found to terminate in the fibrous-tissue coat of the vessels, and in the trabeculae and capsule. The nonmedullated nerve-fibers, no doubt the neuraxes of sympathetic neurones, are very numerous ; they enter the spleen with the artery and mainly follow its branches. By means of the chrome-silver method, Retzius (92) has shown that in the rabbit and mouse these nerve- fibers follow the vessels, forming plexuses which surround them, the terminal branches of these plexuses terminating in free endings in the muscular coat of the arteries. Here and there a nerve-fiber could be traced into the spleen pulp. The mode of ending of such fibers could, however, not be determined. The nonstriated muscle- cells of the trabeculae and capsule no doubt also receive their inner- vation from the nonmedullated nerves (neuraxes of sympathetic neurones). D. THE BONE-MARROW. The ingrowing periosteal bud which ushers in the process of endochondral ossification constitutes the first trace of an embryonal bone-marrow (compare p. 117). It consists mainly of elements from the periosteum which penetrate with the vascular bud and later form the entire adult bone-marrow. The red bone-marrow is formed first. This is present in embryos and young animals, and is devel- oped from the above elements during the process of ossification. As Neumann (82) has shown, the red bone-marrow of the human embryo is first formed in the bones of the extremities and gradually replaced in a proximal direction, so that in the adult it is found only in the proximal epiphyses, in the flat bones and in the bodies of the vertebras. In the remaining bones and parts of bones the red bone-marrow is replaced by the yellow bone-marrow (fat- marrow). As a result of hunger and certain pathologic conditions the yel- low bone-marrow changes into a gelatinous substance, which, how- ever, may again assume its original character. The red bone-marrow, surrounded by a delicate fibrous-tissue membrane, the endosteum, is a tissue consisting of various cellu- lar elements imbedded in a matrix of reticular tissue, which has been demonstrated by Enderlen with the chrome-silver method, and which is similar to the adenoid reticulum. Aside from these cellular elements, the marrow contains numerous vessels (see below), fixed connective-tissue cells, etc. 208 BLOOD AND BLOOD-FORMING ORGANS. The typical cellular elements of red bone-marrow are: I. The Marrow-cells, or Myelocytes. — These are cells, slightly larger than the leucocytes, possessing a relatively large nucleus of round or oval shape, rarely lobular, containing a relatively small amount of chromatin. In the protoplasm of these cells are found (in man) neutrophile granules and now and again small vacuoles. They are said to contain various pigment granules. These cells are not found in normal blood, but are found in circulating blood in certain forms of leukemia, where they may be distinguished from the mononuclear leucocytes partly by their structure, more particu- jT Fig. 167. — Cover-glass preparation from the bone-marrow of dog ; X 1200 (from preparation of H. F. Miiller) : a, Mast-cell ; b, lymphocyte ; c, eosinophile cell ; d, red blood-cell ; e, erythroblast in process of division ; f, f, normoblast ; g, erythroblast. Myelocyte not shown in this figure. larly by the presence of neutrophile granules not found in the mononuclear leucocytes. 2. Nucleated Red Blood-cells containing Hemoglobin. — Two varieties of these cells are recognized structurally, with interme- diary stages, as one variety is developed from the other. The erythroblasts, being genetically the earlier cells, possess relatively large nuclei with distinct chromatin network, surrounded by a protoplasm tinged with hemoglobin, and are often found in a stage of mitosis. The other variety of nucleated red blood-cells, the normoblasts, are developed from the erythroblasts. They contain globular nuclei, staining deeply, in which no chromatin network is recognizable, and surrounded by a layer of protoplasm containing hemoglobin. The normoblasts are changed into the nonnucleated red blood-discs by the extrusion of the nucleus. This process occurs normally in the red bone-marrow, or in the venous spaces THE BONE-MARROW. 2O9 of the bone-marrow (see below). In certain pathologic conditions, nucleated red blood-cells are found in the circulation. 3. Cells with Eosinophile Grannies. — In the red bone-marrow are found numerous eosinophile (acidophile) cells, some with round or oval nuclei (mononuclear eosinophile cells), others with horse- shoe-shaped nuclei (transitional eosinophile cells), and still others with polymorphous nuclei. The latter, which are the most numer- ous, are no doubt the mature cells, and are identical with those elements of the blood having the same structure. /i ••"•O ' ,^7> •• 7 • %^| F ^2i « ,^;f , O)^- ^^ ' terna. v Endothelium of the intima. £• \ Media. Fenestrated elastic mem- brane. Elastica ex- terna. Inner layer of adventitia. Outer layer of adventitia. . Vasa vasorum. Fig. 171. — Cross-section of the human carotid artery ; X J5°' (fenestrated membranes) into a large number of concentric layers containing but few muscle-fibers. Here also the tunica media is separated from the intima by an elastic limiting membrane, the fenestrated membrane of Henle, or the lamina elastica interna. In the aorta this membrane as such is not recognizable. The intima presents three distinct layers — the inner composed of flattened endo- thelial cells, and the other two consisting chiefly of elastic tissue (fibrous layers). Of these latter the inner is the richer in cellular Endothelium of the intima. •Intima. Media. Adventitia with nonstriated mus- cle-fibers in cross- section. Fig. 172. — Section through human artery, one of the smaller of the medium-sized ; X 640. elements and has a longitudinal arrangement of its fibers, while the 218 THE CIRCULATORY SYSTEM. Fig. 173. — Precapillary vessels from mesentery of cat : a, Precapillary artery ; b, precapillary vein possessing no muscu- lar tissue. outer is the looser in structure, possesses few cellular elements, and shows a circular arrangement of its fibers. The adventitia is also made up of fibre-elastic tissue, but in this case with a still looser structure and a longitudinal arrangement of its elastic fibers. In the outer portion of the adventitia the white fibrous tissue is more abundant. The adventitia is rich i'n blood-vessels. The medium-sized arteries differ in structure from the larger in that the elastic elements of the intima and media are replaced to a considerable extent by nonstri- ated muscular fibers. To this type belong the majority of the arterial vessels, ranging in caliber from the brachial, crural, and radial arteries to the supraorbital artery. In these the intima shows, besides its endothelium, only a single connec- tive-tissue layer with numerous longitudinal fibers, the subendo- thelial layer, which is thin and is limited externally by the fenes- trated membrane of Henle (lamina elastica interna). The media no longer gives the impression of being laminated, but consists of circularly arranged muscle-fibers separated from each other by elastic fibers and membranes and a small amount of fibrous connective tissue in such a way that the muscle-cells form more or less clearly defined groups. Here also the media is limited externally by the external elastic membrane. The adventitia, which becomes looser externally, is not so well de- veloped as in the larger vessels, but presents in general the same structure. In certain arteries (renal, splenic, dorsalis penis) it shows in its inner layers scattered longitudinal muscle-cells, which, how- ever, may also occur in other arteries at their points of division. With regard to the elastic tissues, the arteries of the brain differ to some extent from those of the remainder of the body. The elastica interna is much more prominent, the elastic fibers in the circular muscular layer are fewer, and the longitudinal strands are almost entirely lacking (H. Triepel). The walls of the smaller arteries consist mainly of the circular muscular layer of the media. The intima is reduced to the endo- thelium, which rests directly on the elastic internal limiting mem- brane. Outside of the external limiting membrane is the adventitia, which now consists of a small quantity of connective tissue. The vasa vasorum have disappeared. To this type belong the supra- orbital, central artery of the retina, etc. In the so-called precapillary vessels the intima consists only of the endothelial layer. The internal elastic membrane is very delicate. The media no longer forms a continuous layer, but is THE VASCULAR SYSTEM. 219 made up of a few circularly disposed muscular fibers. The adven- titia is composed of a small quantity of connective tissue, and con- tains no vasa vasorum. (<£) Veins. — In the foregoing account of the structure of the arteries we have described the structure of their walls according to the caliber of the vessels. Such a differentiation in the case of the veins would be impossible, since sometimes veins of the same cali- ber present decided differences in structure in various parts of the body. For the sake of convenience, we will commence with the de- scription of a vein of medium size. Its intima consists of three layers : (i) Of an inner layer of endothelium ; (2) of an underly- ing layer of muscle-cells, interrupted here and there by connective tissue ; and (3) of a fibrous connective-tissue layer containing fewer elastic but more white fibrous connective -tissue fibers than is the case in the arteries. Externally, the intima is limited by an in- ,-'Intima. T Elastica interna. Media. Fenestrated elastic membrane. Inner layer of the adventitia with • longitudinally ar- ranged muscle- cells. Connective tissue of the adventitia. '•".:" ;-'«'-V" Nerve. ' Fig. 174. — Cross-section of human internal jugular vein. At the left of the nerve are two large blood-vessels with a smaller one between them (vasa vasorum) ; X I5°- ternal elastic layer. The media is in general less highly developed than that of a corresponding artery, and contains muscle-cells which have a circular arrangement and in some veins form a continuous 22O THE CIRCULATORY SYSTEM. layer, although they sometimes occur as isolated fibers. The adven- titia shows an inner longitudinal muscular layer, which may be quite prominent and even form the bulk of the muscular tissue in the wall of the vein. Otherwise the adventitia of the veins belonging to this class corresponds in general to that of the arteries of the same size ; but here also we have, as in the intima, a preponderance of white fibrous connective-tissue elements. In the crural, brachial, and subcutaneous veins, the muscula- ture of the media is prominent ; while in the jugular, subclavian, and innominate veins, and in those of the dura and pia mater, the muscular tissue of the media is entirely wanting, and, as a conse- quence, the adventitia with its musculature, if present, is joined directly to the intima. In the smaller veins the vascular wall is reduced to an endothe- lial lining, an internal elastic membrane, a media consisting of interrupted circular bands of smooth muscle-fibers (which may be absent), and an adventitia containing a few muscle-fibers. The precapillary veins, which possess in general thinner walls than the corresponding arteries, present a greatly reduced intima and ad- ventitia, while the media has completely disappeared. — Adventitia with nonstriated muscle-cells in cross-sec- tions. Fig- 175- — Section of small vein (human); X 640. The valves of the veins are reduplications of the intima and vary slightly in structure at their two surfaces. The inner surface next to the blood current is covered by elongated endothelial cells, while the outer surface possesses an endothelial lining composed of much shorter cellular elements. The greater part of the valvular structure consists of white fibrous connective-tissue and elastic fibers. Flattened and circularly arranged muscle-cells are met with at the inner surface of many of the larger valves. The elastic fibers are more numerous beneath the endothelium on the inner surface of the valves (Ranvier, 89). (r) The Capillaries. — The capillaries consist solely of a layer of endothelial cells, accompanied here and there by a very delicate struc- tureless membrane, and rarely by stellate connective-tissue cells. The connective tissue in the immediate neighborhood of the capillaries is modified to such an extent that its elements, especially those of a cellular nature, seem to be arranged in a direction parallel with the long axis of the capillaries. When examined in suitable prepara- THE VASCULAR SYSTEM. 221 tions, the endothelium of the capillaries is seen to form a continuous layer, the cells of which are, as a rule, greatly flattened and present very irregular outlines. It is a well-known fact that a migration of the leucocytes occurs from the capillaries and smaller vessels (compare p. 193). In this connection arises the question as to whether or not the cells pass through certain preformed openings in the endothelium of these vessels, the so-called stomata, or through the stigmata and intercel- lular cement uniting the endothelial cells. The latter seems more probable, as stomata do not occur normally in the capillary wall. This subject will be further touched upon in the description of the lymphatic system. The capillaries connect the arterial and venous precapillary ves- sels, and in general accommodate themselves to the shape of the elements of tissues or organs in which they are situated. In the Fig. 176.— Endothelial cells of capillary (a) and precapillary (b) from the mesentery of rabbit ; stained in silver nitrate. muscles and nerves, etc., they form a network with oblong meshes, while in structures having a considerable surface, such as the pul- monary alveoli, the meshes are more inclined to be round or oval ; such small evaginations of tissue as the papillae of the skin contain capillaries arranged in the shape of loops. In certain organs — as, for instance, in the lobules of the liver — the capillaries form a distinct network with small meshes. Sinusoids. — In connection with the description of capillaries we may here insert a brief account of another type of terminal or peripheral blood-channels, described by Minot under the name of sinusoids; his account is here followed. The sinusoids are also composed only of endothelial cells. They differ, however, from blood-capillaries in shape and size, in their relation to the cellular elements of the tissues in which they are found, and in their devel- opment. They are of relatively large size, and vary between wide extremes. They are of very irregular shape and anastomose freely. "A sinusoid has its endothelium closely fitted against the paren- 222 THE CIRCULATORY SYSTEM. chyma of the organ," without the intervention of connective tissue; or, when this is present, usually only in small quantity, it is secondarily acquired, since in the early developmental stages of sinusoids no connective tissue intervenes between them and the parenchyma of the tissue. They develop by the intergrowth and intercrescence of the parenchyma of the organ and venous endothe- lium. Sinusoids are found in the following organs : liver, suprarenal, heart, parathyroid, carotid gland, spleen, and hemolymph glands. (d] Anastomoses, Retia mirabilia, and Sinuses. — In the course of certain vessels, abrupt changes are seen to occur — as, for instance, when a small vessel suddenly breaks up into a network of capillary or precapillary vessels, which, after continuing as such for a short distance, again unite to form a larger blood-channel, the latter then dividing as usual into true capillaries. Such struc- tures are known as retia mirabilia, and occur in man in the kid- Sensory nerve-ending. Plexus of vasomotor nerves. ¥r Fig. 177. — Small artery from the oral submucosa of cat, stained in methylene- blue, and showing a small portion of a sensory nerve-ending and the plexus of vasomotor ney, intestine, etc. Again, instead of breaking up into capillaries, a vessel may empty into a large cavity lined by endothelial cells (blood sinus). The latter is usually surrounded by loose con- nective tissue and is capable of great distention when filled with blood from an afferent vessel, or when the lumen of the efferent vessel is contracted by pressure or otherwise. The cavernous or erectile tissue of certain organs is due to the presence of such sinuses (penis, nasal mucous membrane, etc.). If vessels of larger caliber possess numerous direct communications, a vascular plexus is the result ; but if such communications occur at only a few points, we speak of anastomoses. Especially important are the direct communications between arteries and veins without the mediation of capillaries. Certain structural conditions of the tis- sue appear to favor such anomalies, which occur in certain exposed € THE LYMPHATIC SYSTEM. 223 areas of the skin (ear, tip of nose, toes) and in the meninges, kid- ney, etc. The blood-vessels, and more particularly the arteries, possess a rich nerve supply, comprising both nonmedullated and medullated nerves. The nonmedullated nerves, the neuraxes of sympathetic neurones, the cell-bodies of which are situated as a veiy general rule in some distant ganglion, form plexuses in the adventitia of the vessel-walls ; from this, single nerve-fibers, or small bundles of such, are given off, which enter the media and, after repeated division, end on the involuntary muscle-cells in a manner previously de- scribed. (See p. 166 and Fig. 133.) Through the agency of these nerves, the caliber of the vessel is controlled. They are known as vasomotor nerves. Quite recently Dogiel, Schemetkin, and Huber have shown that many vessels possess also sensory nerve-endings. The medullated nerve-fibers terminating in such endings, branch repeatedly before losing their medullary sheaths. These nerve-fibers with their branches accompany the vessels in the fibrous tissue immediately surrounding the adventitia. The nonmedullated ter- minal branches end in telodendria, consisting of small fibrils, beset with large varicosities and usually terminating in relatively large nodules. The branches and telodendria of a single medullated nerve-fiber (sensory nerve) terminating in a vessel are often spread over a relatively large area, some of the branches of such a nerve often accompanying an arterial branch, to terminate thereon. In the large vessels, the telodendria of the sensory nerves ajre found not only in the adventitia, but also in the intima, as has been shown by Schemetkin. (Seep. 215.) B. THE LYMPHATIC SYSTEM. \. LYMPH-VESSELS. The larger lymph-vessels — the thoracic duct, the lymphatic trunks, and the lymph-vessels — have relatively thin walls, and their structure corresponds in general to that of the veins. They possess numerous valves, and are subject to great variation in cali- ber according to the amount of their contents. When empty, they collapse and the smaller ones are not easily distinguished from the surrounding connective tissue. Tiraofeew and Dogiel (97) have shown that the lymph -vessels are supplied with nerves, which in their arrangement are similar to those found in the arteries and veins, though not so numerous. The latter, who has given the fuller description, states that the nerves supplying the lymph- vessels are varicose, nonmedullated fibers which form plexuses sur- rounding these structures. The terminal branches would appear to end on the nonstriated muscle cells found in the wall of the lymph -vessel. 224 THE CIRCULATORY SYSTEM. 2. LYMPH CAPILLARIES, LYMPH-SPACES, AND SEROUS CAVITIES. The walls of the lymph capillaries consist of very delicate, flat- tened endothelial cells, which are, however, somewhat larger and more irregular in outline than those of the vascular capillaries. The two may also be further differentiated by the fact that the diameter of the lymph capillaries varies greatly within very short distances. From a morphologic standpoint, the relations of the lymph capil- laries to the vascular capillaries and adjacent tissues are among the most difficult to solve. The distribution of the lymph-vessels and capillaries can be studied only in injected preparations, and it is easily seen that structures of such elasticity and delicacy are pecu- liarly liable to injury by bursting under this method of treatment. The resulting extravasations of the injection-mass then spread out in the direction of least resistance and still further obscure the picture, rendering it difficult to determine what spaces are preformed and what are the result of the injection. So much is, however, cer- tain : that the more carefully and skilfully the injection is made, the greater are the areas obtained, showing the injection of true lymph capillaries. The recent work of W. G. MacCallum confirms this, since he has shown quite conclusively that the lymphatics form a system of channels, with continuous walls, and are thus not in direct commu- nication with the so-called intercellular lymph-spaces — the lymph- canalicular spaces. Further confirmation of the fact that the lymphatics form a closed system of channels is found in the excel- lent contribution of Dr. Florence R. Sabin, dealing with the development of the lymphatic system. It is here shown that the lymphatic system begins as two blind ducts, guarded by valves, which bud off from the veins of the neck, and from two similar buds which arise from veins in the inguinal region. These buds grow and enlarge to form lymph-hearts, and from these ducts grow out toward the skin, which they invade and in which they spread out to form anastomosing plexuses. Ducts also grow toward the aorta to form the anlagen for the thoracic ducts, and from these grow out and invade the various organs. In some regions very dense networks of lymph capillaries sur- rounding the smaller blood-vessels have been demonstrated. Larger cleft-like spaces, lined with endothelium and communicating with the lymphatic system, are also found surrounding the vessels, peri- vascular spaces. These are present in man in the Haversian canals of bone tissue, around the vessels of the central nervous system, etc., and are separated from the actual vessel-wall by flattened endo- thelial cells. As in the case of the so-called perilympJiatic spaces, the walls of the perivascular spaces are joined here and there by connective-tissue trabeculae covered by endothelium. Such struc- tures exist in the perilymphatic spaces of the ear, the subdural spaces of the pia, the subarachnoidal space, the lymph-sinuses, etc. The perivascular spaces are better developed in the lower animals (amphibia, reptilia, etc.) than in mammalia. THE CAROTID GLAND. 225 Mention has been made of the migration of leucocytes and, under certain conditions, of red blood-cells through the walls of blood capillaries, and in the case of the former through the walls of lymph capillaries and lymph-vessels and spaces. This diapedesis of leucocytes probably takes place by a wandering of these cells through the intercellular cement uniting the endothelial cells lining these spaces. According to later investigations, it would seem that leucocytes may bore through endothelial cells, and thus migrate from the vessel or space in which they are found previous to such migration. C THE CAROTID GLAND (GLANDULA CAROTICA, GLOMUS CAROTICUM). At the point where the common carotid divides, there lies in man a small oval structure about the size of a grain of wheat, known as the carotid gland or the glomus caroticum. It is imbedded in ^ Septum. Trabecula of cells in cross- section. Distended blood capil- laries. _. Efferent vein. Fig. 178. — Section of a cell-ball from the glomus caroticum of man ; X I^°- (Injected specimen, after Schaper.) connective tissue, surrounded by many nerve-fibers, and on account of its great vascularity has a decidedly red color. The connective- tissue envelope of the gland penetrates into the interior in the form of septa, which divide its substance into small lobules, and these in turn into smaller round masses, the cell-balls. A small branch from the internal or external carotid enters the gland, where it branches, sending off twigs to the lobules, and these in turn still 15 226 THE CIRCULATORY SYSTEM. smaller divisions to the cell-balls. The latter vessels break up into capillaries, which merge at the periphery of each cell-ball to form a small vein, from which the larger trunks that pass from the lobules are derived. Each lobule is thus surrounded by a venous plexus from which the larger veins originate that leave the organ at sev- eral points. The cell-balls are composed of cellular cords, or trabeculae, the elements of which are extremely sensitive to the action of reagents. The cells are round or irregularly polygonal and separated from each other by a scanty reticular connective tissue. The capillaries already mentioned come in direct contact with the cells of the cell-balls. The organ contains a relatively large number of nerve-fibers and a few ganglion cells. As the individual grows older, the organ undergoes changes which finally make it unrecognizable. The former belief that the carotid gland was developed as an evagination of one of the visceral pouches has been replaced by a newer theory which gives it an origin solely from the vessel-wall (vid. Schaper). The structure of the coccygeal gland is in general like that of the carotid gland here described. TECHNIC (BLOOD AND BLOOD-FORMING ORGANS). Red blood-corpuscles may be examined in the blood fluid without special preparation. . The tip of the finger is punctured and a small drop of blood pressed out, placed upon a slide, and immediately covered with a cover-glass and examined. In such preparations the red blood- cells soon become crenated. The evaporation causing the crenation may be prevented by surrounding the cover-glass with oil (olive oil). A fluid having but slight effect upon the red blood-cells is Hayem's solution, which, however, is not adapted to the examination of leucocytes. It consists of sodium chlorid i gm., sulphate of soda 5 gm., corrosive subli- mate 0.5 gm., and water 200 gm. The fresh blood is brought directly into this solution, the amount of which should be at least one hundred times the volume of the blood to be examined. The fixed blood-cells sink to the bottom, and after twenty-four hours the fluid is carefully poured off and replaced by water. The blood-corpuscles are then removed with a pipet and examined in dilute glycerin. They may be stained with eosin and hematoxylin. Fresh red blood-corpuscles may also be fixed in osmic acid and other special fixing agents. This is done by dropping a small quantity of blood into the fixing fluid ; the blood-cells immediately sink and allow the osmic acid to be decanted ; they are then washed with water, drawn up with a pipet, and examined in dilute glycerin. Cover-glass Preparations. — A method almost universally used con- sists in preserving the blood-corpuscles in dry preparations. A drop of fresh blood is placed between two thoroughly cleaned cover-glasses, which are then quickly drawn apart, leaving on the surface of each a thin film of blood which dries in a few moments at ordinary room temperature. The specimens are further dried for several hours at a temperature of 120° C. After they have been subjected to this process, they may be stained, etc. The same results may be obtained by treating specimens dried in the air TECHNIC (BLOOD AND BLOOD-FORMING ORGANS). 227 with a solution of equal parts of alcohol and ether for from one to twenty- four hours, after which they are again dried in the air, and are then ready for further treatment. A cover-glass preparation of fresh blood may also be treated for a quarter of an hour with a concentrated solution of corrosive sublimate in saline solution, then washed with water, stained, dehydrated with alcohol and mounted in Canada balsam. A concentrated aqueous solution of picric acid may also be used, but in this case the specimen should remain in it for from twelve to twenty-four hours. The elements of the blood may also be examined in sections. Small vessels are ligated at both ends, removed, fixed with osmic acid, corrosive sublimate, or picric acid, and imbedded in paraffin. After fixation by any of the above methods the blood-cells may be stained. Eosin brings out very well the hemoglobin in the blood- cells, coloring it a brilliant red ; the stain should be used in very dilute aqueous or alcoholic solutions (i% or less), or in combination with alum (eosin i gm., alum i gm., and absolute alcohol 200 c.c., E. Fischer). Eosin may also be used as a counterstain subsequent to a nuclear stain — for instance, hematoxyiin. The preparation is stained for about ten min- utes, then washed in water or placed in alcohol until the blood-cells alone remain colored ; the cover-glass preparation should then be thoroughly dried between filter-paper and mounted in Canada balsam. Besides eosin, other acid stains — as orange G, indulin, and nigrosin — have the property of coloring blood-cells containing hemoglobin. Blood platelets are best fixed with osmic acid, and may be seen without staining. They may also be stained and preserved in a sodium chlorid solution to which methyl-violet is added in a proportion of i : 20000 (Bizzozero, 82). Afanassiew adds 0.6% of dry peptone to the solution (this fluid must be sterilized before using). Ehrlich's Granulations. — The leucocytes of the circulating blood and those found in certain organs possess granulations which were first studied by Ehrlich and his pupils, and which may be demonstrated by certain methods. The names given to these granulations are based upon Ehrlich's classification of the anilin stains, which differs from that of the chemist. This author distinguishes acid, basic, and neutral stains. By the acid stains he understands those combinations in which the acid is the active staining principle, as in the case of the picrate of ammonia. Among these are congo, eosin, orange G, indulin, and nigrosin. The basic stains are those which, like the acetate of rosanilin, consist of a color base and an indifferent acid. To these belong fuchsin, Bismarck brown, safranin, gentian, dahlia, methyl-violet, methylene-blue, and tolui- din. Finally, the neutral anilins may be considered as those stains which, like the picrate of rosanilin, are formed by the union of a color base with a color acid. The granula may be demonstrated in dry preparations as well as in those fixed with alcohol, corrosive sublimate, glacial acetic acid, and sometimes even Flemming's solution. Five kinds of granules are distinguished, and designated by the Greek letters from alpha to epsilon. The a-granules (acidophile, eosinophile) occur in leucocytes of the normal blood, in the lymph, and in the tissues, and are differen- tiated from the others by their peculiar staining reaction to all acid stains. They are first treated for some hours with a saturated solution of an acid 228 THE CIRCULATORY SYSTEM. stain (preferably eosin) in glycerin, washed with water, subsequently col- ored with a nuclear stain (as hernatoxylin or methylene-blue), and then dried and mounted in Canada balsam. Sections may be treated in the same way, with the exception that after being washed with water, they are first dehydrated with absolute alcohol before mounting in balsam. Another method by which both nuclei and granules are stained consists in the use of Ehrlich's hernatoxylin solution (see page 43), to which 0.5% eosin is added. Before using, the. solution should be permitted to stand exposed to the light for three weeks. This mixture stains in a few hours, after which the preparation is washed with water, treated with alcohol, and then mounted in Canada balsam. The a-granules appear red, the nuclei blue. The /3-granules (amphophile, indulinophile) stain as well in acid as in basic anilins. They do not occur in man, but may be observed in the blood of guinea-pigs, fowl, rabbits, etc. They are demonstrated as follows : Equal parts of saturated glycerin solutions of eosin, naph- thylamin-yellow, and indulin are mixed, and the dried preparations treated with this combination for a few hours, then washed with water, dried between filter-paper, and mounted in Canada balsam. The amphophile granules are stained black, the eosinophile granules red, the nuclei black, and the hemoglobin yellow. The T' -granules, or those of the mast-cells, are found in normal tissues and also in small quantities in normal blood, and are found in larger numbers in leukemic blood. They may be shown by two methods : (i) A mixture is made consisting of concentrated solution of dahlia in glacial acetic acid 12.5 c.c., absolute alcohol 50 c.c. , distilled water 100 c.c. (Ehrlich). The treatment is the same as for the amphophile gran- ules ; (2) Westphal's alum-carmin-dahlia solution (vid. Ehrlich). This mixture is used in staining dry preparations as well as sections of objects fixed for at least one week in alcohol. Alum i gm. is dissolved in dis- tilled water 100 c.c., and carmin i gm. added. The whole is then boiled for one-quarter hour, cooled, filtered, and 0.5 c.c. of carbolic acid added (Grenacher's alum-carmin, see page 42). This solution is now mixed with 100 c.c. of a saturated solution of dahlia in absolute alcohol, glycerin 50 c.c., and glacial acetic acid 10 c.c., the whole stirred and allowed to stand for a time. The specimen is stained for twenty-four hours, decolorized in absolute alcohol for the same length of time, and finally mounted in Canada balsam. The ^-granules are colored a dark blue and the nuclei red. A simpler method of demonstrating the ^-granules consists in overstating dry and fixed cover-glass preparations with a saturated aqueous solution of methylene-blue, decolorizing for some time in absolute alcohol, drying between filter-papers, and mounting in Canada balsam. The ^-granules (basophile) occur in mononuclear leucocytes of the human blood. Their staining may be accomplished in a few min- utes by treating fixed cover-glass preparations with a concentrated aqueous solution of methylene-blue, after which they are washed with water, dried between filter-papers, and mounted in Canada balsam. The e- or neutrophile granules which are found normally in the polynuclear leucocytes of man (as also in pus-cells), in some of the transitional cells, and in the myelocytes, are stained by Ehrlich as follows : 5 vols. of a saturated aqueous solution of acid fuchsin are mixed with i vol. TECHNIC (BLOOD AND BLOOD-FORMING ORGANS). 229 of a concentrated aqueous solution of methylene-blue. To this 5 vols. of water are added, and the whole allowed to stand for a few days, after which the solution is filtered. This mixture stains in five minutes, and the specimen is then washed with water, etc. The neutrophile granules are colored green, the eosinophile granules red and the hemoglobin yellow. Neutrophile and eosinophile granules may also be stained in Ehrlich's neutrophile mixture : Orange G, saturated aqueous solution, . . 130 to 135 c.c. Acid fuchsin, " " " . . 80 to 120 Methyl-green, " " " . . 125 Distilled water, 300 Absolute alcohol, 200 Glycerin, loo • Mix the above quantities of orange G, acid fuchsin, water, and alco- hol in a bottle and add slowly, while shaking the bottle, the methyl-green and finally the glycerin. The cover-glass preparations should be fixed in the ether and alcohol solution for about one hour, or fixed with dry heat at a temperature of no°C. for from fifteen to thirty minutes. Float the preparation on a small quantity of the stain for about fifteen minutes, wash in water, dry and mount in balsam. The red blood-cells are stained a reddish -brown color (brick-color), all nuclei a light blue-gieen, the eosinophile granules a fuchsin-red, and the neutrophile granules a violet- red. Griibler, of Leipzig, has prepared a dry powder, known as the Ehrlich-Biondi-Heidenhain three-color mixture, which is prepared for use by making a 0.4% solution in distilled water, to 100 c.c. of which are added 7 c.c. of a 0.5% aqueous solution of acid fuchsin. Wright's Method of Staining Blood Films. — This excellent and rapid method is especially recommended. Stain. — Make a one-half per cent, aqueous solution of sodium bicar- bonate in an Erlenmeyer flask and add to it one per cent, of methylene- blue. Steam for one hour in an Arnold steam sterilizer and allow mixture to cool, and when it is cold pour in a large dish. To 100 c.c. of this solution add about 500 c.c. of a one-tenth per cent, aqueous solution of eosin (Grubler's yellowish eosin, soluble in water). The quantity of the eosin solution can not be definitely given ; it is added while constantly stirring until the solution becomes of purple color and a yellowish scum with metallic luster forms on the surface and a finely granular black precipitate appears in suspension. The precipitate is col- lected on a filter and allowed to dry thoroughly. Make a saturated solu- tion in pure methylic alcohol (0.3 gm. of precipitate to 100 c.c. of methylic alcohol) and filter. To 80 c.c. of the filtrate 20 c.c. of meth- ylic alcohol is added to complete the stain. Staining of Blood Films. — Allow blood film to dry in the air and pour as much of the stain on the cover-glass or slide as it will hold, allowing it to remain in contact with the preparation for about one minute ; then add, drop by drop, enough water to make the stain semi- transparent, and a reddish tinge appears at the borders and a metallic scum on the surface. This diluted stain remains on the preparation two or three minutes. The preparation is now washed in distilled water until the better parts have a yellowish or reddish color. Dry quickly between filter-papers and mount on balsam. Red cells are orange or 23O THE CIRCULATORY SYSTEM. pink in color ; the nuclei, of blue color of varying intensity, eosinophile granules red, neutrophile granules reddish- lilac, basophile granules dark blue or almost black. The hemoglobin shows itself in the form of crystals. In certain teleosts the crystals are formed in the blood-corpuscles around the nuclei and often within a short time after death. In old alcoholic specimens, hemoglobi"n crystals (blood crystals) are found in the vessels and were first discovered here by Reichert in the blood of the guinea-pig. They have been found in large quantities in the splenic blood of a sturgeon which had been preserved for forty years in alcohol. The hemoglobin crystals belong to the rhombic series of crystallographic classification. The simplest method of demonstrating hemoglobin crystals is probably the following : The blood is first defibrinated by whipping or agitating with mercury, after which process sulphuric ether is added, drop by drop, until the mixture has been made laky ; this change may be detected macroscopically by the sudden change from an opaque to a dark, trans- parent, cherry-red color. No red blood-cells should now be seen under the microscope. The preparation is placed on ice for from twelve to twenty-four hours after which a drop of the blood is placed on a slide. In half an hour it will be seen that the margin of the drop has begun to dry. A cover-slip is now applied and, after a few minutes, numerous crystals are seen to form at the margin of the drop, a process which may be followed under the microscope. Large hemoglobin crystals are ob- tained by Gscheidtlen as follows : Defibrinated blood is placed in a glass tube, which is then hermetically sealed. The blood is now sub- jected to a temperature of about 40° C. for two or three days ; if then the glass be broken and the blood poured into a flat dish, large hemoglobin crystals are immediately formed. Crystals also appear if a drop of laky blood be placed in a thick solution of Canada balsam in chloroform and covered with a cover-slip. Hemin crystals (Teichmann's crystals ; hemin is hematin-chlorid) in the shape of rhombic plates are very easily obtained from the blood. A drop of the latter is placed on a slide and carefully mixed with a small drop of normal salt solution. This is then carefully warmed until the fluid evaporates and leaves a reddish-brown residue, after which a cover- glass is applied and glacial acetic acid added until the space between slide and cover-glass is filled. The preparation is now heated until the acetic acid boils. As soon as the latter evaporates, Canada balsam may be brought under the cover-glass, thus producing a permanent specimen. When fluids or stains suspected of containing blood are to be examined, the hemin crystals become of the utmost importance, as their demonstra- tion is then a positive indication of the presence of blood. Fluids are evaporated and treated with glacial acetic acid as above directed. Sus- pected blood stains on cloth are treated as follows : Small pieces are cut from the cloth in the region of the stain, soaked in normal salt solution, and the resulting fluid treated as above. If the stain is on wood or other solid object, the stain is scraped off and dissolved in normal salt and then tested for hemin crystals. Hemin crystals are almost or entirely insoluble in water, alcohol, ether, ammonia, glacial acetic acid, dilute sulphuric acid, and nitric acid. They are, however, soluble in potassium hydrate. A third form of crystals occasionally found in the blood and fre- quently in the corpora lutea and, under pathologic conditions, also in apo- TECHNIC (BLOOD AND BLOOD-FORMING ORGANS). 231 plectic areas, are the hematoidin crystals first discovered by Virchow. Masses of these crystals have an orange color. Microscopically, they appear as red rhombic plates. As they are soluble in neither alcohol nor chloroform, they are easily preserved in Canada balsam. Their artificial production has as yet never been accomplished. Hematoidin contains no iron. The fibrin thrown down when the blood coagulates may be dem- onstrated upon the slide in the form of very fine particles and fila- ments. A drop of blood is brought upon the slide and kept for a time in a moist chamber or on the table until it begins to clot ; after which a cover-slip is applied and the preparation washed with water by continued irrigation. In this manner most of the red blood-corpuscles are removed. Lugol solution may now be added, which stains brown the filaments of the fibrin network adherent to the slide. In order to see the fibrin net- work in sections, it is better to use specimens previously fixed in alcohol ; the sections are stained for ten minutes in a concentrated solution of gen- tian-violet in anilin water (Weigert), rinsed in normal salt solution, treated for about ten minutes with iodo-iodid of potassium solution, and then spread upon a slide and dried with filter-paper. They are now placed in a solution consisting of 2 parts of anilin oil and i part of xylol until they become perfectly transparent. This solution is then replaced by pure xylol and finally by Canada balsam. The fibrin network is stained a deep violet. Blood Current. — There are different methods and a variety of mate- rial at our disposal for the demonstration of the blood current through the vessels. The best object for this purpose is probably the frog. The proce- dure is as follows : The animal is immobilized by poisoning with curare. ^ gm. of a i % aqueous solution injected into the dorsal lymph-sac will immo- bilize the frog in a short time. The exact dose can not, however, be given, as the commercial curare is not a uniform chemical compound ; the dose must therefore be ascertained by experiment. As is well known, curare affects exclusively the nerve end-organs of striated voluntary muscle, but does not affect either the heart muscle or unstriated muscular tissue ; hence the utility of curare for this purpose. In order to see the blood current, it is only necessary to stretch the transparent web between the frog's toes and fasten it with insect needles to a cork plate having a suitable open- ing. If the cork plate be large enough to accommodate the whole frog, it may be placed in such a position that its opening lies over that in the stage of the microscope. The web thus spread out may be examined with a medium magnification. The tongue of the frog is also used for the same purpose. As the latter is attached to the anterior angle of the lower jaw, it may be conveniently drawn out, suitably stretched, and then placed over the hole in the cork plate. A very good view of the circula- tion may be obtained by examining the mesentery of a frog. The migra- tion of the leucocytes through the vessel-walls can also be studied in such preparations. An incision 0.5 cm. in length is made in the right axillary line through the skin of a frog (best in the male), care being taken not to injure any vessels (which can be seen through the skin in frogs possessing little pigment). The abdominal muscles are then incised and a pair of forceps introduced to grasp one of the presenting intestinal loops. The latter is then attached to the cork plate with needles, and the mesentery carefully stretched over the opening. On examining the specimen it is best to moisten it with normal salt solution and to cover the area to be 232 THE CIRCULATORY SYSTEM. examined with a fragment of a cover-glass. The lung may also be ex- amined, but here the incision must be farther forward. Counting Blood=cells. — The instrument now generally used for this purpose is the Thoma-Zeiss hemocytometer. This apparatus consists of two parts : pipettes by means of which the blood is diluted 100 times, when counting red, or 10 times when white blood-cells are to be counted, and a glass slide, on which there is a small well of known depth, the bot- tom of the well being divided off into small squares. The pipette used when counting the red cells consists of a capillary tube, near the middle of which there is an ampullar enlargement. This is so graduated that the cubical contents of the capillary tube is just one-hundredth part of the cubical contents of the ampulla. The blood to be examined is drawn into the capillary tube to a line marked i (just below the ampulla); the end of the pipette is then inserted into the diluting fluid, and this is sucked up until the diluted blood reaches a line marked 101 (just above Fig. 179. — Thoma-Zeiss hemocytometer: a, Slide used in counting ; b, sectional view ; f, a portion of ruled bottom of the well ; ( counted. J the number of white = blood-cells found in n (number of squares counted). a cubic millimeter. Or, multiply the average number of white corpuscles in each square by 40,000. Lymph-glands. — To obtain a general idea of the structure of lymphatic glands, sections are made of small glands fixed in alcohol or corrosive sublimate. They are then stained with hematoxylin and eosin. In such preparations the cortical and medullary substances can be studied ; the trabeculae and blood take the eosin stain. The flattened endothelial cells covering the trabeculae are brought to view by injecting a o. i% solution of silver nitrate into a fresh lymph- atic gland. After half an hour the gland is fixed with alcohol and car- ried through in the regular way ; the sections should be quite thick (not under 20 //). After the sections have been mounted in Canada balsam and exposed to light for a short time, the endothelial mosaic will be seen wherever the silver nitrate has penetrated. 234 THE CIRCULATORY SYSTEM. Fixing with Flemming's solution and staining with safranin is the best method for studying the germ centers of the lymph-follicles. Other fluids which bring out the mitoses may also be employed. Reticular tissue is best demonstrated by sectioning a fresh gland with a freezing microtome, removing a section to a test-tube one-quarter filled with water, and agitating it. The lymphocytes are thus shaken out of the meshes of the reticulum, leaving the latter free for examination. The same results can be obtained by placing a section prepared in the above-named manner upon a slide, wetting it with water, and carefully going over it with a camel's-hair brush. The lymphocytes ad- here to the brush. Both methods (His, 61) may be applied to hardened sections which have lain in water for a day or so. In this case, how- ever, the removal of the lymphocytes is not so easy as in fresh sections. In thick sections the reticulum is hidden by the lymphocytes. If, on the other hand, very thin sections (not over 3 /*) be made, especially of objects fixed in Flemming's solution, the adenoid reticulum stands out clearly without any further manipulation. The reticular structure may also be demonstrated by an artificial digestion of the sections with trypsin. The sections are then agitated in water, spread on a slide, dried, then moistened with a picric acid solu- tion (i gm. in 15 c.c. of alcohol and 30 c.c. of water), again dried, cov- ered with a few drops of fuchsin S solution (fuchsin S i gm., alcohol 33 c.c., water 66 c.c.), and left to stand for half an hour. The fuchsin solution is then carefully removed, the section washed again for a short time in the same picric acid solution, then treated with absolute alcohol, xylol, and finally mounted in Canada balsam. The reticular tissue of both lymphatic glands and spleen are stained a beautiful red (F. P. Mall). (See also page 129.) The treatment of splenic tissue is practically the same as that of the lymphatic glands. In all these organs (lymph-glands, spleen, and bone-marrow) a certain amount of fluid may be obtained by scraping the surface of the fresh tissue. This may then be examined in the same manner as blood and lymph (see Technic of same). Sections of lymph -glands and spleen previously fixed in alcohol, mercuric chlorid, or even in Flemming's solution may be examined by the granula methods of Ehrlich. By using the chrome-silver method a peculiar network of retic- ular fibers may be seen in the spleen. (Gitterfasern ; Oppel, 91.) The examination of the bone=marrow belongs also to this chap- ter. The marrow of the diaphysis is taken out by splitting the bone longitudinally with a chisel. With a little practice, it is easy to obtain small pieces of the marrow, which are then fixed by the customary methods and cut into sections. In the epiphysis the examination is confined either to the pressing out of a small quantity of fluid with a vice, or to the decalcification of small masses of spongy bone, containing red bone-marrow. In the first case, methods applicable to blood examina- tion are employed ; in the second, section methods (see also the petrifi- cation method, page 132) are used. The methods given for the prepara- tion of lymph -glands and spleen are also applicable in many cases. THE ORAL CAVITY. 235 TECHNIC (CIRCULATORY SYSTEM). To obtain a topographical view of the layers composing the heart and vessels, sections are made of tissues that have been fixed in Miiller's fluid, chromic acid, etc. If the specimens are to be studied in detail, small pieces must be used, and are best fixed in chromic-osmic mixtures or corrosive sublimate. Celloidin imbedding is recommended for general topographic work. The further treatment is elective. The endothelium of the intima may be brought to view by silver nitrate impregnation methods, by injecting silver solutions into the vascu- lar system. The endothelial elements of the smallest vessels and capil- laries are then clearly defined by lines of silver. Larger vessels must be cut open, the intima separated, and pieces of its lamellae examined. Elastic elements, plates and networks are best observed in the tunica media of the vessels, very small pieces of which are treated for some hours with 33% potassium hydrate. The appropriate stains for sectionwork are those which bring out the elastic elements and the smooth muscle-cells. For the former, orcein is used. For demonstrating the distribution of the capillaries, the reader is referred to the injection methods. The lymph-capillaries are injected by puncture ; compare also the methods of Altmann. III. THE DIGESTIVE ORGANS. THE intestinal canal with the glands derived therefrom originates from the inner layer of the blastoderm, the entoderm. The latter, however, does not extend to the external openings of the body, as the ectoderm forms depressions at these points which grow inward toward the still imperforate fore and hind gut to communicate finally with its lumen. This applies as well to the formation of the primitive oral cavity, which is separated only secondarily into oral and nasal cavities, as to the anus. The anterior boundary between the ectodermal and entodermal portions of the digestive tube consists of a plane passing through the opening of the pos- terior nares and continued downward along the palatopharyngeal arch. Everything lying anterior to this is of ectodermal origin, therefore the entire oral and nasal cavities with their derivatives. The lining of these cavities consists, however, of a true mucous membrane, closely resembling in its structure that of the intestinal tract. A. THE ORAL CAVITY. The epithelium of the oral mucous membrane is of the stratified squamous type, differing from the epithelium of the epidermis in that the stratum granulosum does not appear here as an inde- pendent layer. The stratum lucidum is also wanting, and the 236 THE DIGESTIVE ORGANS. cornification of the layer analogous to the stratum comeum of the skin is not complete (compare Skin). In the mucous membrane the cells of even the most superficial layers contain nuclei, which, although partly atrophied, still show chromatin, and as a conse- quence are easily demonstrated. Beneath the epithelium lies a tissue of mesodermic origin, also belonging to the mucous membrane and known as the mucosa or stratum proprium (lamina propria, tunica propria), in which nu- merous glands are situated. The mucosa consists of a fibrillar connective tissue with few elastic fibers, and of adenoid tissue containing numerous lymphoid cells ; essentially, therefore, a diffusely distributed adenoid tissue with occasional lymph-follicles imbedded in its substance. The mucosa presents numerous papillae, which are either simple or compound (branched) eleva- tions of the mucosa, varying in length and density, according to their location and extending for variable distances into the over- lying epithelium. As in the papillary layer of the corium (see Skin), so also here the superficial layer of the stratum proprium contains very fine elastic and connective -tissue elements which con- tribute to the structure of the papillae. All these papillae contain capillaries and arterioles which are derived from an arterial network in the mucosa. The lymphatics are similarly arranged. At the red margin of the lips the papillae are unusually high and are covered at their summits by a very thin epithelial layer (Fig. 1 80). Besides the sebaceous glands which lie at the angles of the mouth, and whose ducts open at the surface, there are here no other glandular structures. In the mucosa of the mucous membrane of the lips and cheeks the papillae are low and broad ; here also open the ducts of compound lobular, alveolar glands, the glandules labiales and buccales whose structure is similar to that of the large salivary glands (see these). The gums possess very long and attenuated papillae, covered by a very thin layer of epithelium, therefore bleeding at the slightest injury. That part of the gum covering the tooth has no papillae. The gums contain no glands. The papillae of the hard palate are arranged obliquely, with their points directed toward the opening of the mouth. The papillae of the soft palate are very low and may even be absent. They are somewhat higher on the anterior surface of the uvula. On the posterior surface of the latter occur ciliated epithelia distributed in islands between the areas of stratified squamous epithelium. In the soft palate and uvula are found small mucous glands. Under the mucous membrane there is a layer consisting princi- pally of connective tissue and elastic fibers, the submucosa (stratum submucosum, tela submucosa). In the mucous membrane of the mouth the transition of the tissue of the mucosa into that of the submucosa is very gradual. The submucosa of the hard palate is closely connected with the periosteum and contains, especially at its posterior portion, numerous glands. In other regions of the THE ORAL CAVITY. 237 mouth (lip) the glands extend also into the submucosa. The mucosa and epithelium lining the mouth cavity are richly supplied with nerves which terminate either in special sensory nerve-endings or in free sensory nerve-endings, or on the blood- vessels. In the papillae of the mucosa are found corpuscles of Krause. (See p. 169.) The nerves terminating in free sensory endings are the dendrites of sensory neurones (medullated sensory nerves), which, while yet medullated, branch and form plexuses with large meshes, situated in the submucosa and deeper portion of Transitional zone with irregular papillae. Mucous ; — ""~-~,M o d i - / fied ep- / iderm- — '- Striated v / muscle. Duct of j-^ gland. Epithe- — 1 i u m of mu- cous mem- brane. Gland. Fig. 180. — Section through the lower lip of man ; X l&- the mucosa. The medullated branches of the nerve-fibers constitut- ing these plexuses proceed toward the epithelium, dividing further in their course. Immediately under the epithelium the medullated branches lose their medullary sheaths, divide further, and form the subepithelial plexuses. The nonmedullated branches enter the epithelium, where they form telodendria (end-brushes), the terminal branches of which surround the epithelial cells, between which they end either in very fine granules or in small groups of such, or, again, in variously shaped end-discs. (See Fig. 135.) The blood- 238 THE DIGESTIVE ORGANS. vessels are richly supplied with vasomotor nerves, the neuraxes of sympathetic neurones, which terminate on the muscle-cells of the vessels. In the adventitia are also found free sensory nerve- endings. (See Fig. 177.) I. THE TEETH. The human dentition comprises twenty temporary or milk teeth, namely, above and below, four incisors, two canines, and four molars, which are replaced by thirty-two permanent teeth, con- sisting of four incisors, two canines, four premolars, and six molars for each jaw. Each tooth consists of a crown, which projects above the gums, a relatively short and narrowed portion known as the neck, and a portion which fits accurately into the alveolus and is known as the root. For the variations in shape which the different kinds of teeth present, the reader is referred to the text- books of anatomy or to special works dealing with this subject. Structure of the Adult Tooth. — The adult tooth is made up of three substances — the enamel, the dentin, and the cementum. The latter covers that part of the tooth within the alveolar process of the jaw and also the root of the tooth. The enamel caps that part of the tooth projecting into the oral cavity, the crown of the tooth. The neck of the tooth is the region where the cementum and enamel come in contact. The greater part of the tooth consists of dentin, which is present in the crown as well as in the root. All the substances of the tooth just mentioned become very hard from the deposition of lime-salts. Every tooth contains a cavity sur- rounded by dentin, the pulp cavity, or dental cavity. This is filled with a soft tissue, the pulp, consisting of white fibrous tissue, ves- sels, and nerves. That part of the pulp cavity lying in the axis of the fang is called the root-canal ; by an opening in the latter (fora- men apicis dentis) the pulp is connected with the periosteal con- nective tissue of the dental alveolus. The enamel is a very hard substance, the hardest in the body, and may be compared to quartz. In uninjured teeth the enamel is covered by an exceedingly thin, structureless membrane, the cuti- cula dentis or Nasmyth's membrane, which varies in thickness, measuring from 0.9 fj. to 1.8 //. It is very resistant to acids and alkalies. On its under surface it often shows small pits, into which project the ends of the enamel prisms. The enamel contains very little organic substance (from 3^ to 5^), in consequence of which it is soluble in acids with scarcely any residue. The elements composing it are prismatic columns, the enamel prisms, which probably occupy the whole thickness of the enamel from the superficial membrane to the dentin. They are slightly thicker at the surface of the tooth than at the dentin, and in transverse section show a hexagonal or polygonal shape, and measure from 3 /j. to 6 // in diameter. They often show quite regular THE TEETH. 239 — Enamel. — Pulp cavity. transverse markings which express, however, no structural pecu- liarity, but are due to irregularities in the prisms. They are joined to each other by a cement-substance which is somewhat more resistant than the substance of the prisms themselves. In the adult they are entirely homogeneous, but in embryos and even in the new-born they show a (fibrillar) longitudinal striation. In their course through the thickness of the enamel they change their direction by a series of symmetrical curves, and f cross each other in groups in a typical manner. There are also seen in the enamel the parallel lines known as the lines of Retzius (see Fig. 181), which pass obliquely through the enamel and which are to be regarded as traces of the strata caused by the periodic deposition of lime- salts ; they are very vari- able, as their structure depends on the nutritive condition during the depo- sition of the lime - salts (Berten). Another series of parallel or nearly par- allel stripes or lines, known as Schrdgcrs lines, are also observed. Those in the lateral portions of the enamel have a direction nearly perpendicular to the surface. They are thought to be due to a difference in the refraction of the light, presented by bundles or layers of enamel prisms so disposed as to be -' X___^ cut in different directions. The dentin is, next to the enamel, the hardest tissue of the tooth. After decalcification it presents a ground substance in which are found numerous very fine fibrils, which do not branch nor anastomose, and are in their behavior toward acids and alkalies like the fibrils of white fibrous (collagenous) tissue. They yield gelatin on boiling. The fibrils are separated by Dentin. Cementum. Fig. 181. — Scheme of a longitudinal section through a human tooth. In the enamel are seen the "lines of Retzius." 240 THE DIGESTIVE ORGANS. an interfibrillar substance, in which the mineral salts are deposited. The course of the fibrils is, in the main, parallel to the surface of the dentin. They are often grouped in small bundles (v. Ebner). The dentin is permeated by a system of canals having usually a transverse direction, the so-called dentinal tubules, which are from 1.3 fjt to 4.5 // in diameter. These originate in the pulp cavity, and during their course become slightly curved, like the letter S- The dentinal tubules usually present several dichotomous divisions near their origin, then pass to the outer third of the dentin without conspicuous divisions; here they again branch, becoming constantly smaller. In their course they give off numerous side twigs which m •llllli ifc — Enamel. Branching of the dentinal tubules. — Dentinal tubules. Interglobular space. Fig. 182. — A portion of a ground tooth from man, showing enamel and dentin ; X 17°- anastomose with those of neighboring tubules. The general course of these tubules is shown in figure 181. Certain of the tubules pass for a short distance into the enamel, where they are found between the prisms. In the human tooth the majority end just before reach- ing the enamel. In the root of the tooth they end near the surface, or in the interglobular spaces (see below), or, again, they may be joined to form loops. The dentinal tubules possess sheaths, the sheaths of Neumann, which may be isolated, analogous to the sheaths of the canaliculi of bone. They may be regarded as differ- entiated and more resistant ground substance. The dentinal tubules contain throughout their entire length filiform prolongations of cer- tain pulp-cells (odontoblasts), the dentinal fibers. Peculiar, irregu- THE TEETH. 241 larly branched spaces are often seen in the dentin. These are the interglobular spaces of Czermak. They represent areas in which calcification has not taken place. Their number is variable ; when relatively small and numerous, they appear, in dry preparations seen under low magnification, as a granular layer — the granular layer of Tomes. The cementum is closely adherent to the dentin, and consists of Fig. 183. — A, Longitudinal section through a human molar from the center of the enamel layer, decalcified with dilute hydrochloric acid ; B, tangential, C, radiate, and D, transverse section through the dentin of a human tooth, showing the fibrillar struc- ture of the ground-substance (taken from v. Ebner, 91) : a and b, Two layers in which the direction of the enamel prisms changes ; in c is seen a dentinal fiber with its sheath ; e, groups of fibrils ; d, dentinal tubules. bone tissue, the parallel lamellae of which contain, as a rule, no Haversian canals. There occur, however, cement lamellae, which in places lose their bone-cells. A peculiarity of the cementum is the presence of a large number of Sharpey's fibers, which are especially abundant in those areas containing no bone-corpuscles. These fibers are usually found in an uncalcified condition. The tooth=pulp is a tissue resembling embryonic connective tissue, consisting of connective-tissue fibrils, branched connective- tissue cells, and a semifluid, interfibrillar ground-substance. It is characteristic of this tissue that the fibrils never join to form con- nective-tissue fibers. It is probable that the fibrils are similar to those of white fibrous (collagenous) connective tissue (possibly retic- ular fibers), although there is a difference of opinion as concerns this point, by observers who have given it special attention (see v. Ebner, Rose). At the surface of the pulp is a continuous layer of cells, the odontoblasts. These are columnar cells with basal 16 242 THE DIGESTIVE ORGANS. nuclei and two or three processes extending into the canaliculi of the dentin, forming here the dentinal fibers already described. As a rule, the odontoblasts also send a single fiber into the pulp. These may intertwine and give rise to a network within its sub- stance. Peridental Membrane, Alveolar Periosteum. — The tooth is joined to the alveolus by a fibrous tissue membrane, the peridental mem- brane or alveolar periosteum, which represents the periosteum of the alveolus and the cementum of the tooth. This consists of bundles of connective tissue (elastic fibers are here absent) directly continuous with Sharpey's fibers in the cementum and the alveolus. Between these coarser bundles of fibers, which have a direction nearly horizontal in the upper portions of the peridental membrane and incline toward the lower end of the tooth in its lower portion, there is found a looser connective tissue, containing numerous nerve-fibers, blood-vessels, and peculiar masses of epithelial cells representing the remains of the enamel organs, to be described later. These epithelial remains have by some observers been regarded as glandular in nature; further observation is, however, needed before this can be accepted as proved. At the apex of the root there is found a less dense connective tissue, continuous with that of the tooth pulp. At the neck of the tooth the peridental membrane disappears in the submucosa of the gum. The blood-vessels of the teeth have been fully described by Lepowsky, who has studied them in a number of mammals, and in man in embryos and in full development ; his account is here fol- lowed. The artery, accompanied by the veins, enters through the apical foramen, passes up through the pulp, dividing into branches as it reaches the upper portion of the pulp cavity; these branches are spread out fan-shaped and after further division and the forma- tion of capillaries, end in capillaries which are situated between the layer of odontoblasts and the dentin, forming here a capillary plexus which presents narrow meshes, in regions where the odontoblasts are most active. There are in all probability no lymphatic vessels in the pulp. Numerous medullated nerve-fibers (dendrites of sensory neurones) enter the pulp cavity through the apical foramen. Some of these lose their medullary sheaths soon after entering, or just before entering, the pulp, and divide into long, fine, varicose fibers which interlace to form a loose plexus under the odontoblasts. Other medullated fibers, grouped into small bundles, ascend in the pulp for variable distances ; the nerve-fibers of the bundles then sepa- rate and as single fibers approach the superficial portion of the pulp, and, after losing their medullary sheaths, divide into fine varicose fibers forming under the odontoblasts a plexus continuous with the plexus above mentioned. From the varicose nerve-fibers THE TEETH. 243 Cementum. of this plexus small terminal branches are given off which termi- nate between the odontoblasts, or pass through the layer of odontoblasts, to end between these and the dentin (Retzius, 94 ; Huber, 98; Rygge, 1902). Medullated nerve-fibers also terminate in free endings in the peridental membrane. Development of the Teeth. — In the second month of fetal life the first traces of the teeth are seen in the development of a groove along the inner edge of the fetal jaw, the dentinal or en- amel groove. From the floor of the latter an epithelial ridge is formed constituting the anlage of the enamel organs and known as the dentinal ridge, or enamel ledge. At those points at which the milk-teeth later appear, the enamel ledge develops solid protuberances corre- sponding in number to the temporary teeth. These are known as the dentinal bulbs or enamel germs. In their first stage of development the enamel germs are knob- like, but later their bases spread, and they become flattened and finally cup- shaped by the pushing up into them of connective - tissue projections, the den- tinal papilla. At the same time they gradually sink deeper into the underlying tissue, but still remain con- nected, by means of a thin cord, with the epithelium of the enamel ledge, which now lies on the inner side of the enamel germs. The enamel germs now differentiate into enamel organs. In this stage they consist of an outer layer of columnar epithelial cells, which are to be regarded as a direct continuation of the basal cells from the epithelium of the oral mucous membrane, or still better, of the enamel ledge ; the epithelium in the interior of the organ is derived from the stratum Malpighii of the oral epithe- lium. The cells of this layer, however, undergo a change in shape and structure, in that an increased quantity of lymph-plasma or intercellular substance collects in the interspinous spaces between the cells, pushing the cells apart, and allowing their processes to develop until the cells finally assume a stellate shape. In this way the enamel pulp is gradually formed. The next stage is character- ized by a vertical growth of the dentinal papillae, which soon be- come surrounded on all sides by the cap-like enamel organs. The cylindric cells (enamel cells) of the enamel organs lying next to Dentin. Fig. 184. — Cross-section of human tooth, showing cement and dentin; X 2I2- At a are seen small interglobular spaces (Tomes' granular layer). 244 THE DIGESTIVE ORGANS. the papillae become lengthened, and after passing through further changes, finally develop into the enamel prisms of the teeth. At the periphery of the dentinal papillae, there is differentiated a layer of columnar cells, the odontoblasts, which have a connective-tissue origin, and later form the dentin. During these processes a connective -tissue mantle, the dental sac, rich in cellular and fibrous elements, is formed around each tooth anlage. The earliest appearance of the enamel is in the form of a cuticu- lar membrane, developed from the ends of the enamel cells resting on the dentinal papilla, this cuticular membrane appearing in the form of a thin layer covering the top of the dentinal papilla. Sometime later, short striated processes — Tomes' processes — appear on the Odontoblasts. Terminal nerve- fiber. Odontoblasts. - - Terminal nerve- fiber. Fig. 185. — Nerve termination in the pulp of a rabbit's molar, stained in methylene- blue (intra vi(am) : a, Odontoblasts seen in side view ; b, a number of odontoblasts seen in end view, showing a terminal branch of a nerve-fiber situated between the odonto- blasts and the dentin (Huber, "Dental Cosmos," October, 1898). lower end of each of the enamel cells (the end toward the dentinal papilla). These are imbedded in a cement-substance, forming a continuous layer. The Tomes' processes are regarded as the be- ginnings of the enamel prisms. Calcification begins in the middle of these processes ; they thicken at the expense of the cement- substance surrounding them, which later also calcifies. The enamel as a whole thickens by the elongation of the Tomes' processes of the enamel cells and by their subsequent calcification. The process ends finally in the death and partial absorption of the enamel cells and the remaining elements of the enamel organs ; these structures persist for a short time after the eruption of the tooth as a cuticular sheath. The dentin is developed by the odontoblasts by a process analogous to that observed in the formation of bone by the osteo- blasts. These epithelioid cells secrete at their outer surfaces a homogeneous substance which fuses to form a continuous layer, the membrana pr&formativa. The further development of the dentin is as follows : Its ground-substance is deposited at the cost of the lateral portions of the odontoblasts (under the membrana praeforma- Fig. 186. Fig. 187. Fig. 188. Fig. 189. Figs. 186-189. — Four stages in the development of a tooth in a sheep embryo (from the lower jaw) ; Fig. 186, Anlage of the enamel germ connected with the oral epithelium by the enamel ledge ; Fig. 187, first trace of the dentinal papilla ; Fig. 188, advanced stage with larger papilla and differentiating enamel pulp ; Fig. 189, budding from the enamel ledge of the anlage of the enamel germ, which later goes to form the enamel of a permanent tooth ; at the periphery of the papilla the odontoblasts are beginning to differentiate. Figs. iS6, 187, and 188, X IIO5 Fig. 189, X 4°- rt> a> a> a< Epithelium of the oral cavity ; b, b, b, l>, its basal layer ; c, t, c, the superficial cells of the enamel organ ; d, d, d, d, enamel pulp ; /, />, p, dentinal papilla ; .r, s, enamel-forming elements (enamel cells) ; Fundus gland; b, three pyloric glands; the one at the left is a simple tubular gland, the middle one a branched tubulo-alveolar gland ; the one at the right a typical pyloric gland of the branched tubular variety ; <-, leaf-shaped villi and crypts of Lieberkiihn of the duodenum ; d, crypts of Lieberkiihn of the large intestine. 18 2/4 THE DIGESTIVE ORGANS. while the nuclei return to the center of the cells. Since chemic examination has shown that the amount of pepsin found in the gas- tric mucous membrane increases with the enlargement of the chief cells of the body of the fundus glands, and decreases with their diminution in size, there can be hardly any doubt that this ferment is elaborated by these cells. It is assumed that the parietal cells secrete the acid of the gastric juice, although, in spite of all efforts, it has not yet been definitely proved that these cells possess an acid reaction. The vascular and lymph-vessels of the stomach, and also its nerve supply, will be considered in a general discussion of these structures pertaining to the entire intestinal canal. 3. THE SMALL INTESTINE. The mucous membrane of the small intestine is characterized by the presence of villi. The villi vary in size and shape in the different mammals. In man, in the upper portion of the small in- testine, they are distinctly leaf-shaped, being three to four times as broad in one direction as they are in the other, often showing a narrowing at their bases. This has been shown by reconstruction of the mucosa and a number of villi from the duodenal region of a well-preserved human intestine. The villi are of columnar shape in the jejunum, and club-shaped in the ileum. The mucous mem- brane also forms permanent folds in both the duodenum and the remainder of the small intestine, the valvulae conniventes (Kerk- ring). Upon these the villi rest, and, indeed, it is probable that the very existence of the plicae is due to the blending of the basilar ends of the villi. The epithelium of the intestinal mucous membrane covers the villi in a continuous layer, and penetrates into the mucosa to form the glands. Its structure is essentially the same in all regions of the small intestine, the cells being of the high columnar variety with free surfaces covered by wide, striated cuticular borders. The basilar portions of these cuticular borders are nearly always homo- geneous, and upon vertical section give the appearance of a fine line. The cuticular borders of adjacent cells blend with each other, form- ing a continuous membrane, large areas of which may be detached from the villi (cuticula). The body of the ce.U consists of a fine fibrillar structure (spongioplasm) with the main threads parallel to long axis of the cell. This is more distinct in the free portions of the cell. In the interfibrillar substance are found fine granules- The nuclei lie usually in the basilar third of the cells, and only where they show mitoses, as, for instance, in the tubular intestinal glands, do they pass to the free ends of the cells. The basal ends of the epithelial cells in the small intestine are also seen to be pointed, and the probability is that they rest upon the basement membrane. The question has, however, not been fully settled. THE STOMACH AND INTESTINE. 275 The epithelial cells undergo a special metamorphosis, after \vhich, by an increased production of mucus, they change into gob- let cells. From recent investigations it would seem that any epithelial cell, whether it be situated upon the upper surface of a villus or deep down in one of the tubules of the intestinal glands, is capable of transformation into a goblet cell. The number of goblet cells is subject to great variation ; they are found singly in small numbers, or are very numerous, according to the stage of digestion and quantity of food in the intestine. The manner in which an ordinary epithelial cell changes into a goblet cell is very easily explained if the mechanical action on the cell caused by an accumu- lation of secretion be taken into consideration. As the secretion increases in quantity the upper portion of the cell becomes distended, and the remains of the protoplasm, together with the nucleus, are pushed toward the nar- row base of the cell ; the cuticular zone is stretched, bulges into the lumen of the intes- tine, and is finally perfor- ated, and perhaps even thrown off. In this way the cell loses its mucous secretion, collapses, and then appears as a thin, almost rod - like struc- ture, with a long nu- cleus. It is the gener- ally accepted theory that such an empty goblet cell may again assume the shape of an ordinary epithelial cell and repeat the process just de- scribed. Leucocytes are some- times found within the epithelial cells, but more usually between them, and according to Stohr (84, 89, 94), when seen in these positions, are in the act of migrating into the lumen of the intestine. That some of these cells actually pass into the lumen is probably true ; but as yet no leucocytes have ever been observed in the cuticula itself, and neither is the number Gland (crypi) of Lieber- kuhn. Mucosa. Muscularis mucosae. Fig. 216. — Section through mucous membrane of human small intestine ; X 88. At a is a col- lapsed chyle-vessel in the axis of the villus. 276 THE DIGESTIVE ORGANS. the large intestine and rectum. of cells found in the lumen of the intestine proportionate to the leuco cytes present in the epithelium. Since many are seen in the epithe- lium undergoing karyokinetic division, it is more probable that a part of them actually wander into the epithelium for the purpose of division (chemotaxis ?), only to return to the mucosa after the com- pletion of the process (compare p. 61). Into the spaces between the villi open numerous tubular glands. These are seldom branched, and are known as Lieberkiiliri 's glands, or crypts. Their length varies from 320 // to 450 //. They are regularly arranged in a continuous row, and often have an ampulla- like widening of their lumina extending almost to the muscularis mucosae, but never quite reaching it. They are uniformly distrib- uted not only throughout the small intestine, but also throughout The cells lining the crypts of the small intestine are about one- half as long as those covering the villi ; a cuticu- lar border is seen on the cells lining the upper part of the glands, but is ab- sent in the cells lining the fundus of the glands. The cells are conical in shape, — a condition probably due to the curvature of the glandular wall, — the base of each cone lying toward the basement membrane, the apex toward the lumen of the gland — a condition opposite to that found in the villi. Numerous goblet cells are also present. They vary only slightly in shape during mucous secretion, and do not, as in the villi, assume the form of goblets with distinct pedicles. Mito- ses are always seen in the intestinal glands, especially in cells which do not con- tain mucin. They are readily distinguished, since the nuclei in process of division, as we have seen, lie outside of the row formed by the remaining nuclei. The plane of division in these cells lies horizontal to the long axis of the gland, so that an increase in the number of cells results in an increase in the area of the glandular walls. Mitoses are very rarely observed in the epi- Fig. 217. — Longitudinal section through sum- mit of villus from human small intestine ; X 9°° (Flemming's solution) : At a is the tissue of the villus axis ; b, epithelial cells ; c, goblet cell ; d, cuticular zone. THE STOMACH AND INTESTINE. thelium covering the villi. If, therefore, any cells be destroyed on the surface of the villi, it must be assumed that the loss is replaced by new elements pushed up from the glands below (Bizzozero, 89, 92, I). In the fundus of the crypts of Lieberkuhn of the small intes- tine are also found a variety of cells first described by Paneth, and known as the cells of Paneth. These cells contain granules which stain readily in eosin and in iron-lac-hematoxylin, and are no doubt cells which contain zymogen granules, cells which elaborate an enzyme. In the opossum the cells of Paneth are found not only in the crypts but also in epithelium of the villi intermixed with the columnar cells and goblet cells (Sidney Klein). The entire duodenum, as well as that part of the pylorus in the immediate vicinity of the pyloric valve, is characterized by the presence of glands of a second type. In the duodenum these are seen intermingled with the glands of Lieberkuhn, and in the pylorus with the pyloric glands. These glands, Brnnner's glands, have a diameter of from 0.5 to I mm., and are branched tubulo-alveolar glands, with tubules provided with alveoli, especially along their lower portions. The bodies of the glands are situated principally in the submucosa, although a part may be in the mucosa. In the stomach they open into the gastric crypts, in the intestine either in- dependently between the villi, or into the glands of Lieberkuhn. Here the glandular cells are in general similar to those of the pyloric glands, although, as a rule, somewhat smaller than the latter. The secretion of these glands is mucus (Bensley). Just as the duodenal glands extend into the stomach, so also the pyloric glands of the latter are found in the upper portion of the duodenum. Besides short villi, there are also present in the duodenum depres- sions of the mucous membrane analogous to the gastric crypts. The glands of Lieberkuhn begin at a certain distance from the pylorus ; at first they are short, and do not attain their customary length until a point is reached at which the pyloric glands extending into the duodenum finally disappear (yid. Fig. 218). It is therefore obvious that a transition zone exists between pylorus and duodenum, and that a distinct boundary line can not be drawn between the two, at least so far as the mucous membrane is concerned. The duodenal glands, as their name would indicate, are present only in the duod- enum. Between the jejunum and ileum there is no distinct boundary, not even when microscopically examined. The differences are mostly of a quantitative nature ; in the jejunum the valvulae conniventes are more numerous than in the ileum, ancj the villi more slender and closer together. The glands of Lieberkuhn also appear to be more numerous in the jejunum. The mucosa of the small intestine consists of reticular adenoid tissue containing mononuclear lymphocytes, polymorphonuclear leucocytes, and leucocytes with granular protoplasm. It sup- ports the glands and extends into the villi whose axes it 2/8 THE DIGESTIVE ORGANS. forms. The mucosa is separated from the glands, from the epithelium of the villi, as well as from that of the remain- ing surface of the intestine by a peculiar basement membrane. Longitudinal muscular layer. Sphincter »' pylori. • Submucosa. Muscularis mucosae. Pyloric glands. Brunner's glands. Longitudinal muscular layer. Circular mus- cular layer. Lymph- Villus. nodule. "- Muscularis mucosae. — Submucosa. Brunner's glands. \Blood-vessel. '"•••Glands of Lieberkiihn. Fig. ai8. — Section through the junction of the human pylorus and duodenum ; X about 15 : At a the pyloric glands extend into the duodenum. The latter somewhat complicates a proper histologic analysis, and as a consequence opinions regarding its structure and significance vary considerably. By some it has been described as a homo- geneous, hyaline, and exceedingly fine membrane containing nuclei, by others as a lamella consisting entirely of endothelial cells. At all events, there are certainly nuclei in the basement membrane. Beneath the basement membrane is a marginal layer of a more fibrillar character. This is closelv associated with the mucosa, and THE STOMACH AND INTESTINE. 279 may be regarded as a differentiation of the latter. Toward the muscularis mucosae the mucosa is terminated by a reticulated elastic membrane (F. P. Mall, in the dog), containing openings for the entrance of vessels, nerves, and muscle-fibers. The muscularis mucosce consists of two layers of unstriped muscular fibers arranged in a manner similar to that in the external muscular tunic — i. e., having an inner circular and an outer longi- tudinal layer. The fibers are frequently gathered into bundles, which appear to be separated from each other by connective tissue. From both layers, but more especially from the inner, muscle-fibers are given off at right angles, which enter the tunica propria and pass between the glands of Lieberkiihn, and even into the villi. In the latter these muscle-fibers are arranged in bundles, and lie Leucocyte inepithe- f/Ts^'- Hum. Epithelium. •—-'/-/ Intermedi- . ary zone. Submucosa. - Fig. 219. — Section of solitary lymph-nodule from vermiform appendix of guinea- pig, showing crypt ; X about 400 (Flemming's fluid). near their axes around the lacteal vessels. The contraction of these fibers causes a contraction of the entire villus. Lymph-nodules are distributed throughout the mucosa of the small intestine, occurring either singly, as solitary follicles, or aggregated, as Peyer's patches. At the points where they occur, the villi are absent and a lateral displacement of the glands of Lieberkiihn is observed. The lymph-nodule is usually pyriform in shape. The thinner portion protrudes somewhat in the direction of the lumen of the intestine, while the thicker portion extends outward to the muscularis mucosae, the latter being frequently in- dented or even perforated if the lymph-nodules be markedly devel- oped. Their structure is similar to that of the lymph-follicles (see under these), and consists of reticular adenoid tissue, supporting 280 THE DIGESTIVE ORGANS. lymph-cells. It should be remembered that every nodule may possess a germ center. Peyer's patches are collections of these lymph-follicles. The surface of the nodule presenting toward the lumen of the intestine is covered with a continuous layer of intestinal epithelium. In man the summit of that portion of the — ^ Intestinal epithelium. — Lumen of gland. — Goblet cell. Mucosa. — Mucosa. — Muscularis mucosae. Fig. 220. — From colon of man, showing glands of Lieberkiihn ; X 2O°- nodule projecting into the lumen of the intestine presents but a slight depression of the intestinal epithelium, while in some animals (guinea - pigs), and especially in the nodules composing Peyer's patches, there is a deeper depression, even leading to the formation of a so-called "crypt" or "lacuna" (vid. Fig. 219). At the summit, the intestinal epithelium where it cornes in contact with the lymph-nodule, is peculiarly altered. In most cases there is an absence of a basement membrane, the epithelium resting directly upon the lymphoid tissue. No clearly defined boundary between the two is distinguishable (intermediate zone of v. David- off) ; they are therefore in the closest relationship to each other. The basal surfaces of the epithelial cells are fibrillar, the fibrils seeming to penetrate into the adenoid reticulum of the follicles. THE STOMACH AND INTESTINE. 28l 4. THE LARGE INTESTINE, RECTUM, AND ANUS. The small intestine ends at the ileocecal valve. At some dis- tance from the margin of the valve the villi of the ileum become broad and low. In the immediate vicinity of the valve their basilar portions become confluent, forming a honeycomb structure which supports a few villi. At the base of the honeycomb open the glands of Lieberkuhn. On the cecal side of the valve the villi become fewer in number and finally disappear, while the folds which give the honeycomb appearance persist for a considerable distance. In Fig. 221. — Transverse section of human vermiform appendix; X 20. Observe the numerous lymph nodules. The clear spaces in the submucosa are adipose tissue. the adult cecum the villi are absent. The mucosa and glands pre- sent a structure similar to that of the remainder of the large intes- tine. In the mucosa of the vermiform appendix is found a relatively large number of solitary lymph-follicles, occasionally forming a continuous layer. The marked development of the lymph-follicles encroaches upon the glands of Lieberkuhn, so that many are obliterated ; they are penetrated by the adenoid tissue, the epithe- lial cells of the glands mingling with the lymph-cells. What finally becomes of the secretory cells has not been definitely ascertained (Riidinger, 91). 282 THE DIGESTIVE ORGANS. In the colon the villi are wanting, while the glands of the mucosa are densely placed and distributed with regularity. The glands of Lieberkiihn in the colon are somewhat longer, and as a rule contain many more goblet cells than those in the small intestine. Only the neck and fundus of the glands show cells de- void of mucus. Transitional stages between the latter and the goblet cells have been observed in man (Schaffer, 91). Solitary lymph-follicles are found throughout the colon. They are situated in the mucosa, only the larger ones extending into the submucosa. The glands of Lieberkiihn are displaced in the regions of the lymph- follicles. Gland. Submu- cosa. Fig. 222. — A solitary lymph-follicle from the human colon : At a is seen a pronounced concentric arrangement of the lymph-cells. The tanice and plica semilunarcs cease at the sigmoid flexure, and are replaced in the rectum by the plica transversales recti. Permanent longitudinal folds, the so-called columns rectales Mor- gagni, are present only in the lower portion of the rectum. Here the intestinal glands are longest but disappear simultaneously with the rectal columns. At the anus the mucous membrane of the rectum forms a narrow ring devoid of glands, covered by stratified pavement epithelium, and terminating in the skin in an irregular line. The transition from the mucous membrane to the skin is gradual, yet reminding one of the appearance presented at the junction of the esophagus with the cardiac end of the stomach. External to the anus, and at a distance of about one centimeter from it, are numerous highly developed sweat-glands, the circum- anal glands of Gay, which are almost as large as the axillary glands ; also sweat-glands of a peculiar type, in that they show a branching of the tubules (see Sweat-glands, under Skin). THE STOMACH AND INTESTINE. 283 5. BLOOD, LYMPH, AND NERVE SUPPLY OF THE INTESTINE. In general, the following holds true with regard to the blood- vessels of the intestinal tract (further details will be discussed in dealing with the vessels of the various regions of the intestine) : The arteries enter along the line of the mesenteric attachment and penetrate the longitudinal muscular layer. Between the two mus- cular layers branches are given off which form an intermuscular plexus, from which, in turn, smaller branches pass out to supply the muscles themselves. The arterial trunks penetrate the circu- lar muscular layer and form an extensive network of larger vessels in the deeper layer of the submucosa. This is known as Hellers plexus (F. P. Mall). From this, radiating branches are ; Epithelium of stomach. • — Region of the bodies of the gastric glands. - - Muscularis mucosae. Fig. 223- — Section through fundus of cat's stomach. The blood-vessels are injected ; X °°- given off which supply the muscularis mucosae, forming under the latter a close network of finer vessels. This plexus, together with that of Heller, gives rise to vessels which penetrate the mus- cularis mucosae and break up into capillaries in the mucous mem- brane. The veins of the mucous membrane form beneath the muscularis mucosae a plexus with small meshes, giving off many radiating branches ; these in turn unite to form an extensive net- work of coarser vessels. Veins extend from the latter and unite to form larger trunks, which then lie side by side with the arteries. According to F. P. Mall, delicate retia mirabilia occur here and there in the venous network in the submucosa of the intestine of the dog. In the esophagus the arteries end in a capillary network situated 284 THE DIGESTIVE ORGANS. in the mucosa and extending into the connective-tissue papillae of the mucosa. The vessels of the stomach are arranged in plexuses in the muscular coat, submucosa, and beneath the muscularis mucosae, as previously described. From the plexus beneath the muscularis mu- cosse, small branches are given off which pass through this layer and in the mucosa form a capillary network, consisting of relatively small capillaries, which surround the gastric glands, this plexus being par- ticularly well developed in the region around the body and neck of the glands, where the parietal cells are most numerous. The capil- laries of this network are continuous with capillaries of a much larger size, forming a network surrounding the gastric crypts and situated immediately under the epithelium lining the mucosa of the stomach. The blood is collected from this capillary plexus by small veins which pass nearly perpendicularly through the mucosa, forming a plexus above the muscularis mucosae, from which small veins pass through the muscularis mucosae to the venous plexus in the sub- mucosa. The blood-vessels of the mucosa of the small intestine may be divided into (i) the arteries of the villi and (2) the arteries of the intestinal glands. The former arise principally from the deep arterial network in the submucosa, then penetrate the muscularis mucosae and give off branches at acute angles which continue without further branching into the summits of the villi. Within the villi themselves the arteries lie in the axes. The broader villi may contain two arteries. The circular muscle-fibers of the arteries gradually disappear inside of the villi (dog), and at the summit of the latter the vessels break up into a large number of capillaries. These form a dense network extending beneath the basement mem- brane and into its marginal layer. These networks give rise to venous capillaries which unite to form small vessels and finally end in two or more larger veins inside of the villi. These latter are con- nected with the venous network in the mucosa. The glandular arteries, derived principally from the superficial network of the submucosa, also pass through the muscularis mucosae and break up internally into capillary nets which encircle the intestinal glands ; these give rise to small veins which empty into the venous plexus of the mucosa. The veins of the plexus in the mucosa unite to form larger branches, which connect with the plexus in the submucosa (compare Fig. 224). In the dog these trunks inside of the muscularis mucosae are encircled by bundles of muscle-fibers (sphincters, F. P. Mall). The capillaries of the solitary lymph-nodules do not always penetrate into the centers of the latter, but often leave a central nonvascular area. The blood-vessels of the mucosa of the large intestine are, in their distribution, similar to the glandular vessels of the small intes- tine and stomach. The lymph-vessels begin in the mucosa near the epithelium, pass THE STOMACH AND INTESTINE. 285 down between the glands, and are arranged in the form of a net- work just above the muscularis mucosae, but with coarser meshes than that formed by the blood-vessels. Here the valves begin to make their appearance. The lymph-vessels pass through the mus- cularis mucosae and in the outer portion of the submucosa form a plexus with open meshes, from which are derived the efferent ves- sels which penetrate the muscular coat and thus gain access to the mesentery. In their course through the muscular coat they com- municate with the branches of a plexus of lymph -vessels situated between the two muscular layers, and also with lymph-vessels found in the serous coat. Epithelium of villus. Central chyle- vessel of vil- lus. Vein. Fig. 224. — Schematic transverse section of the human small intestine (after F. P. Mall). The lymphatics of the small intestine begin in the axes of th'e villi. When filled, these lymph-vessels are conspicuous, irregularly cylindric capillary tubules, lined by endothelial cells, and known as the axial canals, the chyle-vessels, or the lacteals of the villi. They are hardly discernible when collapsed. If the villus be broad, it may contain two chyle-vessels, which then join at the apex of the villus, and may also be connected with each other by a few anasto- moses. At the base of the villus the chyle-vessel enters a lymphatic capillary network, the structure of which is due to the confluence 286 THE DIGESTIVE ORGANS. of similar canals. Numerous lymph-vessels from this network penetrate the mucous membrane in a vertical direction, uniting at the bases of the intestinal glands to form a second plexus — sub- glandular plexus of the mucosa. A few of the lymph-vessels pene- trating the mucous membrane directly perforate the muscularis mucosae to join the lymphatic network of the submucosa. The subglandular plexus also communicates with the submucous lymphatic plexus by means of small radiating branches (vid. Fig. 224). The solitary lymph-nodules themselves contain no lymphatic vessels, but are encircled at their periphery by a network of lymph capillaries. The same is true of the nodules in Peyer's patches. It is an interesting fact that in the rabbit lymph-sinuses exist around Peyer's patches, giving to the latter a still greater similarity to the nodules of lymph-glands. The solitary nodules of the same Fig. 225. — A portion of the plexus of Auerbach from stomach of cat, stained with methylene-blue (infra vitani), as seen under low magnification. animal are not surrounded by the sinuses just mentioned (Stohr, 94)- The structures of the alimentary canal receive their innervation mainly from sympathetic neurones, the cell-bodies of which are grouped to form small ganglia, located at the nodal points of two plexuses, one of which is situated between the two layers of the muscular coat, the other in the submucosa. These two plexuses are found in the entire digestive tract, although not equally well developed in its different regions. The outer plexus, the more prominent of the two, situated between the two layers of the muscu- lar coat, is known as the plexus myentericus, or the plexus of Auer- bach. It consists of innumerable small sympathetic ganglia, united by small bundles of nonmedullated fibers, containing here and there a much smaller number of medullated nerve-fibers. The cell-bodies of the sympathetic neurones of this plexus are grouped to form the THE STOMACH AND INTESTINE. 28; sympathetic ganglia. The dendrites, the number of which varies for the different cells, divide and redivide in the ganglia, some ex- tending into the nerve bundles uniting the ganglia. The neuraxes of the sympathetic neurones of the ganglia form nonmedullated nerve-fibers, which leave the ganglia by one of the several roots possessed by each ganglion, and, after repeated division and forming intricate plexuses in the circular and longitudinal layers of the mus- cular coat, terminate on the involuntary muscle-cells of these layers. The plexus in the submucosa, known as the plexus of ' Meissner, is similarly constructed, although it contains fewer and much smaller ganglia and the meshes of the plexus are much finer. It commu- nicates by numerous anastomoses with the plexus of Auerbach. The neuraxes of the sympathetic neurones of this plexus have not been traced, with any degree of certainty, to their terminations. Numerous nonmedullated nerves enter the muscularis mucosae and, according to Berkley (93, I), form in the dog terminal bulbs and nodules which perhaps rep- resent the endings of motor (sympathetic) nerves in this layer. Nerve-fibers have also been traced into the mucosa, and in the vicinity of the glands and in the villi are found numerous exceedingly fine nerve-fibers which inter- lace, but in the greater por- "tion of the intestinal tract the endings of these fibers have not been fully worked out. That they end on the gland- cells seems very probable from observations made by Kytmanow (96), who was able, by means of the methylene-blue method, to stain plexuses of fine nerve-fibrils surrounding the gastric glands of the cat, some of these fibrils being traced through the basement membrane of the glands and to and between the gland-cells, where they ter- minated in clusters of small nodules on both the chief and parietal cells. The plexus of Meissner is not so well developed in the esophagus as in the remaining portions of the digestive tract. That the cell-bodies of many of the sympathetic neurones of Auerbach's and Meissner's plexuses are capable of being stimulated by cerebrospinal nerves seems certain from observations made by Dogiel (95), who has shown that many small medullated nerve- fibers which enter the digestive tract through the mesentery (small and large intestines) terminate after repeated division in terminal end-baskets which surround the cell-bodies of many of the sympa- thetic neurones of these plexuses. Similar nerve-fibers ending in Fig. 226. — From thin section of esophagus of cat, showing the epithelium and a portion of the mucosa and the terminal nerve-fibrils in the epithelium (from preparation of Dr. DeWitt). 288 THE DIGESTIVE ORGANS. baskets have also been observed in the ganglia of the plexuses of the stomach and esophagus. Large medullated nerve-fibers, the dendrites of sensory neurones, have also been traced to the alimen- tary canal. In the esophagus these pass to the mucosa, where, after repeated division, they lose their medullary sheaths, the non- medullated terminal branches forming a subepithelial plexus from which terminal, varicose branches, further dividing, enter the strati- fied epithelium and may be traced to near the surface of the epithe- lium. Large medullated nerve-fibers may be traced through the ganglia of Auerbach's and Meissner's plexuses in the stomach and intestinal canal and through the nerve bundles uniting these ganglia (Dogiel, 99), but the termination of these fibers has not been deter- mined. In the large intestine of the cat they have been traced to the epithelium and between the epithelial cells covering the mucosa (Huber). 6. THE SECRETION OF THE INTESTINE AND THE ABSORPTION OF FAT. The cells of Brunner's glands are similar in many respects to those of the pyloric glands. They form, as has been shown, a mucous secretion, and present in their various physiological activi- ties, structural changes which are similar to the structural changes presented by the cells of other mucous glands under similar condi- tions (Bensley). It is well known that the goblet cells of the in- testinal glands are very numerous during starvation, and that they nearly disappear after continued functional activity ; furthermore, they entirely disappear in certain portions of the rabbit's intestine after pilocarpin-poisoning. It would therefore appear that t*he prin- cipal physiologic function of the glands of Lieberkuhn is to secrete mucus, although the possibility of the production of another secre- tion, especially in the small intestine, must not be excluded (compare R. Heidenhain, 83), especially since it has been shown that the cells of Paneth probably elaborate an enzyme. Until recently it was believed that the fat contained in the food was emulsified in the intestine, and furthermore that the bile acted upon the cuticular margins of the epithelial cells in the villi in such a manner that an assimilation of the emulsified fat by the cells of the villi (not by the goblet cells) was made possible. It has been re- peatedly observed that the epithelial cells contained fat granules during absorption. Hence a mechanism was sought for which would account for an assimilation of globules of emulsified fat on the part of the cells. It seemed most probable that protoplasmic threads (pseudopodia) were thrown out from each cell through its cuticular zone, which, after taking up the fat, withdrew with it again into the cell. But when it was shown that, after feeding with fatty acids or soaps, globules of fat still appeared in the epithelial cells as before, and that the chyle also contained fat, the hypothesis was THE LIVER. 289 suggested that the fat is split up by the pancreatic juice into glycerin and fatty acids, and that the fatty acids are then dissolved by the bile and the alkalies of the intestinal juice, only again to combine with the glycerin to form fat within the epithelial cells. It remains for the histologist to ascertain the exact mechanism in the cell which changes the fatty acids into fat. Altmahn (94) claims that certain granules of the cells (elementary organisms) offer a solution to this problem. The manner in which the fat globules gain access to the lacteal vessels of the villi is a question which has not as yet been settled definitely ; it would appear, however, that the leucocytes play an important part in this transfer, since in preparations of the intestinal mucosa, taken from an animal fed on a diet rich in fat — milk diet — and stained in osmic acid, numerous leucocytes contain- ing black granules or globules may be observed in the lacteal vessels and in the spaces of the adenoid reticulum of the villi. D. THE LIVER, In the adult the liver is a lobular, tubular gland with anastomos- ing tubules. When viewed with the unaided eye or under low magnification the liver is seen to be composed of a large number Intralobular vein. Branch of portal vein. Bile-duct. Branch of hepatic artery. Interlobular connective tissue. Fig. 227. — Section through liver of pig, showing chains of liver-cells ; X 7°« of nearly spheric divisions of equal size ; this is particularly notice- able in some animals, especially in the pig. These divisions are the liver lobules and have a diameter of from 0.7 to 2.2 mm. They are separated from each other by a varying amount of interlobular con- nective tissue, which is a continuation of the capsule of Glisson, a fibro-elastic layer surrounding the entire liver and covered for the greater portion by a layer of mesothelium. In the interlobular septa are found the larger blood-vessels, bile passages, nerves and 19 290 THE DIGESTIVE ORGANS. lymph-vessels. On examining a thick section of the liver with a low power, a radiate structure of the lobule is noticeable, and an open space is seen in its center, which according to the direction of the section, is either completely surrounded by liver tissue or con* nected with the periphery of the lobule by a canal. This open space represents the central or intralobidar vein of the lobule which belongs to the system of the inferior vena cava. From the center of the lobule toward its periphery extend numerous radiating strands of cells, which branch freely and anastomose with each other, and are known as the trabeculce, or cords of hepatic cells. Be- tween the latter are small, clear spaces occupied partly by blood capillaries and partly by the intralobular connective tissue. The above description is in some respects not a true statement of the appear- ance presented by the human liver, as in the latter one or more lobules may blend with each other, thus rendering the individual lobules less distinct. The hepatic cords consist of rows of hepatic cells. The cells fortal inter- lobular branch, cut longitudi- nally. The same, cut transversely. Fig. 228. — Section through injected liver of rabbit. The boundaries of the lobules are indistinct ; X about 35. are usually polyhedral in form, with surfaces so approximated that a cylindric capillary space, known as the bile capillary remains be- tween them. The angles of the cells also show grooves which join those of the neighboring cells to form canals in which lie the blood capillaries. A closer examination of the hepatic cells reveals the fact that they possess no distinct membrane, and, in a resting state, usually contain a single nucleus, although some possess two. It is an interesting fact that nearly all the hepatic cells of some THE LIVER. 291 animals — as, for instance, the rabbit — contain two nuclei. The cell-bodies of the hepatic cells, which average from 18 p to 26 // in diameter, show a differentiation into protoplasm and paraplasm. This is especially manifest in a state of hunger. In this condition it is seen that the network of protoplasm around the nucleus is un- usually dense, and becomes looser in arrangement as it extends toward the periphery of the cell-body. The paraplasm is slightly granular, and contains glycogen and bile drops during the func- tional activity of the cell (secretion vacuoles). The vacuoles in the paraplasm play an important part in the secretion of the cell, and are Intralobular vein. Fig. 229. — Human bile capillaries. The capillaries of one lobule are seen to anas- tomose with those of the adjoining lobule (below, in the figure) ; X JI° (chrome-silver method). Vacuole of secretion. Tubule of same. Bile capillary. Fig. 230. — Human bile capillaries as seen in section ; X 4^o (chrome-silver method). due to the confluence of minute drops of bile into a large globule. As soon as the vacuole has attained a certain size it tends to empty THE DIGESTIVE ORGANS. its contents into the bile capillary through a small tubule connect- ing the vacuole with the bile capillary (Kupffer, 73, 89). The bile capillaries are, as we have remarked, nothing but tubu- lar, capillary spaces between the hepatic cells, with no distinct indi- vidual walls, although the outer portions of the liver cells (exc- plasm) are somewhat denser than the remainder of the cells, and serve to form a wall for the bile-capillaries. They may be compared to the lumen of a tubular gland, although in the human liver their walls consist of. only two rows of hepatic cells. In the lower vertebrates the walls of the bile capillaries appear in transverse section to consist of several cells (in the frog generally three, in the viper as many as five). The bile capillaries naturally follow the course of the hepatic cords — i. c., in man extending radially. They form networks, the meshes of which correspond to the size of the hepatic cells. At the periphery of the lobule the hepatic cells pass directly over into the epithelial cells of the smaller interlobular bile-ducts. The epithelium of the latter is of the cubical variety, its cells being considerably smaller than the hepatic cells. At the point where the hepatic cells become ^-cxN ^S\ Bile capillaries, continuous with the walls of the smaller passages we find a few cells of gradually decreasing size which represent a transition stage from the cells of the bile capil- Fig. 231. — Schematic diagram of he- patic cord in transverse section. At the left the bile capillary is formed by four cells, at the right by two ; the latter type occurs in the human adult. Fig. 232. — From the human liver, showing the beginning of the bile-ducts ; X 90 (chrome-silver). laries (hepatic cells) to those of the interlobular bile passages. The vascular system of the liver is peculiar in that, besides the usual arterial and venous vessels common to all organs, there is found another large afferent vein — the portal vein. It arises from a confluence of the superior and inferior mesenteric, the splenic, coronary veins from the stomach, and cystic veins. It divides into two branches, the right supplying the right lobe of the liver, the left the remaining lobes. These branches again divide into numerous smaller branches, the smallest of which finally reach the individual lobules. Along its whole course through the inter- THE LIVER. 293 lobular connective tissue the portal vein and its branches are accom- panied by divisions of the hepatic artery and bile passages. In a transverse section of the liver the arrangement of these structures in the interlobular tissue is such that the cross-sections of the vessels belonging to the hepatic vein are seen to be at some distance from the closely approximated branches of the portal vein and bile pas- sages. Branches of the portal vein encircle the liver lobules at different points, and while they remain within the interlobular con- nective tissue, are known as interlobular veins. From these, small offshoots are given off to the lobules which, on entering, divide into capillaries and form a closely reticulated network between the hepatic cords. The meshes of this network are about as large as an hepatic cell, each cell coming in repeated contact with the blood capillaries. All of these capillaries pass toward the central or intralobular vein of the lobule, which during its efferent passage through the lobule continues to receive capillaries from the portal Blood capillaries. Intralobular vein. - . Cord of hepatic cells. Interlobular vessel. Fig. 233. — Injected blood-vessels in liver lobule of rabbit ; X Ioa system. The intralobular veins unite to form the sublobular veins,- situated in the interlobular connective tissue, and these unite to form the larger hepatic veins which empty into the inferior vena cava. The hepatic artery is of much smaller size than the portal vein. It is distributed in the main to the connective tissue of the liver and to the bile-ducts, breaking up into branches which are situated in the interlobular connective tissue. The terminal capillaries form small venules which communicate with the interlobular THE DIGESTIVE ORGANS. branches of the portal system. Whether the capillaries of the hepatic artery pass as such into the hepatic lobules is difficult to say, since injection masses forced into the hepatic artery pass over into the terminal branches of the portal vein and vice versa. This question needs, therefore, further investigation. The smaller divisions of the hepatic artery constitute, therefore, internal radi- cals of the portal vein, since they are within the liver itself. The relations of the various blood-vessels within the lobule are in themselves somewhat difficult of comprehension, but the whole be- comes still more complicated when the reciprocal relations of the vessels and bile capillaries are taken into consideration. In order to understand the structure of the liver lobule, with its hepatic cords, vessels, and bile capillaries, the following points should be borne in mind : The course of the bile capillaries is along the sur- faces, and that of the blood-vessels along the angles of the hepatic cells ; every cell comes in contact with a bile capillary and a blood - Intralobular vein. Boundary of - lobule. Fig. 235. — Reticulum (Gitterfasern) of dog's liver; X I2° (gold-chlorid method). capillary. The latter, however, do not come in contact with the former, but in man are separated by at least half the breadth of a hepatic cell. In animals in which the bile capillaries are bounded by more than two cells, the blood-vessels extend along the outer .sides of the hepatic cells ; here the bile and blood capillaries are separated from each other by the breadth of a whole cell. The connective tissue within the hepatic lobules presents points of interest which, however, are not demonstrable in organs treated by ordinary methods. But when the liver tissue is treated by a certain special method (see page 307), an astounding number of fibers are seen extending in regular arrangement from the periphery toward the central vein. These fibers are extremely delicate, of nearly THE LIVER. equal size, and intermingle in such a manner as to form an envel- oping network about the blood capillaries (Gitterfasern ; Kupffer ; Oppel, 91 ; vid. Fig. 235). A few coarser fibers (radiate fibers, Kupffer, 73) seem to enter in a less degree into the formation of the sheath around the blood capillaries ; they also extend from the periphery toward the center of the lobule and form a coarse reticu- lum, the meshes of which are drawn out radially. The radiate fibers are less prominent in man, but are numerous and well devel- oped in animals (rat, dog). With what exuberance the intralobular connective tissue may develop, is seen in the accompanying sketch of a sturgeon's liver, which is taken from one of Kupffer's prepara- Connective-tissue fibers. Fig. 236. — Connective tissue from liver of sturgeon. At a is an open space from which the hepatic cells were mechanically removed during treatment. tions. The Gitterfasern of Kupffer are, as has been shown by F. P. Mall, reticular fibrils, presenting the same characteristics as similar fibrils found in other regions. Certain peculiar cells — the so-called stellate cells of Kupffer (76) — occur in the lobule, and are seen only after a special method of treatment. They are uniformly distributed, of differ- ent shapes, elongated, and end in two or three pointed projec- tions. They are smaller than the hepatic cells, and contain one or two nuclei. In a recent communication Kupffer (99) states that the stellate cells belong to the endothelium of the intralobular capillaries of the portal vein. These capillaries, which are, according to their devel- opment, sinusoids (Minot), form in all probability a syncytial lining (Kupffer) consisting of thin continuous lamellae, the proto- plasm appearing as a network of threads, with nucleated masses of protoplasm at nodal points of this network. In places where this protoplasm is present in larger quantity and contains round or 296 THE DIGESTIVE ORGANS. oval nuclei it is more readily brought out with special stains, and appears in such preparations in the form of structures to which the name stellate cells has been given. In such cells blood corpuscles and fragments of such were often found. The endothelium of these capillaries possesses, therefore, a phagocytic function, taking up par- ticles of foreign matter, blood-corpuscles, etc. The efferent ducts of the liver, the bile-ducts, are lined by col- umnar epithelium, varying in height in direct proportion to the cal- iber of the passage. The smallest ducts possess a low, the medium sized a cubical, and the larger a columnar epithelium. The smaller bile-ducts have no clearly defined external walls other than the membrana propria ; the larger ones, on the other hand, possess a connective-tissue sheath which may even present two layers in the larger passages. Unstriped muscular fibers occur in the large Fig. 237. — From preparation from the liver of a rabbit, showing the so-called stellate cells of Kupffer : a, Stellate cells; b, liver cells. ducts, and also small mucous glands. The gall-bladder consists of a mucous, fibre-muscular, and, where covered by the peritoneum, of a subserous and serous coats, as has recently been shown by Sudler, whose account is here followed. The mucous coat is covered by a single layer of columnar epi- thelium, with nuclei situated in the basal portions of the cells. The epithelial cells rest on a poorly developed muscularis mucosa::. The mucosa presents folds, covering ridges of connective tissue of the fibre-muscular layer, and contains small lymph-nodules, and a varying number of small mucous glands. The fibro- muscular layer consists of interlacing bands of nonstriated muscle and fibrous connective tissue, and is not arranged in distinct layers. The subserous and serous coats present the same appearance as in other regions of the peritoneum. The artery or arteries going to the gall-bladder divide into branches which form capillaries in the mucosa under the epithelium ; these are most numerous in the THE LIVER. 297 folds above mentioned. The lymphatics form a subserous and submucous plexus. The lymphatics accompany the portal vein and hepatic artery, also the branches of the hepatic vein (Wittich). They form a net- work in the interlobular connective tissue. The lymphatics form further a superficial network in subserous layer of the peritoneum. The superficial lymphatics and the lymphatics accompanying the vessels are in communication. Within the lobules, the lymphatics occur as perivascular spaces, as was first shown by MacGillavry. F. P. Mall, who has recently studied the origin of the lymphatics in the liver, summarizes his results as follows : The lymphatics of the liver arise from peri- lobular lymph-spaces, and these communicate directly with peri- vascular lymph-spaces ; the lymph reaches these spaces by a process of filtration through openings which are normally present in the capillary walls of the liver. Intralobular vein. -IBB Interlobular con- nective tissue. Stellate cells. Fig. 238.— Part of a section through liver lobule from dog, showing stellate cells; Xi68. Berkley (94) has described several divisions of the intrinsic nerves of the liver, all connected and morphologically alike. These nerves are no doubt the neuraxes of sympathetic neurones, the cell-bodies of which are located in ganglia outside of this organ. No medul- lated fibers were found by him, although it seems probable that the nerve-fibrils terminating between the cells of the bile-ducts (see be- low) are terminal branches of sensory nerve-fibers. The nerves of the liver accompany the portal vessels, the hepatic arteries, and the bile-ducts. The' first division of the nerves, embracing the larger number of the intrinsic hepatic nerves, accompany the branches of the portal vessels, form plexuses about them, and end in inter- lobular and intralobular ramifications, the latter showing here and there knob-like terminations on the liver-cells, and, in their course, give off here and there branches which end on the portal vessels. 298 THE DIGESTIVE ORGANS. The nerve-fibers following the hepatic arteries are in every respect like the vascular nerves in other glands. Some of the terminal branches seem, however, to end on hepatic cells. The nerve-fibers following the bile-ducts may be traced to the smaller and medium-sized ducts, forming a network about them, and ending here and there in small twigs on the outer surface of the cells, and occasionally, it would seem, between the epithelial cells lining the ducts. The suggestion seems warranted that these terminal fibrils are the end- ings of sensory nerves. Some of the nerve-fibers following the bile-ducts may be traced into the hepatic lobules. The intralobu- lar plexus is formed, therefore, by the terminal branches of the non- medullated nerve-fibers accompanying the portal and hepatic ves- sels and the bile-ducts. In the wall of the gall-bladder are found numerous small sympathetic ganglia formed by the grouping of the cell-bodies of sympathetic neurones (Dogiel). The neuraxes of these innervate the nonstriated muscle of this structure. Large, medul- lated nerve-fibers may be traced through these ganglia which appear to end in free sensory endings in and under the epithelium lining the gall-bladder (Huber). In the human embryo the liver originates from the intestine during the second month as a double ventral diverticulum. Later solid trabecular masses are developed which then unite and become hollow. At this stage the whole gland is uniform in structure, as a division into lobules does not take place until later. The bile capillaries are surrounded by more than two rows of cells. In this stage the embryonal liver suggests a condition which is permanent during the life of certain animals. Only later when the venae ad- vehentes, which later represent the branches of the portal vein, penetrate the liver, is there a secondary division into lobules (about the fourth month), by which process the primitive type gradually changes to that characteristic of the adult. E. THE PANCREAS, Like the liver, the pancreas is an accessory intestinal gland, and originates as a diverticulum of the intestine. It remains in perma- nent communication with the intestine by means of its duct — the pancreatic or Wirsungian duct. The pancreas is composed of numerous microscopic lobules, surrounded by connective tissue which penetrates into the lobules and between the alveoli and is accompanied by vessels and nerves. The secretory portion of the organ may be regarded as a compound, branched alveo- lar gland, the general structure of which is shown in Fig. 240, the alveoli forming the principal portion of the gland. The epithelial walls of the alveoli consist of a number of secretory cells, whose appearance varies according to the func- tional state of the organ. The basilar portions of the cells present THE PANCREAS. 299 a homogeneous protoplasm, while those parts of the cells border- ing upon the lumen are granular. The relation of these zones to Nucleus and outer zone. Fig. 239. — Transverse section through alveolus of frog's pancreas. each other depends upon the physiologic condition of the gland ; during starvation the internal or granular zone is wide and promi- nent ; after moderate secretion the cells become as a whole some- what smaller, the granules decrease in number, and the outer or protoplasmic zone increases in size. After prolonged secretion there is an entire absence of the granules, and the whole cell appar- ently consists of homogeneous protoplasm. It is therefore probable Fig. 240. — Model of lobule of human pancreas (Maziarski, "Anatomische Hefte," 1901). that during a state of rest peculiar granules (zymogen granules) are formed at the expense of the protoplasm, and that these granules represent a preliminary stage of the finished secretion. During the 3°° THE DIGESTIVE ORGANS. functional activity of the gland the granules gradually disappear, while the fluid secretion simultaneously makes its appearance in the lumen, although the granules have as yet never been observed in the lumen itself. After secretion the cell grows again until it reaches its original size, only again to begin the formation of zymo- gen granules. Whether the cells of the gland are destroyed or not during secretion is still a matter of uncertainty, but does not seem probable. An intermediate tubule similar to those of the salivary glands connects with each alveolus, and then passes over into a short in- tralobular duct. This is lined, as in the salivary glands, with columnar epithelial cells, which are not, however (at least in man), striated at their basal ends. The intralobular ducts merge Centro-acinal cell. Intermediary duct. Intralobular _ duct. Alveolus. _ Intermediary duct. Fig. 241. — From section through human pancreas ; X about 200 (sublimate). into excretory ducts, which finally empty into the pancreatic duct. The epithelium of the excretory ducts is simple columnar in type. Goblet cells are seen only in the pancreatic duct. In the secreting alveoli small protoplasmic, polygonal, and even stellate cells are often seen, the so-called centro-acinal cells, or cells of Langerhans. The significance of these structures is not fully understood. Langerhans himself supposed that they belonged to the walls of the excretory ducts. This interpretation seems war- ranted by the fact that it has been found that the secreting cells of the alveoli are directly joined to the low cells of the intermediate tubules. When the alveoli lie closely packed together, the ad- joining intermediate tubules fuse and are reduced to one or, at most, a few cells. As a result a condition is seen within the alveolar complexus, especially when the excretory ducts are in a collapsed state, closely resembling the structures seen by Langer- THE PANCREAS. 301 bans. Peculiar cells, wedged in here and there between the secre- tory cells, but resting on the membrana propria, have also been observed. They undoubtedly are sustentacular cells of the gland (cuneate cells, Podwyssotzki, 82). The incinbrana propria of the alveoli is probably homogenous. Immediately adjoining it is another delicate but firm membrane, consisting of fibrils whose structure in many respects resembles that of the reticular fibers (Gitterfasern) in the liver and spleen, but which are here in relation to the alveoli (Podwyssotzki, 82). In warm- and cold-blooded animals, groups of cells differing in arrangement, size, and structure from the secretory cells, are found among the gland tubules and alveoli of the pancreas ; these are known as the intcrtubular cell-masses, or areas of Langerhans. They are most numerous in the splenic end of the pancreas (Opie). They Outer zone of a secretory- cell. Connective . tissue. Larger gland — duct. - Centro-acinal cell. — Centro-acinal cell. — - Ivi.ermediate tubule. — Inner granular zone of secre- tory cells. Fig. 242. — From section through human pancreas ; X45° (sublimate). consist of slightly granular cells, smaller than the secretory cells of the alveoli, arranged in the form of anastomosing trabeculae, with irregular spaces, varying in size, separating the trabeculae. Dogiel (93) has shown that in a well-preserved human pancreas treated by the chrome-silver method, in which the gland ducts even to their finest intra-alveolar branches were well stained, no ducts were found in the areas of Langerhans. Such areas are, in the human pancreas, usually separated from the surrounding gland tissue by a small amount of connective tissue. They possess a blood supply, consisting of relatively large capillaries found in the spaces formed by the trabeculae of cells above mentioned. The areas of Langer- hans have been variously interpreted. They have been looked upon as small areas of gland tissue in process of degeneration, or 302 THE DIGESTIVE ORGANS. again as areas of embryonic gland tissue. From their structure and distinct blood supply, and the fact that no ducts have been traced into these areas, it seems probable that they are small masses of cells forming a secretion which passes into the blood-vessels — in- ternal secretion. The blood-vessels after entering the gland, divide into smaller branches in the lobules, and finally break up into capillaries which Fig. 243. — Scheme showing relation of three adjoining alveoli to excretory duct, illustrating origin of centro-acinal cells. < ,. ? .<$£: 0 .>,',*. ?«••'.> . Blood capillary. Alveolus or gland. Area of Langer- hans. Fig. 244. — From section of human pancreas, showing gland alveoli surrounding an area of Langerhans. encircle the secreting alveoli. The blood-vessels do not follow the course of the ducts so regularly as in the salivary glands (Flint). The meshes of the capillary network are not all of the same size. In some regions they are so wide that quite large areas of the alveoli are without blood-vessels. The nerves of the pancreas have been investigated by Cajal and Sala (91) and Erik Muller (92), who find in this gland large num- bers of nonmedullated nerve-fibers, some coming from sympathetic TECHNIC. 303 ganglion cells situated in the pancreas and others entering from without. The nonmedullated nerve-fibers form plexuses surround- ing the excretory ducts and end in periacinal networks. Fibrils from the network about the alveoli were traced to the secretory cells. A portion of the nonmedullated nerves in the pancreas form perivascular plexuses. The development of the pancreas is peculiar in that the larger portion, together with the duct of Santorini, originates from the dorsal intestinal wall, and a smaller portion from the ductus chole- dochus. The latter part, with its accessory pancreatic duct, fuses with the former, after which there is a gradual retrogression of the duct of Santorini, so that finally the entire secretion of the pancreas almost invariably flows into the pancreatic or Wirsungian duct. TECHNIC The oral mucous membrane may be fixed with corrosive sublimate or alcohol, stained in bulk, and examined in cross-section. If special structures, such as glands, nerves, or the distribution of mitoses, are to be examined, special methods must be adopted. Teeth. — In order to obtain a general- view of the structure of the teeth, the latter must be macerated and ground as in the case of bone. The relations of the hard and soft parts in undecalcified teeth are best studied by the use of Koch's petrifaction method. The teeth may also be examined in section, and when decalcified are treated as bone. Hydrochloric acid, dilute chromic acid, and picric acid dissolve the enamel prisms, their cement-substance being the first to disappear (von Ebner, 91). The enamel of young teeth stains brown in a solution of chromic acid . or its salts, and blackens in osmic acid. In the enamel cells, globules are seen, which are stained in osmic acid. If longitudinal sections of the enamel be corroded with hydrochloric acid, the cruciform arrangement of the enamel prisms is plainly seen. The fibrils of the dentin may be demonstrated by decalcifying a tooth in the fluid recommended by von Ebner, the teeth of young individuals being well adapted for this purpose. Occasionally carious teeth also show the fibrils plainly. Corrosion with hydrochloric acid produces the same result. The cementum, especially that part lacking in cells, contains a large number of Sharpey's fibers. The development of the teeth is studied in the embryo ; the jaw-bone is fixed, decalcified, and cut in serial sections. The most convenient material is a sheep embryo, which can almost always be had from the slaughter-house. Taste-buds. — To study the taste-buds of the tongue and the rela- tions which their constituent cells bear to each other, fixation in Flem- ming's fluid is recommended. The orientation of the taste-buds must be very carefully done, after which exactly longitudinal or transverse serial sections are made (not thicker than 5 /j.) and stained with safranin- gentian-violet. THE DIGESTIVE ORGANS. The nerves in the taste-buds are brought out either by Golgi's method, the methylene-blue method, or by the use of gold chlorid. If the last be used the procedure is as follows : A papilla foliata of a rabbit is removed with a sharp razor and placed for ten minutes in lemon juice, then in gold chlorid for from three-quarters of an hour to one hour, after which the specimen is placed in water weakly acidulated with acetic acid (5 drops to 100 c.c. of water) and exposed to the light. As soon as reduction has taken place the specimen is treated with alcohol and cut in vertical sections. These may be treated for a short time with formic acid (in which they swell slightly), washed with water, and mounted in glycerin. In certain objects, such as the nictitating membrane of the frog, certain lobules of the rabbit's pancreas (the latter being so thin as to be especially well adapted for microscopic examination), etc., the glandular structure may be examined in normal salt solution. Glands of the Digestive Tract. — Microscopically, the glands pre- sent varying pictures according to the phase of secretion in which they are fixed. Specimens in the different stages may be obtained either by feeding and then killing the animal after a definite period, or by irritating certain nerves, or finally by the use of certain poisons especially adapted to this purpose, such as atropin and pilocarpin. In the rabbit, for in- stance, i c.c. of a 5% solution of pilocarpin hydrochlorate or i c.c. of a 0.5% solution of atropin sulphate is used for each kilogram of the ani- mal's weight. In atropin- intoxication secretion is suppressed, while in pilocarpin -poisoning it is increased. By this method cells are obtained either full of secretion or containing no secretion at all. Sections should be made from carefully selected material which has been fixed either in Flemming's solution or corrosive subli- mate, although fixation with strong alcohol also gives instructive pictures. In preparations fixed with Flemming's solution the crescents of Gianuzzi stain somewhat more deeply than the remaining cells of the alveoli, and in objects that have been treated with alcohol or corrosive sublimate and then stained with hematoxylin the crescents take on a very deep color. The intermediate tubules of the salivary glands also assume a deeper stain with hematoxylin and carmin. The intralobular tubes are particularly well defined by certain stains, as for instance when Congo red is used after staining with hematoxylin ; other acid anilin stains may also be used. The intralobular tubes of most salivary glands (not, how- ever, of the parotid of the rabbit nor of the sublingual of the dog) are stained a dark -brown color (calcareous reaction) by agitating small, fresh pieces of tissue in order to facilitate the entrance of air, and then treating them with a dilute aqueous solution of pyrogallic acid. The stain persists for some time in specimens preserved in alcohol. Sec- tions made by free hand from tissues treated by this method give excel- lent results. Mucin is soluble in dilute alkalies, as for instance lime-water, and may be precipitated from these solutions by the addition of acetic acid, although the precipitate does not redissolve in an excess of acetic acid ; mucin is also precipitated by alcohol, but not by heat. Mucin- ogen does not stain with hematoxylin, as does mucin. By this latter test a gland in a state of functional activity may be differentiated from one at rest (R. Heidenhain, 83). After treatment with alcohol, safranin TECH NIC. 305 stains mucin orange-yellow. For the demonstration of mucin, more es- pecially in alcoholic preparations, H. Hoyer (90) has recommended thionin or its substitute, toluidin-blue. Indeed, the basic anilin dyes in general seem to have a particular affinity for mucin. P. Mayer (96) recommends the following two solutions for the staining of mucin : (i) Mucicarmin — Carmin i gm., aluminium chlorid 0.5 gm., and distilled water 2 c.c. are stirred together and heated over a small flame till the mixture becomes quite dark. As soon as the mixture has attained the consistency of thick syrup, 50% alcohol is added and the whole transferred to a bottle in which it is shaken after the addition of more alcohol. Finally, still more 50% alcohol is added until the whole amounts to too c.c. Before using, this stock solution is diluted tenfold with tap-water rich in lime-salts. (2) Muchematein : (#) Aqueous solution — 0.2 gm. of hematein is ground in a mortar con- taining a few drops of glycerin ; to this are added o. i gm. aluminium chlorid, 40 c.c. glycerin, and 60 c.c. distilled water. (t>~) Alcoholic solution — 0.2 gm. hematein, o.i gm. aluminium chlorid, 100 c.c. 70% alcohol, and i or 2 drops of nitric acid. Both of these solutions are used for staining mucin in sections and thin membranes. By the use of these methods the mucous acini of mixed glands are shown with ease and pre- cision. Under favorable conditions the whole secretory and excretory system of the gland may be brought out by Golgi's method (see this). In order to obtain a general structural view of the esophagus a small animal may be selected, in which case small pieces of tissue are fixed and imbedded in paraffin. If a large animal is used, the tissue is imbedded in celloidin. The mucous membrane of the stomach should be fixed while still fresh and warm, the best fixative for this purpose being corrosive sub- limate. Mixtures of osmic acid are also serviceable, but fixing with cor- rosive sublimate increases the staining power of the tissue. In order to preserve the stomach and intestine in a dilated condition, they should be filled with the fixing fluid and after ligation placed whole in the fixing agent. In gastric mucous membrane that has been fixed either with corrosive sublimate or alcohol, the parietal cells are easily differentiated from the chief cells by staining. The most reliable and convenient method is as follows : Sections fastened to the slide by the water-albumin fixative method are stained with hematoxylin and then placed in a dilute aqueous solution of Congo red until they assume a red color (minutes); they are then washed with dilute alcohol until the parietal cells appear red and the chief cells bluish (Stintzing). Almost all acid anilin dyes have an affinity for the parietal cells ; hence the red stains may be com- bined with hematoxylin and the blue ones with carmin. The chief cells then take the color of the carmin or hematoxylin, and the parietal cells that of the anilins. An accurate fixation of that portion of the small intestine possessing villi is attended with great difficulty, since the axial tissue of the villi shows a tendency to retract from the epithelial layer surrounding it (the latter becoming fixed first); and as a consequence spaces are formed at the summits of the villi which undoubtedly represent artefacts. A good method is to cut pieces from tissue while still warm and fix in osmic acid. If portions of the intestine be filled with alcohol or corrosive sub- limate and thus dilated, both the glands and villi are shortened. The 306 . THE DIGESTIVE ORGANS. methods above mentioned for staining mucin may be used to stain the goblet cells. The villi may also be examined in a fresh condition in one of the indifferent fluids. For this purpose the intestine of the mouse is especially well adapted. The absorption of fat is best studied in preparations fixed in osmic acid, and especially in those treated by Altmann's method. The technic for the solitary lymph-follicles and Peyer's patches is the same as that for lymph-glands. For this purpose the cecum of a rabbit or guinea-pig is the best material. The nerves of the intestinal mucous membrane are best demon- strated by means of the methylene-blue method or Golgi's method (vid. Technic), and the coarser filaments of Auerbach' sand Meissner's plexuses may also be stained by the gold method (Lowit's procedure, p. 48). Good results are also obtained by staining with hematoxylin such speci- mens as have been previously fixed and distended with alcohol. The plexuses then appear somewhat darker than the remaining tissue of the isolated mucous membrane or muscular layer. Liver. — The arrangement of the liver lobules is best seen in the pig's liver. In the human liver and in most domestic animals the lobules are not sharply defined, two or three adjacent lobules merging into each other. In the liver of the fetus, of the new-born, and of children, the lobules are seen indistinctly or not at all, although the perivascular spaces of the blood-vessels are better seen than in the adult. The liver-cells are best examined by treating small pieces of tissue with i cjo osmic acid or osmic mixtures ; in the latter case subsequent treatment with pyroligneous acid is necessary. Good results can also be obtained by fixing with corrosive sublimate and staining with hematoxylin (after M. Heidenhain). In order to see the glycogen in the liver-cells Ranvier (89) proceeds as follows : A dog is fed on boiled potatoes for two days, after which sections of its liver are cut with a freezing microtome and examined in iodized serum. In a short time the glycogen is stained a wine-red. If the preparation be now exposed to osmic acid vapor, the stain will remain fixed for from twenty-four to forty-eight hours. Glycogen is insoluble in alcohol and ether, and stains a port wine-red in iodin solutions ; the color disappears when the specimen is warmed, but returns again on cool- ing. The distribution of the hepatic blood-vessels is usually demon- strated by injection of the portal vein, as the injection of the hepatic artery does not, as a rule, give such satisfactory results. The injection method is also employed for the demonstration of the bile capillaries. Chrzonszczewsky recommends the following so-called physiologic autoinjection : A saturated aqueous solution oif indigo-carmin is injected into the external jugular vein three times in the course of one and one-half hours (dog 50 c.c. each time, cat 30 c.c., full-grown rabbit 20 c.c.). The animal is then killed and small pieces of its liver fixed in absolute alcohol or in potassium chlorate ; in the latter case a saturated solution of the salt may be injected into the blood-ves- sels. A subsequent injection of the blood-vessels with carmin -gelatin may also be employed and the whole liver then hardened in alcohol. By TECHNIC. 307 this method the bile capillaries finally become filled with the indigo-car- min by a gradual elimination of the substance from the blood- and lymph- vessels and passage through the cells into the biliary system, while the blood-vessels themselves are distended by the carmin-gelatin. In the frog, the demonstration of the biliary passages is more easily accomplished by injecting 2 c.c. of the indigo-carmin solution into the large lymph- sac and killing it after a few hours. The liver is then fixed in the manner described above and is then ready for further treatment. The bile passages may also be injected directly through the hepatic duct or the ductus choledochus. For this purpose it is best to use a concentrated aqueous solution of Berlin blue (Berlin blue that is soluble in water). The results obtained by this method are not, however, always satisfactory, and even in the best of cases only the peripheral por- tions of the liver lobules are successfully injected. The bile capillaries may be impregnated with chrome-silver. Fresh pieces of liver tissue are placed for two or three days in a potas- sium bichromate-osmic acid solution (4 vols. of a 3% bichromate of potassium solution and i vol. of i% osmic acid) and then transferred to a °-75% aqueous solution of silver nitrate. After rinsing in distilled water the specimens are cut with a razor, the sections again washed with distilled water, placed for a short time in absolute alcohol, cleared in xylol, and finally preserved in hard Canada balsam. Both celloidin and paraffin imbedding are admissible, but either process must be hurried, as the preparation always suffers under such treatment. In the finished specimen, the bile capillaries appear black by direct light. Another method which brings to view more extensive areas of the bile capillaries is as follows : A piece of liver tissue from a freshly killed animal is fixed in rapidly ascending strengths of potassium bichromate solution (from 2% to 5%). After three weeks the specimen is placed in a 0.75% silver nitrate solution, when after a few days (very marked after eight days) the bile capillaries, if examined in sections, will appear black by direct light (Oppel, 90). Sometimes the bile capillaries are brought out in preparations treated by the method of R. Heidenhain, although only small areas are colored and these not constantly. The application of other stains, as for instance the method of M. Heidenhain following the gold chlorid treat- ment, often results in the staining of small areas of bile capillaries. In all the methods used for the demonstration of the bile capil- laries, whether physiologic autoinjection, direct injection, or impregna- tion, the secretion vacuoles of the liver-cells are clearly brought to view. By treating pieces of liver tissue according to the method of Kupffer (76) the connective tissue of the liver, especially the reticular structure {Gitterfaserri) , is shown. Fresh liver tissue is cut with the double knife and the thinnest sections placed for a short time in a 0.6% sodium chlorid solution or in a 0.05% solution of chromic acid. From this they are transferred to a very dilute solution of gold chlorid (Gerlach) (gold chlorid i gm., hydrochloric acid i c.c., water 10 liters), and kept for one to several days in the dark until they assume a reddish or violet color. If the staining has been satisfactory (which is by no means always the case), the reticular fibers, and occasionally also the stellate cells, are 308 THE DIGESTIVE ORGANS. seen. Instead of the double knife the freezing microtome may be used and the method continued as stated (Rothe). The reticular fibers are seen under more favorable conditions by using the following method, recommended by Oppel (91): Fresh pieces of tissue fixed in alcohol are placed for twenty-four hours in a o. 5 % aque- ous solution of yellow chromate of potassium (larger pieces in stronger solutions up to 5%), then washed with a very dilute solution of nitrate of silver (a few drops of a 0.75% solution to 30 c.c. distilled water), and transferred to a 0.75% solution of silver nitrate. In twenty-four hours the intralobular network surrounding the blood capillaries will have be- come stained. The best areas lie at the periphery of the specimen, and extend about i mm. into the parenchyma. Free-hand sections are made, or the specimens are quickly imbedded in celloidin or paraffin, to be cut afterward by means of the microtome. The same results are obtained by placing small fresh pieces of the tissue for two or three days in a 0.5% chromic acid solution and then one or two days in a 0.5% solution of silver nitrate. The further treatment is as in the preceding method. The method of F. P. Mall is also employed in the examination of the hepatic connective tissue. The following method is recommended by Berkley for demon- strating the nerves of the liver : Small pieces of liver tissue from 0.5 to i mm. in breadth are placed in a half-saturated aqueous solution of picric acid for from fifteen to thirty minutes, and .then in TOO c.c. of potassium bi- chromate solution that has been saturated in the sunlight and to which 16 c.c. of 2% osmic acid has been added. The specimens now remain in this fluid for forty-eight hours in a dark place, and at a temperature of 25° C. After this the tissue is treated with a 0.25% to 0.75% aqueous solution of silver nitrate for five or six days, washed (quick imbedding may be employed), cut, cleared in oil of bergamot, and mounted in xylol-Canada balsam. The cellular elements of the pancreas may be examined without further manipulation in very thin lobules from the rabbit (Kiihne and Lea). There are various methods of differentiating the inner and outer zones of the cells. In sections of the tissue fixed in alcohol, car- min stains the outer zone of the cells more intensely than the inner (R. Heidenhain, 83). For the staining of the inner zone, fixation in Flem- ming's fluid is to be recommended, then staining with safranin, and finally washing in an alcoholic solution of picric acid. The granules of the inner zone (zymogen granules) appear red. These also stain red with the Biondi-Ehrlich mixture. The simplest and most precise method of demonstrating the zymogen granules is that of Altmann. The secretory and excretory ducts of the pancreas are shown, as in the case of the salivary glands, by the chrome-silver method. THE LARYNX. 309 IV. ORGANS OF RESPIRATION. A. THE LARYNX. THE greater portion of the laryngeal mucous membrane is cov- ered by a stratified columnar ciliated epithelium containing goblet cells, and resting on a thick basement membrane. The epithelium covering the free margin of the epiglottis, the true vocal cords, and Glands in false vocal cord. Stratified pavement ._ epithelium of true \ vocal cord. \ I Stratified ciliated col- umnar epithelium. Glands. __ -Muscle. Muscle. Fig- 245. — Vertical section through the mucous membrane of the human larynx ; X 5- 3IO ORGANS OF RESPIRATION. part of the arytenoid cartilage as far as the cavity between these cartilages, is of the stratified squamous variety, and is provided with connective-tissue ridges and papillae. The mucosa consists of fi- brous connective tissue, contains many elastic fibers, which become larger and more prominent as the deeper layers of the mucosa are approached, and is rather firmly connected with the structures underneath it, but is somewhat more loosely connected in the re- gions supplied with squamous epithelium. The mucosa contains numerous lymphocytes and leucocytes, which now and then, espe- cially in the region of the Ventricles, form simple follicles. In it are found branched tubulo-alveolar glands, which may be single or arranged in groups. These are found at the free posterior portion of the epiglottis, in the region of the latter' s point of attachment — i. e., in the so-called cushion of the epiglottis. Larger collections of glands are found in the false vocal cords, and on the cartilages of Wrisberg (cuneiform cartilages), which appear almost imbedded in the glandular tissue and in the ventricles. In the remaining parts of the larynx glands are found only at isolated points. The true vocal cords have no glands. The glands of the larynx are of the mucous variety, containing crescents of Gianuzzi. The cartilages of the larynx are of the hyaline variety, with the exception of the epiglottis, the cartilages of Santorini (the latter are derivatives of the epiglottis, Goppert), the cuneiform cartilages, the processus vocalis, and a small portion of the thyroid at the points of attachment of the vocal cords, which consist of elastic car- tilage. The vascular supply of the larynx is arranged in three super- imposed networks of blood-vessels. The capillaries are very fine, and lie directly beneath the epithelium. The lymphatic network is arranged in two layers, the superficial being very fine and di- rectly beneath the network of blood capillaries. The nerves of the laryngeal mucous membrane will be de- scribed in connection with those found in the trachea. B. THE TRACHEA. The trachea is lined by a stratified ciliated columnar epithelium containing goblet cells and resting on a well-developed basement membrane. The mucosa is rich in elastic tissue. In the super- ficial portion of the mucosa the elastic fibers form dense strands, which usually take a longitudinal direction. The deeper layer of the mucosa is more loosely constructed, and passes over into the perichondrium of the semilunar cartilages of the trachea without any sharp line of demarcation. Numerous leucocytes are scattered throughout the mucosa, and are also frequently found in the epi- thelium. Connecting the free ends of the semilunar cartilages, which are of the hyaline variety, are found bundles of nonstriated muscle tissue, the direction of which is nearly transverse. THE BRONCHI, THEIR BRANCHES, AND THE BRONCHIOLES. 3! I The trachea contains numerous branched tubulo-alveolar glands of the mucous variety containing here and there crescents of Gianuzzi. The glands are especially numerous where the tracheal wall is devoid of cartilage. The larynx and trachea receive their nerve supply from sensory nerve-fibers and sympathetic neurones. These have been described by Ploschko (97) working in Arnstein's laboratory. According to this observer, the sensory fibers divide in the mucosa, forming sub- epithelial plexuses from which fibrils are given off which enter the epithelium of the larynx and trachea and, after further division, end on the epithelial cells in small nodules, or small clusters of nodules. In the trachea of the dog, such fibrils were traced to the ciliary border of the columnar ciliated cells before terminating. Numerous sympathetic ganglia are found in the larynx and trachea. In the latter they are especially numerous in the posterior wall. The neuraxes of the sympathetic neurones forming these ganglia were traced to the nonstriated muscular tissue of the trachea. The cell- bodies of these sympathetic neurones are surrounded by end-baskets of small medullated fibers terminating in the ganglia. Medullated Fig. 246. — From longitudinal section of human trachea, stained in orcein: a, Layer of elastic fibers ; i>, cartilage. nerve-fibers, ending in the musculature of the trachea in peculiar end-brushes, were also described by Ploschko. C THE BRONCHI, THEIR BRANCHES, AND THE BRONCHIOLES. The primary bronchi and their, branches show the same general structure as the trachea, showing, however, irregular plates and platelets of cartilage instead of half-rings, which surround the bronchi. The cartilage is absent in bronchial twigs of less than 3I2 ORGANS OF RESPIRATION. 0.85 mm. in diameter. The epithelium of the bronchi of medium size (up to 0.5 mm. in diameter) consists of a ciliated epithelium having three strata of nuclei. Kolliker (81) distinguishes a deep layer of basilar cells, a middle layer of replacing cells, and a super- ficial zone consisting of ciliated and goblet cells. The number of the last varies greatly. Glands are found only in bronchial twigs that are not less than I mm. in diameter ; as in the trachea, they are branched tubulo-alveolar glands of the mucous variety. In these structures the mucosa contains a large number of elastic fibers, the greater part of which have a longitudinal direction. Furthermore, numerous lymph-cells are found, and here and there a lymph-nodule. The muscularis presents, as a rule, circular fibers, which do not, however, form a continuous layer. The smaller bronchi subdivide into still finer tubules of less than 0.5 mm. in diameter (bronchioles), which contain neither car- stratified cili- .__ ated columnat epithelium. — Elastic fibers, cut trans- versely. - Gland. •5vr«— Mucosa. --8a — Cartilage. Connective tissue. Fig. 247. — Transverse section through human bronchus ; X 27- tilage nor glands. The stratum proprium, as well as the external connective-tissue sheath, becomes very thin ; and the epithelium now consists of but one layer,, but is still ciliated. TERMINAL DIVISIONS OF BRONCHI AND ULTIMATE AIR-SPACES. 313 D. TERMINAL DIVISIONS OF BRONCHI AND ULTIMATE AIR-SPACES. The bronchioles are continued as the respiratory bronchioles. — Lung tissue. Bronchiole. Fig. 248. Respiratory _ bronchiole. Alveolar duct. — -—Lung tissue. Fig. 249. Figs. 248 and 249. — Two sections of cat's lung : Fig. 248, X 52 > Fig. 249, X 35- The epithelium of the latter is ciliated in patches, but soon becomes nonciliated and assumes the character of respiratory epithelium. ORGANS OF RESPIRATION. (See below.) The walls of the respiratory bronchioles are rela- tively thin, consisting of fibro-elastic connective tissue and nonstri- ated muscle. Our knowledge of the further divisions of the bronchioles and of their relation to the terminal air-spaces has been increased greatly by Miller, who has made use of Born's method of wax-plate reconstruction in the study of these structures. His account is here followed. According to Miller, the respiratory bronchioles divide into or become the terminal bronchioles or alveo- Section of al veolus of lung. _ — Respiratory bronchiole with two kinds of epithelium. — Respiratory bronchiole. Fig. 250. — Internal surface of a human respiratory bronchiole, treated with silver nitrate ; X 234 (after Kolliker). lar ducts. These are somewhat dilated at their distal ends and communicate, by means of three to six round openings, with a cor- responding number of spherical cavities, known as atria. Each atrjum communicates with a variable number of somewhat irregu- lar spaces or cavities, the air-sacs, the walls of which are beset with numerous somewhat irregular hemispheric bulgings, the air-cells or lung alveoli. The air-cells or alveoli are also numerous in the walls of the atria and the terminal bronchioles or alveolar ducts, TERMINAL DIVISIONS OF BRONCHI AND ULTIMATE AIR-SPACES. 315 and may even be found in the walls of the respiratory bronchioles. The terminal bronchioles or alveolar ducts have an epithelium which is of the cubic variety in their proximal portions, and which changes to a squamous epithelium in their distal portions. The epithelium of the distal portions of the terminal bronchi- oles or alveolar ducts, atria, and air-sacs (i I fj. to 15 fj. in diameter) and of the alveoli (the so-called respiratory epithelium) consists of two varieties of cells (F. E. Schulze) — smaller nucleated elements and larger nonnucleated platelets (the latter derived very probably from the former). The arrangement of the epithelial cells is gen- erally such that the nonnucleated platelets rest directly upon the blood capillaries, while nucleated cells lie between them. In am- phibia the epithelium of the alveoli consists of cells, of which the portion containing the nucleus forms a broad cylindric base; from ^jjjgrjj Nonnucleated epi- thelial cell. Nucleated epithelial cell. Fig. 251. — Inner surface of human alveolus treated with silver nitrate, showing respira- tory epithelium ; X 24° (after Kolliker). the free end of each cell a lateral process extends over the adjoin- ing capillary to meet a similar process from the neighboring cell. When viewed from above, the basal portion of the cell appears dark and granular, while the processes are clear and transparent. These cells, together with their prolongations, are about 50 [J. in diameter. The surface view greatly resembles that of the human respiratory epithelium (Duval, Oppel, 89). The terminal bronchioles or alveolar ducts have a distinct layer of nonstriated muscle having annular thickenings about the openings which lead to the atria. Muscular tissue is not found in the walls of the atria, air-sacs, and air-cells or alveoli (Miller). Beneath the respiratory epithelium in the atria, air-sacs, and air- cells, there is found a thin basement membrane, which is apparently homogeneous. Here and there are found some fibrils of fibrous ORGANS OF RESPIRATION. tissue and fixed connective-tissue cells. Elastic fibers are, however, numerous, forming networks beneath the basement membrane. The work of Miller has given a clearer conception of what may be regarded as the units of lung structure, namely, the lobules. Such a unit or lobule is composed of a terminal bronchiole or alveolar duct, with the air-spaces — atria, air-sacs, and air-cells — connected with it, and their blood- and lymph-vessels and nerves. The general arrangement of these structures may be observed in Fig. 253, which gives a diagram of a lung lobule. The shape of the atria, air-sacs, and air-cells may be seen in Fig. 254, which is from a wax reconstruction of these structures. The blood-vessels of the lung, including their relation to the structures of the lung lobules, have been investigated by Miller ; his account is closely followed in the following description: The pul- monary artery follows closely the bronchi through their entire length. An arterial branch enters each lobule of the lung at its apex in close proximity to the terminal bronchiole. After entering the lobule the artery divides quite abruptly, a branch going to each atrium ; from these branches the small arterioles arise which supply the alveoli of the lung. " On reaching the air-sac the artery breaks up into small radicals which pass to the central side of the sac in the sulci between the air-cells, and are finally lost in the rich system of capil- laries to which they give rise. This network surrounds the whole air- sac and communicates freely with that of the surrounding sacs." This capillary network is exceedingly fine and is sunken into the epithelium of the air-sacs so that between the epithelium and the capillary there is only the extremely delicate basement membrane. Only one capillary network is found between any two contiguous air-cells or air- sacs. The atria, the alveolar ducts and their alveoli, and the alveoli of the respirator}- bron- chioles are supplied with similar capillary networks. The veins collecting the blood from the lobules lie at the periphery of the lobules in the interlobular con- nective tissue, and are as far dis- tant from the intralobular arteries as possible. These veins unite to form the larger pulmonary veins. The bronchi, both large and small, as well as the bronchioles, derive their blood supply from the bronchial arteries, which also partly supply the lung itself. Capillaries derived from these ar- teries surround the bronchial system, their caliber varying according Fig. 252. — Scheme of the respiratory epithelium in amphibia : The upper figure gives a surface view : f>, Basilar portion ; a, the thin process. The lower figure is a sec- tion : a> The irregular interlacing pro- jections. iV^'-iP^*1-- -"••••*&- ^ Fig. 265. — From cortical portion of longitudinal section of kidney of young child. giving to the lumen a zigzag outline corresponding to the length of the cell. The thick portion of the loop, for the most part repre- sented by the ascending limb, but generally embracing the loop itself, THE URINARY ORGANS. 329 from 23 fi. to 28 fji in diameter, possesses a columnar epithelium similar to that of the proximal convoluted portion. Here, however, the basal striation of the cells is not so distinct, the lumen is some- what larger than that of the descending limb, and by treatment with certain reagents the epithelium may often be separated as a whole from the underlying basement membrane. The distal convoluted or intercalated portion (segment of Schweigger-Seidel), from 39 /j. to 45 f* in diameter, is only slightly curved (2 to 4 convolutions). Its epithelium is relatively high, though not so high as that lining the proximal convoluted portion and not so distinctly striated, though containing numerous granules. The cells are provided with large nuclei and their basal portions are joined by interlacing projections. a—. £5asSf;«£0*jv .-, g* ^•itf^vW _J $•&- M -•• J»v '*>?'"• 4/-"'^c;i^' i.^^gSr'i > vCvp:p^ ©<---• .9g^'.™£D^>. sa«'./!* 4 ,-,"J? •. .. EW^34&=-,. -^"' ••&'• : -® ^X^£^W;^ e) ^^ & &-*? * ¥ G>^ © ;=- c^ Fig. 266. — Section of medulla of .human kidney; X about 300: a, 0, a, Ascending limb of Henle's loop ; b, l>, b, blood-vessels ; c, c, c, descending limb of Henle's loop. The next important segment is the short arched collecting portion, which has nearly cubical epithelial cells and a lumen somewhat wider than that of the intercalated tubule. The smaller straight collecting tubules have a low columnar epithelium with cells of somewhat ir- regular shape, the basal portions of which are provided with short, irregular, intertwining processes, which serve to hold the cells in place. The diameter of the collecting tubules measures from 45 // to . 330 THE GENITOURINARY ORGANS. In the larger collecting tubules the epithelium is more regular and becomes higher as the tube widens. These tubules gradually unite within the Malpighian pyramid and the regions adjacent to the columns of Bertini to form 1 5 to 20 papillary ducts from 200 fj. to 300 // in diameter. The latter have a high columnar epithelium, and empty into the pelvis of the kidney at the apex of the papilla, forming the foramina papillaria in an area known as the area cribrosa. Besides the epithelium, the uriniferous tubules possess an ap- parently structureless membrana propria, that of the collecting tubules being very thin. This membrane may be isolated, as has been shown by F. P. Mall, by macerating frozen sections in a cold saturated solution of bichromate of soda for several days. This membrane is digested in pancreatin. Papillary duct. Blood-vessel. Fig. 267. — From longitudinal section through papilla of injected kidney ; X 4° : a> Epi- thelium of collecting tubule under greater magnification. Between the Malpighian pyramids are found the columns of Bertini, presenting a structure similar to that of the cortex of the kidney, and extending to the hilum of the kidney. Between the uriniferous tubules and surrounding the blood- vessels of the kidney there is found normally a small amount of stroma tissue, consisting of white fibrous and reticular fibers, elastic fibers being found in connection with the blood-vessels (F. P. Mall, Riihle). Between the convoluted portions of the tubules this is present only in small quantity, the fibrils being felted to form sheaths for the tubules ; a somewhat greater amount being found in the neighborhood of the Malpighian corpuscles, in the boundary zone between the cortex and medulla and between the larger col- lecting tubules in the apices of the Malpighian pyramids. From wrmt has been said concerning the uriniferous tubule it must be evident that its course is a very tortuous one. Beginning THE URINARY ORGANS. 331 with the Malpighian corpuscles, situated in the cortex between the medullary rays, the tubule winds from the cortex to the medulla and back again into the cortex, where it ends in a collecting tubule, which passes to the medulla to terminate at the apex of a Malpig- hian pyramid. The different portions of the tubules have the following positions in the kidney : In the cortex between the medul- lary rays are found the Malpighian corpuscles, the neck, the proxi- mal and distal convoluted portions of the uriniferous tubule, and the arched collecting tubules. The medullary rays are formed by the cortical portions of the straight collecting tubules and a portion of " Boundary line between two Malpighian pyramids. <~-*—~ Uriniferous tubules. ^-Glomerulus. Fig. 268. — Section through junction of two lobules of kidney, showing their coalescence ; from new-born infant. the descending and ascending limbs of Henle's loops. The me- dulla is made up mainly of straight collecting tubules of various sizes and of the descending and ascending limbs and loops of Henle's loops, the latter being often found in the boundary zone between the cortex and medulla. (See Fig. 266.) The ascending limb of Henle's loop of each uriniferous tubule, after it enters the cortex, comes into close proximity with the Malpighian corpuscle of the respective uriniferous tubule. 332 THE GENITOURINARY ORGANS. The blood-vessels of the kidney have a characteristic distribu- tion, and are in the closest relationship to the uriniferous tubules. The renal artery, as has been shown by Brodel, divides at the hilum on an average into four or five branches, about three-fourths of the blood-supply passing in front of the pelvis, while one-fourth runs posteriorly. The portion of the kidney supplied by the anterior branches is in its blood-supply quite distinct from that supplied by the posterior branches ; the one set of branches do not cross over to the other. The two ends of the kidney are supplied by an anterior and a posterior branch, each of which generally divides into three branches, which pass respectively, one anteriorly, one posteriorly, and one around the end of the uppermost and the lowest calyx. The main branches of the renal artery give off lateral branches to the renal pelvis, supplying its mucous membrane and then breaking up into capillaries which extend as far as the "area crib- rosa." The venous capillaries of this region empty into veins which accompany the arteries. Besides these, other arteries origi- nate from the principal branches, or from their immediate offshoots, and pass backward to supply the walls of the renal pelvis, the renal capsule, and the ureter. The main trunks themselves pene- trate at the hilum, and divide in the columns of Bertini to form arterial arches (arteriae arciformes) which extend between the cortical and medullary substances. Numerous vessels, the intralobular arteries, originate from the arteriae arciformes and penetrate into the cortical pyramids between the medullary rays. Here they give off numerous twigs, each of which ends in the glomerulus of a Mal- pighian corpuscle. These short lateral twigs are the vasa affcrcntia. Each glomerulus is formed by the breaking down of its afferent vessel, which, on entering the Malpighian corpuscle, divides into a number of branches, five in a glomerulus of a child three months old reconstructed by W. B. Johnston, each in turn subdividing into a capillary net. From each of these nets the blood passes into a somewhat larger vessel constituting one of the branches of the efferent vessel which carries the blood away from the glomerulus. Since the afferent and efferent vessels lie in close proximity, the capillary nets connecting them are necessarily bent in the form of loops. The groups of capillaries in a glomerulus are separated from each other by a larger amount of connective tissue than separates the capillaries themselves, so that the glomerulus may be divided into lobules. In shape the glomerulus is spheric, and is covered by a thin layer of connective tissue over which lies the inner mem- brane of the capsule, the glomerular epithelium. On its exit from the glomerulus the vas efferens separates into a new system of capillaries, which gradually becomes venous in character. Thus, the capillaries which form the glomerulus, together with the vas efferens, are arterial, and may be included in the category of the so-called arterial retia mirabilia. Those capillaries formed by the vas efferens after its exit from the Malpighian corpuscle lie both in the medullary THE URINARY ORGANS. 333 rays and in the cortical pyramids. The meshes of the capillary net- works distributed throughout the medullary rays are considerably longer than those of the networks supplying the cortical pyramids and labyrinth, the latter being quadrate in shape. The glomeruli nearest the renal papillae give off longer vasa efferentia which extend into the papillary region of the Malpighian pyramids (arteriolae rectae spuriae) and form there capillaries which ramify throughout the papillae with oblong meshes. Artery cf capsule. Arched collecting . tubule. '""*---. Straight collect- ing tubule. '"* Distal convoluted — tubule. Malpighian cor- puscle. Proximal convo- luted tubule. "•-„-•-- — Loop of Henle. ;.-^,_ Collecting tubule. Arteria arcuata. ___„.» [ Glomerulus. Vena arcuata. Large collecting tubule. Papillary duct. Fig. 269. — Diagrammatic scheme of uriniferous tubules and blood-vessels of kidney. Drawn in part from the descriptions of Golubew. Arterial retia mirabilia also occur in the course of the vasa afferentia between the intralobular arteries and the glomeruli, but nearer the latter. Each is formed by the breaking down of the small afferent vessels into from two to four smaller branches, which then reunite to pass on as a single vessel. In structure these retia differ greatly from the glomeruli in that here the resulting twigs are not capillaries and have nothing to do with the secretion of urine (Golubew). From the vasa afferentia arterial twigs are occasionally given 334 THE GENITOURINARY ORGANS. off, which break down into capillaries within the cortical substance. Other arteries originate from the lower portion of the intralob- ular arteries or from the arciform arteries themselves and enter the medullary substance, where they form capillaries. These vessels constitute the so-called "arteriolae rectae verae." Their capillary system is in direct communication with the capillaries of the vasa afferentia and "vasa recta spuria." The intralobular arteries are not entirely exhausted in supplying the vasa afferentia which pass to the glomeruli. A few extend to the surface of the kidney and penetrate into the renal capsule, where they termin- ate in capillaries which communicate with those of the recur- rent, suprarenal, and phrenic arteries, etc. Smaller branches from these latter vessels may penetrate the cortex and form glomeruli of their own in the renal parenchyma (arteriae capsulares glomeruliferae). These relations, first described by Golubew, are of importance not only in the establishment of a collateral circula- tion, but also as a partial functional substitute in case of injury to the renal arteries. The same author also confirms the statements of Hoyer (77) and Geberg, that between the arteries and veins of the kidney, in the cortical substance, in the columns of Bertini, and at the bases of the Malpighian pyramids, etc., direct anastomoses exist by means of precapillary twigs. From the capillaries the venous blood is gathered into small veins which pass out from the region of the medullary rays and cortical pyramids and unite to form the "intralobular veins." These have an arrangement similar to that of the corresponding arteries. The venous blood of the labyrinthian capillaries also flows into the intralobular veins, and as a result a peculiar arrangement of these vessels is seen at the surface of the kidney where the capillaries pass radially toward the terminal branches of the intralobular veins and form the stellate figures known as the vence stellatce. This sys- tem is also connected with those venous capillaries of the capsule which do not empty into the veins accompanying the arteries of the capsule. The capillary system of the Malpighian pyramids unites to form veins, the "venulae rectae," which empty into the venous arches (venae arciformes) which lie parallel with and adjacent to the corresponding arteries. The larger veins are found side by side with the arteries and pass out at the hilum of the organ. Lymphatics of the kidney may be divided into superficial lymphatic vessels, situated in the capsule, and deep ones, found in the substance of the kidney. The deep lymphatic vessels need to be investigated further. They form a network of closed lymphatic vessels throughout the cortex. These empty, according to Rin- dowsky, into larger lymphatics, which follow the intralobular ves- sels ; and, according to Stahr, into larger vessels situated in the medullary rays. The lymphatic vessels of the kidney proper (deep vessels) leave this organ at the hilum. The kidneys receive their innervation through nonmedullated THE URINARY ORGANS. 335 and medullated nerve-fibers. The former accompany the arteries and may be traced along these to the Malpighian corpuscles. From the plexuses surrounding the vessels small branches are given off, which end on the muscle-cells of the media. According to Berkley, small nerve-fibrils may be traced to the uriniferous tubules, which pierce the membrana propria and end on the epithelial cells. Smirnow has also traced nerve-fibers to the epithelial cells of the uriniferous tubules and the Malpighian corpuscles. Dogiel has shown that medullary (sensory) nerve-fibers terminate in the adventitia of the arteries of the capsule. • The secretory processes of the kidney can be considered only briefly in this connection. The theories concerning uriniferous secretion may be grouped under two heads : namely, the theory of C. Ludwig, who believed that all the constituents of the urine — A Fig. 270. — A, Direct anastomosis between an artery and vein in a column of Berlin of child ; B, bipolar rete mirabile inserted in the course of an arterial twig. Dog's kidney (after Golubew). leave the blood through the glomeruli, entering the uriniferous tubules as a urine containing a large percentage of water, which is concentrated in its passage through the uriniferous tubule by the absorption of water ; while according to the theory of Bowman, and later Heidenhain, only the water and inorganic salts leave the blood through the glomerulus, and that in the proportion found in the urine, while the urea is secreted by the epithelial cells of the uriniferous tubules, and mainly in those portions of the tubules possessing a striated epithelium. The majority of writers who have considered the question of urinary excretion have directly or indirectly expressed themselves as adherents to one or the other of the above theories. A number of recent observers have departed somewhat from either of the above theories, and of these we may 336 THE GENITOURINARY ORGANS. mention especially the careful researches of Cushny, who brings forth strong proof to show that with the fluid passing through the glomerular epithelium there are carried certain salts and urea, the salts and urea in the proportion in which they occur in the blood- plasma, and that in passage through the uriniferous tubules a cer- tain percentage of the fluids and certain salts are again absorbed, the salts in proportion to their diffusibility or their permeability of the renal cells. The permanent kidney is developed as early as the fifth week of embryonic life. The renal anlagen, from which the epithelium of the ureter, renal pelvis, and a portion of the uriniferous tubules is formed, originate from the median portion of the posterior wall of the Wolfrian duct. These buds grow with their blind ends ex- tending anteriorly, and are soon surrounded by cellular areas, the blastema of the kidneys. After the renal bud has become differ- entiated into a narrow tube (the ureter) and a wider central cavity (the renal pelvis) hollow epithelial buds are developed from the latter. These extend radially toward the surface of the renal anlagen, where they undergo a T-shaped division. These latter are the first traces of the papillary ducts and collecting tubules. The ends of these T-shaped divisions are surrounded by a cellular tissue, derived from the mesoderm, which is known as the renal blastema or the nephrogenic tissue. In this tissue there are differentiated spheric masses of cells, which in their further growth differentiate into S-shaped structures one end of which unites with the ends of the epithelial buds, developed as above described. The S-shaped structures acquire a lumen and form the anlagen of the uriniferous tubules, from the arched collecting tubules to and including Bowman's capsule. The ducts of the kidneys, from the papillary ducts to the collecting tubules of the medullary rays, have their origin from the epithelial buds which develop from the side of the Wolfrian ducts, while the uriniferous tubules proper have their origin in the nephrogenic tissues. 2. THE PELVIS OF THE KIDNEY, URETER, AND BLADDER. The renal pelvis, ureter, and urinary bladder are lined by strati- fied transitional epithelium. Its basal cells are nearly cubical ; these support from two to five rows of cells of varying shape. They may be spindle-shaped, irregularly polygonal, conical, or sharply angular, and provided with processes. Their variation in form is probably due to mutual pressure. The superficial cells are large and cylindric, a condition characteristic of the ureter and bladder. Their free ends and lateral surfaces are smooth, but their bases pre- sent indentations and projections due to the irregular outlines of the underlying cells. The superficial cells often possess two or more nuclei. THE URINARY ORGANS. 337 The mucosa often contains diffuse lymphoid tissue, which is more highly developed in the region of the renal pelvis. Here also there are found folds or ridges of mucosa which extend into the epithelium and present the appearance of papillae when seen in cross-section. A few mucous glands are also met with in the pelvis and in the upper portion of the ureter in certain mammals; in man, however, no typical glands are found, although solid Superficial epi- thelial cells Epithelium. Mucosa. Inner longitud- inal muscular layer. Middle circular muscular layer. Outer muscular layer. Fig. 271. — Section of lower part of human ureter ; X I4°- epithelial buds, which extend into the mucosa for a distance, have been described. The ureter possesses two layers of nonstriated muscle-fibers — the inner longitudinal, the outer circular. From the middle of the ureter downward a third external muscular layer is found with nearly longitudinal fibers. The urinary bladder has no glands, and its musculature appar- ently consists of a feltwork of nonstriated muscle bundles, a condi- 338 THE GENITOURINARY ORGANS. tion particularly well seen in sections of the dilated organ. But even here three indistinct muscle layers may be distinguished, the outer and inner layers being longitudinal and the middle circular. A remarkable peculiarity of these structures is the extreme elasticity of their epithelium, the cells flattening or retaining their natural shape according to the amount of fluid in the cavities which they *•••'**'**• • -•!,.:< .'..1 '•* '"ff* •*' ' 5 1 V~ A\ ^ v-\ fu'M iU\Ui V? ff-lVVAl 1 ; I- •»« '*! ' v»l fV'i™ .':.- • 'fW:'. ' •/ « ' ' "*' '^"tf' ' »i » ' >f • ^«; — , s.2ijK *\'r£p>;; .,;• vp*^: I \*. ' . • •. . X. aim. 77W Fig. 272. — Transverse section of the wall of the human bladder, giving a general view of its structure. X 15- ePi Epithelium; tp, tunica propria or mucosa; sw, sub- mucosa ; ilm, inner longitudinal layer of muscle ; rin, circular layer of muscle ; aim, ex- ternal longitudinal layer of muscle ; fa, tunica adventitia. line (compare London, Kann). The terminal blood-vessels of the mucosa of the pelvis of the kidney deserve special mention. The capillaries arise from arterioles which are situated in the ridges of the mucosa above mentioned. The capillaries are peculiar in that they are not completely surrounded by connective tissue, but are in part embedded in the epithelium, the epithelial cells resting on the endothelial wall of the capillaries (Disse). The blood-vessels of the bladder anastomose in the tunica adventitia, smaller branches pass to the muscular tissue. The main stems of the vessels form a plexus in the submucosa, from which arise the capillaries of the mucosa. The veins form submucous, muscular, and subperitoneal plexuses (Fenwick). Lymphatic vessels are found only in the muscular coat and not in the mucosa. THE SUPRARENAL GLANDS. 339 The nerve supply of the bladder has been studied by Retzius, Huber, and Griinstein in the frog and a number of the smaller mammalia. Numerous sympathetic ganglia are observed, situated outside of the muscular coat, at the base and sides of the bladder. The neuraxes of the sympathetic neurones of these ganglia are grouped into smaller or larger bundles which interlace and form plexuses surrounding the bundles of nonstriated muscle-cells. From these plexuses nerve-fibers are given off, which penetrate the muscle bundles and end on the muscle-cells. The cell-bodies of the sym- pathetic neurones are surrounded by the telodendria of small medullated fibers, which terminate in the ganglia. Passing through the ganglia large medullated fibers (sensory nerves) may be ob- served which pass through the muscular coat, branch repeatedly in the mucosa, and lose their medullary sheat'hs on approaching the epithelium in which they end in numerous telodendria, the small branches of which terminate between the epithelial cells. The ureters are surrounded by a nerve plexus containing non- medullated and medullated nerve-fibers. The former end on cells of the muscular layers ; the latter pass through the muscular layer, and on reaching the mucosa branch a number of times before losing their medullary sheaths. The nonmedullated terminal branches form telodendria, the terminal fibers of which have been traced between the cells of the lining epithelium (Huber). B. THE SUPRARENAL GLANDS. The suprarenal gland is surrounded by a fibrous-tissue capsule containing nonstriated muscle-cells, blood- and lymph-vessels, nerves, and sympathetic ganglia. The glandular structure is divided into a cortical and a medullary portion. In the former are distin- guished three layers, according to the arrangement, shape, and structure of its cells — an outer glomerular zone, a middle broad fas- cicular zone, and an inner reticular zone. According to Flint, who worked in F. P. Mall's laboratory, and whose account will here be followed, the framework of the gland is made up of reticulum. In the glomerular zone this reticulum is arranged in the form of septa, derived from the capsule, which divide this zone into more or less regular spaces of oval or oblong shape. In the fascicular zone the reticulum is arranged in processes and fibrils running at right angles to the capsule. In the reticular zone the fibrils form a dense network, while in the medulla the reticular fibrils are arranged in processes and septa which outline numerous spaces. The gland-cells of the glomerular zone are arranged in coiled col- umns of cells found in the compartments formed by the septa of reticulum above mentioned. The cells composing these columns are irregularly columnar, with granular protoplasm and deeply stain- ing nuclei. In the fascicular zone the cells are arranged in regular 340 THE GENITOURINARY ORGANS. columns, consisting usually of two rows of cells, and situated be- tween the reticular processes, which run at right angles to the cap- sule. The cells of this zone are polyhedral in shape, with gran- ular protoplasm often containing fat droplets and with nuclei containing little chromatin. Similar cells are found in the reticular zone, but here they are found in small groups situated in the meshes of the reticulum. The cells of the medullary substance are less granular and smaller in size than those of the cortex, and are grouped in irregular, round, or oval masses bounded by the septa of reticulum. These cells stain a deep brown with chromic acid and its . Zona fasciculate. Capsule. Zona glomerulosa. Zona reticularis. Fig. 273. — Section of suprarenal cortex of dog ; X 1 2O' salts are therefore known as chromaffin cells ; the color cannot be washed out with water — a peculiarity which shows itself even during the development of these elements, and which is possessed by few other types of cells. Numerous ganglion cells, isolated and in groups, and many nerve-fibers occur in this portion of the organ. THE SUPRARENAL GLANDS. 341 The blood-vessels of the suprarenal glands are of special interest, since it has been shown that the secretion of the glands passes directly or indirectly into the vessels. The following statements we take from Flint : The blood-vessels, derived from various sources, form in the dog a poorly developed plexus, situated in the capsule. From this plexus three sets of vessels are derived, which are distributed respectively in the capsule, the cortex, and the medulla of the gland. The vessels of the capsule divide into Fig. 274. — Arrangement of the intrinsic blood-vessels in the cortex and medulla of the dog's adrenal (Fig. 17, Plate V, of Flint's article in " Contributions to the Science of Medicine," dedicated to Professor Welch, 1900). capillaries, which empty into a venous pldxus situated in the deeper portion of the capsule. The cortical arteries divide into capillaries which form networks, the meshes of which correspond to the arrangement of the cells in the different parts of the cortex, encircling the coiled columns of cells in the glomerular zone, while in the fascicular zone the capillaries are parallel with occa- sional anastomoses. These capillaries form a fine-meshed plexus in the reticular zone and unite in the peripheral portion of the 342 THE GENITOURINARY ORGANS. medulla to form small anastomosing veins, from which the larger veins are derived. The latter do not anastomose, and are therefore terminal veins. The arteries of the medulla pass through the cortex without giving off any branches until the medulla is reached, where they break up into a capillary network surrounding the cell masses situated here. The blood from this plexus may be col- lected into veins of the medulla which empty into the terminal vein or some of its larger branches, or .may flow directly into branches of the venous tree. The endothelial walls of the capil- laries rest directly on the specific gland cells, with the intervention here and there of a few reticular fibrils. According to Pfaundler, the walls of the blood-vessels of the entire suprarenal body consist solely of the tunica intima. The nerves of the suprarenal glands have been studied recently by Fusari and Dogiel (94) ; the description given by the latter will here be followed. Numerous nerve-fibers, both nonmedullated and medullated, arranged in the form of a plexus containing sym- pathetic ganglia, are found in the capsule. From this plexus numerous small bundles and varicose fibers enter the cortex, where they form plexuses surrounding the columns of cells or groups of cells found in the three zones of the cortex and about the vessels and capillaries of the cortex. The nerve-fibers of these plexuses are on the outside of the columns and cell groups and do not give off branches which pass between the cells. The nerve supply of the medullary substance is very rich, and is derived mainly from large nerve bundles which pass from the plexus in the capsule to the medulla, where they divide and form dense plexuses which surround the groups of gland-cells and veins ; from these plexuses fine varicose fibers pass between the gland-cells, forming intercel- lular plexuses. In the medulla there are found in many animals large numbers of sympathetic cells, some isolated, others grouped to form small ganglia. Pericellular networks surround the cell- bodies of certain of these sympathetic cells. (For further informa- tion concerning the suprarenal glands consult Gottschau, Weldon, Hans Rabl, C. K. Hoffmann (92), Pfaundler, Flint, and Dogiel.) TECHNIC. Kidney. — The arrangement of the cortical and medullary portions of the kidney is best seen in sections of the kidney of small mammalia, cut in the proper direction, and, if possible, embracing the whole organ. If, on the other hand, the finer epithelial structures are to be examined, small pieces are first fixed in osmic acid mixtures or in corrosive sublimate. Impregnation with silver nitrate (method of Golgi or Cox) reveals some points as to the relation of the cells of the uriniferous tubules to each other. In order to isolate the tubules, thin strips of kidney tissue are treated for from fifteen to twenty hours with pure hydrochloric acid TECHNIC. 343 having a specific gravity of 1.12 (for this purpose kidney tissue is used taken from an animal killed twenty-four hours previously). It is then washed, teased, and examined in glycerin (Schweiger-Seidel). Fuming nitric acid (40%), applied for a few hours to small pieces of tissue, occa- sionally isolates the uriniferous tubules very extensively. The further treatment is then the same as after hydrochloric acid. A 35% potassium hydrate solution may also be employed. The isolated pieces are, however, not easily preserved permanently. The epithelium of the uriniferous tubules may be isolated either in YI alcohol or, according to R. Heidenhain (83), in a 5^ aqueous solu- tion of neutral ammonium chromate. The latter method shows clearly the striation of the epithelium. The autophysiologic injection with indigo-carmin, applied as in the case of the liver, fills the uriniferous tubules, which may then be further examined in sections. The blood-vessels are examined in injected specimens (injection of the kidney is easily accomplished). In larger animals the injection is made into the renal artery, while in smaller ones the whole posterior half of the body is injected through the abdominal aorta. The ureter and bladder are cut open, fixed, and then sectioned. In this way the organs are shown in a collapsed condition, in which the arrangement of the epithelium is totally different from that found in the distended organs. In order to observe them in the latter condition the fix- ing agent is injected into the ureter or bladder, when, after proper liga- tion, they are placed in the same fixing agent. The usual fixing fluids are employed in the demonstration of the suprarenal capsule; but mixtures containing chromic acid, whether Flemming's fluid, chromic acid, or its salts, are of special importance in the examination of the organ, since the medullary substance of the supra- renal capsule stains a specific brown when treated by these mixtures (a con- dition only reduplicated in certain cells of the hypophysis). This brown staining also occurs when the cortical and medullary portions are entirely separated, as is the case in certain animals and during the development of the suprarenal capsule. The fat found in the cells of the suprarenal cortex is not identical with that of the rest of the body, as it may be dis- solved by chloroform and oil of bergamot out of tissue fixed with osmic acid (Hans Rabl). 344 THE GENITOURINARY ORGANS. C THE FEMALE GENITAL ORGANS. 1. THE OVUM. The product of the ovaries is the matured " ovum," or egg, hav- ing a diameter of from 0.22 to 0.32 mm. It forms a single cell with a thick membrane, from 7 /j. to 1 1 // in thickness, known as the zona pellucida. The ovum consists of a cell-body known as the yolk or vitellus, and a nucleus, from 30^ to 40 /J. in diameter, termed the germinal vesicle. The vitellus consists of two sub- stances— a protoplasmic network, with a somewhat denser arrange- ment at the periphery of the cell and in the neighborhood of the germinal vesicle, and of small, highly refractive, and mostly oval bodies imbedded between the meshes of the protoplasm — the yolk globules. These latter, as a rule, are merely browned on being treated with osmic acid, although occasionally a true fatty reaction may be obtained. The germinal vesicle is surrounded by a distinct membrane having a double contour. In its interior we find a scanty lining framework containing very little chromatin, and one or two relatively large false nucleoli, or germinal spots, from 7// to lOfJt in diameter, due to a nodal thickening of the chromatin. In the latter a further very distinct differentiation is sometimes seen in the shape of a small body (vacuole?) of doubtful origin, which has been called Schron's granule. The germinal vesicle and spot were formerly known as " Purkinje's vesicle" and "Wagner's spot," respectively, from their discoverers. 2. THE OVARY. The ovaries are almost entirely covered by peritoneum. The mesothelial cells of the latter, however, undergo here a differentia- tion, to form the germinal epithelium. At the hilum the peritoneal covering is absent, and it is here that the connective -tissue elements of the ovarian ligament penetrate into the organ to form its con- nective-tissue framework, the so-called stroma of the ovary. At an early period in the development of the ovaries, the germinal epithe- lium begins a process of imagination into the stroma of the ovary, so that at the periphery of the organ a zone is soon formed which consists of both connective tissue and epithelial (mesothelial) ele- ments. This zone is called the cortex, or parenchymatous zone. That portion of the organ in the neighborhood of the hilum (aside from the rudimentary structure known as the epoophoron) consists of connective tissue containing numerous elastic fibers and unstriped muscle-cells, and is known as the medullary substance, or vascular zone. This connective tissue penetrates here and there into the cor- tex, separates the epithelial elements of the latter from each other, and is in direct continuation with a stratum immediately beneath the germinal epithelium, called the tunica albuginea. This latter layer of connective tissue is generally distinct in the adult ovary, although THE FEMALE GENITAL ORGANS. 345 its structure and thickness vary to a considerable extent. In young ovaries it is irregular, but shows in its highest development three layers distinguishable from each other by the different direction of the fibers. In the medullary substance the connective-tissue fibers are long, in the cortex short, and in the zone containing the follicles (see below) are mingled with numerous connective-tissue cells. Nonstriated muscle-fibers occur exclusively in the medulla. Here they are gathered in bundles which accompany the blood-vessels, and may even form sheaths around the latter. They are especially prominent in mammalia. The germinal epithelium is distinguished from that of the re- maining peritoneum by the greater height of its cells, which are Young follicle with ovum. Primordial ova Ovum with fol- licular epithe- lium. Fig. 275. — Section from ovary of adult dog. At the right the stellate figure repre- sents a collapsed follicle with its contents. Below and at the right are seen the tubules of the parovarium (copied from Waldeyer). cubic or even cylindric in shape. At an early period in the devel- opment of the ovaries this epithelium pushes into the underlying embryonic connective tissue in solid projections, to form the primary egg tubes of Pfluger, the cells of which very soon begin to show differentiation. Some retain their original characteristics and shape, while others increase in size, become rounded, and develop into the young ova. Those retaining their indifferent type become the fol- licular cells surrounding the egg. This differentiation into ova and follicular elements may even occur in the germinal epithelium itself, in which case the larger round cells are known as the primitive or primordial ova. In the further development of the ovarian cortex 346 THE GENITO-URINARY ORGANS. the primitive egg tubes are penetrated throughout by connective tissue, so that each egg tube is separated into a number of irregular divisions. In this way a number of distinct epithelial nests are formed, which lose their continuity with the germinal epithelium and finally lie imbedded in the connective tissue. According to the shape and other characteristics of these epithelial nests, we may distinguish several different groups: (i) The primitive egg tubes Germinal epi- thelium. •»•» Tunica albu- ginea. — Follicular _^ epithelium. Ovum. *** O P ~( •Sv«^' WronSfr** • °**fr°'a ™i%f/f$i$ *» 9 v-Qfj ~0 "o " ">T»*5 *»*ij3X0 ef'S yGrttJ^OOO feSS/^s^Sw8^?^ Granular layer of large Graafian follicle. Fig. 276. — From ovary of young girl ; X I9°- of Pfluger ; (2) the typical primitive follicles — i. e., those which contain only a single egg-cell (present in the twenty-eighth week of fetal life) ; (3) the atypic follicles — i. e., those containing from two to three egg-cells ; (4) the so-called nests of follicles, in which a large number of follicles possess only a single connective-tissue en- velope ; (5) follicles of the last-named type which may assume the form of an elongated tube, and which are then known as the con- THE FEMALE GENITAL ORGANS. 347 stricted tubes of Pfliiger. The fourth, fifth, and possibly the third types are further divided by connective-tissue septa, until they finally form distinct and typical follicles (Schottlander, 91, 93). In the adult ovary true egg tubes are no longer developed. Isolated imaginations of the germinal epithelium sometimes occur, but apparently lead merely to the formation of epithelial cysts (Schottlander). The theories as to when the formation of new epithelial nests or follicles ceases are, however, very conflicting, some authors believing that cessation takes place at birth, others that it continues into childhood and even into middle age. The typical primitive follicle consists of a relatively large egg- cell surrounded by a single layer of smaller cubical or cylindric follicular cells. The growth of the follicle takes place by means of mitotic division in the follicular cells and increase in size of the ovum. The egg-cell is soon surrounded by several layers of cells, and gradually assumes an eccentric position in the cell complex. At a certain distance from the ovum and nearly in the center of the follicle one or more cavities form in the follicular epithelium. These become confluent, and the resulting space is filled by a fluid derived, on the one hand, from a process of secretion and, on the other hand, from the destruction of some of the follicular cells. The cavity is called the antrum of the follicle, and such a follicle has received the name of Graafian follicle. Its diameter varies from 0.5 to 6 mm. The follicle in- creases in size through cell-proliferation, the cavity increasing and gradually inclosing the egg together with the follicular cells imme- diately surrounding it, although the latter always remain connected with the wall of the vesicle at some point. The egg now lies imbedded in a cell-mass, the discus proligerus, which is composed of follicular epithelium, and projects into the follicular cavity. The follicular epithelium forming the wall of the cavity is known as the stratum granulosum, the cavity as the antrum, and the fluid which it contains as the liquor folliculi. Those follicular cells which immediately surround and rest upon the ovum are some- what higher than the rest and constitute the egg epithelium, or corona radiata. During the growth of the follicle the connective tissue surround- ing it becomes differentiated into a special envelope, called the tJicca folliculi. In it two layers may be distinguished — the outer, the tunica externa, consisting of fibrous connective tissue, is continu- ous with the inner, or tunica interna, rich in blood-vessels and cellular elements. The follicle gradually extends to the surface of the ovary, at which point it finally bursts (see below), allowing the ovum to escape into the body cavity and thus into the oviduct. During the growth and development of the ovarian follicles the ova undergo certain changes of size and structure which may receive further consideration. These have been described for the human ovary by Nagel (96), whose account will here be followed. The Fig. 280. Figs. 277, 278, 279, and 280. — From sections of cat's ovary, showing ova and follicles in different stages of development ; X 225 • ai a> a-> a> Germinal spots ; />, i>, b, 6, germinal vesicles ; t, c, c, c, ova ; •- Mucosa. Fig. 285. — From uterus of young woman ; X 34- (From a preparation by Dr. J. Amann.) entering the mucosa, where they form capillary networks surround- ing the glands and a dense capillary network situated under the uterine epithelium. The veins form a venous plexus in the deeper portion of the mucosa, especially well developed in the cervix and os uteri. From this plexus the blood passes to a second well- developed venous plexus situated in the stratum vasculosum of the muscular layer, whence the blood passes to the plexus of uterine and ovarian veins. The lymphatics begin in numerous clefts in the uterine mucosa ; 358 THE GENITOURINARY ORGANS. from here the lymph passes by way of lymph-vessels to the mus- cular coat, between the bundles of which are found numerous lymph-vessels especially in the middle or vascular layer. These lymph-vessels terminate in larger vessels found in the subserous connective tissue. The uterus receives numerous medullated and nonmedullated nerves. The latter terminate in the muscular layers. Medullated fibers have been traced into the mucosa, where they form plexuses under the epithelium, from which branches have been traced between the epithelial cells and between the gland cells. In the course of the nerves ganglion cells of the sympathetic type have been observed. Fig. 286. — From section of human vagina. In the vagina we distinguish also three coats — the mucous membrane, the muscular layer, and the outer fibrous covering. The epithelium of the mucous membrane is of the stratified squamous type, and possesses, as usual, a basal layer of cylindric cells. The mucosa of the vagina consists of numerous connective- tissue fibers mingled with a number of exceptionally coarse elastic fibers. Papillae containing blood-vessels are present everywhere ex- cept in the depressions between the columnar rugarum. It is generally stated that the vagina has no glands, but according to the observa- tions of von Preuschen and C. Ruge, a few isolated glands occur in THE FEMALE GENITAL ORGANS. 359 the vagina. They are relatively simple in structure, form irregular tubes, and are lined by ciliated columnar epithelium. The excre- tory ducts are lined by stratified squamous epithelium. Diffuse adenoid tissue is met with in the mucosa, which sometimes assumes the form of lymphatic nodules. The muscular coat, which in the lower region is quite prominent, may be separated indistinctly into an outer longitudinal and an in- ner circular layer ; the latter is, as a rule, poorly developed, and may be entirely absent. The muscular coat is especially well developed anteriorly in the neighborhood of the bladder. Fig. 287. — From section of human labia minora. The outer fibrous layer consists of dense connective tissue loosely connected with the adjacent structures. At its lower end the vagina is partially closed by the hymen which must be regarded as a rudiment of the membrane which in the embryo separates the lower segment of the united Miillerian ducts from the ectoderm of the sinus urogenitalis. Accordingly, the epithelium on the inner surface of the hymen partakes of the character of the vaginal epithelium ; that on the outer surface re- sembling the skin in structure (G. Klein). 360 THE GENITO-URINARY ORGANS. The epithelium of the vestibulnm gradually assumes the char- acteristics of the epidermis ; its outer cells lose their nuclei and sebaceous glands occur here and there in the neighborhood of the urethral orifice and on the labia minora. Hair begins to appear on the outer surface of the labia majora. The clitoris is covered by a thin epithelial layer, resembling the epidermis. This rests on a fibrous-tissue mucosa having numerous papillae, some of which contain capillaries, others special nerve- endings. In the clitoris of the adult no glands are found. The greater portion of the clitoris consists of cavernous tissue, homol- ogous to the corpora cavernosa of the penis ; the corpus spongi- osum is not present in the clitoris. The glands of Bartholin, the homologues of the glands of Cowper in the male, are mucous glands situated in the lateral walls of the vestibule of the vagina. The terminal portions of their ducts are lined by stratified squamous epithelium. Free sensory nerve-endings, with or without terminal enlarge- ments, have been demonstrated in the epithelium of the vagina (Gawronski). The sensory nerve-fibers form plexuses in the mucosa, and lose their medullary sheaths as they approach the epithelium. Sympathetic ganglia are met with along the course of these nerves, and nonmedullated nerves terminate in the involuntary muscular tissue of the vaginal wall. In*the connective-tissue papillae and in the deeper portions of the mucosa of the glans clitoridis are found, besides the ordinary type of tactile corpuscles and the spherical end-bulbs of Krause, the so- called genital corpuscles (see p. 171). Numerous Pacinian cor- puscles have been observed in close proximity to the nerve-fibers of the clitoris and the labia minora. In varying regions of the medullary substance of the ovary, but more usually in the neighborhood of the hilum, there occur irregular epithelial cords or tubules provided with columnar epithe- lium, ciliated or nonciliated, which constitute the paroophoron. These are the remains of the mesonephros, and are continuations of that rudimentary organ — the epoophoron — of similar structure which lies within the broad ligament. The separate tubules of the epoophoron communicate with the duct of Gartner (Wolfifian duct), which in the human being is short, ends blindly, and never, as in certain animals, opens into the lower portion of the vagina. These derivatives of the primitive kidney consist of blindly ending tubules of varying length lined by a ciliated epithelium, the cells of which are often found in process of degeneration. The hydatids of Morgagni are duplications of the peritoneum. THE MALE GENITAL ORGANS. 361 D. THE MALE GENITAL ORGANS. J. THE SPERMATOZOON. The semen, or sperma, is a fluid that, as a whole, consists of the secretion of several sets of glands in which the sexual cells, the spermatosomes, or spermatozoa, which are formed in the testes, are suspended. We shall first consider the structure of the typical adult sperma- tosome, taking up consecutively its component parts. Three prin- cipal parts may be distinguished — the head, the middle piece, and the tail or flagellnm. The round or oval body of the head termi- nates in a lanceolate extremity. The former consists of chromatin, and is most intimately associated with the phenomenon of fertiliza- tion. The middle piece, which is attached to the posterior end of the head, is composed of a protoplasmic envelop which surrounds a portion of the so-called axial thread. The latter is enlarged ante- riorly just behind the head to form the terminal nodule, which fits into a depression in the head. From the middle piece on, the axial thread Fig. 288. — Diagram showing the general characteristics of the spermatozoa of various vertebrates : a, Lance ; b, segments of the accessory thread ; c, accessory thread ; d, body of the head ; e, terminal nodule ; f, middle piece ; g, marginal thread ; h, axial thread ; i, undulating membrane ; k, fibrils of the axial thread ; /, fibrils of the marginal thread ; m, end piece of Retzius ; ;/, rudder-membrane. is continued into the tail of the spermatozoon, and is here sur- rounded by a transparent substance — the sheatJi of the axial thread. The envelop is lacking at the posterior extremity of the tail, where the axial thread extends for a short distance as a naked filament called the end-piece of Retzius. From the middle piece a still finer thread is given off", the marginal thread, which extends at a certain distance from the axial thread as far as the end-piece of Retzius. In its course it crosses and recrosses the axial thread at various points, and may even wind around it in a spiral manner. In all in- stances it is connected with the sheath of the axial thread by a delicate membrane — the undulating membrane. Another and still more delicate filament — the accessory thread — runs parallel with the axial thread along the surface of its sheath and terminates at a cer- tain distance from the end-piece of Retzius. Near the extremity of the flagellum and immediately in front of the end-piece is another and shorter membrane, — the rudder membrane, — which is continu- ous with the undulating membrane. Maceration reveals a fibrillar 362 THE GENITOURINARY ORGANS. I- a structure of both the axial and marginal threads (Ballowitz), while the accessory thread is separated into a number of short segments. In mammalia, and especially in man, the spermatozoa seem to be" more simply con- structed. Here the head is pyriform, and somewhat flattened, with a slight ridge along the depression at either side of its anterior thinner portion (Fig. 289). In some mammalia (mouse), the head is provided with a so- called cap, which corresponds to the lance previously mentioned. The middle piece is relatively long and shows a distinct cross- striation, which may be attributed to its spiral structure. Here also the middle piece is tra- versed by the axial thread, which ends at the head in a terminal nodule, and may be sep- arated as in other mammalia into a number of fibrils. Some years ago Gibbes described an undulating membrane in the human sper- matozoon, an observation which was confirmed by W. Krause (81). The head of the .human spermatosome is from 3 /j. to 5 [i long, and from 2 u to 3 u in breadth ; the middle piece Fig. 289. — Human ' J ' spermatozoa. The two is 6 // long and I fj. in breadth ; the tail is from at the left after Retzius 40 // to 60 /J. long, and the end-piece 6 fj. long. extreme left0^ seen* in ^he spermatozoa are actively motile, a phe- profile; the other in nomenon due to the flagella, which give them surface view; the one a Spira1 boring motion. They are character- at the right is drawn as , described by Jensen -. a, lzed by great longevity and are very resistant Head ; l>, terminal nod- to the action of low temperatures (vid. Pier- ule; c, middle piece; gol g \ jn SQme spedes of bat the Sper- a, tail ; e, end-piece of . L r Retzius. matozoa penetrate into the oviduct of the female in the fall, but do not contribute to im- pregnation until the spring, when the ova mature. (For the structure of the spermatosomes see Jensen, Ballowitz.) 2. THE TESTES. The testis is inclosed within a dense fibrous capsule, — the tunica albuginea, — about one-sixteenth of an inch in thickness, and surrounded by a closed serous sac, derived from the peritoneum during the descent of the testes, and therefore lined by mesothelial cells. This serous sac — the tunica vaginalis — consists of a visceral layer attached to the tunica albuginea, and a parietal layer which blends with the scrotum. The cavity contains normally a small amount of serous fluid. On the inner surface of the tunica albuginea is found a thin layer of loose fibrous tissue containing blood-vessels — the tunica vasculosa. The tunica albuginea is thickened in its THE MALE GENITAL ORGANS. posterior portion to form the mediastinum testis, or the corpus Highmori, which projects as a fibrous-tissue ridge for a variable distance into the substance of the testis. The gross structure of the testis is best seen in a sagittal longitudinal section. Even a low magnification will show that the testis is composed of lobules. These are produced by septa which extend into the substance of the organ and are derived from the investing tunics of the testis and diverge in a radiate manner from the mediastinum testis. The lobules are of pyramidal shape, with their bases directed toward the capsule and their apices toward the mediastinum. They consist principally of the seminiferous tubules, whose transverse, oblique, and longitudinal Lobule of testis. Tunica albuginea. _Caput epidi- dymidis. Corpus Highmori and rete testis. — Blood-vessel. Tubuli recti. J, flattened sustentacular cell ; <-, :„:* Fig. 293. — From section of human testis, showing convoluted seminiferous tubules. the tubuli recti to an epithelium consisting of a single layer of short columnar or cubical cells resting on a thin basement membrane. The canals of the rete testis (Haller) are lined by nonciliated epithelium, which varies in type from flat to cubical. Communicat- ing with the rete testis is a blind canal, the vas aberrans of the rete testis, lined with ciliated epithelium. The vasa efferentia are lined partly by ciliated columnar and partly by nonciliated cubical epithelium. The two varieties form groups which alternate, giving rise to nonciliated depressions, which represent gland-like structures (Schaffer, 92), but do not 366 THE GENITOURINARY ORGANS. cause corresponding evaginations of the mucosa. The mucosa, which consists of fibrous connective tissue, contains flattened endo- thelioid cells, which resemble nonstriated muscle-cells. The latter are found only at the end of the vasa efferentia, just before reaching the vas epididymidis. Fig. 294. — Section through human vasa efferentia : a, Glands ; b, ciliated epithelium ; f, glandular structure ; d, connective tissue. Fig. 295. — Cross-section of vas epididymidis of human testis. The vas epididymidis is lined by stratified ciliated columnar epithelium, resting on a thin mucosa, outside of which there is found an inner circular and an outer, though thin and not continuous, longitudinal layer of nonstriated muscular tissue. An aberrant canaliculus also communicates with the vas epi- didymidis, and is here known as the vas aberrans Halleri. Num- THE MALE GENITAL ORGANS. 367 bers of convoluted and blindly ending canaliculi are frequently found imbedded in the connective tissue around the epididymis. These constitute the paradidymis, or organ of Giraldes. The blood-vessels of the testis spread out in the corpus High- mori and in the tunica vasculosa of the connective-tissue septa and of the tunica albuginea, their capillaries encircling the seminal tu- bules in well-marked networks. The lymphatic vessels begin in clefts in the tunica albuginea and in the connective tissue between the convoluted tubules. They con- verge toward the corpus Highmori and pass thence to the spermatic cord. Retzius (93) and Tim- ofeew (94) have described plexuses of nonmedul- lated, varicose nerve-fibers surrounding the blood- vessels of the testis. From such plexuses single fibers, or small bundles of such, could be traced to the seminiferous tubules, about which they also form plexuses. Such Fig. 296. — Section of dog's testis with in- jected blood-vessels (low power) : a, Seminifer- ous tubule ; b, connective-tissue septum ; c, blood- vessel. fibers have not been traced into the epithelium lining the tubules. In the epididymis Timofeew found numerous sympa- thetic ganglia, the cell- bodies of the sympathetic neurones of which were surrounded by pericellular plexuses. In the wall of the vas epididymidis and the vasa efferentia were observed numerous varicose nerve-fibers, arranged in the form of a plexus, many of which seemed to terminate on the nonstriated muscle cells found in these tubes. Some of the nerve-fibers were traced into the mucosa, but not into its epithelial lining. 3. THE EXCRETORY DUCTS. The vas deferens possesses a relatively thick muscular wall, con- sisting of three layers, of which the middle is circular and the other two longitudinal. The subepithelial mucosa is abundantly supplied with elastic fibers and presents longitudinal folds. The lining epi- thelium is in part simple ciliated columnar and in part stratified ciliated columnar, with two rows of nuclei. The cilia are, however, often absent, beginning with the lower portion of the vas epidi- 368 THE GENITOURINARY ORGANS. dymidis. According to Steiner, the epithelium of the vas deferens varies. It may be provided with cilia in the lower segments, or it may even be similar to that found in the bladder and ureters. The inner muscular layer is wanting in the ampulla of the vas deferens ; here the epithelium is mostly simple columnar and pig- mented. Besides the folds, there are also evaginations and tubules which sometimes form anastomoses — structures which may be re- garded as glands. The seminal vesicles are also lined, at least when in a distended condition, by simple, nonciliated columnar epithelium containing yellow pigment. In a collapsed condition the epithelium is pseudo- stratified, with two or even three layers of nuclei. The arrange- ment of the epithelial cells in a single layer would therefore seem to be the result of distention. The mucous membrane shows Epithelium. Outer lon- gitudinal muscular layer. Fig. 297. — Cross-section of vas deferens near the epididymis (human). numerous folds, which, in the guinea-pig for instance, present a delicate axial connective -tissue stroma. Besides scanty subepithe- lial connective tissue, the seminal vesicles are provided with an inner circular and an outer longitudinal layer of muscle-fibers. Sperma- tozoa are, as a rule, not met with in the seminal vesicles. The epithelium of the ejaculatory ducts is composed of a single layer of cells ; the inner circular muscle-layer is very poorly devel- oped. In the prostatic portion of the ejaculatory ducts the longi- tudinal muscle-layer mingles with the musculature of the prostate and loses its individuality. The ejaculatory ducts empty either directly into the urethra at the colliculus seminalis, or indirectly into the prostatic portion of the urethra through the vesicula prostatica. 1\i& prostate is a compound branched tubulo-alveolar gland. Its THE MALE GENITAL ORGANS. 369 capsule consists of dense layers of nonstriated muscle-fibers, connec- tive tissue, and yellow elastic fibers. Processes and lamellae com- posed of all these elements extend into the interior of the gland, con- verging toward the base of the colliculus seminalis. Between the larger trabeculae are situated numerous glands, consisting of large, Fig. 298. — Cross-section of wall of seminal vesicle, showing the folds of the mucosa (human). Fig. 299. — From section of prostate gland of man. irregular alveoli, separated by fibromuscular septa and trabeculas. The alveoli are lined by simple columnar epithelium, the inner portion of the cells often showing acidophile granules. Now and then the alveoli present a pseudostratified epithelium, with two rows of nuclei (Rudinger, 83). A basement membrane, although 24 3/O THE GENITOURINARY ORGANS. present, is difficult to demonstrate and consists of a network of deli- cate connective-tissue threads, as was shown by Walker. The numerous excretory ducts, lined by simple columnar epithelium, be- come confluent and form from 1 5 to 30 collecting ducts which empty, as a rule, either at the colliculus seminalis or into the sulcus prosta- ticus. Near their terminations the larger ducts are lined by transi- tional epithelium similar to that lining the prostatic portion of the urethra. In the alveoli of the glands, peculiar concentrically laminated concrements are found, known as prostatic bodies or concretions (corpora amylacea). They are more numerous in old men, but are found in the prostates of young men and also of young boys. The secretion of the prostate (succus prostaticus) is not mucous in character, but resembles a serous secretion and has an acid reac- tion. The vesicula prostatica (sinus pocularis) is lined by stratified epithelium, consisting of two layers of cells and provided with a dis- tinct cuticular margin upon which rest cilia. In its urethral region occur short alveolar glands. The glands of Cowper are branched tubular alveolar glands, the alveoli being lined by mucous cells. The smaller excretory ducts, lined by cubical epithelium, unite to form two ducts, one on each side of the urethra ; these are I ^ inches long, and are lined by strat- ified epithelium consisting of two or three layers of cells. The blood-vessels of the prostate ramify in the fibromuscular trabeculae and form capillary networks surrounding the alveoli. The veins collecting the blood pass to the periphery of the gland, where they form a plexus in the capsule. The lymphatics begin in clefts in the trabeculae and follow the veins. The terminal branches of the vessels supplying Cowper's glands are, in their arrangement, like those of other mucous glands. Numerous sympathetic ganglia are found in the prostate under the capsule and in the larger trabeculse near the capsule. The neuraxes of the sympathetic cells of these ganglia may be traced to the vessels and into the trabeculae ; their mode of ending has, however, not been determined. Small medullated nerve-fibers terminate in these ganglia in pericellular baskets. Timofeew has described peculiar encapsulated sensory nerve-endings, found in the prostatic and membranous portions of the urethra of certain mam- malia. They consist of the terminal branches of two kinds of nerves, inclosed within nucleated laminated capsules : one large medul- lated nerve -fiber, after losing its medullary sheath, breaks up into a small number of ribbon-shaped branches with serrated edges, which may pass more or less directly to the end of the nerve-ending or may be bent upon themselves ; and very much smaller medullated nerve-fibers which, after losing their medullary sheaths, divide into a large number of varicose fibers which form a dense network en- circling the ribbon-shaped fibers previously mentioned. The penis consists of three cylindric masses of erectile tissue — the two corpora cavernosa, forming the greater part of the penis THE MALE GENITAL ORGANS. and lying side by side, and the corpus spongiosum, surrounding the urethra and lying below and between the corpora cavernosa. The two latter are surrounded by a dense connective-tissue sheath, the tunica albuginea. These erectile bodies are surrounded by a thin layer of skin, containing no adipose tissue and no hair-follicles. The corpus spongiosum is enlarged anteriorly to form the glans penis. The principal substance of the erectile bodies is the so-called erectile tissue : septa and trabeculae, consisting of connective tissue, elastic fibers, and smooth muscle-cells inclosing a sys- tem of communicating spaces. These latter may be regarded as venous sinuses, the walls of which, lined by endothelial cells, are in apposition to the erectile tissue. Under certain conditions the venous sinuses are distended with blood, but normally they are in a collapsed state and form fissures which simulate the clefts found in ordinary connective tissue. In other words, there is here such an arrangement of the blood-vessels within the erectile tissue that the circulation may be carried on with or without the aid of the cavernous spaces. The arteries of the corpora cavernosa possess an especially well-developed musculature. They ramify through- out the trabeculas and septa of the erectile tissue and break up within the septa into a coarsely meshed plexus of capillaries. A few of these arteries empty directly into the cavernous spaces. On the other hand, the arteries give off a rich and narrow-meshed capillary network immediately beneath the tunica albuginea. This is in com- munication with a deeper and denser venous network, which, in turn, gradually empties into the venous sinuses. Aside from these there are anastomoses between the arterial and venous capillaries, which later communicate with the venous network just mentioned. The blood current, regulated as it thus is, may pass either through the capillaries alone, or may divide and flow through both these and the venous sinuses. These conditions explain both the erec- tile and quiescent state of the penis. The relations are somewhat different in the corpus spongiosum urethrae and in the glans penis. In the corpus spongiosum the arteries do not open directly into the venous spaces, but break up first into capillaries. In the sub- mucosa of the urethra there is found a rich venous plexus. In the glands the arteries end in capillaries which pass over into veins with well-developed muscular walls. The blood is collected by means of the venae emissariae which empty into the vena dorsalis penis and into the venae profundae. The epithelium of the urethra varies in the several regions. The prostatic portion possesses an epithelium similar to that of the bladder. In the membranous portion, the epithelium may be simi- lar to that found in the prostatic portion, but more often pre- sents the appearance of a pseudostratified epithelium with two or three layers of nuclei. The cavernous region is lined by pseudo- stratified epithelium, except in the fossa navicularis, where a stratified squamous epithelium is found. Between the fibre-elastic 372 THE GENITOURINARY ORGANS. mucosa and the epithelium there is a basement membrane. There occur in the urethra, beginning with the membranous portion, ir- regularly scattered epithelial sacculations of different shapes. Some of these show alveolar branching, and are then known as the glands of Littre. The submucosa of the cavernous portion of the urethra, which contains nonstriated muscle-tissue arranged circularly, is richly sup- plied with veins, and contains pronounced plexuses communicating with cavernous sinuses, which correspond in general to those of the corpora cavernosa penis. The glans is covered by a layer of stratified squamous epithe- lium, often possessing a thin stratum corneum (see Skin). Near the corona of the glans penis there are now and then found small sebaceous glands (see Hair), known as glands of Tyson. The pre- puce is a duplication of the skin, the inner surface presenting the appearance of a mucous membrane. The nerves terminating in the glans penis have recently been studied by Dogiel, who made use of the methylene-blue method in his investigation. He finds Meissner's corpuscles in the connective- tissue papillae under the epithelium, Krause's spheric end-bulbs somewhat deeper in the connective tissue, and the genital corpuscles situated still deeper (see Sensory Nerve-endings). In the epithelium are found free sensory nerve-endings. Pacinian corpuscles have also been found in this region. 4. SPERMATOGENESIS. In order that the student may obtain an understanding of the com- plicated process of spermatogenesis we shall give a description of it as it occurs in salamandra maculosa, which of all vertebrate animals presents the phenomena in their simplest and best known form. The student should understand, however, that many of the details here described have not been observed in the testes of mammalia ; and, since the spermatozoa of many of the mammalia are of simpler structure than those of the salamander, the development of the spermatozoa of the former is consequently simpler. It should also be noticed that the general structure of the testes of the salamander differs in some respects from that of the testes of mammalia, as given in the preceding pages. At first the seminiferous tubules consist of solid cellular cords, and it is only during active production of spermatozoa that a central lumen is formed, in which the spermatosomes then lie. The cells which compose these solid cords may be early differentiated into two classes — those of the one class being directly concerned in the pro- duction of the spermatosomes ; those of the other appearing to have a more passive role. The cells of the first class — the spcrmatogo- nia, or primitive seminal cells — undergo a process of division accom- panied by an increase in size. In this way they soon commence to press upon the cells of the second class — \\\& follicular o\~ sustentacu- SPERMATOGENESIS. 373 lar cells. The result is that the nuclei of the latter are forced more or less toward the wall of the seminal tubule, while their proto- plasm is so indented by the adjacent spermatogonia that the cells assume a flattened cylindric shape presenting indentations and processes on all sides. In this stage the spermatogonia have a radiate arrangement and entirely surround the elongated susten- tacular cells. At present three periods are distinguished in the development of the male sexual cells (spermatosomes) from the spermatogonia. The first period embraces a repeated mitotic divi- sion of the spermatogonia — the period of proliferation. In the sec- ond, the spermatogonia, which have naturally become smaller from repeated division, begin to increase in size — the period of growth. The third is characterized by a modified double mitotic division without intervening period of rest, and results in the matured sper- matozoa— the period of maturation, figure 300. During the third period, a very important and significant process takes place — the Primordial sexual cell. Spermatogonia. ,, \ \ Spermatocyte I order. Spermatocytes II o Sperma Zone of proliferation. (The generations are much larger.) Zone of growth. Zone of maturation. Fig. 300. — Schematic diagram of spermatogenesis as it occurs in ascaris (after Boveri). ("Ergebn. d. Anat. u. Entw.," Bd. I.) reduction in the number of chromosomes, so that in the spermatids, the chromosomes are reduced to half the number present in a somatic cell of the same animal. The manner in which this reduc- tion in the number of chromosomes takes place will be described as it occurs in salamandra maculosa. After the cells composing the last generation of spermato- gonia have attained a certain size (period of growth), they under- go karyokinetic division. First, the usual skein or spirem is formed, but instead of dividing into twenty-four chromosomes, as in the somatic cell, the filament of the skein segments into only twelve loops. The cell thus provided with twelve chromosomes now enters upon the period of maturation, and is known as a 374 THE GENITOURINARY ORGANS. spermatocyte of the first order, or a "mother cell" (O. Hert- wig, 90). The division of these cells is heterotypic (vid. p. 70); the chromosomes split longitudinally and in such a way that the division begins at the crown of the loops, extending gradually toward their free ends. In this case the daughter chromosomes remain for some time in contact, so that the metakinetic figure resembles a barrel in shape. Finally, the daughter chromosomes separate and wander toward the poles. As soon as the daughter stars (diaster) are developed, the number of chromosomes is again doubled by a process of longitudinal division. The spermatocyte of the first order thus divides into two spermatocytes of the second order, or daughter cells (O. Hertwig, 90). The nuclei of the daughter cells now contain twenty-four chromosomes, as is the case in the somatic cell, and, without undergoing longitudinal split- ting, the daughter chromosomes are distributed to the two nuclei of the spermatids. In other words, the latter contain only twelve chromosomes. The spermatozoa are formed from the spermatids by a rearrangement of the constituent elements of these cells. It may thus be said that even in the stage of the segmenting skein in the mother cells, the spermatocytes of the first degree contain twice as many chromosomes as a somatic cell, a condition which is first clearly seen in the stage of the diaster (here only an apparent duplication in the diaster stage). As a result, there is, first, a de- crease in the double number of chromosomes found in the sperma- tocytes of the second degree to the normal number ; second, a decrease in the number of chromosomes in the spermatocytes of the third degree (spermatids) to one-half the number present in a somatic cell, a condition probably due to the fact that here there is no stage of rest nor longitudinal splitting of the chromosomes. This is the general process in heterotypic division. Besides the heterotypic form, there occurs in the division of the spermatocytes another (homeotypic) form of karyokinetic cell-division. This dif- fers from the heterotypic in the shortness of the chromosomes, the absence of the barrel phase, the late disappearance of the aster, and the absence of duplication in the chromosomes of the diaster. According to Meves (96), the spermatocytes of the first degree undergo heterotypic, those of the second degree, homeotypic division. The spermatids develop into the spermatozoa, beginning imme- diately after the close of the second division of maturation. This process has been fully described for salamandra maculosa by Her- mann, Flemming, Benda, and others, but need not engage our attention at this point beyond the statement that the chromatin of the nuclei of the spermatids develops into the heads of the sperma- tozoa, while the remaining structures are developed from the proto- plasm. " The mature spermatozoon of the salamander represents a completely metamorphosed rell ; in the course of its develop- ment no portion of the original cell is cast off" (Meves, 97). Spermatogenesis in mammalia may be compared to the foregoing SPERMATOGENESIS. 375 process, with the exception that here the different stages are seen side by side in the seminiferous tubule and without any apparent sequence, making the successive stages more difficult to demon- strate. The various generations of cells form columns, and are arranged in such a manner that the younger are found near the lumen and the older close to the wall of the tubule. (Figs. 301 and 9 Fig. 301. — Schematic diagram of section through convoluted seminiferous tubule of mammal, showing the development of the spermatosomes. The number of chromo- somes is not shown in the various generations of the spermatogenic cells. The pro- gressive development of the spermatogenic elements is illustrated in the eight sectors of the circle : a, Young sustentacular cell ; b, spermatogonium ; c, spermatocyte ; d, spermatid. In I, 2, 3, and 4 the spermatids rest on the enlarged sustentacular cell in the center of the sector ; on both sides of the sustentacular cells are the spermatogenic or mother cells in mitosis. In the sectors 5, 6, 7, and 8 spermatozoa are seen in ad- vanced stages resting on the sustentacular cells, with new generations of spermatids on each side. [From Rauber (after Brown) with changes (after Hermann).] 302.) These columns are separated from each other by high sus- tentacular cells, or Sertoli's cells or columns. The metamorphosis of the cells into spermatids and spermatosomes is accomplished by the changing of the cells bordering upon the lumen and then of those in the deeper layers, etc., into spermatids and then into spermatosomes. During this process the spermatids arrange 3/6 THE GENITOURINARY ORGANS. themselves around the ends of Sertoli's columns, a phenomenon which was formerly regarded as representing a copulation of the two elements, although it was clearly understood that no real fusion or interchange of chromatin occurred, but that the close relations of the two were for the purpose of furnishing nourishment to the developing spermatosomes. The whole forms a spermato- blast of von Ebner. Since the spermatids lining the lumen are changed into spermatozoa, and the process is repeated in the cells of the deeper layers as they come to the surface, the result is that the entire column is finally used up. The compensatory elements are supplied by the proliferation of the adjacent spermatogonia. The resulting products again divide, and thus build up an entirely new generation of spermatogenic cells. Hand in hand with these progressive phenomena occurs an extensive destruction of the cells taking part in spermatogenesis. This is shown by the presence of so-called karyolytic figures in the cells, which later suffer complete demolition. These developmental changes are represented in the preced- Fig. 302. — Section of convoluted tubule from rat's testicle (after von Ebner, 88). The pyramidal structures are the sustentacular cells, together with spermatids and spermatosomes. Between these are spermatogenic cells, some of which are in process of mitotic division. Below, on the basement membrane and concealing the spermato- gonia, are black points representing fat-globules, a characteristic of the rat's testicle. Fixation with Flemming' s fluid. ing schematic figure (Fig. 301), and may in part be observed in figure 302. In mammalia it has been possible to trace the development of the spermatids into the spermatosomes. These phenomena have been studied and described by numerous writers, and although many conflicting views have been expressed, the essential steps of this process seem quite clearly established. The account here given is based in part on the recent observations of v. Lenhossek and the observations of Benda. Before considering the method o/ development of the spermatosomes from the spermatids, a few words concerning the structure of the latter may be useful. The sharply outlined spermatid possesses a slightly granular protoplasm and a round or slightly oval nucleus with a delicate chromatic network. In the protoplasm there is found a sharply defined globule, known as the sphere or sphere substance, which lies near the nucleus and SPERMATOGENESIS. 377 presents throughout a nearly homogeneous structure. This sub- stance is first noticed in the spermatocytes, disappears during the cell-divisions resulting in the spermatids, and reappears in the latter. In the protoplasm of the spermatid, lying near the nucleus, there is further found a small globular body, the chromatoid accessory nucleus of Benda, smaller than the sphere and staining very deeply in Heidenhain's hematoxylin. A true centrosome may also be found in the spermatid. The nucleus of the spermatid develops into the head of the spermatosome, during which change the originally spheric nucleus becomes somewhat flattened and at the same time assumes a denser structure and moves toward that portion of the spermatid pointing away from the lumen of the seminiferous tubule. Accompanying these changes in the nucleus, marked changes are observed in the shape and structure of the sphere, which marks the position of the future anterior end of the head of the spermatosome, and applies itself to the nucleus on the side pointing away from the lumen of the tubule. In this position it differentiates into an outer clear homogeneous zone and a central portion which stains more deeply and to which v. Lenhossek has given the name akrosome. From these structures are developed the head-cap and the lance of the spermatosomes, which differ in shape and relative size in the sper- matosomes of the different vertebrates. Recent investigation seems to establish quite clearly that the axial thread of the tail is devel- oped from the centrosome (from the larger, if two are present), which is situated at some distance from the nucleus. Soon after the begin- ning of the development of the axial thread the centrosome wanders to the posterior part of the future head of the spermatosome (the pole of the nucleus opposite the head-cap) and becomes firmly attached to the nuclear membrane in this position (observations made on the rat by v. Lenhossek, and on the salamander by Meves). The middle piece and the undulating membrane, it would appear, are differentiated from the protoplasm, although the question of the mode of their development is still open to discussion. The chro- matoid body assumes a position near the axial thread at its junc- tion with the cell membrane ; its fate has not, however, been fully determined. According to Hermann (97), the end-piece in the selachia is derived from the centrosome, the ring-shaped body from the invagi- nated half of the intermediate body of the spermatid formed during the last spermatocytic division, and the axial thread from filaments of the proximal half of the central spindle. The lance, according to him, represents a modified portion of the nuclear membrane of the spermatid. For further particulars regarding spermatogenesis see the in- vestigations of v. la Valette St. George, 67-87 ; v. Brunn, 84 ; Biondi, Benda, Meves, and v. Lenhossek. 378 THE GENITOURINARY ORGANS. TECHNIC The ovaries of the smaller animals are better adapted to study than those of the human being, since the former are more easily fixed. The germinal epithelium and its relations to the egg-tubes of Pfliiger are best studied in the ovaries of young or newly born animals — cats, for instance, being especially well adapted to this purpose. Normal human ovaries are usually not easily obtainable. Human ovaries very often show pathologic changes, and in middle life frequently contain but few follicles. Fresh ova may be easily procured from the ovaries of sheep, pig, or cow in the slaughter-houses. On their surfaces are prominent trans- parent areas — the larger follicles. If a needle be inserted into one of these follicles and the liquor folliculi be caught upon a slide, the ovum may as a rule be found, together with its corona radiata. That part of the preparation containing the ovum should be covered with a cover-glass under the edges of which strips of cardboard are laid. If no such strips are employed, the zona pellucida of the ovum is likely to burst in the field of vision, giving rise to a funnel-shaped tear. These tears have often been pictured and described as preformed canals (micropyles). The best fixing fluid for ovarian tissue is Flemming's or Her- mann's, either of which may be used for small ovaries or pieces of large ovaries ; safranin is then used for staining. Good results are also ob- tained with corrosive sublimate (staining with hematoxylin according to M. Heidenhain), and also with picric acid (staining with borax - carmin). The treatment of the Fallopian tubes is the same as that of the intestine ; in order to obtain cross -sections of a tube it is advisable to dis- sect away the peritoneum near its line of attachment and then distend the tube before fixing. It is instructive to dilate the tube by filling it with the fixing agent, thus causing many of the folds to disappear. No special technic is necessary in fixing the uterus and vagina. The epithelium is, however, best isolated with one-third alcohol. Seminal fluid to which normal salt solution has been added may be examined in a fresh condition. The effect upon the spermatozoa of a very dilute solution of potassium hydrate ( i % or weaker) or of a very dilute acid (acetic acid) is worth noticing. The spermatozoa of sala- mandra maculosa show the different structural parts very clearly (lance, undulating membrane, marginal thread, etc.). In macerated prepara- tions (very dilute chromic acid), or in those left for some time in a moist chamber, the fibrillar structure of the marginal and axial threads may be seen quite distinctly. The spermatozoa may also be examined in the form of dry preparations (treatment as for blood), stained, for instance, with safranin. Osmic acid, its mixtures, and osmic vapors are useful as fixing agents, certain structures being better brought out so than by employing the dry methods. In examining the testicle (spermatogenesis) it is advisable to begin with the testis of the salamander, which does not show such com- plicated structures as do the testes of mammalia. Here also either Flem- ming's or Hermann's fluid may be used as a fixing agent, the latter being THE SKIN. 379 followed by treatment with crude pyroligneous acid. For the salaman- der Hermann recommends a mixture composed of i % platinum chlorid 15 c.c. , 2% osmic acid 2 c.c., and glacial acetic acid i c.c., and for mammalia the same solution with double the amount of osmic acid. The fluid is allowed to act for some days, the specimen then being washed for twenty-four hours in running water and carried over into alco- hols of ascending strengths. Paraffin sections are treated as follows : Place for from twenty-four to forty-eight hours in safranin (safranin i gm. is dissolved in 10 c.c. of absolute alcohol and diluted with 90 c.c. of anilin water). After decolorizing with pure or acidulated absolute alcohol the sections are placed for three or four hours in gentian-violet (saturated alcoholic solution of gentian -violet 5 c.c. and anilin water 100 c.c.), and are then placed for a few hours in iodo-iodid of potassium solution until they have become entirely black (iodin i, iodid of potas- sium 2, water 300); finally, they are washed in absolute alcohol, until they become violet with a dash of brown. The various structures appear differently stained : for instance, the chromatin of the resting nucleus and of the dispirem, bluish-violet ; the true nucleoli, red ; while, on the other hand, in the aster and diaster stages the chromatin stains red. It is of especial importance that small testicles should not be cut into pieces before fixing, as this causes the seminal tubules to swell up and show marked changes, even in regions at some distance from the cut (Hermann, 93, I). The treatment of the remaining parts of the male reproductive organs requires no special technic. VI. THE SKIN AND ITS APPENDAGES. A. THE SKIN (CUTIS). THE skin consists of two intimately connected structures — the one, of mesodermic origin, is the true skin, corium or dermis ; the other, of ectodermic origin, is the epidermis or cuticle. The super- ficial layer of the corium is raised into ridges and papillae which penetrate into the epidermis, the spaces between the papillae being filled with epidermal elements. Thus, the lower surface of the epidermis is alternately indented and raised into a system of furrows and elevations corresponding to the molding of the corium. In the epidermis two layers of cells may be observed — the stratum Malpighii, or stratum germinativum (Flemming), and the horny layer, or stratum corneum. According to the shape and characteristics of its cells, the stratum germinativum may also be divided into three layers — first, the deep or basal layer, consisting of columnar cells resting immediately upon the corium ; second, the middle layer, consisting of polygonal cells arranged in several strata, the number of the latter varying according to the region of the body ; and third, the upper layer, or stratum granulosum, which is composed, at most, of two or three strata of gradually flattening cells characterized by their peculiar granular contents. 380 THE SKIN AND ITS APPENDAGES. All these cell layers consist of prickle cells, and for this reason the stratum Malpighii is sometimes known as the stratum spinosum. When these cells are isolated by certain methods, their surfaces are seen to be provided with short, thread-like processes. In section the cells appear to be joined together by their processes. Since it has been proved that the processes of adjacent cells do not lie side by side, but meet and fuse, they must be regarded as belonging alike to both cells. Between the fused processes, which are known as intercellular bridges, there exists a system of channels which is in communication with the lymphatic system of the corium. The prickles just mentioned are variously regarded by different investi- gators ; some considering them to be exclusively protoplasmic Fig. 303. — Under surface of the epidermis, separated from the cutis by boiling. The sweat-glands may be traced for a considerable part of their length ; X 4° : a> Sweat- gland ; b, longitudinal ridge ; c, depression ; d, cross-ridge. processes of the cells, others regarding them as derived from the membranes of the cells composing the stratum Malpighii. Ranvier and others ascribe a fibrillar structure to the peripheral portion of the cellular protoplasm, and, according to them, these fibrillae, surrounded by a small quantity of indifferent protoplasm, form the processes. Ranvier has also shown that such fibrillae may extend from one cell around several others before reaching their ultimate destination in other cells at some distance. (Fig. 305.) The cells of the stratum granulosum contain peculiar deposits of a sub- stance to which Waldeyer has given the name of keratohyalin. This substance occurs in the form of irregular bodies varying in size and imbedded in the protoplasm. The nuclei of such cells always THE SKIN. 381 show degenerative processes, which are possibly due to the forma- tion of the keratohyalin (Mertsching, Tettenhamer). These karyo- lytic figures and keratohyalin possess in common many apparently identical microchemic peculiarities, and it is very probable that karyolysis and the formation of keratohyalin are processes origin- ally very closely allied — i. e., that the keratohyalin is derived from the fragments of the dying nucleus. The stratum corneum forms the outer layer of the epidermis and presents, as a rule, a somewhat differentiated lower stratum. This | Stratu .jJ- m corneum. - Stratum Malpighii. Duct of sweat- gland. Corium. Subcutis. , ~~ Blood-vessel. - -Sweat-gland. Fig- 3°4- — Cross-section of skin of child, with blood-vessels injected ; X 3°- latter is more especially noticeable in those regions in which the stratum corneum is highly developed, and is known as the stratum lucidiim. It is quite transparent, this property being due to the presence in its cells of a homogeneous substance, which is in all probability a derivative of the more solid keratohyalin of the stratum granulosum. The cells of the stratum corneum are more or less flattened and cornified, especially at their periphery. This applies more particularly to the superficial cells. In the inte- rior of each cell a more or less degenerated nucleus may be seen, but otherwise its contents are homogeneous, or, at most, arranged THE SKIN AND ITS APPENDAGES. in concentric lamellae (Kolliker, 89). Here and there between the cornified cells structures may be seen which probably represent the remains of intercellular bridges. The thickness of the epidermis varies greatly according to the locality, and is directly proportionate to the number of its cell layers. As a rule, the stratum Malpighii is thicker than the stratum corneum, but in the palm of the hand and the sole of the foot the latter is considerably the thicker. The various layers of the epidermis are in close genetic relation- ship to one another. The constant loss to which the epidermis is subjected by desquamation is compensated by a continuous upward pushing of its lower elements ; cell-proliferation occurs in the basal cells and adjacent cellular strata of the stratum germinativum (Malpighii), where the elements are often seen in process of mitotic division. The young cells are gradually pushed outward, and dur- ing their course assume the general characteristics of the elements composing the layers through which theypass. For instance, such a cell changes first into a cell of the stratum germina- tivum ; then, when it commences the forma- tion of keratohyalin, into a cell of the stratum granulosum ; later, into a cell of the stratum lu- cidum, and finally into an element of the stra- tum corneum, where it loses its nucleus, corni- fies, and at last drops off. The mesodermic por- tion of the skin, the co- rium, consists of a loose, subcutaneous connective tissue containing fat, the subcutaneous layer, with the panniculus adiposus, and of the true skin, or corium proper. The amount of adipose tissue in the subcutaneous layer is subject to great variation ; there are, however, a few re- gions in which there is normally very little or no fat (external ear, eyelids, scrotum, etc.). To the subcutaneous connective tissue is due the mobility of the skin. The corium may be compared to the mucosa of a mucous membrane, and consists of two layers — of a deeper and looser pars reticularis, and of a superficial pars papillaris supporting the papillae. The transition from the one to the other is very gradual. Elastic fibers are present in the connective tissue of both layers. The pars reticularis is made of bundles of connective-tissue fibers arranged in a network, nearly all of the strands of which have a direo» Fibrils which pass from one __ cell to another. Nucleolus. -- Intercellular bridges. Nucleus of cell. Fig. 305. — Prickle cells from the stratum Malpighii of man ; X 4^°- THE SKIN. tion parallel with the surface of the skin and are surrounded by a retic- ulum of rather coarse elastic fibers. In that portion of the pars papil- laris bordering upon the epidermis, the interlacing strands of con- nective tissue, as well as the surrounding reticulum of elastic fibers, are finer, so that the whole tissue is denser. This stratum supports the papillae — knob-like or conical elevations of still denser tissue end- ing in one or more points. We accordingly speak of simple or com- pound papillae. These structures are especially numerous and well developed in the palm of the hand and sole of the foot, where they are from 1 10 // to 220 fi long. Here they rest upon ridges of the corium, which are nearly always arranged in double rows. Accord- ing to whether the papillae contain blood-vessels alone, or special nerve-endings also, they are known as vascular or tactile papillae. Stratum corneum. Lower border of stratum lucidum. Stratum granu- losum. Stratum Mal- pighii. Fig. 306. — Cross-section of human epidermis ; the deeper layers of the stratum Malpighii are not represented ; X 75°- The smallest papillae are found in the mammae and scrotum — from 30 fj. to 50 ft long. The surface of the pars papillaris is covered by an extremely delicate membrane — the basement membrane. Accord- ing to most authors, the basal cells of the epidermis are simply cemented to this structure. Others believe that the epithelial cells are provided with short basilar processes which penetrate into the basement membrane and meet here with similar structures from the connective-tissue cells of the corium. This would give the base- ment membrane a fibrillar structure (Schuberg). The subcutaneous layer contains numerous more or less verti- cal strands of connective tissue, containing numerous large elastic- tissue fibers and joining the stratum reticulare of the corium to the THE SKIN AND ITS APPENDAGES. superficial fascia of the body or underlying structure, whatever that may be. These strands are the retinaculce cutis, and inclose in their meshes masses of fatty tissue which form the panniculu* adiposus. The latter varies greatly in thickness in different parts of the body. The vertically arranged cords of connective tissue are accompanied by blood-vessels, nerves, and the excretory ducts of glands. Smooth muscle-fibers are also present in the skin, and around the hair follicles are grouped into bundles. Nearly continuous layers of smooth muscle tissue are found in the subcutaneous layer of the scrotum (forming here the tunica dartos), in the perineum, in the areolae of the mammae, etc. In the face and neck striated muscle-fibers also extend outward into the corium. Even in the white race certain regions of the epidermis always contain pigment — as, for instance, the areolae and mammillae of the • Stratum corneum. Pigment -- cell with two pro- cesses. Pigmented basal cell. Fig. 307. — Cross-section of negro's skin, showing the intimate relationship of the pigment cells of the corium to the basilar cells of the epidermis. The latter are more deeply pigmented at their outer ends. The pigment granules may be traced into the outermost layers of the stratum corneum ; X 525« mammary glands, the scrotum, labia majora, around the anus, etc. In these regions the epithelial cells and the connective-tissue cells of the pars papillaris corii contain a variable number of small pigment granules. The latter occur chiefly in the basal cells of the epider- mis and diminish perceptibly in the cells of the overlying layers, so that in those of the stratum corneum few, if any, are left. In negroes and other colored races the deep pigmentation is due to a similar distribution of the pigment granules in the entire epidermis ; but even here the pigmentation decreases toward the surface, although the uppermost cells of the stratum corneum always con- tain some pigment. The nuclei of the cells are always free from the coloring-matter. The question as to the origin of the pigment is as yet unsolved. This much is known : that in those regions where pigment is present certain branched and deeply pigmented connec- THE SKIN. 385 tive-tissue cells are found immediately beneath the epiderni's, sending out processes which may be traced outward between the cells of the stratum Malpighii (Aeby). This fact has led some authors to believe that the connective tissue is in reality the source of the pig- ment, and that by some unknown process the latter is taken up and conveyed to the cells of the epidermis. This theory would preclude a direct production of pigment granules in the epidermal cells. But although it can not be denied that the pigment may be derived from the connective tissue, it is hardly logical to assume a priori that epithelial cells are not capable of pigment production, since, in other regions of the body, pigment formation may be observed in cells of undoubted epithelial origin, as, for instance, in ganglion cells and in Fig. 308. — A reconstruction showing the arrangement of the blood-vessels in the skin of the sole of the foot (Spalteholz): a, Stratum Malpighii and corium; b, boundary between cutis and subcutis, in the region of the coiled portions of the sweat-glands ; f, subcutis ; J, subpapillary arterial network ; e, cutaneous arterial network ; f, g, //, and t, first, second, third, and fourth venous plexuses. the pigment epithelium of the retina. An interesting proof that the processes of pigmented connective-tissue cells actually penetrate the epidermis is afforded by the case reported by Karg, of transplanta- tion of a piece of skin from a white man to a negro. After some time the piece of white skin became pigmented. Reinkehas demon- strated that the pigment in certain cells is in combination with certain definite bodies. The latter have been given the botanical name of trophoplasts. If the pigment be removed, colorless tropho- plasts are left. They may be tinged with certain stains. In the epidermis of the white race trophoplasts are also constantly present, although they are only slightly or not at all pigmented (Barlow). 25 386 THE SKIN AND ITS APPENDAGES. Stratum corneum. Nerve-fibers in the epi- dermis. Stratum Malpighii. The following may be said concerning the vascular system of the skin : The arteries which supply the skin with nutriment penetrate the corium and form a characteristic network in its lowest stratum. They also anastomose freely in the fascia and the subcutaneous layer. From this plexus branches pass outward to form a second or subpapillary plexus. From the latter, branches are again given off which, without further anastomoses, pass along beneath the rows of papillae and supply each separate papilla with capillary twigs. These in turn pass over into venous capillaries which unite and form four venous plexuses, one over the other and in general parallel to the surface of the skin. The upper- most venous plexus lies beneath the pap- illae, each venule cor- responding to a single row of papillae and anastomosing with its neighbors. The sec- ond plexus is found immediately beneath the first, the third in Papillae. the lower portion of the corium, and the fourth at the junction of the cutis and sub- cutis. Near the mid- dle of the subcutis the arteries show a circu- lar musculature, but the veins are already thus provided in the network between the cutis and subcutis, where they also seem to pos- sess valves. As already stated, the subcutaneous fat is divided into lobes by transverse and longitudinal bundles of connective tissue ; a second system of bundles midway between the cutis and fascia separates the panniculus adiposus into an upper and a lower layer. The former is supplied by direct arterial branches ; the latter, by branches passing backward from the cutaneous network. Those regions which are subjected to great external pressure are supplied by a greater number of afferent vessels the caliber of which is increased. In regions where the skin is very mobile the arteries are greatly convoluted. All these vascular peculiarities are present in the new- born (Spalteholz). The lymph-vessels of the true skin are also distributed in two layers — a deep and wide-meshed plexus in the subcutis, and a Nerve-fiber. Fig. 309. — Nerves of epidermis and papillae from ball of cat's foot ; X 75°- THE SKIN. 387 superficial narrow-meshed plexus immediately beneath the papillae. Into the latter empty the lymph -vessels coming from the papillae. After treating the skin by certain methods, a fine precipitate may be noticed here and there in the papillary region of the corium, a proof that lymph clefts are present. These are regarded as the beginnings of the cutaneous lymphatic system. They may also be traced into the epithelium, where they are in direct communication with the interspinal spaces between the epithelial cells (Unna). Cells are also met with in the interspinal spaces of the epidermis ; these are migratory cells, or cells of Langerhans. The skin owes its great sensitiveness to the numerous nerves and special nerve-endings present, not only in the epithelium, but also in the corium and subcutis. In certain regions of the skin the nerves have been traced into the epithelium. In the finger-tip, for instance, numerous nerves are seen in the epidermis, where they branch and end in telodendria with or without small terminal swell- ings. There is no direct communication between the terminal — Nerve-fiber. h- Nerve-fiber. \ — Nerve-fiber. Fig. 310. — Meissner' s corpuscle from man ; Fig. 311. — Meissner's corpuscle from man; x 750. x 750. nerve filaments and the epithelial cells. (Fig. 309.) In certain peculiarly sensitive regions, as the end of the pig's snout, the nerve- fibers end in distinct saucer-like discs (tactile menisci) which, as a rule, clasp the lower ends of the basal Malpighian cells. The special sensory nerve -endings are situated in the corium and subcutis. Of these, we may mention the tactile corpuscles of Meissner, the end-bulbs of Krause, the Pacinian corpuscles, Ruf- fini's nerve-endings, and the Golgi-Mazzoni corpuscles. All these special sensory nerve-endings with the exception of the two last mentioned have been discussed in a former chapter (p. 169). Meiss- ner's tactile corpuscles are situated in the tactile papillae of the true skin. They are especially numerous in the hand and foot. 388 THE SKIN AND ITS APPENDAGES. In the distal phalanx of the index-finger every fourth papilla is a tactile papilla, containing one or sometimes two corpuscles of Meissner. They are, however, not nearly so numerous in other parts of the hand or in the foot. These corpuscles are further found on the dorsal surface of the hand and volar surface of the forearm, in the nipple and external genitals, in the eyelids (border), and in the lips. In figures 310 and 311 are shown two Meissner's corpuscles, giving the appearance presented by these end-organs when not stained with special reference to nerve terminations. For the latter see figure 137. The Krause's end-bulbs, both spheric and cylindric, are, as a rule, situated a short distance below the papillary layer, although they are frequently found in the papillae. They occur in man in the conjunctiva, lips, and external genitals, and in the mucous mem- branes previously mentioned (p. 170). See page 170 and figure 136 for their structure. In the palm of the hand and sole of the foot, the subcutaneous connective tissue contains numerous Pacinian corpuscles. They occur also along the nerve-fibers of the joints and in the periosteum of the extremities. Very recently Ruffini demonstrated in the human corium the existence of peculiar nerve end-organs, which consist of a connec- tive-tissue framework supporting a rich arborization of telodendria. They occur side by side with the Pacinian corpuscles and in appar- ently equal numbers. These nerve terminations resemble in many respects the neurotendinous spindles (see Fig. 145), although they present certain structural differences. Instead of intrafusal tendon fasciculi, the Ruffini end-organ is composed of white fibrous and elastic tissue. In this end-organ the medullated nerves make long and tortuous turns before becoming nonmedul- lated, and the terminations of these nerve-fibers occupy the whole of the cross-section. The Golgi-Mazzoni corpuscle re- sembles in structure the Pacinian corpuscle, although it possesses fewer lamellae and a relatively larger core, and the nerve - fibers terminating therein are more extensively branched than in the Pacinian corpuscle. Ruf- fini has found these nerve-endings in the subcutaneous tissue of the finger- tips. The blood-vessels of the skin are richly supplied with the vaso- rnotor nerves, which terminate in the nonstriated muscle of the Terminal disc of nerve-fibers. Epithelial cell. Connective-tissue capsule. •- — Nerve-fiber. .Fig. 312. — Grandry's corpuscles from bill of duck ; X 5°°- THE HAIR. 389 vessel walls. These vasomotor nerve-fibers are neuraxes of sym- pathetic neurones. In aquatic birds, and more especially in ducks, the waxy skin of the beak and the cornified portion of the tongue contain the so- called corpuscles of Herbst, which resemble the Pacinian corpuscles in general structure, but have cubical cells in the core. In the same tissues are also found the corpuscles of Grandry, 60 fj. long and 40 IJL broad. They consist of a thin connective-tissue capsule, con- taining two or three large cells. The nerve-fiber retains its medul- lary sheath for some distance within the capsule. The axis-cylinder ends in discs situated between the cells inclosed by the capsule. B. THE HAIR. The hair and nails are regarded as special differentiations of the skin. Hair is found distributed over almost the entire extent of the skin, varying, however, in quantity and arrangement in different regions. None whatever is present in the palm of the hand and sole of the foot. In the third fetal month small papillary elevations of the skin are seen to develop in those areas in which the hairy growth later appears. Under each of these elevations there occurs a proliferation of the cells of the Malpighian layer downward into the corium. Although the elevations soon disappear, the epithelial ingrowth continues and finally forms the hair germ. This is soon surrounded by a connective-tissue sheath from the corium, in which two layers may be distinguished. At the lower end of the hair germ the corium is pushed upward, forming a papilla which pene- trates into the thickened bulb of the germ. This is called the hair papilla. In the mean time the hair germ itself is undergoing marked differentiation. An axial portion, forming later the hair and inner root-sheath, and a peripheral, constituting later the outer root- sheath, are developed. From the latter are derived also the first traces of the sebaceous glands, which in the adult state are in close relationship to the hair and empty their secretion into the space between the hair and its sheath. As soon as the various layers of the hair are complete it grows outward, breaking through the over- lying layers of the epidermis. The visible portion of the hair is called the hair shaft, and that portion below the skin is the hair root. The lower portion of the hair resting upon the papilla is known as the hair bulb, and the sheaths encircling the root and bulb are called the root- sheaths, the entire structure constituting the hair follicle. The adult hair is covered by a thin cuticle, consisting of over- lying plate-like cells, i.i ft thick, most of which possess no nuclei. Beneath the cuticle is the cortical layer, composed of several strata of long, flattened cells from 4. 5 (i to 1 1 ft broad and provided with nuclei. These are also known as the cortical fibers of the hair. Upon treatment with ammonia the fibers separate into delicate 390 THE SKIN AND ITS APPENDAGES. fibrils, the hair fibrils (Waldeyer, 82). Scattered between and within the cells of the cortical layer are varying quantities of pigment granules. The axial region of the hair is occupied by the medullary substance, from 1 6 // to 20 // in diameter. This may be lacking ; but if present, consists of from 2 to 4 strata of polygonal, nucleated and pigmented cells. The hair shaft often contains air vesicles. The inner root-sheatJi consists of three concentric layers — first, of an outer single layer of clear nonnucleated cells, the so-called Fig- 3T3- — Transverse section of human scalp; X I2: AP, Musculus arrector pili ; c, corium ; ep, epidermis; fp, hair follicle ; Gap, aponeurosis ; gls, sweat-gland ; glse, seba- ceous glands; KH, club-hair; //, papilla of hair; Re, retinacula cutis; Rp, root of hair; Sp, shaft of hair; ts, subcutaneous layer (Sobotta, "Atlas and Epitome of Histology" ). layer of Henle ; second, of a thicker middle layer, made up of a stratum of nucleated cells containing keratohyalin, the layer of Hux- ley ; and, third, of an inner cuticle, bordering upon the hair. The outer root-sheath is made up of elements from the stratum germinativum. Here we have to do with prickle cells, surrounded by an outer layer of columnar elements. The connective-tissue portion of the hair follicle is composed of an outer, looser layer of longitudinal fibrous bundles ; of an inner, compacter layer of circu- THE HAIR. 391 lar fibers ; and of an innermost well-developed basement mem- brane— the glassy membrane. At a certain distance above the root bulb all the layers of the The hair. Stratum Malpighii of outer root-sheath. ^L Hair papilla. Inner root- sheath. Glassy layer of hair bulb. Connective tis- sue of the cutis. Fig. 314. — Longitudinal section of human hair and its follicle ; X about 300. epithelial portion of the hair follicle are well developed and distinct from each other. This condition changes toward the hair papilla 392 THE SKIN AND ITS APPENDAGES. as well as toward the hair shaft. Below, in the region of the thick- ened hair bulb, the root-sheaths begin to lessen in thickness, their layers becoming more and more indistinct toward the base of the hair papilla. Finally, all differentiation is lost in the region where they encircle the neck of the papilla. Toward the shaft of the hair, the root-sheath also undergoes changes. In the region into which the sebaceous glands empty, the inner root-sheath disappears, while the outer becomes continuous with the stratum germinativum of the epidermis ; the outer layers of the latter — the stratum granu- losum, stratum lucidum, and stratum corneum — push downward between the outer root-sheath and the hair to the openings of the sebaceous glands. Regarding the growth of the hair, two theories are prevalent. Glassy layer. _ Cortex of hair. _ Medulla of hair. _ Cuticle of inner root- sheath. — Henle's layer. Fibrous-tis- sue sheath. Fig. 3T5 — Cross-section of human hair with its follicle ; X about 300. The one theory assumes that the elements destined to form the epithelial root-sheaths are derived from the epidermis by a constant process of invagination. The component parts of the hair would thus be continuous with the layers of the root-sheaths, and conse- quently with those of the epidermis. Thus the basal cells of the external root-sheath would extend over the papilla, and be continu- ous with the cells of the medulla of the hair (these relations are especially well defined in the rabbit), and the stratum spinosum (middle layer of stratum Malpighii) of the outer root-sheath would be continuous with the cortical substance of the hair. According to this theory also, the layer of Henle would correspond to the stratum lucidum of the epidermis, and at the base of the hair THE HAIR. 393 Nerve- — • plexus of Bonnet. would become its cuticle, while the layer of Huxley would form the cuticle of the inner root-sheath (Mertsching). The other theory assumes that the hair is derived from a matrix, consisting of proliferating cells situated on the surface of the papilla. From these germinal cells would be derived the medullary and cortical substance of the hair, its cuticle, and the inner root-sheath (Unna). The shedding of hair is common to all mammalia, a phenomenon occurring periodically in the majority of species. In man the pro- cess is continuous. Microscopic examination shows that the hair destined to be shed becomes loosened from its papilla by a cornifi- cation of the cells of its bulb. At the same time the cortical por- tion of the hair bulb breaks up into a brush-like mass. Such hairs are called club hairs or bulb hairs, in contradistinction to papillary hairs. In the region of the former papilla there arises, by a prolifera- tion of the external root-sheath, a bud which grows downward, from which a new hair with its sheaths and connective-tissue papilla is developed. The result is that the developing new hair gradually pushes the old hair outward until the latter finally drops out. The exact details of this process have given rise to considerable discus- sion (yid. Gotte and Stieda, 87). Adjacent to the hair follicles are bundles of smooth muscle- fibers, known as the arrectores pi- lorum. They originate from the papillary layer of the corium and extend to the lower part of the connective-tissue sheath of the hair follicles. In their course they not infrequently encircle the sebace- ous glands of the follicle. Since the hair follicles have a direction oblique to the skin surface, forming with it an acute and an obtuse angle, and since the muscle is situated within the obtuse angle, its function may easily be conceived as being that of an erector of the hair. The hair papillae are very vascular. The nerve-fibers of the hair follicles have recently been studied by a number of investigators, with both the Golgi and the methylene- blue methods. It has been shown that the hair follicles receive their nerve supply from the nerve-fibers which terminate in the immediate skin area. Each follicle receives, as a rule, only one nerve-fiber, which reaches the follicle a short distance below the mouth of the sebaceous gland. The nerve-fiber, on reaching the Fig. 316 — Longitudinal section through hair and hair follicle of cat ; X 1 60. 394 THE SKIN AND ITS APPENDAGES. follicle, loses its medullary sheath and divides into two branches, which surround it in the form of a ring. From this complete or partial ring of nerve-fibers numerous varicose fibers proceed upward parallel to the axis of the follicle for a distance about equal to the cross-diameter of the follicle, to terminate, it would seem, largely outside of the glassy layer (Retzius). In certain mammalia the nerve-fibers end in tactile discs, found in the external root-sheaths of the so-called tactile hairs. The muscles of the hairs receive their innervation through the neuraxes of sympathetic neurones, which reach the periphery from the chain ganglia through the gray rami communicantes. These nerves are known as pilomotor nerves, and when stimulated, excite contraction of the erector muscles of the hairs, causing these to assume an upright position and producing the appearance termed goose skin, or cutis anserina. Langley and Sherrington have made interesting and important observations on the course and distribution of the pilomotor nerves. C. THE NAILS. The nails are a peculiar modification of the epidermis. The external arched portion is called the body of the nail ; that area upon Nail wall.--- — _^ Nail.-M ^'"-, • Stratum -i—:. Malpighii. ;- Stratum cor--""""1-" neum of the nail groove. < -Blood- vessel. Fig. 3'7- — Longitudinal section through human nail and its nail groove (sulcus) ; X 34- which the latter rests, the nail bed, or matrix ; and the two folds of epidermis which overlap the nail, the nail walls. The groove which exists between the nail wall and nail bed is known as the sulcus of the matrix, and the proximal imbedded portion of the nail as the nail root, since all growth of the nail takes place in this region. The nail bed consists of the corium, which is here made up of a dense felt-work of coarse connective-tissue fibers. Immediately beneath the nail the corium is raised into a number of more or less symmetric longitudinal ridges, which again become con- tinuous with the connective-tissue papillae of the skin at the line where the nail projects beyond its bed. The depressions between the ridges are occupied by epidermal cells, which also form a thin covering over the ridges themselves. THE NAILS. 395 These cells correspond here to the basilar layer of the stratum Mal- pighii. The stratum granulosum is not uniformly present, although occurring as isolated areas in the region of the nail root and lunula, the white area of demilunar shape at the proximal portion of the nail. Unna has demonstrated that the pale color of the lunula and root of the nail is due to the presence of keratohyalin. Formerly, this peculiarity was attributed to a difference in the distribution of the vessels in the various portions of the nail bed. The body of the nail, with the exception of the lunula, is transparent — a con- dition which may be explained by the fact that the elements of the nail correspond to those of the stratum lucidum. As a consequence, the vessels of the matrix shine through, except at the lunula, where the keratohyalin granules render the nail opaque. The nail itself consists of elements homologous to those of the stratum lucidum. They are flat, transparent cells, closely approxi- mated, and all contain nuclei. The cells overlie each other like tiles, and are so arranged that each succeeding lower layer projects Corium. . J — Blood-vessel . Fig. 318. — Transverse section through human r.ail and its sulcus ; X 34- a little further distalward than the preceding. At the period when the nails are formed, about the fourth month of fetal life, sulci are already present. The first trace of the nail is seen as a marked thickening of the stratum lucidum in the region which later be- comes the body of the nail ; in this stage the structure is still cov- ered by the remaining layers of the stratum corneum, constituting the eponychium. The embryonal nail then spreads in all directions until it finally reaches the sulcus. Henceforward the growth is uniform. The eleidin normally present in the stratum lucidum of the skin also occurs in the nail, and is derived, as we have already seen, from the keratohyalin. It may readily be conceived that later, when growth is confined to the root of the nail, keratohyalin is also present. As soon as the nail begins to grow forward, in the ninth month, the greater, part of the eponychium is thrown off; but during the entire extrauterine life, a portion of the eponychium is retained at the nail wall, and as hyponychium on the anterior and under surface of the nail. 396 THE SKIN AND ITS APPENDAGES. D. THE GLANDS OF THE SKIN. The glands in the skin are of two kinds — sweat-glands and sebaceous glands. In this connection we may also consider the mammary glands, which may be regarded as a modified skin gland. I. The Sweat-glands. — The sweat-glands, or sudoriparous Fig. 319. — A, B, Two views of a model of the coiled portion of a sweat-gland from the plantar region of a man, reconstructed by .Bern's wax-plate method ; X Io° ( Huber-Adamson). glands, are distributed throughout the entire skin, but are especially numerous in certain regions — as, for instance, the axilla, palm of the hand, and sole of the foot. They lie imbedded either in the adipose tissue of the true skin, or still deeper in the subcutaneous connective tissue. The sweat-glands are simple tubular in type, and their secreting portion is coiled ; hence the name coil-glands. The coiled portion of these glands measures 0.3 to 0.4 mm. The excretory duct is nearly straight in its course through the corium. From here on its course is spiral, and it should be borne in mind that in its passage through the epidermis it has no other wall than the epidermal cells of the various lay- ers through which it passes, although these cells are arranged con- centrically around the lumen of the duct. The duct takes part in the formation of the coiled portion of the gland, forming about one- fourth of the length of the tubule which takes part in the formation Basement membrane. Nonstriated muscle-cell. Gland-cell. Fig. 320. — Cross - section of tubule of coiled portion of sweat-gland of human axilla. Fixation with sublimate ; X 600. THE GLANDS OF THE SKIN. 397 — Nucleus of nonstriated muscle-cell. Nucleus of gland-cell. of the coil. In figure 319 are shown two views of a model of the coiled portion of a sweat-gland from the plantar region of the foot of a man. The length of the tubule in the coiled portion of this gland measures 4.25 mm., of which 1.25 mm. fall to the excretory duct and 3.0 mm. to the secretory tubule. The blind end of the secreting portion of the tubule and the ex- cretory duct as it enters the coil are usually in close proximity. The secretory portion of the tubule of sweat-glands is lined by a single layer of cubic or columnar cells, with finely granular proto- plasm and round or oval nuclei possessing one or two nucleoli. Be- tween this layer of cells and the basement membrane there is found a layer of nonstriated muscle-cells, longitudinally disposed. The portion of the excretory duct found within the coil of the glands is lined by a single layer of short cubic cells, with cuticular border, outside of which there is a delicate basement membrane. The muscular layer is lacking in this and the remaining portion of the duct The excretory portion of the duct passing through the corium is lined by short, somewhat irregu- larly cubic cells arranged in two layers. Capillary networks sur- round the secreting portions of the sweat-glands. The nerves of the sweat- glands have been studied with the aid of the methylene-blue method by Ostroumow, work- ing under Arnstein's direction. These glands receive their in- nervation through the neur- axes of sympathetic neurones, the terminal branches of which form an intricate network just out- side of the basement membrane, known as the epilamellar plexus, From this plexus fine varicose nerve-fibers pass through the base- ment membrane, and, after coursing a shorter or longer distance with or without further division, end on the gland-cells, often in clusters of small terminal granules united by delicate threads. The development of the sweat-glands begins in the fifth month of fetal life. At first solid cords grow from the stratum germi- nativum of the epidermis into the corium. Later, in the seventh month, these become hollow. Joseph has shown a structural change in the secretory cells 01" the sweat-glands when perspiration was induced by electrical stimulation or by drugs. With the sweat-glands as here described, and which have, as Fig. 321. — Tangential section through coiled portion of sweat-gland from human axilla. Sublimate fixation ; X 7°°- 398 THE SKIN AND ITS APPENDAGES. has been stated, a very wide distribution, we may also class certain skin glands, grouped under the term of "modified sweat-glands," which show certain structural and morphologic peculiarities and are found in special regions of the body. To these belong the axillary glands, the circumanal glands, the ciliary glands or glands of Moll of the eyelid, and the ceruminous glands of the external auditory canal. The axillary glands resemble the sweat-glands in shape and structure, possessing, however, larger and longer tubules. The coiled portions of these glands measure 1.5 to 2 mm., the tubule of the coil attaining a length of 30 mm. In the circumanal region are found several types of sweat-glands, especially in an area having the form of an elliptical ring with a width of about 1.5 cm. and situated about 1.5 cm. from the anus. In this region there Fig. 322. — Model of a sebaceous gland with a portion of the hair follicle, reconstructed by Born's wax-plate method. A, Hair follicle. are found large sweat-glands, known as the circumanal glands of Gay ; branched sweat-glands of the type of tubulo-alveolar glands ; sweat-glands with relatively straight ducts, ending in a relatively' large saccule or vesicle, from which arise secondary tubules or alveoli ; and, finally, sweat-glands of the type as found in other regions of the body. The ciliary glands or glands of Moll may also be classed as branched glands of the type of tubulo -alveolar glands, with relatively large vesicles. The ceruminous glands are branched tubulo-alveolar glands. 2. The Sebaceous Glands. — The distribution of the sebaceous glands in the skin is closely connected with that of the hair follicles into which they pour their contents. Exceptions to this rule occur THE GLANDS OF THE SKIN. 399 in only a few regions of the body, as, for instance, in the glans penis and foreskin (Tyson's glands), in the labia minora, angle of the mouth, glandule tarsales, and the Meibomian glands of the eyelids, etc. As a rule the sebaceous gland empties by a wide excretory duct into the upper third of the hair follicle. The walls of the duct also produce secretion, and can therefore hardly be differentiated from the rest of the gland. At its base the duct widens and is pro- vided with a number of simple or branched alveoli. The sebaceous glands are therefore of the type of simple branched alveolar glands, varying in length from 0.2 mm. to 0.5 mm. They are surrounded by connective-tissue sheaths, which at the same time cover the hair follicles. Inside of the sheath is the membrana propria, which is a continuation of the glassy membrane of the follicle. The two or three basal strata of glandular cells must be regarded as a direct continuation of the elements of the external root-sheath. In the Fig. 323. — Section of alveoli from sebaceous gland of human scalp. more centrally placed strata the cells are distinctly changed in char- acter ; their contents consist of fat globules, varying in size and distributed throughout the protoplasm, giving this a reticular appearance, while the nuclei suffer compression from the accumu- lation of the fat globules and gradually become smaller and more angular. Finally, the cells change directly into secretion, which is then poured into the hair follicle as sebum. It is thus seen that in the secretion of sebum the cells are consumed and must be re- placed. This renewal takes place by the constant proliferation of the basilar cells, which push the remains of the secreting cells upward and finally take their places. The final disintegration of the cells occurs either within the gland itself or between the hair follicle 4OO THE SKIN AND ITS APPENDAGES. and the hair. The secretion contains fatty globules of varying size, which occur either free or attached to cellular detritus. 3. The Mammary Glands. — The mammary glands are also included among the cutaneous glandular structures. They are developed early, but not until the fifth month is it possible to dis- tinguish a solid central portion, with radially arranged tubules terminating in dilatations. The structures are all derived from the basal layers of the epidermis. From birth to the age of puberry Fig. 324. — Model of a small portion of a secreting mammary gland ; X 2O°- (Maziarski, Anatomische Hefte, vol. xvm.J the organs are in a state of constant growth, and are early sur- rounded by a connective-tissue sheath. The alveoli, which have been developed in the mean time, are still solid and relatively small. Up to the twelfth year the glands remain identical in structure in boys and girls. In the female the mammary glands continue to develop from the age of puberty ; in the male, on the other hand, they undergo a retrograde metamorphosis, ending, finally, in the atrophy of all except the excretory ducts. The mammary glands do not attain their full stage of development in women until the last months of pregnancy, and are functionally active at parturition. The human mammary gland when fully developed has the fol- lowing structure : It consists of about twenty lobes, separated from each other by connective-tissue septa. These lobes are again divided into a larger number of lobules, and these in turn are com- posed of numerous irregularly round or oval or even tubular al- veoli. The alveoli are provided with small excretory passages, which unite to form the smaller ducts, these in turn uniting to form the larger ducts. Shortly before terminating at the surface of the mammilla, each mammary duct widens into a vesicle, the sinus lactiferus. The number of excretory ducts corresponds to that of the larger lobes. The ducts are lined by simple cubical epithe- THE GLANDS OF THE SKIN. 4OI Hum, except near their termination in the nipple, where they are lined by stratified pavement epithelium, and surrounded by a fibrous tissue sheath. The epithelium of the alveoli differs according to the state of functional activity. In a state of rest it consists of a single layer of glandular cells of nearly cubical shape which stain deeply, the internal surfaces now and then projecting slightly into the lumen. At the beginning of secretion fat globules make their appearance in the distal ends of the cells. At the same time a corresponding Alveolus. Connective- tissue stroma. )uct and alveoli. Adipose tissue. Fig. 325. — From section of mammary gland of nullipara. (From Nagel's "Die weiblichen Geschlechtsorgane," in " Handbuch der Anatomic des Menschen," 1896.) increase in size occurs throughout the entire alveolus. There are as yet current two quite contradictory views as to the manner in which the milk is secreted. According to certain observers, the free ends of the cells, which contain the most fat globules, are constricted off, after which the fat globules are freed in the lumen. The secretory portion of the alveolus is then composed of low epithelial cells, in which the process begins anew. The process of milk secretion therefore consists in throwing off the inner halves of the cells con- taining the fat globules, and in regeneration of the cells from the 26 4O2 THE SKIN AND ITS APPENDAGES. nucleated remains of the glandular epithelium. Whether a karyokin- etic division of the nuclei occurs in this process is not known, and how often the process of regeneration may be repeated in a single cell is not capable of demonstration. It is -certain, however, that entire cells are destroyed, to be replaced later by new elements. Other observers regard the secretion of milk as occurring without a partial or total destruction of the secretory cells, but after the manner of the secretion of other glands. This latter view seems more in accord with the more recent observations. The membrana propria of the alveoli appears homogeneous. Between it and the glandular cells are so-called basket cells, similar to those in the salivary glands. Benda regards the basket cells as nonstriated muscle elements having a longitudinal direction, making the structure of the alveoli of the mammary gland similar in this respect to that of the secreting portion of the sweat-glands. The skin of the mammilla is pigmented, and the papillae of its corium are very narrow and long. In the corium are also found large numbers of smooth muscle-fibers, which form circular bun- dles around the excretory ducts. In the areolse of the mammae are the so-called glands of Montgomery, which very probably repre- sent accessory mammary glands. These are especially noticeable during lactation. The blood-vessels of the mammary gland, the larger branches of which are situated mainly in the subcutaneous tissue, form rich capillary networks about the alveoli. The mammary glands possess many lymphatics. These are especially numerous in the connective-tissue stroma between the lobules. The lymph-vessels collect to form two or three larger vessels, which empty into the axillary glands. The mammary gland receives its nerve supply from the sympathetic and cerebro- spinal nervous systems through the fourth, fifth, and sixth inter- costal nerves. The terminations of the nerves in the mammary gland have been studied by means of the methylene-blue method by Dmitrewsky, working in the Arnstein laboratory, who finds that the terminal branches form epilamellar plexuses outside of the basement membrane of the alveoli, from which fine nerve branches pass through the basement membrane and end on the gland cells in clusters of terminal granules united by fine filaments. The nipple has a rich sensory nerve supply. In the connective-tissue papillae are found tactile corpuscles of Meissner. The milk consists of fat globules of varying size, which, how- ever, do not coalesce — an attribute due to the presence of albu- minous haptogenic membranes surrounding the globules. Shortly before, and for some days after, parturition the milk contains true nucleated cells in which are fat globules ; these are known as the colostrum corpuscles. They probably represent leucocytes which have migrated into the lumen of the gland and have taken up the fat globules of the milk. This milk is known as colostrum. TECHNIC. 403 TECHNIC. Good general views of the skin can be obtained only from sections. Any fixation method may be employed, although alcohol is preferable on account of the better subsequent staining. For detail work Flamming' s solution, corrosive sublimate, or osmic acid is the best. Sectioning of the skin is attended with many difficulties, and large pieces can be cut only in celloidin. Small and medium-sized pieces may be cut in paraffin ; but even in this case the skin must be rapidly imbedded in the paraffin — /'. e., it must not remain too long in either alcohol or toluol — and the paraffin must have only the consistency necessary to cut well (about 50° C. melting- point). In order to obtain good paraffin sections of the skin the follow- ing procedure is recommended : Pieces fixed in Flemming's solution or osmic acid are kept in 96% alcohol, then placed for not more than twenty- four hours in absolute alcohol and imbedded in paraffin by means of the chloroform method. In the chloroform, chloroform -paraffin, and pure paraffin they remain for one hour each. The paraffin used should consist of two parts paraffin of 42° C. , and one part paraffin of 50° C. melting-point. The thermostat must be kept at 50° C. (R. Barlow). The sections should not be mounted by the water-albumen method. In sections of epidermis which have been freshly fixed with osmic acid, the stratum corneum may be clearly differentiated into three layers (probably because of the defective penetration of the reagent) — into a blackened superficial, a middle transparent, and a still lower black layer (vid. Fig. 326). In tissue fixed in alcohol or corrosive sublimate the stratum lucidum stains yellow with picrocarmin, but is very weakly colored by basic anilin stains. In unstained preparations the stratum lucidum is glass-like and transparent. Eleidin is diffusely scattered throughout both the stratum lucidum and stratum corneum. Like keratohyalin, it stains with osmic acid and also with picrocarmin, but not with hematoxylin. Nigrosin stains eleidin, but not keratohyalin. Keratohyalin is insoluble in boiling water and is not attacked by weak organic acids. It dissolves, however, in boiling acetic acid, but is not changed by the action of pepsin or trypsin. The keratohyalin granules of the stratum granulosum swell in from ic/f to 5 c/o potassium- hydrate solution ; under the influence of heat these granules together with the cells containing them are finally dissolved. They are not attacked by ammonia, and remain unaffected for a long time in strong acetic acid. As ammonia and acetic acid render the remaining portions of the tissue transparent, these reagents may be employed for the rapid identification of keratohyalin. The larger flakes of keratohyalin swell in sodium car- bonate solution (i%), but not the smaller granules, and it would seem that the larger granules have less power of resistance than the smaller. Keratohyalin remains unchanged in alcohol, chloroform, and ether, but is digested in trypsin and pepsin (not, however, the keratin). Kerato- hyalin can be stained with hematoxylin and most of the basic anilin dyes. The prickles of the cells composing the stratum Malpighii may be seen in very thin sections (not over 3 // in thickness) of skin previ- ously fixed in osmic acid. In this case it is best to employ not Canada balsam, but glycerin, which does not have so strong a clearing action. Isolation of the prickle cells is best accomplished as follows (Schieffer- 404 THE SKIN AND ITS APPENDAGES. decker): A fresh piece of epidermis is macerated for a few hours in filtered, cold-saturated, aqueous solution of dry pancreatin ; the whole may then be preserved for any length of time in equal parts of glycerin, Outer dark layer. Stratum corneum. Middle light layer. Inner dark layer. Stratum lucidum. Stratum Malpighii. Cut is and sub- cutis. Fat cell. Fig. 326. — Transverse section through the human skin. Treated with osmic acid ; X 30 : a, Part of the tortuous duct of a sweat-gland in the epidermis ; b, duct of same sweat-gland in the corium. water, and alcohol. Small pieces taken from such specimens are readily teased and show both isolated and small groups of attached prickle cells. The distribution of the pigment in the skin is best studied in unstained sections. With a nearly closed diaphragm and under medium magnification the pigment granules appear darker on raising the tube and lighter upon lowering it. In sections of skin treated with Flemming's fluid, the structure of the cutis also may be studied. The medullary sheaths of the nerve- fibers and the fat appear black. In preparations stained with safranin the elastic fibers are colored red and are very distinct (Stohr and O. Schultze). For the orcein method according to Unna, see p. 128. Hair may be examined in water without further manipulation. The cuticle is then seen to consist of polygonal areas, the border-lines of which correspond to the limits of the flattened cells. By slightly lower- ing the objective the cortical substance comes into view with its indistinct TECHNIC. 405 striation and occasional pigmentation. The medullary substance, if pres- ent, may also be seen with its vesicles containing air. Both the cortical and cuticular cells may be isolated, the process consisting in treating the hairs for several days with 33 c/c potassium hydrate solution at room tem- perature, or in heating the whole for a few minutes. Concentrated or weak sulphuric acid produces the same result. On warming a hair in sul- phuric acid until it begins to curl and then examining it in water, we find that the cortical and medullary layers as well as the cuticle are separated into their elements. Treatment of the skin with Miiller's fluid, alco- hol, or sublimate is recommended for the examination of hair and hair follicles. The orientation of the specimen should be very precise, in order to obtain exact longitudinal or cross-sections of the hair. There is hardly a structure of the body which is more suitable for staining with the numerous coal-tar colors than the hair and its follicle (Merkel). The corpuscles of Meissner may be best obtained from the end of the finger. After boiling a piece of fresh skin from the finger-tip for about a quarter of an hour, the epidermis may be easily removed ; the papillae are now seen on the free surface of the cuds. A portion of the latter is cut away with a razor and examined in a 3 % solution of acetic acid. The corpuscles are readily distinguished. Their relations to the nerves should be studied in specimens fixed with osmic acid or gold chlorid. The terminations of the nerves in these end-organs are best seen in preparations stained after the infra vitam methylene-blue method. The corpuscles of Herbst and Grandry are found in the waxy skin covering the bill, and in the palate of the duck (especially numerous in the tongue of the woodpecker). For the study of the nervous ele- ments the following method is useful : Pieces of the waxy skin are removed with a razor and placed for twenty minutes in 50% formic acid. After washing the specimens for a short time in distilled water they are transferred to a small quantity of i c/c gold chlorid solution (twenty min- utes), then again rinsed in distilled water, and placed for from twenty- four to thirty-six hours in the dark in a large quantity (^ liter) of Pichard's solution (amyl alcohol i part, formic acid i part, water 100 parts). After again washing in water the specimens are transferred to alcohols of gradually increasing strengths and finally imbedded in celloidin or celloidin-paraffin. The Pacinian corpuscles occur in the mesentery of the cat and may be examined in physiologic saline solution. The nerves of the epidermis are demonstrated by the gold- chlorid method (see p. 48). But even here the chrome-silver method and the intra vitam methylene-blue method yield extremely good results, and may be used with great advantage in the study of the nerves in the cutis. The so-called tactile menisci are very numerous in the snout of the pig and the mole. Bonnet recommends for these structures fixation in °-33% chromic acid solution, overstaining with hematoxylin, and differ- entiation in an alcoholic solution of potassium ferricyanid. 406 THE CENTRAL NERVOUS SYSTEM. VII. THE CENTRAL NERVOUS SYSTEM. IN a study of the minute anatomy of the central nervous system consideration should be given to the arrangement of the nerve-cells and nerve-fibers in the various regions, and to the mutual relations which the elements of the nervous system bear to one another. In a text-book of this scope, however, we shall be unable to enter into the consideration of these subjects in detail, but must content our- selves with a very general discussion of the structure of certain regions of the central nervous system and an account of a few typical examples illustrating the mutual relationship of the nerve-elements to one another. We shall, therefore, give a general description of the structure of the spinal cord, cerebellum, cerebrum, olfactory lobes, and ganglia. In this description we have drawn freely from the results of the researches of Golgi (94), Ramon y Cajal (93» l)> von Lenhossek (95), Kolliker (93), and van Gehuchten (96). ' A. THE SPINAL CORD. The spinal cord extends from the upper border of the atlas to about the lower border of the first lumbar vertebra. It has the form of a cylindric column, which at its lower end becomes quite abruptly smaller, to form the conns medullaris, and terminates in an attenu- ated portion — the filum terminate. It presents two fusiform enlarge- ments, known as the cervical and lumbar enlargements respectively. The spinal cord is partly divided into two symmetric halves by an anterior median fissure and by a septum of connective tissue, extend- ing into the substance of the cord from the pia mater (one of the fibrous tissue membranes surrounding the cord), and known as the posterior median septitm. Structurally considered, the spinal cord consists of white matter (mainly medullated nerve-fibers) and gray matter (mainly nerve-cells and medullated nerve-fibers). The white and the gray matter present essentially the same general features at all levels of the spinal cord, although the relative proportion of the two substances varies somewhat at different levels. The different portions of the cord present also certain structural peculiarities. The distribution of the gray and the white substances of the spinal cord is best seen in transverse sections. The varying shape of the spinal cord in the several regions and the changing relations of the gray to the white substance are shown in the illustrations of cross-sections of the adult human spinal cord (see p. 407). The gray substance is arranged in the form of two crescents, one in each half of the cord, united by a median portion extending from one half of the cord to the other, the whole presenting some- what the form of an H. The horizontal part contains the commis- THE SPINAL CORD. 407 Fig. 327. — Four cross-sections of the human spinal cord ; X 7 : ^» Cervical region in the plane of the sixth spinal nerve-root ; J3, lumbar region ; C, thoracic region ; Z>, sacral region (compare with Fig. 328). (From preparations of H. Schmaus.) 408 THE CENTRAL NERVOUS SYSTEM. sures and the central canal of the spinal cord, while the vertical limbs or crescents extend to the ventral and dorsal nerve-roots, forming the anterior and posterior horns. The former are, as a rule, the larger, and at their sides (laterally) the so-called lateral horns may be seen, varying in size in different regions. In each anterior horn are three main groups of ganglion cells : the ventro- lateral, made up of root or motor nerve-cells ; the ventromesial, composed of commissural cells ; and the lateral (in the lateral horn), containing column cells. At the median side of the base of each posterior horn we find a group of cells and fibers known as the column of Clark, most clearly defined in the dorsal region, while in the posterior horn itself is the gelatinous substance of Rolando. Aside from these, numerous cells and fibers are scat- tered throughout the entire gray substance. The motor nerve-cells lie in the ventrolateral portion of the ante- rior horn, their neuraxes extending into the anterior nerve-root. Their dendrites are distributed in a lateral, dorsal, and mesial direc- tion, the two former groups ending in the anterior and lateral col- umns, the mesial in the region of the anterior commissure. Some of the mesial dendrites extend beyond the median line and form a sort of commissure with the corresponding processes of the other side. The commissural cells lie principally in the mesial group of the anterior horn, but occur here and there in other portions of the gray substance. Their neuraxes form the anterior gray commis- sure with the corresponding processes from the other side. After entering the white substance of the other side, these neuraxes undergo a T-shaped division, one branch passing upward and the other downward. The column cells are small multipolar elements, represented by the cells of the lateral horns, although they are also found throughout the entire gray mass. Their neuraxes pass directly into the anterior, lateral ^ and posterior horns. The cells of the column of Clark, or micleus dorsalis, are of two kinds — those in which the neuraxes pass to the anterior commis- sure (commissural cells) and those in which the neuraxes pass into the direct cerebellar tract of the same side. The plurifunicular cells are cells the neuraxes of which divide two or three times in the gray substance, the branches then passing to different columns of the white matter on the same or opposite side of the cord. In the latter case the branches must necessarily extend through the commissure. The cells of the substantia gclatinosa (Rolando) are cells with short, freely branching neuraxes, which end after a short course in the gray mass (Golgi's cells). The posterior horn con- tains marginal cells, spindle-shaped cells, and stellate cells. The first are situated superficially near the extremity of the posterior horn, their neuraxes extending for some distance through the gela- tinous substance of Rolando and then into the lateral column. The spindle-shaped cells are the smallest in the spinal cord and possess a rich arborization of dendrites extending to the nerve-root of the pos- THE SPINAL CORD. 409 terior horn. Their neuraxes, which originate either from the cell- body or from a dendrite, pass over into the posterior column. The stellate cells are supplied with dendrites, which either branch in the substance of Rolando or extend into the column of Burdach. The gray matter contains, further, numerous medullated nerve- fibers, in part the neuraxes of the nerve-cells previously mentioned, and in part collateral and terminal branches of the nerve-fibers of the white matter with their telodendria ; also supporting cells, known as neurogliar cells (to be discussed later), and blood-vessels. The white matter of the spinal cord consists of medullated fibers, which are devoid of a neurilemma, of neurogliar tissue, and of fibrous connective tissue. In each half of the cord the white substance, which surrounds the gray, is separated by the gray matter and its nerve-roots into Posterior horn cell. Crossed pyram- idal column. Golgi cell of posterior horn. Direct cerebel- lar column. Column cells. Golgi'scommis- sural cells. Gowers' column. Motor cells. Collaterals of crossed pyramidal column. Collaterals ending in the gray matter. Direct pyramidal column. Fig. 328. — Schematic diagram of the spinal cord in cross-section after von Lenhos- sek, showing in the left half the cells of the gray matter, in the right half the collateral branches ending in the gray matter. three main divisions or columns: The first division, lying between the anterior median fissure and the anterior horn, is the anterior column ; the second, lying between the anterior and posterior horns, is the lateral column (since the anterior and lateral columns belong genetically to each other, the term anterolat- eral column is often used) ; and the third, lying between the poste- rior nerve-root and the posterior median septum, is the posterior column. By means of certain methods it has been possible to separate the white substance into still smaller divisions, the most important of which may here be described. In each anterior column is found a narrow median zone extend- ing along the entire length of the anterior median fissure and con- 4io THE CENTRAL NERVOUS SYSTEM. THE SPINAL CORD. 411 taining nerve-fibers which come from the pyramids of the medulla. The majority of the pyramidal fibers cross from one side of the cord to the other in the lower portion of the medulla, at the crossing of the pyramids, and form a large bundle of nerve-fibers found in each lateral column, which will receive attention later. Some of the pyramidal fibers descend into the cord on the same side, to cross to the opposite side at different levels in the cord. These latter fibers constitute the narrow median zone, on each side of the anterior median fissure previously mentioned, forming the anterior or direct pyramidal tract, or the column of Tiirck. Between the direct pyramidal tract and the anterior horn lies the anterior ground bundle. In the lateral columns are found a number of secondary col- umns, which may now be mentioned. In front of and by the side of the posterior horn in each lateral column lies a large group of nerve-fibers, forming a bundle which varies somewhat in size and shape in the several regions of the spinal cord, but which has in general an irregularly oval outline. These nerve-fibers are the pyramidal fibers, previously mentioned, which in the lower part of the medulla cross from one side to the other, and for this reason are known as the crossed pyramidal fibers, forming the crossed pyramidal columns. External to these columns and to the poste- rior horns, and extending from the posterior horns half-way around the periphery of the lateral columns, lie the direct cerebellar col- umns, consisting of the neuraxes of the cells of the columns of Clark, which have an ascending course. Lying just external to and between the anterior and posterior horns is a somewhat irregular zone, the mixed lateral column, containing several short bundles of fibers, the anterior of which are probably motor ; the posterior, sensory. In the ventrolateral portions of the lateral columns, between the mixed lateral and the direct cerebellar columns and extending as far backward as the crossed pyramidal columns, lie two not well-defined columns, known as the ascending anterolat- eral or Gowers's columns and the descending anterolateral col- umns ; the former are nearer the outer portion of the cord. In the posterior column we distinguish a median and a lateral column. The former lies along the posterior median septum, and may even be distinguished externally by an indentation ; its upper portion tapers into the fasciculus gracilis. This is the column of Goll, and it contains ascending or centripetal fibers. The lateral tract lies between the column of Goll and the posterior horn, and is known as the column of Burdach, posterior ground-bundle, or posterolateral column. It contains principally the shorter tracts, or bundles of longitudinal fibers connecting the adjacent parts of the spinal cord with one another. Many of the nerve-fibers of the posterior column are the neu- raxes of spinal ganglion cells which enter the spinal cord through the posterior roots. The cell-bodies of the spinal ganglion or sen- 412 THE CENTRAL NERVOUS SYSTEM. sory neurones are situated in the spinal ganglia found on the pos- terior roots of the spinal nerves. In the embryo they are distinctly bipolar, but during further development their two processes approach each other, and then fuse for a certain distance, forming finally single processes which branch like the letter T. In reality, then, there are two processes which are fused for a certain distance from the cell-body of each neurone. The peripherally directed process is regarded as the dendrite of the cell, and the proximal as the neuraxis passing to the spinal cord. The neuraxes enter the spinal cord through the posterior roots and pass to the posterior columns, where they divide, Y-shaped, into ascending and much shorter descending branches, from each of which numerous collateral branches are given off. From the preceding account of the white matter of the spinal cord, it may be seen that it consists of longitudinally directed neu- raxes arranged in so-called short and long tracts or columns. The neuraxes constituting the former, after a short course through the gray matter, emerge from it, and after giving off various collaterals, again penetrate into the gray matter, where their telodendria enter into contact with the ganglion cells. The long columns consist of the neuraxes of neurones the cell -bodies of which are situated in the cerebrum or cerebellum, and of neurones the cell-bodies of which are in the spinal cord or spinal ganglia and the neuraxes of which terminate in the medulla or cerebellum. The nerve-fibers of the various columns give off numerous collaterals which enter the gray matter to end in telodendria. The collaterals of the posterior col- umns end : (i) between the cells of the gelatinous substance of the posterior horns ; (2) in the columns of Clark ; (3) in the anterior horns, these constituting the principal portion of the so-called reflex bundles ; (4) in the posterior horn of the opposite side. The col- laterals of the lateral columns pass horizontally toward the central canal, some ending in the anterior horn, others closely arranged near the columns of Clark, and some arching around the central canal, forming with the corresponding fibers of the other side the anterior bundles of the posterior commissure. The collaterals of the anterior columns form well-marked plexuses in the anterior horns of the same and opposite sides. We have still to describe the two commissures. The anterior consists of: first, neuraxes from the commissural cells ; second, dendrites from the lateral group of the anterior horn cells ; and, third, the collaterals of the anterolateral column, which end in the gray substance of the other side of the cord. The posterior com- missure is probably composed of the collaterals from all the remain- ing columns. The posterior bundle of this commissure comes from the posterior column ; the middle, from the posterior portion of the lateral column ; and the anterior, or least developed, from the anterior portion of the lateral column, possibly also from the anterior column. THE CEREBELLAR CORTEX. 413 In the gray commissure, nearer its anterior border, is situated the central canal of the spinal cord, continuous above with the ventricular cavity of the medulla and terminating caudally in the filum terminale. This canal is not patent in the majority of adults, being occluded from place to place. The canal is lined by a layer of columnar cells, developed from columnar cells, known as spongio- blasts, lining the relatively larger canal of the embryonic spinal cord. In young individuals these cells are ciliated and their basal portions terminate in long, slender processes in which are embedded neuroglia fibers. B. THE CEREBELLAR CORTEX, In the cerebellar cortex we distinguish three general layers — the outer molecular, the middle granular (rust-colored layer), and the inner medullary tract. Blood-vessel.- V- ~ ** « » . " --' »* - 41 ^> i ... — Nerve-fiber layer. • - - * ... -j^_ _-^ Fig. $30. — Section through the human cerebellar cortex vertical to the surface of the con- volution. Treatment with M tiller's fluid ; X XI5- The molecular layer contains three varieties of nerve-cells, those of Purkinje, which border upon the granular layer, the stel- 0 -8 • »„ v?; ••»•£•»•«» « I THE CEREBELLAR CORTEX. 415 late cells, and the small cortical cells. The cells of Purkinje pos- sess a large flask-shaped body (about 60 p. in diameter), from which one or more well -developed dendrites pass toward the periphery. The latter branch freely and the main arborization has in each case the general shape of a pair of deer's antlers. These dendrites extend nearly to the periphery of the cerebellar cortex. In a section horizontal to the surface of the organ the dendrites of the Purkinje's cells are seen to lie in a plane very nearly vertical to the surface of the convolutions, so that a longitudinal section through the latter would show a profile view of the cells. In other words, they have an appearance much like that of a vine trained upon a trellis. The neuraxes of the cells of Purkinje arise from their basal -••*• — Dendrite. Cell-body. Neuraxis. Fig. 332. — Cell of Purkinje from the human cerebel- lar cortex. Chrome-silver method ; X I2°- — Neuraxis. — Claw-like telo- dendrion of dendrite. Fig. 333. — Granular cell from the granular layer of the hu- man cerebellar cortex. Chrome- silver method ; X IO°- (inner) ends and extend through the granular layer into the medul- lary substance. During their course they give off a few collaterals, which pass backward to the molecular layer and end in telodendria near the bodies of the cells of Purkinje. The stellate cells lie in various planes of the molecular layer. Their peculiar interest lies in the character of their neuraxes. The latter are situated in the same plane as the dendrites of the cells of Purkinje, run parallel to the surface of the convolution, and possess two types of collaterals. Those of the first are short and branched ; those of the second branch at a level with the cells of Purkinje, and form, together with their telodendria, basket-like nets around the bodies of these cells. The small cortical cells of the molecular layer are found 4l6 THE CENTRAL NERVOUS SYSTEM. in all parts of this layer, but are more numerous in its peripheral portion. They are multipolar cells with neuraxes which are not readily stained and concerning the fate of which little is known. The granular layer contains two varieties of ganglion ele- ments, the so-called granular cells (small ganglion cells) and the large stellate cells. The dendrites of the granular cells are short, few in number (from three to six), branch but slightly, and end in short, claw-like telodendria. Their neuraxes ascend vertically to the surface and reach the molecular layer. At various points some of them are seen to undergo a T-shaped division, the two branches then running parallel to the surface of the cerebellum in a plane vertical to that of the dendrites of the cells of Purkinje. Large numbers of these T-shaped neuraxes produce the striation of the molecular layer of the cerebellum. It is very probable that during their course these parallel fibers come in contact with the dendrites of the cells of Purkinje. The large stellate cells are fewer in number and lie close to the molecular layer, some of them even within this layer. Their dendrites branch in all directions, but extend principally into the molecular layer. Their short neuraxes give off numerous collaterals which end in telodendria among the granular cells. The medullary substance is composed of the centrifugal neu- raxes of the cells of Purkinje and of two types of centripetal neu- raxes, the mossy and the climbing fibers. The position of their corresponding nerve-cells is not definitely known. The mossy fibers branch in the granular layer into numerous twigs, and are not uniform in diameter, but are provided at different points with typical nodular swellings. These fibers do not extend beyond the granular layer. The climbing fibers pass horizontally through the granular layer, giving off in their course numbers of collaterals, which extend to the cells of Purkinje, up the dendrites of which they seem to climb. In the medullary portion of the cerebellum are found a number of groups of ganglion cells known as central gray nuclei. The nerve-cells of these nuclei are multipolar, with numerous, oft- branching dendrites and a single neuraxis. C THE CEREBRAL CORTEX. The cell-bodies of the neurones of the cerebrum are grouped in a thin layer of gray matter, varying in thickness from 2 to 4 mm., — which, as a continuous sheet, completely covers the white matter of the hemispheres, — and in larger and smaller masses of gray mat- ter, known as basal nuclei. In our account of the histologic struc- ture of the cerebral hemispheres we shall confine ourselves in the main to a consideration of the cerebral cortex, the thin layer of gray matter investing the white matter. THE CEREBRAL CORTEX. 417 From without inward the following layers may be differentiated in the cerebral cortex : (i) a molecular layer ; (2) a layer of small pyramidal cells ; (3) a layer of large pyramidal cells ; (4) a layer of polymorphous cells ; and (5) medullary substance or underlying nerve-fibers. Aside from neurogliar tissue, we find in the molecular layer a large number of nerve-fibers, which cross one another in all direc- tions, but, as a whole, have a direction parallel with the surface of the brain. Within this layer there are found : (i) the tuft-like telo- dendria of the chief dendritic processes of the pyramidal cells ; (2) the terminations of the ascending neuraxes, arising mostly from the polymorphous cells ; and (3) autochthonous fibers — /. e., those which arise from the cells of the molecular layer and terminate in this layer. The cells of the molecular layer may be classed in three general types — polygonal cells, spindle-shaped cells, and triangular or stellate cells. The polygonal cells have from four to six den- drites, which branch out into the molecular layer and may even penetrate into the underlying layer of small pyramidal cells. Their neuraxes originate either from the bodies of the cells or from one of their dendrites, and take a horizontal or an oblique direction, giving off in their course a large number of branching collaterals, which terminate in knob-like thickenings. The spindle=shaped cells give off from their long pointed ends dendrites which extend for some distance parallel with the surface of the brain. These branch, their offshoots leaving them at nearly right angles, the majority passing upward, assuming as they go the characteristics of neuraxes having collaterals. The arborization is entirely within the molecular layer. The triangular or stellate cells are similar to those just described, but possess not two, but three, dendrites. The triangular and spindle-shaped cells, with their numerous den- dritic processes resembling neuraxes, are characteristic of the cere- bral cortex. The elements which are peculiar to the second and third layers of the cerebral cortex are the small (about 10 // in diameter) and large pyramidal cells (from 20 //to 30 ,« in diameter). They are composed of a triangular body, the base of the triangle being down- ward and parallel to the surface of the brain, of a chief, principal, or primordial dendrite ascending toward the brain-surface, of several basilar dendrites arising from the basal surface of the cell-body, and of a neuraxis which passes toward the medullary substance and which has its origin either from the base of the cell or from one of the basilar dendrites. The ascending or chief dendrite gives off a number of lateral offshoots which branch freely and end in terminal filaments. The main stem of the dendrite extends upward to the molecular layer, in which its final branches spread out in the form of a tuft. The neuraxis, during its course to the white substance, gives off in the gray substance from six to twelve collaterals, which divide two or three times before terminating. 27 4i8 THE CENTRAL NERVOUS SYSTEM. Aside from the fact that the layer of polymorphous cellt con- tains a few large pyramidal cells, it consists principally of (i) mul- tipolar cells with short neuraxes (Golgi's cells) and (2) of cells with Fig. 334. — Portions of vertical section of human cerebral cortex, treated by the Golgi method; X 7°- The figure shows the arrangement of the different cells of the cerebral cortex : gP, Layer of large pyramidal cells ; kF, layer of small pyramidal cells ; pZ, layer of polymorphous cells (Sobotta, "Atlas and Epitome of Histology"). only slightly branched dendrites and with neuraxes passing toward the surface of the brain (Martinotti's cells). Both these types of cells are, however, not found exclusively in the layer of polymorphous THE CEREBRAL CORTEX. 419 cells, but may be met with here and there in the layers of the small and large pyramidal cells. The dendrites of the cells of Golgi are projected in all directions, those in the neighborhood of the medul- lary substance even penetrating into this layer. The neuraxes break up into numerous collaterals, the telodendria of which lie ad- jacent to the neighboring ganglion cells. The cells of Martinotti, which, as we have seen, occur also in the second and third layers, are either triangular or spindle-shaped. The neuraxis of each cell originates either from the cell-body or from one of its dendrites, and Brush-like telodendrion Main dendrite. • - Secondary dendrite Basal dendrite. -^3^ Neuraxis with collaterals. Fig. 335. — Large pyramidal cell from the human cerebral cortex. Chrome-silver method ; X I5°- ascends (giving off collaterals) to the molecular layer, in which it finally divides into two or three main branches ending in telo- dendria. Occasionally it divides in a similar manner in the layer of small pyramidal cells. In the medullary substance the following four classes of fibers are recognized : (i) The projection fibers (centrifugal) — i. e., those which indirectly connect the elements of the cerebral cortex with the* periphery of the body ; their course may or may not be interrupted 420 THE CENTRAL NERVOUS SYSTEM. - d during their passage through the basal nuclei ; (2) the commissural fibers, which, according to the original definition, pass througli the corpus callo- sum and anterior commis- sure, thus joining corre- sponding parts of the two hemispheres ; (3) the asso- ciation fibers, which con- nect different parts of the gray substance of the same hemispheres ; and (4) the centripetal or terminal fibers — i.e., the terminal arborizations of those neu- raxes, the cells of which lie in some other region of the same or opposite hemi- sphere, or even in some more distant portion of the nervous system. The pro- jection fibers originate from the pyramidal cells, some of them perhaps from the polymorphous cells. The commissural fibers are also derived from the pyramidal cells, and lie somewhat deeper in the white sub- stance than the association fibers. With the exception of those which join the cunei and those which lie in the anterior commissure, all the commissural fibers are situated in the corpus callosum. They give off during their passage through the hemispheres large num- bers of collaterals, which penetrate at various points into the gray substance and end there in terminal filaments. In this respect their arborization is contrary to the old definition of these fibers, and the latter must be completed by the statement that, besides joining symmetric points of the two hemispheres, they also, by means of their collaterals, may connect other areas of the gray substance with the peripheral regions supplied by their end-tufts (Ramon y Cajal, 93). The association fibers have their origin also in the pyramidal cells. In the medullary substance their neuraxes divide T-shaped, and after a longer or shorter course penetrate into the •gray substance of the same hemisphere, where they end as ter- minal fibers. A few collaterals are, however, previously given off. Fig. 336. — Schematic diagram of the cerebral cortex : a, Molecular layer with superficial (tan- gential) fibers ; b, striation of Bechtereff-Kaes ; c, layer of small pyramidal cells; d, stripe of Bail- larger; e, radial bundles of the medullary sub- stance ; y, layer of polymorphous cells. THE OLFACTORY BULB. 421 which also terminate in the same manner in the gray substance. The association fibers form the bulk of the medullary rays. On examining a vertical section through one of the cerebral convolutions a number of successive striations may be seen. These are more or less distinct, according to the region, and consist of strands of medullated nerve-fibers between the layers of cells, and parallel with the surface of the convolution. The most superficial form a layer of tangential fibers. Between the molecular layer and the layer of small pyramidal cells is the striation of Bechtereff and Kaes, and in the region of the large pyramidal cells the striation of Baillarger (Gennari) corresponding to the striation of Vicq d'Azyr in the cuneus. In figure 336 the medullary substance is seen below, \vith rays, composed of parallel bundles of fibers, passing upward into the gray substance ; in reality these fibers penetrate much higher than is shown in the illustration. D. THE OLFACTORY BULB. The olfactory bulb is composed of five layers, which are espe- cially well marked on its ventral side : first, the layer of peripheral nerve-fibers ; second, the layer of olfactory glomeruli ; third, the stratum gelatinosum, or molecular layer ; fourth, the layer of pyr- amidal cells (mitral cells) ; and, fifjth, the granular layer with the deeper nerve-fibers. The layer of peripheral fibers is composed of the nerve- bundles of the olfactory nerve which cross one another in various directions and form a nerve-plexus. The glomerular layer con- tains peculiar, regularly arranged, round or oval, and sharply defined structures, which were first accurately studied by Golgi. They are known as glomeruli (from 100 [J. to 300 /JL in diameter), and are in reality complexes of intertwining telodendria. As we shall see, the epithelial cells of the olfactory region of the nose must be regarded as peripheral ganglion cells and their centripetal (basal) processes as neuraxes. The telodendria of these neuraxes, together with those of the dendrites from the mitral or other cells, come in contact with each other within the olfactory glomeruli. The molec- ular layer consists of small, spindle-shaped ganglion cells. Their neuraxes enter the fifth layer and their short dendrites end in ter- minal ramifications in the glomeruli. The mitral cells give off neuraxes from their dorsal surfaces which also enter the granular layer, but the majority of their dendrites break up into terminal ramifications in the olfactory glomeruli, as just described. The granular layer (absent in the illustration) is made up of nerve-cells and nerve-fibers ; but, aside from these, we find also large numbers of peculiar cells with a long peripherally and several short centrally directed dendrites. No neuraxes can be demonstrated in these 422 THE CENTRAL NERVOUS SYSTEM. cells (granular cells). This layer also contains the stellate ganglion cells. The latter are not numerous, but lie scattered, and each pos- sesses several short dendrites and a peripherally directed neuraxis which ends in the molecular layer in a rich arborization. The deep nerve-fibers are grouped into bundles which inclose between them the granular and stellate cells just mentioned. These nerve-fibers Mitral cells. Layer of olfactory glomeruli. Peripheral nerve-, fibers. Fig. 337. — The olfactory bulb, after Golgi and Ram6n y Cajal. The not shown. granular layer is are derived partly from the neuraxes of the pyramidal or mitral cells and partly from the cells of the molecular layer, while some of them are centripetal fibers from the periphery, which end between the granules of the fifth layer. E. EPIPHYSIS AND HYPOPHYSIS. In mammalia the epiphysis, or pineal gland, consists of a fibrous capsule derived from the pia mater, from which numerous fibrous tissue septa and processes pass into the gland, uniting to form quite regular round or oval compartments in which closed follicles or alveoli, whose walls consist of epithelial cells, are found. In the lower portion of the epiphysis there is found a relatively large amount of neuroglia tissue, consisting of coarse fibers, as has been shown by Weigert. The epithelial cells forming the walls of the follicles are of cubic or short columnar shape, and may be arranged in a single layer or may be pseudostratified or stratified. Follicles EPIPHYSIS AND HYPOPHYSIS. 423 completely filled with cellular elements are found. Other follicles contain peculiar concretions, known as brain-sand or acervulus, of irregular round or oval or mulberry shape. Medullated nerve-fibers have been traced into the epiphysis, but their mode of termination is not known. The hypophysis, or pituitary body, consists of two lobes. The posterior or infundibular lobe is developed from the floor of the first primary brain-vesicle, and remains attached to the floor of the third ventricle by a stalk, known as the infundibulura ; the anterior or glandular lobe develops from a hollow protrusion derived from the primary oral ectoderm. The distal end of this protrusion or pouch comes in contact with the anterior surface of the lower portion of the infundibulum, and becomes loosely attached to it. As the bones at the base of the skull develop, the attenuated oral end of this pouch atrophies, the distal end becoming finally completely severed from the buccal cavity. In the infundibular lobe of the hypophysis of the dog, Berkley (94) described three portions presenting different microscopic struc- ture. His account will here be followed :. (i) An outer stratum consisting of three or four layers of cells resembling ependymal cells, which are separated into groups by thin strands of fibrous tissue entering from the fibrous covering of this lobe. (2) A zone consisting of glandular epithelial cells which in certain places are arranged in the form of alveoli, often containing a colloid substance. This zone merges into the central portion, (3), containing variously shaped cells and connective-tissue partitions with blood-vessels. In this portion neurogliar cells (see these) and nerve-cells were stained by the chrome-silver method. The glandular or anterior lobe resembles slightly in structure the parathyroid. This lobe is surrounded by a fibrous tissue capsule and within it are found variously shaped alveoli or follicles, or, again, columns or trabeculae of cells separated by a very vascular connective tissue. In the alveoli or columns of cells are found two varieties of glandular cells, which may be differentiated more by their staining reaction than by their size and structure, although they present slight structural differences. One variety of cells pos- sesses a protoplasm which shows affinity for acid stains ; these are known as chromophilic cells. They are of nearly round or oval shape, with nuclei centrally placed, and have a protoplasm present- ing coarse granules. The other variety of cells, known as chief cells, are more numerous than the chromophilic. They are of cubic or short columnar shape, with nuclei placed in the basal portions of the cells and with protoplasm showing a fine granulation and with an affinity for basic stains. Now and then alveoli containing a colloid substance, similar to that found in the alveoli of the thy- roid gland, may be observed. The blood-vessels of the glandular portion are relatively large, the majority of them having only an endothelial lining which comes in contact with the glandular cells. 424 THE CENTRAL NERVOUS SYSTEM. The circulation of the hypophysis must be regarded as sinusoidal. In the glandular portion of the hypophysis of the dog, Berkley (94) found small varicose nerve-fibers belonging to the sympathetic sys- tem. From the larger bundles, which follow the blood-vessels, are given off single fibers, or small bundles of such, which end on the glandular elements in numerous small nodules. F. GANGLIA* In the course of peripheral nerves are found numerous larger and smaller groups of nerve-cells, known as ganglia. The neurones of these ganglia are in intimate relation with the neurones of the cen- Fig. 338. — Longitudinal section of spinal ganglion of cat. tral nervous system, and may, therefore, be discussed with the lat- ter. According to the structure and function of their neurones, the ganglia are divided into two groups — (i) spinal or sensory ganglia and (2) sympathetic ganglia. The spinal ganglia are situated on the posterior roots of the spinal nerves. Certain cranial ganglia — namely, the Gasserian, geniculate, and auditory ganglia, the jugular and petrosal gan- glia of the glossopharyngeal nerves, and the root and trunk ganglia of the vagi — are classed with the spinal ganglia, since they present the same structure. The spinal and sensory cranial ganglia are surrounded by firm connective- tissue capsules, continuous with the perineural sheaths of the incoming and outgoing nerve-roots. From GANGLIA. 425 these capsules connective-tissue septa and trabeculae pass into the interior of the ganglia, giving support to the nerve-elements. The cell-bodies (ganglion cells) of the neurones constituting these ganglia are arranged in layers under the capsule and in rows and groups or clusters between the nerve-fibers in the interior of the ganglia. More recent investigations have shown that several types of neurones are to be found in the spinal and cranial sensory gan- glia ; of these, we may mention the following : (i) Large and small unipolar cells with T- or Y-shaped division of the process. These neurones, which constitute the greater number of all the neurones of the ganglia under discussion, consist of a round or oval cell-body, from which arises by means of an implantation cone Fig. 339. — Ganglion cell from the Gasserian ganglion of a rabbit ; stained in methylene- blue (intra vitani). a single process, which, soon after it leaves the cell, becomes in- vested with a medullary sheath and usually makes a variable num- ber of spiral turns near the cell-body. According to Dogiel, this process divides into two branches, usually at the second or third node of Ranvier, sometimes not until the seventh node is reached. Of these two branches, the peripheral is the larger, and enters a peripheral nerve-trunk as a medullated sensory nerve-fiber, termi- nating in one of the peripheral sensory nerve-endings previously .described. The central process, the smaller of the two, becomes a medullated nerve-fiber, which enters the spinal cord or medulla in a manner described in a former section. The cell-body of each of these neurones is surrounded by a nucleated capsule, continuous with 426 THE CENTRAL NERVOUS SYSTEM. the neurilemma of the single process. (2) Type II spinal ganglion cell of Dogiel. Dogiel has recently described a second type of spinal ganglion cell which differs materially from the type just described. The cell-bodies of these neurones resemble closely those of the typ- ical spinal ganglion neurones. Their single medullated processes divide, however, soon after leaving the cells into branches which divide further and which do not pass beyond the bounds of the gan- glia but terminate, after losing their medullary sheaths, in compli- cated pericapsular and pericellular end-plexuses surrounding the capsules and cell-bodies of the typical spinal ganglion cells. (3) Mul- tipolar ganglion cells ; in nearly all spinal and cranial ganglia there are found a few multipolar nerve-cells, which in shape and struc- ture resemble the nerve-cells of the sympathetic system. Fig. 340. — Diagram showing the relations of the neurones of a spinal ganglion ; p. r., posterior root; a. r., anterior root; /. s., posterior branch and a. s., anterior branch of spinal nerve ; w. r., white ramus communicans ; a, large, and 6, small spinal ganglion cells with T-shaped division of process ; c, type II spinal ganglion cells (Dogiel); s, multipolar cell ; d, nerve-fiber from sympathetic ganglion terminating in pericellular plexuses (slightly modified from diagram given by Dogiel). Entering the spinal ganglia from the periphery are found a rel- atively small number of small, medullated or nonmedullated nerve- fibers, probably derived from sympathetic ganglia. These nerve- fibers, medullated and nonmedullated, the former losing their medullary sheaths within the ganglia, approach a spinal ganglion cell, and after making a few spiral turns about its process, termi- nate in pericapsular and pericellular end-plexuses. Dogiel believes that the cell-bodies and capsules thus surrounded by the terminal branches of the sympathetic fibers terminating in the spinal ganglia belong to the spinal ganglion cells of the second type first described by him. In figure 340 is represented by way of diagram the structure of a spinal ganglion. In the medium-sized cells (from 30 fJ. to 45 fj. in diameter) of the GANGLIA. 427 spinal ganglia of the frog, von Lenhossek (95) found centrosomes surrounded by a clear substance (centrospheres). The entire struc- ture lay in a depression of the nucleus and contained more than twelve extremely minute granules (centrosomes), which showed a staining reaction different from that of the numerous concentrically laminated granules present in the protoplasm. This observation is interesting in that it proves that centrosome and sphere occur also in the protoplasm of cells which have not for a long time under- gone division and in which there is no prospect of future division. Sympathetic Ganglia. — The ganglia, of the sympathetic ner- vous system comprise those of the two great ganglionated cords, found on each side of the vertebral column and extending from its cephalic to its caudal end, with which may be grouped certain cranial ganglia having the same structure, — namely, the sphenopalatine, otic, ciliary, sublingual, and submaxillary ganglia ; also three un- Fig. 341. — Neurone from inferior cervical sympathetic ganglion of a rabbit; methylene- blue stain. paired aggregations of ganglia, found in front of the spinal column, of which the cardiac is in the thorax, the semilunar in the abdomen, and the hypogastric in the pelvis ; and further, large numbers of smaller ganglia, the greater number of which are of microscopic size and are found in the walls of the intestinal canal and bladder, in the respiratory passages, in the heart, and in or near the majority of the glands of the body. The sympathetic ganglia are inclosed in fibrous tissue capsules continuous with the perineural sheaths of their nerve-roots. The thickness of the capsule bears relation to the size of the ganglion, being thicker in the larger and thinner in the smaller ones. From these capsules thin connective-tissue septa or processes pass into the interior of the ganglia, supporting the nerve elements. The sympathetic neurones, the cell-bodies and dendritic processes of which are grouped to form the sympathetic ganglia, are variously 428 THE CENTRAL NERVOUS SYSTEM. shaped unipolar, bipolar, and multipolar cells, the cell-bodies of which are surrounded by nucleated capsules, continuous with the neurilemma of their neuraxes. In the sympathetic ganglia of mam- malia and birds the great majority of sympathetic neurones are multipolar, although in nearly all ganglia a small number of bipolar and unipolar cells are to be found, usually near the poles of the ganglia. The dendrites of the sympathetic neurones in any one ganglion branch repeatedly. Of these branches, some extend to the per- iphery of the ganglion, where they interlace to form a peripheral subcapsular plexus, while others interlace to form plexuses between the cell-bodies of the neurones in the interior of the ganglion — O O pericellular plexuses. These pericellular plexuses are external to the capsules surrounding the cell-bodies of the sympathetic neurones. Fig. 342. — From section of semilunar ganglion of cat ; stained in methylene-blue, intra •vitam (Huber, Journal of Morphology, 1899). The neuraxes of the sympathetic neurones, the majority of which are nonmedullated, the remainder surrounded by delicate medullary sheaths, arise from the cell-bodies either from implanta- tion cones or from dendrites at variable distances from the cell- bodies, leave the ganglion by way of one of its nerve-roots, and terminate in heart muscle tissue, nonstriated muscle, and glandular tissue, and to some extent in other ganglia, both sympathetic and spinal. Terminating in all sympathetic ganglia are found certain small medullated nerve-fibers, varying in size from about 1.5 // to 3 fjt. The researches of Gaskell, Langley, and Sherrington have shown that these small medullated nerve-fibers leave the spinal cord through the anterior roots of the spinal nerves from the first dorsal to the third or fourth lumbar and reach the sympathetic GANGLIA. 429 ganglia through the white rand comnmnicantes. Similar small medullated nerve-fibers are found in certain cranial nerves. These small medullated nerve-fibers, which may be spoken of as white rami fibers, after a longer or shorter course, in which they may pass through one or several ganglia without making special con- nection with the neurones contained therein, terminate in some sympathetic ganglion in a very characteristic manner. After enter- ing the sympathetic ganglion in which they terminate, they branch repeatedly while yet medullated. The resulting branches then lose their medullary sheaths and divide into numerous small, varicose nerve -fibers, which interlace to form intracapsular plexuses, which surround the cell-bodies of the sympathetic neurones. In the sympathetic ganglia of mammalia such intracapsular pericellular Fig. 343. — From section of stellate ganglion of dog, stained in methylene-blue and alum carmin : a, white ramus fiber ( Huber, Journal of Morphology, 1899). plexuses may be very simple, consisting of only a few varicose nerve-fibers, or very complicated, consisting of many such fibers. In the sympathetic ganglia of reptilia, in which are found very large sympathetic neurones, the white rami fibers are wound spirally about the cell-bodies of such neurones before terminating in com- plicated pericellular plexuses. In the frog and other amphibia the sympathetic neurones are unipolar nerve-cells. The white rami fibers terminating in the sympathetic ganglia of amphibia are wound spirally about the single processes of these unipolar cells while yet medullated fibers, but they lose their medullary sheaths before ter- minating in the intracapsular pericellular plexuses. From what has been said concerning the white rami fibers and their relation to the sympathetic neurones, it is evident that the sympathetic neu- 430 THE CENTRAL NERVOUS SYSTEM. rones, the cell-bodies and dendrites of which are grouped to form the sympathetic ganglia, form terminal links in nerve or neurone chains ; the second link of these chains is formed by neurones the cell-bodies of which are situated in the spinal cord or medulla, the Fig. 344. — From section of sympathetic ganglion of turtle, showing white rami fibers wound spirally about a large process of a unipolar cell, and ending in pericellular plexus (Huber, Journal of Morphology, 1899). neuraxes leaving the cerebrospinal axis through the white rami as small medullated nerve-fibers, which terminate in pericellular plex- uses inclosing the cell-bodies of the sympathetic neurones. Large medullated nerve -fibers, the dendrites of spinal ganglion neurones, reach the sympathetic ganglia through the white rami. Fig. 345. — From section of sympathetic ganglion of frog, showing spiral fiber (white ramus fiber) and pericellular plexus (Huber, Journal of Morphology, 1899). They make, however, no connection with the sympathetic neurones, but pass through the ganglia to reach the viscera, where they ter- minate in special sensory nerve-endings or in free sensory nerve- endings. RELATIONSHIP OF NEURONES. 43 I G. GENERAL SURVEY OF THE RELATIONS OF THE NEURONES TO ONE ANOTHER IN THE CENTRAL NERVOUS SYSTEM. The following figures illustrate the modern theories with re- gard to the relationship of the neurones in a sensorimotor reflex cycle. The pathway along which the impulse from the stimulated area of the body is transmitted to the motor nerve end-organ tra- verses two neurones (primary neurones) which are in contact by means of their telodendria situated within the gray matter of the spinal cord. The cell-body of the sensory neurone lies within the spinal ganglion ; that of the motor neurone, in the anterior horn of the spinal cord. The dendrite of the sensory neurone commences mN Fig- 346. — Schematic diagram of a sensorimotor reflex arc according to the modern neurone theory ; transverse section of spinal cord : mN, Motor neurone ; sN, sensory neurone ; C1, nerve-cell of the motor neurone ; C2, nerve-cell of the sensory neurone ; d, dendrite ; n, neuraxis of both neurones ; f, telodendria ; M, muscle-fiber ; A, skin with peripheral telodendrion of sensory neurone. as a telodendrion in the skin or perhaps also in more deeply seated structures, and transmits a cellulipetal impulse, while its cellulifugal neuraxis and telodendrion (the latter in the gray matter of the cord) transfer the impulse to the cellulipetal telodendrion of the motor neurone. The cellulifugal neuraxis of the latter finally ends as a telodendrion in the muscle. (Figs. 346 and 347.) In the case of longer tracts the conditions are somewhat more complicated, as, for instance, in tracing the impulse along the sen- sory fibers to the cortex of the brain, and from there along the motor fibers to the responding muscle. In such cases secondary neurones are called into play by means of their telodendria, which are necessarily in contact with the primary neurones just described. 432 THE CENTRAL NERVOUS SYSTEM. When we take into consideration the simplest possible case, that of the motor segment of such a neurone-chain, we find, for instance (Fig. 348), that the neuraxis of a pyramidal cell in the brain cortex (psychic cell) enters the white substance and traverses it as a nerve- fiber through the peduncle and the pyramid into the crossed pyramidal tract of the opposite side. Here its telodendria come in contact with those of the motor neurone of the anterior horn. In the foregoing instance the motor nerve tract is composed of two neurones — of a motor neurone of the first order, extending from the cortex of the brain to the anterior cornua of the spinal cord, and of a motor neurone of the second order, the elements of which extend from the anterior cornua to the telodendria in the muscle. Fig. 347. — Schematic diagram of a sensorimotor reflex cycle ; sagittal section of the spinal cord: C1, Motor cells of the anterior cornua; «, «, neuraxes ; sN, sensory neurone ; C2, spinal ganglion cell ; C, collaterals of the sensory neuraxes ; 2>°% alcohol 100 c.c.) ; rinse in distilled water and dehydrate quickly, clear in oil of bergamot, and mount in xylol-balsam. Benda' s Selective Neuroglia Staining Method. — Benda has for some years concerned himself with perfecting selective staining methods for differentiating certain constituents of the protoplasm of cells, and has recently published a number of staining methods, by all of which neuroglia fibers may be more or less successfully differentiated. According to him, certain hematoxylin solutions, used after proper fixation and mordanting of the tissues, maybe used for neuroglia stains; also hematoxylin staining, followed by staining with an acid-anilin water crystal violet solution. These will not be considered here. We wish, however, to call especial atten- tion to the following method for staining neuroglia tissue, suggested by Benda, since it has certain advantages not possessed by other selective neu- roglia stains. Fix small pieces of tissue in 10% formalin; place in Weigert's chrome-alum solution (formula given above), four days in warm oven at 38° C. ; wash in water twenty-four hours ; dehydrate in graded alcohols ; imbed in paraffin ; cut thin sections and fix these to slides with the albumin -glycerin fixative ; remove paraffin and place sections in mor- dant consisting of a 4% aqueous solution of ferric alum ; rinse thoroughly in two tap waters and one distilled water ; place in a sodium sulphaliz- arate solution (add to distilled water a sufficient quantity of a saturated solution of sodium sulphalizarate in 70% alcohol to give it a sulphur-yellow color) twenty-four hours ; rinse in distilled water ; stain for fifteen min- utes in a o. i % aqueous solution toluidin blue, which should be heated after 446 THE EYE. the sections are in the stain until the solution steams ; allow the stain to cool ; rinse in distilled water ; wash in a i CJC aqueous solution of glacial acetic acid for a few seconds or in acid alcohol (six drops of hydrochloric acid ; 70% alcohol looc.c. ) for a few seconds ; dry sections with filter- paper ; dip sections a few times in absolute alcohol ; differentiate in cre- osote, ten minutes to an hour — control now and then under the micro- scope ; wash in several xylols and mount in xylol -balsam. Neuroglia fibers blue, chromatin of neuroglia cell nuclei a purplish blue, protoplasm of neuroglia cells brownish red to bluish red. VIII. THE EYE. A. GENERAL STRUCTURE. THE organ of vision consists of the eyeball, or bulbus oculi, and the entering optic nerve. In the eyeball we distinguish three tunics : (i) a dense external coat, the tunica fibrosa or externa, which may be regarded as a continuation of the dura mater, consisting of an anterior transparent structure, called the cornea, and the remaining portion, known as the tunica sclerotica, or, briefly, the sclera ; (2) within the tunica fibrosa a vascular tunic, the tunica vasculosa or media, subdivided into the choroid, ciliary body, and iris ; (3) an inner coat, the tunica interna, which consists of two layers, the inner being the retina ; the outer, the pigment membrane. The latter lines the internal surface of the tunica vasculosa throughout. Within the eyeball are the aqueous humor, the lens, and the vitreous body. The lens is attached to the ciliary body by a special accessory apparatus — the zomda ciliaris. These two structures — the lens and its fixation apparatus — divide the cavity of the eyeball into two principal cham- bers, the one containing the aqueous humor and the other the vitreous. The former is further subdivided by the iris into an anterior and a posterior chamber. During life the latter is only a narrow capillary cleft. B. DEVELOPMENT OF THE EYE, In man the eyes begin to develop during the fourth week of embryonic life, and at first consist of a pair of ventrolateral diver- ticula, projecting from the anterior brain vesicle. These evaginations gradually push outward toward the ectoderm, and are then known as the primary optic vesicles. The slender commissural segments connecting the vesicles with the developing brain are termed the optic stalks. Very soon a process of invagination takes place ; that portion of the vesicular wall nearest the ectoderm is pushed inward, thus DEVELOPMENT OF THE EYE. 447 forming a double-walled cup — the secondary optic vesicle, or optic cup. An internal and an external wall may now be differentiated, continuous at the margin of the cup. At the same time a disc-like thickening of the adjacent ectoderm sinks inward toward the mouth of the cup-shaped optic vesicle, forming the first trace of the lens. During the development of the secondary optic vesicle a groove Blood-vessels Sphincter Vein. Canal of Petit, of the iris. Cornea, pupillae. Iris. Fontana's spaces. — Pigment layer. Physiologic excavation. Macula lutea. Fig. 352. — Schematic diagram of the eye (after Leber and Flemming) : a, Vena vorti- cosa ; b, choroid ; /, lens. is formed on its ventral side, extending from the marginal ring into the optic stalk. This is the embryonic optic fissure, or the choroi- dal fissure. At the edges of the groove the two layers of the optic cup are continuous. This groove serves for the penetration of mesoblastic tissue and blood-vessels into the interior of the optic cup, and in its wall the fibers of the optic nerve develop. The outer layer of the secondary optic vesicle becomes the pig- ment membrane ; the inner, the retina. The optic nerve-fibers con- sist not only of the centripetal neuraxes of certain ganglion cells in the retina, but also of centrifugal neuraxes, which pass out from the brain (Froriep). The invaginating ectoderm which later constitutes the lens is constricted off from the remaining ectoderm in the shape of a vesi- 448 THE EYE. cle, the mesial half of which forms the lens fibers by a longitudinal growth of its cells, while the lateral portion forms the thin anterior epithelial capsule of the lens. The epithelium of the ectoderm external to the lens differentiates later into the external epithelium of the cornea and conjunctiva, neither of which structures is at this stage sharply defined from the remaining ectoderm. It is only during the development of the eyelids that a distinct demarcation is established. All the remaining portions of the eye, as the vitre- ous body, the vascular tunic with the iris, the sclera with the substantia propria of the cornea and the cells of Descemet's layer, are products of the mesoderm. C TUNICA FIBROSA OCULL J. THE SCLERA. The sclera is the dense fibrous tissue covering of the eyeball, and is directly continuous with the transparent cornea. At the poste- rior mesial portion of the eyeball, the sclera is perforated for the en- trance of the optic nerve, this region being known as the lamina cribrosa. The sclera consists of bundles of connective-tissue fibers arranged in equatorial and meridional layers. At the external scleral sulcus, in the vicinity of the cornea, the arrangement of the fibers is principally equatorial. The tendons of the ocular muscles are continuous with the scleral fibers in such a manner that those of the straight muscles fuse with the meridional fibers, while those of the oblique muscles are continuous with the equatorial fibers. In the sclera are many lymph-channels communicating with those of the cornea. They are much coarser and more irregularly arranged than those of the cornea, and in this respect simulate the lymph- channels found in aponeuroses. Pigmentation is constantly present at the corneal margin, in the vicinity of the optic nerve entrance, and also on the surface next the choroid. The innermost pigment layer of the sclera is lined by a layer of flattened endothelial cells, and is regarded by some as a separate membrane, known as the lamina fusca ; generally, however, it is regarded as forming a part of the outermost layer of the choroid (lamina suprachoroidea). The external surface of the sclera also presents a layer of flattened endo- thelial cells, belonging to the capsule of Tenon. Anteriorly, the mobile scleral conjunctiva is attached to the sclera by a loose con- nective tissue containing elastic fibers. The cornea is inserted into the sclera very much as a watch- crystal is fitted into its frame. At the sclerocorneal junction is found an annular venous sinus, the canal of Schlemm, which may appear as a single canal or as several canals separated by incom- plete fibrous septa. Anteriorly and externally this canal is bounded by the cornea and sclera ; internally, it is partly bounded by the origin of the ciliary muscle. The sclera comprises, therefore, one- TUNICA FIBROSA OCULI. 449 half of the canal-wall, and presents a corresponding circular sulcus, the so-called inner scleral sulcus. The blood-vessels of the sclera are derived from the anterior and posterior ciliary vessels. The capillaries enter either into the ciliary veins or into the venae vorticosae. The numerous remaining vessels traverse the sclera, extending to the choroid, iris, or scleral margin. At the corneal margin the capillaries form loops. Cornea! epithelium. Basal cells. Anterior elastic membrane. Substantia propria. 2. THE CORNEA. The cornea is made up of the following layers : (i) the ante- rior or corneal epithelium ; (2) the anterior elastic membrane, or Bowman's membrane ; (3) the ground-substance of the cornea, or substantia propria ; (4) Des- cemet's membrane; (5) the endothelium of Descemet's membrane. At the center of the human cornea the epithe- lium consists of from six to eight layers of cells, being somewhat thicker near the corneal margin. Its basilar surface is smooth and there are no connective-tissue pa- pillae. The basal epithelial layer is composed of cylin- dric cells of irregular height ; the following layers contain irregular polygonal cells, Fig. 353. — Section through the anterior portion while the two or three most of human cornea > X 5°o. superficial layers consist of flattened cells. The cells of the corneal epithelium are all provided with short prickles, which are, however, very difficult to demon- strate, and between are found lymph-canaliculi. The lower surfaces of the basal cells also possess short processes which penetrate into the anterior basement membrane. In man the anterior elastic or Bowman's membrane is quite thick, measuring from 6 to 8 tj. in thickness and is apparently homo- geneous, but may be separated into fibrils by means of certain reagents. In structure it belongs neither to the elastic nor to the white fibrous type of connective tissue, and may be regarded as a basement membrane. Numerous nerve-fibers penetrate its pores to enter the epithelium. The thickness of this membrane decreases toward the sclera, and it finally disappears about I mm. from the latter. The substantia propria consists of connective-tissue fibrils grouped into bundles and lamellae. Chemically they do not differ 29 450 THE EYE. from true connective-tissue fibers (Morochowetz), but are doubly refracting, although the cornea as a whole yields chondrin and not glutin on boiling. There are about sixty lamellae in the human cornea. The fibrils composing each lamella are cemented together and run parallel to one another as well as to the surface of the cornea, but they are so arranged that the fibrils of each lamella cross those of the immediately preceding one at an angle of about twelve degrees. The lamellae themselves are likewise closely cemented to one another. The most superficial lamella, lying im- mediately beneath the anterior elastic membrane, is composed of finer fibers, the course of which is oblique to the surface of the cornea. Between the anterior and posterior elastic membranes are bundles of fibers, which perforate the various lamellae of the cornea and are consequently known as the perforating or arcuate fibers. Between the lamellae are peculiar, flattened cells, possessing Lymph-canaliculi. Corneal space. Fig. 354. — Corneal spaces of a dog ; X 640. irregular or lamella-like processes, the fixed corneal corpuscles ; these lie in special cavities in the ground substance of the substantia propria, which are known as corneal spaces. In these spaces there are also found a varying number of leucocytes. By means of vari- ous methods (silver nitrate and gold chlorid treatment), these corneal spaces may be shown to be part of a complicated lymphatic system, comparable to the lymph-canalicular system of fibrous connective tissue. This system of canals is also in communication with the lymph-channels at the corneal margin. The posterior elastic or Descemet's membrane is not so inti- mately connected with the substantia propria as Bowman's mem- brane. It is thinnest at the center of the cornea, and becomes thicker toward the margin. It may be separated into finer lamellae, is very elastic, resists acids and alkalies, but is digested by trypsin. TUNICA FIBROSA OCULI. 451 At the periphery — that is, at the edge of the cornea — Descemet's membrane goes over into the fibers of the ligamentum pectinatum. The endothelium of Descemet's membrane consists of low, quite regular hexagonal cells, which in certain vertebrates (dove, duck, rabbit) are peculiar in that a fibrillar structure may be seen in that portion of each cell nearest the posterior elastic membrane. By means of these fibers, not only adjacent cells, but also those further apart, are joined together. Thus we have here to a marked degree the formation of fibers which penetrate the cells and connect them with one another, conditions already met with in the prickle-cells of the epidermis. The cornea is nonvascular. In fetal life, however, the capil- laries from the anterior ciliary arteries form a precorneal vascular network immediately beneath the epithelium, a structure which is obliterated shortly before birth and only rarely seen in the new- born. Its remains are found at the corneal limbus either as an episcleral or conjunctival network of marginal capillary loops. Fine branches of the anterior ciliary arteries extend superficially along the sclera to the corneal margin, and form here a network of capil- laries also ending in loops, from which numerous veins arise, con- stituting a corresponding network emptying into the anterior ciliary veins. The conjunctival vessels likewise form a network of mar- ginal loops at the corneal limbus, and are connected with the epi- scleral vessels (Leber). Under pathologic conditions the cornea may become vascularized from the marginal episcleral network. The nerves of the cornea are derived from the sensory fibers of the ciliary nerves, which form a plexus at the corneal margin ; from this, nonmedullated fibers penetrate the cornea itself and form two plexuses, a superficial and a ground plexus ; the latter is distributed throughout the whole substantia propria with the exception of its inner third (Ranvier, 81). The two plexuses are connected by numerous anastomoses. At one time it was supposed that direct communication existed between the corneal corpuscles and the nerve- fibers of both plexuses. This view, however, contradicts the gen- erally accepted neurone theory. Nerve-fibers from the superficial plexus pass through the ante- rior, elastic membrane and form a plexus over the posterior surface of the epithelium, known as the subcpithelial plexus. From the lat- ter nerve-fibers extend between the epithelial cells, terminating in telodendria with long slender nerve-fibrils, which end in small nodules. Many of the fibrils reach almost to the surface of the epithelium (Rollet, 71 ; Ranvier, 81 ; Dogiel, 90). Smirnow (1900) has described a rich nerve -supply for the sclera, consisting of both medullated sensory fibers and nonmedullated sympathetic fibers, derived mainly from the ciliary nerves. The sympathetic fibers supply the blood-vessels; the sensory fibers ter- minate in free endings between the connective-tissue lamellae. 452 THE EYE. D. THE VASCULAR TUNIC OF THE EYE. THE CHOROID, THE CILIARY BODY, AND THE IRIS. From without inward the following layers may be differentiated in the choroid : (i) the lamina suprachoroidea ; (2) the lamina vas- culosa Halleri ; (3) the lamina choriocapillaris ; and (4) the glassy layer, or vitreous membrane. The lamina suprachoroidea consists of a number of loosely arranged, branching and anastomosing bundles and lamellae of fibrous tissue, joined directly to the sclera. These bundles and lamellae consist of white fibrous connective tissue containing numer- ous elastic fibers, among which a few connective-tissue cells are dis- tributed. Pigment cells are also present in varying numbers. The bundles and lamellae are covered by endothelial cells, and the spaces and clefts between them, and between the lamina suprachoroidea and the lamina fusca, constitute a system of lymph-channels — the peric /toroidal lymph-spaces. Sclera. Lamina supra- choroidea. Lamina vascu- __, losa Halleri. Lamina chorio- capillaris. Glassy layer. -UaSa Fig. 355. — Section through the human choroid ; X I3°- The lamina vasculosa of the choroid is also composed of simi- lar lamellae, which, however, are more closely arranged. The blood- vessels constitute the principal portion of this layer, the vessels being of considerable caliber, not capillaries. They are so distrib- uted that the larger vessels, the veins, occupy the outer layer of the lamina vasculosa. The venous vessels converge toward four points of the eyeball, forming at the center of each quadrant one of the four vence vorticosa. The arteries, on the other hand, describe a more meridional course. THE VASCULAR TUNIC OF THE EYE. 453 In the inner portion of this layer is found a narrow zone, — in the human eye only about 10 fj. in thickness, — consisting largely of elastic fibers and free from pigment cells, known as the boundary zone. This zone is somewhat thicker in many mammals, and in some of these presents a characteristic structure. In the eyes of ruminants and horses this zone consists of several layers of con- nective-tissue bundles, and is known as the tapetwn fibrositm. It gives the peculiar luster often seen in the eyes of these animals. In the eyes of carnivora this zone consists of several layers of endothe- lioid cells, containing in their protoplasm numerous small crystals and forming the iridescent layer known as the tapetum cellulosinn. The lamina choriocapillaris contains no pigment and consists principally of capillary vessels, which form an especially dense net- work in the neighborhood of the macula lutea. As the venous cap- illaries become confluent and form smaller veins, the latter arrange themselves in long, radially directed networks, and form in this way the more or less pronounced stellulce vasculosce (Winslowii). The vitreous or glassy membrane is a very thin (2 /*) homo- geneous membrane which shows on its outer surface the impressions of the vessels composing the lamina choriocapillaris, and on its inner surface those of the pigment epithelium of the retina. At the ora serrata the choroid changes in character ; from this region forward, the choroidal tissue assumes more the appearance of ordinary connective tissue, and the choriocapillary layer is wanting. The region of the vascular coat extending from the ora serrata to the base of the iris is known as the ciliary body. Its posterior portion, about 4 mm. broad, the orbiculus ciliaris, is slightly thicker than the choroid, and presents on its inner surface numerous small folds, meridionally placed, consisting of connective tissue and blood- vessels. Anterior to the orbiculus ciliaris the ciliary body is thick- ened by a development of nonstriated muscle — the ciliary muscle (see below) ; and on the inner surface of this annular thickening are placed about seventy triangular folds, meridionally arranged — the ciliary processes. The attached border of these processes measures from 2 to 3 mm. The anterior border attains a height of about I mm. On and between these folds are found numerous small secondary folds or processes of irregular shape. The ciliary pro- cesses consist of fibrous connective tissue and numerous smaller and larger vessels, which have in the main a meridional arrange- ment. The vitreous membrane extends over the ciliary body, attain- ing in the region of the ciliary processes a thickness of 3 /J. or 4 fj.. Internal to the vitreous membrane, the ciliary body is covered by a double layer of epithelial cells, the continuation forward of the retina {pars ciliaris retince). Of these, the outer layer is composed of cells, which are deeply pigmented, and are of cubic or short columnar shape, and derived from the outer layer of the secondary optic vesicle, while the cells of the inner layer are nonpigmented and of columnar shape, and are developed from the inner layer of the secondary optic vesicle. In the region of the ciliary processes 454 THE EYE. their epithelial lining presents here and there evaginations of glan- dular appearance, lined by the unpigmented cells. These evagina- tions are known as ciliary glands, and to them is attributed — in part, at least — the secretion of the fluid found in the anterior cham- ber of the eye ; it is, however, still a question as to whether these structures are to be regarded as true glands or simply as depressions or crypts in the epithelium. The ciliary muscle is bounded anteriorly (toward the anterior chamber) by the ligamentum pectinatum iridis, externally by the cornea and sclera, posteriorly by the orbiculus ciliaris, and inter- nally by the ciliary processes. It consists of nonstriated muscle- fibers in the majority of vertebrates. This muscle is divided into three portions. The outer or meridional division extends from the posterior elastic lamina of the cornea and its continuation, forming the inner wall of the sinus venosus sclerae, to the posterior portion of the ciliary ring. The origin of the middle division is identical with Cornea! epithe- lium. Substantia pro- pria. — Descemet's membrane. Canal of Schlemm. Iris. .-.. Pigment layer. Loose connec- tive tissue of the conjunc- tiva. Conjunctiva. Meridional fibers. Radial fibers. —»• Miiller's fibers. Sclera. Processus ciliares. Fig. 356. — Meridional section of the human ciliary body ; X 2O- that of the outer, but its fibers (assuming that we have before us a meridional section) spread out like a fan, and occupy a large area at their insertion into the ciliary ring and ciliary processes. The radial course of these fibers is mterrupted by circular bundles. The third or inner division {fibrce circulares, fibers of Mutter} is situated between the ligamentum pectinatum, the ciliary processes, and the middle portion of the muscle just mentioned, and is thus near the base of the iris. Between the ciliary muscle and the posterior elastic membrane of the cornea is an intermediate, richly cellular tissue, which maybe regarded as a continuation of this elastic membrane, and which forms a part of the wall of the sinus venosus. Another structure internal to the foregoing and directed posteriorly is foe.' ligamentum pectinatum iridis, which encircles the anterior chamber and is a con- tinuation of Descemet's membrane to the base of the iris. It con- THE VASCULAR TUNIC OF THE EYE. 455 sists of fibers and lamellae lined by endothelial cells, and bounds certain intercommunicating spaces lying in the ligament, known as the spaces of Fontana. The latter communicate on the one side with the perivascular spaces of the sinus venosus sclerae (canal of Schlemm), and on the other with the anterior chamber. The iris must be looked upon as a continuation of the choroid, and is connected at its anterior peripheral portion with the ligamen- tum pectinatum. The iris possesses the following layers, beginning anteriorly : (i) the anterior endothelium; (2) the ground layer, or stroma of iris, together with the sphincter muscle of the pupil ; and (3) the two-layered, pigmented epithelium — the pars iridica retinae, of which the anterior is in part replaced by a peculiar muscle tissue, developed from the ectoderm and forming the dilator of the pupil. The anterior endothelium is a single layer of irregularly polyg- onal, nonpigmented cells, and is directly continuous with the endothelium of the pectinate ligament. The ground-layer or stroma of iris consists anteriorly of a fine reticulate tissue rich in cellular elements (reticulate layer). The remaining strata which form the bulk of the ground-layer consti- tute its vascular layer. The vessels are here peculiar in that they are covered by coarse, circular, connective -tissue fibers forming vas- cular sheaths. There is also an entire absence of muscular tissue in the vessel walls. The nerves, too, are enveloped by a dense con- nective tissue. In all eyes (except the albinotic) pigment is found in the connective tissue. On the posterior inner surface of the ground-layer is a band of smooth muscle-fibers encircling the pupil — the sphincter muscle of the pupil. Posterior to this and in intimate relation with the layer of pigmented epithelium covering the posterior surface of the iris is a layer of spindle-shaped cells having a radial arrangement and contain- ing pigment. Closer microscopic inspection reveals the fact that in all probability these elements represent muscular tissue. Here, there- fore, we have to deal with a dilator muscle of the pupil. There has been much discussion as to the existence and structure of this muscle. Recent investigations (Szili) indicate that it is developed from the outer layer of the secondary optic vesicle. The posterior epithelium is the direct continuation of the two epithelial layers of the ciliary body, and represents the anterior por- tion of the secondary optic vesicle, the two layers being continuous at the margin of the pupil. In the iris both layers of cells, so far as they exist, are pigmented. The arteries of the choroid are derived from the short posterior ciliary, the long ciliary, and the anterior ciliary arteries. The short posterior ciliary arteries penetrate the sclera in the vicinity of the optic nerve, where they anastomose with branches from the retinal vessels, and spread through the choroid, where they form the choriocapillary layer. The long posterior ciliary arteries (a mesial and a lateral) penetrate the sclera and course forward between choroid and sclera to the ciliary body, forming there the circulus arteriosus iridis major; they 456 THE EYE. also supply the ciliary muscle, the ciliary processes, and the iris, and anastomose in the ciliary ring with the branches of the short pos- terior and anterior ciliary arteries. The latter lie beside and partly within the straight ocular muscles, penetrating the latter at the an- terior margin of the sclera ; they give off branches to the circulus arteriosus iridis major and to the ciliary muscles, anastomosing at the same time with the posterior ciliary arteries. (Compare Figs. 352 and 357.) Within the iris the blood-vessels generally take a radial direction, but also anastomose with one another, forming capillaries, and subsequently the circulus arteriosus iridis minor at the inner pupillary margin. From the region supplied by the posterior ciliary arteries most of the blood is carried toward the vorticose veins. The anterior ciliary veins convey the blood com- ing from the arteries of the same name. Into these veins is also poured the blood from the veins lying in the canal of Schlemm, the canal itself being in reality an open venous sinus. Besides this, these veins convey also venous blood from the conjunctiva (Leber). The nonstriated muscle of the ciliary body and iris receives its innervation through sympathetic nerve-fibers, neuraxes of sympa- thetic neurones, the cell- bodies of which are situated either in the ciliary ganglia or in the superior cervical ganglia. The neuraxes of the sympathetic cells forming the ciliary ganglia form the short ciliary nerves, which pierce the sclera in the neighbor- hood of the optic nerve and pass forward, to terminate in the muscle of the ciliary body and the sphincter muscle of the pupil. Stimulation of these nerves causes a con- traction of the ciliary muscle and a closure of the pupil'. The cell-bodies of the sympa- thetic neurones forming the ciliary ganglia are surrounded by pericellular plexuses, the terminations of small medul- lated nerve -fibers (white rami fibers) which reach the ciliary ganglia through the oculo- motor nerves. Neuraxes of sympathetic neurones, the cell-bodies of which are sit- uated in the superior cervical ganglia, reach the eye through the cavernous plexuses, to ter- minate, it is thought, — in part, at least, — in the dilator of the iris, since stimulation of these nerves causes a dilatation of the Margin of pupil. Choroid. Fig. 357. — Injected blood-vessels of the human choroid and iris ; X 7- THE INTERNAL OR NERVOUS TUNIC OF THE EVE. 457 pupils. The cell-bodies of these sympathetic neurones are sur- rounded by pericellular plexuses, the terminations of white rami fibers which leave the spinal cord through the first, second, and third thoracic nerves (Langley), and which reach the superior cer- vical ganglia through the cervical sympathetic. Melkirch and Agababow have shown that numerous sensory nerves terminate in free sensory endings in the connective tissue of the ciliary body and iris. The sensory nerve-supply of the iris is especially rich. E. THE INTERNAL OR NERVOUS TUNIC OF THE EYE. This tunic is composed of two layers : the outer, or stratum pig- menti ; and the inner, or retina. \. THE PIGMENT LAYER. The pigment layer develops, as we have seen, from the outer layer of the secondary optic vesicle. It consists of regular hexa- gonal cells, 12 fj. to 1 8 ii in length and 9 // in breadth, which con- tain black pigment in the form of granules. The inner surfaces of these cells possess long, thread-like and fringe-like processes, between which project the external segments of the rods and cones of the retina, yet to be described. The nuclei of the pigment cells lie in the outer ends of the cells, the so-called basal plates, and are not pigmented. The distribution of the pigment varies according to the illumination of the retina. If the latter be darkened, the pig- ment collects at the outer portion of each cell ; if illuminated, the pigment is evenly distributed throughout the whole cell. The pig- ment granules are therefore mobile (Kiihne, 79). 2. THE RETINA. The retina has not the same structure throughout. In certain areas peculiarities are noticeable which must be described in detail ; such areas are : (i) the macula lutea ; (2) the region of the papilla (papilla nervi optici) ; (3) the ora serrata ; (4) the pars ciliaris retinae ; and (5) the pars iridica retinae. We shall begin with the consideration of that portion of the retina lying between the ora serrata and the optic papilla (exclusive of the macula lutea). From without inward, we differentiate: (i) the layer of vis- ual cells, including the outer nuclear layer ; (2) the outer molecu- lar (plexiform) layer ; (3) the inner nuclear or granular layer ; (4) the inner molecular (plexiform) layer ; (5) the ganglion-cell layer ; (6) the nerve-fiber layer. Besides these, we must also consider the 458 THE EYE. supporting tissue of the retina and Miiller's fibers, together with the internal and external limiting membranes. The visual cells are either rod-visual cells or cone-visual cells. The rod-visual cells consist of a rod and a rod-fiber with its nucleus. The rod (40 // to 50 // in length) consists of two seg- ments, an outer and an inner, the former of which is doubly refrac- tive and may be separated into numerous transverse discs by the action of certain reagents. The inner is less transparent than the outer segment, and its inner end shows a fine superficial longitu- dinal striation due to impressions from the fiber-baskets formed by Miiller's fibers. In the lower classes of vertebrates a rod-ellipsoid Layer of nerve- ' fibers. Ganglion-cell layer. Inner molecular ., layer. Inner nuclear layer. — i Outer molecular layer. Outer nuclear layer. Ext. limiting mem- brane. Inner segment of rod. Inner segment of cone. Outer segment of cone. Outer segment of rod. Fig. 358. — Section of the human retina ; X 7°°- (a fibrillar structure) may easily be demonstrated in the outer region of each inner portion ; in many mammalia and in man the demon- stration of this is more difficult. This structure is a planoconvex, longitudinally striated body, the plane surface of which is coincident with the external surface of the inner segment, its inner convex sur- face lying at the junction of the outer and middle thirds of the inner segment. The rod-fibers extend as far as the outer molecular layer of the retina, where they end in small spheric swellings. The nuclei of the rod-visual cells are found at varying points within the rod- fibers, but rarely close to the inner segment. When treated with certain fixing agents and stains, the rod-nuclei of certain animals (cat and rabbit) are seen to show several zones, which stain alternately THE INTERNAL OR NERVOUS TUNIC OF THE EYE. 45Q light and dark (striation of the rod-nuclei). This striation is not gen erally observed in the rod-nuclei of the human retina. The cone-visual cells consist, similarly to the rod-visual cells, of a cone and a cone-fiber with its nucleus. The cone (15 fi to 25 fJL in length) is, as a whole, shorter than the rod, and its inner segment is considerably broader than that of the rod. The cone ellipsoid comprises the outer two-thirds of the inner segment, and the outer segment has a more conical shape. The cone-fiber like- wise extends as far as the outer molecular layer, where it ends in a branched basal plate. Its somewhat larger nucleus is always found in the vicinity of the inner segment of the cone. The inner surfaces of the inner segments, not only of the cone-cells, but also of the rod-visual cells, lie in one plane, corresponding to the external limiting membrane, a structure composed of the sustenta- cular fibers of Miiller. The rod-fibers and cone-fibers, with the nuclei of the rod- and cone-visual cells, lie between the external limiting membrane and the outer molecular layer. It will be observed, therefore, that the visual cells include the layer of rods and cones and the outer nuclear layer. The outer molecular layer consists : (i) of the ramifications of Miiller's fibers ; (2) of the knob and tuft-like endings of the visual cells ; and (3) of the dendritic processes of the bipolar cells of the inner nuclear layer. These structures will be considered more in detail in discussing the relations of the elements comprising the retina. The inner nuclear layer contains: (i) the nucleated stratum of Miiller's sustentacular fibers ; (2) ganglion cells situated in the outer region of the layer and extending in a horizontal direction ; (3) bipolar ganglion cells with oval nuclei, densely placed at various depths of the layer and vertical to it ; (4) amacrine cells (neurones, apparently without neuraxes) lying close to the inner margin of the layer and forming with their larger nuclei a nearly continuous layer of so-called spongioblasts. The numerous processes of these spongioblasts lie in the inner molecular layer, the composition of which will be further discussed later. The ganglion-cell layer of the optic nerve consists, aside from centrifugal neuraxes and the fibers of Miiller, which are here present, of multipolar ganglion cells, the dendrites of which extend outward and the neuraxes of which are directed toward the optic nerve-fiber layer. These cells vary in size, and their nuclei are typical, being relatively large, deficient in chromatin, and always provided with large, distinct nucleoli. In man the optic nerve- fibers of the retina are nonmedullated. All these structures are typical of that portion of the retina lying behind the ora serrata. The retina in the vicinity of the optic papilla and macula lutea must be taken up separately. 460 THE EYE. 3. REGION OF THE OPTIC PAPILLA. The optic papilla is the point of entrance of the optic nerve into the retina. At the center of the papilla, in the region where the nerve-fibers spread out radially in order to supply the various areas of the retina, is a small, funnel-shaped depression, the physi- ologic excavation. The fibers of the optic nerve lose their medullary sheaths during their passage through the sclera and choroid, and then continue to the inner surface of the retina, over which they spread in a layer which gradually becomes thinner toward the ora serrata. On account of the deflection of the nerve- fibers, and because, during Physiologic excavation. Layer of nerve-fibers.... Inner nuclear layer. "" Outer molecular lay, T. - Outer nuclear layer..- Rods and cones. ••' Pigment layer. .-•' Sclera.— Lamina cribrosa.--' Fig. 359. — Section through point of entrance of human optic nerve ; X 4°- their passage through the sclera, they lose their medullary sheaths at one and the same point, the optic nerve becomes suddenly thinner. The result is a deeply indented circular depression in this region. On this depression border the three ocular tunics. At this point the retina is interrupted, the outer layers extending to the bot- tom of the depression, while the inner cease at its margin. In many cases the outer layers of the retina are separated from the optic nerve by a thin lamina of supporting tissue (intermediate tissue). 4. REGION OF THE MACULA LUTEA. At the center of the macula lutea is a trough-like depression, the fovea centralis, the deepest part of which, \hzfnndits, lies very close to the visual axis. Here the layers of the retina are practic- ally reduced to the cone-visual cells. The margin of this depression is somewhat thickened, owing to an increase in the thickness of the nerve-fiber and ganglion-cell layers. Toward the fundus of the fovea each of the four inner retinal layers becomes reduced in thickness, the inner layer first and the three others in their order : the inner molecular layer, however, seems to extend as far as the fundus. As we have seen, only the cone-visual cells are found in the fovea cen- tralis, there being an entire absence of the rod-visual cells. Since the nuclei of the cone -visual cells are in the immediate neighborhood THE INTERNAL OR NERVOUS TUNIC OF THE EYE. 461 of the cones, and since the cone-fibers, in order to reach the outer molecular layer, must here describe a curve, there arises a peculiar 1ayer, composed of obliquely directed fibers, known as the outer fiber-layer or Henle's fiber layer. In other words, the fibers of this region are more distinctly seen because they are not covered by the rod-nuclei and rod-fibers. Fovea centralis. Layer of "TSHW9^BO!ftfc^ i nerve-fibers. ! Q- £ Ganglion-cell . ?•'• ~^ layer. Inner molecu- L " lar layer. Inner nuclear . layer. Outer molec-..^ ular layer. -" ><• Outer fibrous g layer. Outer nuclear -4H layer. Cones. ... I Fig. 360. — Section through human macula lutea and fovea centralis ; X 1S°- As a result of treatment with certain reagents, the fovea centralis is deeper and the margin more precipitous than during life. The yellowish color of the fovea centralis is due to pigment held in solution within the layers of' the retina. The cone-visual cells themselves contain no pigment. 5. ORA SERRATA, PARS CILIARIS RETINAE, AND PARS IRIDICA RETINAE. In the region of the ora serrata the retina suddenly becomes thinner. As seen from the inner surface of the retina, its decrease presents the appearance of an irregular curve rather than of the segment of a sphere. Shortly before the retina terminates, its layers become markedly reduced, certain ones disappearing entirely ; first the nerve-fiber layer, then the ganglion-cell layer and cone- and rod- visual cells, their place being taken by an indifferent epithelium. The inner molecular layer of the retina gradually loses the pro- cesses which penetrate inward. In the region of the ora serrata the sustentacular fibers are markedly developed. Relatively large hol- low spaces are often found in the retina at the ora serrata ; they are thought to be due to edema. The pars ciliaris retinae consists essentially of two simple layers of cells, of which the external represents the pigment layer and the internal the inner epithelium of the secondary optic vesicle. In the pars iridica retinae the arrangement is similar ; here both layers are pigmented. 462 THE EYE. 6. MULLER'S FIBERS OF THE RETINA. Genetically, the sustentacular fibers, or fibers of Miiller, in the retina are, like the whole retina, of ectodermic origin, and repre- sent a highly developed form of neurogliar tissue. They penetrate the retina from within and extend as far as the inner segments of the rods and cones. Each fiber represents a long, greatly modified epithelial cell, terminating in one or more broad basal plates, which come in contact with those of adjacent fibers, thus forming a sort of membrane — the internal limiting membrane. Owing to its marked plasticity, each fiber presents certain peculiarities within the various layers of the retina through which it penetrates. Thus, within the molecular layers the fiber is provided with trans- versely directed processes and platelets. Within the nuclear layers, on .the other hand, are numerous lateral indentations, which corre- spond to the impressions produced by the cells of these layers. At the inner surface of the cones and rods the fibers terminate in end- plates, which represent cuticular formations, and, blending with one another, form a single membrane — the external limiting membrane. This membrane is perforated by the rod-fibers and cone-fibers. The end-plates of the fibers give off externally short, inflexible fibrils, which form the fiber-baskets containing the basilar portions of the inner segments of the rods and cones. (Via. Fig. 361.) Miiller's fibers do not appear as fibers in chrome-silver preparations, but as complicated cellular structures, as above depicted. In preparations of the retina, stained in a differential neuroglia stain (Benda's method), clearly defined fibers, stained after the manner of neuroglia fibers, may be differentiated. These fibers are in contact with or are imbedded in the protoplasm of the Miiller's fibers. 7. THE RELATIONS OF THE ELEMENTS OF THE RETINA TO ONE ANOTHER. We shall now take up the relationships existing between the various elements of the retinal strata, giving the theories now generally accepted and based on observations made with the Golgi and methylene-blue methods, and more particularly on the investi- gations of Ramon y Cajal (see diagram, Fig. 361) : 1. The inner processes of the rod-visual cells end, as a rule, in small expansions within the outer molecular layer, in which also the processes of the cone-visual cells terminate in broader branched pedicles. In this layer also are situated the terminal arborizations of the dendrites and neuraxes of certain cells belonging to the inner nuclear layer. 2. The inner nuclear layer consists, as we have seen, (a} of bipolar cells, which constitute the principal portion of this layer, [b] of hori- zontally placed cells lying immediately beneath the outer molecular layer, and (f) of the layer of spongioblasts situated at the junction THE INTERNAL OR NERVOUS TUNIC OF THE EYE. 463 of the inner nuclear with the inner molecular layer. The bipolar cells comprise the following : (a) Bipolar cells of the rod-visual cells the dendrites of which intertwine around the basilar portions of the rod- visual cells, and the neu raxes of which end in telodendria in the neigh- borhood of the cell-bodies of the nerve-cells of the ganglion-cell layer. (/9) Bipolar cells of the cone-visual cells. The dendrites of these cells, sr which also end in the outer molecular layer, are there in relation to the basilar processes of the cone-fibers. Their neuraxes come in contact, by means of terminal arborizations, with the dendrites of the ganglion cells of the ganglion-cell layer at varying depths of the inner molecular layer, (j] Besides these, there are also bipolar cells which, as in the case of a and ft. form contact with the rod- and 464 THE EYE. cone-visual cells, but end on the cell-bodies of the ganglion ceils of the ganglion-cell layer. The horizontal cells send their dendrites into the outer molecular layer, while their neuraxes extend hori- zontally and give off numerous collaterals to the same layer, ending there in telodendria. These cells are of two varieties: the smaller, in- directly connecting the cone-visual cells with one another by means of their dendrites and neuraxes ; and the larger, more deeply situated cells, connecting in a similar manner the basilar ends of the rod- visual cells. A few cells of the second variety give off one or two dendrites each, which penetrate through the inner nuclear layer into the inner molecular layer. 3. The inner molecular layer. This is composed of five strata. The majority of the spongioblasts (amacrine or parareticular cells) in the inner nuclear layer send their processes upward into the inner molecular layer, in which some end in fine arborizations in the first, others in the second, and still others in the third interstice, separat- ing the strata of the inner molecular layer from one another. Be- sides these so-called stratum spongioblasts, there are also others in the inner nuclear layer, the diffuse spongioblasts, whose ramifications end simultaneously in several or in all of the strata of the inner molecu- lar layer. Besides the ramifications of the spongioblasts just men- tioned, autochthonous cells are also present. These lie in one of the interstices of the molecular layers, their ramifications spreading out in a horizontal direction. Besides all these structures the den- drites of the cells in the ganglion-cell layer also ramify throughout the inner molecular layer. 4. The ganglion - cell layer. The cell-bodies are irregularly oval ; their dendrites extend into the inner molecular layer, and their neuraxes into the nerve-fiber layer. According to the manner of their dendritic termination, the ganglion cells may be divided into three groups : (i) those the dendrites of which ex- tend into but one stratum of the molecular layer ; (2) those the dendrites of which extend into several strata of the molecular layer ; and (3) those the dendrites of which are distributed throughout the entire thickness of the molecular layer. Thus, these three groups are made up of the so-called mono-stratified, poly-stratified, and diffuse cells ; by means of their dendrites they come in contact with one or several of the neuraxes of the bipolar cells of the inner nuclear layer. 5. The nerve-fiber layer of the retina. This layer consists of centripetal neuraxes from the ganglion cells of the ganglion-cell layer, and of centrifugal nerve -fibers ending in various layers of the retina, including the outer molecular layer. 8. THE OPTIC NERVE. Within the orbit the optic nerve possesses an external sheath, which is an extension of the dura mater and is continuous with the scleral tissue, and an inner sheath, which is a prolongation of the pia THE INTERNAL OR NERVOUS TUNIC OF THE EYE. 465 mater. Between these two sheaths is a fissure, divided into two smaller clefts by a continuation of the arachnoid. Both these clefts are traversed by connective-tissue trabeculae. The inner cleft com- municates with the subarachnoid space ; and the outer narrower cleft, with the subdural space. The fibers of the optic nerve are medullated, but they possess no neurilemma. They are grouped into small bundles by septa and bands of fibrous tissue penetrating the optic nerve from the inner or pial sheath. Within these bundles the nerves are separated by neu- roglia tissue, — neuroglia cells and fibers, — which further forms a thin sheath about each bundle. In the region of the sclera and cho- roid the optic nerve-fibers lose their myelin, and the septa of the inner or pial sheath become better developed and relatively more numer- ous. Connective-tissue fibers from the sclera and choroid also trav- erse this region of the optic nerve, giving rise to what is known as the lamina cribrosa. At from I y2 to 2 cm. from the eyeball there enter into the optic nerve laterally and ventrally (according to J. Deyl, mesially) the central artery and vein of the retina, which very soon come to lie within the axis of the nerve. Here they are surrounded by a common connective-tissue sheath which is in direct connection with the pial sheath. The optic nerve-fibers extend through the lamina cribrosa into the retina, where they spread out as the nerve- fiber layer in the manner previously described. - Vein. 9. BLOOD-VESSELS OF THE OPTIC NERVE AND RETINA. The blood-vessels of the optic nerve are principally derived from the vessels of the pial sheath. In that portion of the nerve con- taining the central vessels of the retina the latter anastomose with the pial vessels, so that this por- tion of the optic nerve is also supplied by the central vessels. At their entrance through the / sclera the short posterior ciliary arteries form a plexus around the optic nerve, the arterial circle of Zinn, which communicates, on the one hand, with the vessels of the pial sheath, and, on the other, with those of the optic nerve. At the level of the choroid the vessels of the latter communicate by means of capillaries with the central vessels of the optic nerve. The central artery and vein of the retina enter and leave the retina at the optic papilla, 30 -- — Artery. the Zone sur- rounding artery free from capil- laries. • Fig. 362. — Injected blood-vessels of human retina ; surface preparation ; dividing here, or even within 466 THE EYE. the nerve itself, into the superior and inferior papillary artery and vein. Both the latter again divide into two branches, the nasal and temporal arteriole and venule, known, according to their posi- tions, as the superior and inferior nasal and temporal artery and vein. Besides these vessels, two small arteries also arise from the trunk of the central artery itself, and extend to the macula. Two similar vessels extend toward the nasal side as the superior and inferior median branches. Within the retina itself the larger ves- sels spread out in the nerve-fiber layer, forming there a coarsely meshed capillary network connected by numerous branches with a finer and more closely meshed network lying within the inner - - Vascular plexus of macula lutea with wide meshes. Fovea centra- 1 is, free from vessels. Fig. 363. — Injected blood-vessels of human macula lutea ; surface preparation ; X 28. nuclear layer. The venous capillaries of this network return as small venous branches to the nerve-fiber layer, in which they form a venous plexus, side by side with the arterial plexus. The arteries of the retina are of smaller caliber than the veins. The larger arteries possess a muscular layer ; the smaller, only an adventitia. All the vessels possess highly developed perivascular sheaths. The visual-cell 'layer is nonvascular, as are also the fovea centralis and the rudimentary retinal layers lying anterior to the ora serrata. The arteries of the retina anastomose with one another solely by means of capillaries (end-arteries), and it is only in the ora serrata that coarser venous anastomoses exist. THE CRYSTALLINE LENS. 467 F. THE VITREOUS BODY. The vitreous body is a tissue which consists almost entirely of fluid, containing very few fixed cellular elements and only a small number of leucocytes, which are found more particularly in its outer- most portion. Thin structureless lamellae and fibers occur through- out the entire vitreous body, with the exception of the hyaloid canal. These fibrils form an interlacing network with wide meshes. They differ chemically from both the white fibrous tissue and yellow elastic fibers, resembling in some respects cuticular formations (von Ebner). These are particularly numerous at the periphery and especially in the region of the ciliary body. Toward the surface the fibrils are more densely arranged, forming the hyaloid membrane of the vit- reous body, separating the latter from the retina. This membrane is somewhat thicker in the region of its close attachment around the physiologic excavation of the optic nerve and to the internal limiting membrane of the retina in the ciliary region. In the latter region the hyaloid membrane is in close relation with the epithelium of the pars ciliaris retinas. It does not, however, penetrate into and between the ciliary processes, but extends like a bridge over the furrows be- tween them. This arrangement gives rise to spaces, the recessus cam- era posterioris, which form a division of the posterior chamber, and are inclosed between the hyaloid membrane, the ciliary processes, the suspensory ligament of the lens, and the lens itself; these spaces are filled with aqueous humor. In the region of the ciliary pro- cesses the hyaloid membrane is closely associated with numerous fibers, which diverge fan-like toward the lens and become blended with the outer lamella of the lens-capsule. These fibers appear to arise from the epithelium of the pars ciliaris retinas, and may be regarded as cuticular formations. Those coming from the free ends of the ciliary processes become attached along the equator of the lens and to the adjacent posterior portion of the lens-capsule. On the other hand, the fibers originating between the ciliary processes attach themselves to the anterior surface of the lens-capsule in the imme- diate vicinity of the equator. Together these fibers constitute the zonula ciliaris, zonulc of Zinn, or the suspensory ligament of the lens. Between these fibers of the zonula and the lens itself there is, conse- quently, a circular canal divided by septa, the canal of Petit, which communicates by openings with the anterior chamber. G. THE CRYSTALLINE LENS. As we have already, seen, the crystalline lens originates as an ectodermic invagination, which then frees itself from the remaining ectoderm in the shape of a vesicle and becomes transformed into the finished lens. In this process the cells of the inner wall of the vesicle become the lens-fibers, while those of the outer portion re- 468 THE EYE. main as the anterior epithelium of the lens. The lens is surrounded on all sides by the lens-capsule. The lens capsule is a homogeneous membrane, nearly twice as thick on the anterior surface of the lens as on the posterior. Its chemic reactions differ from those of connective tissue, and in this respect it may be compared with the membranae propriae of glands. In sections the lens capsule appears to possess a tangen- tial striation ; under the influence of certain reagents, and under proper preliminary treatment, lamellae may be detached from its surface which are found to be directly connected with the fibers of the suspensory ligament. The anterior epithelium consists, in the fetus, of columnar cells , in children, of cells approaching the cubic type ; and in the adult, of decidedly flattened cells. Toward the equator of the lens, in the so-called transitional zone, the cells increase in height and gradually pass over into the lens fibers. The lens fibers are also derivatives of epithelial cells ; they are long, flattened, hexagonal prisms, which extend through the entire thickness of the lens. In the adult the lens may be differentiated into a resistant peripheral and a softer axial substance. The sur- faces of the fibers present irregularities, and it is with the help of these serrations and a cement substance that the fibers are bound together. Each fiber possesses one or more nuclei, which, although they have no constant position, are usually found in the middle of the fibers situated near the lens-axis, and in the anterior third of those at some distance from the axis. The course of the fibers in the lens is extremely complicated. R THE FETAL BLOOD-VESSELS OF THE EYE. In the eye of the embryo the vitreous body and the capsule of the lens contain blood-vessels. The vessel which later becomes the central artery of the retina passes through the space sub- sequently occupied by the vitreous body as far as the posterior sur- face of the lens (anterior hyaloid artery) and branches in the region of the posterior and anterior lens-capsule. The anterior vascular membrane of the lens capsule of the embryo is known as the membrana capsulopupillaris, and that portion corresponding to the pupil, as the membrana pupillaris. In the embryo numerous other vessels arise at the papilla and extend over the surface of the vitreous body close to the hyaloid membrane ; these are the pos- terior Jiyaloid arteries. These vessels later disappear. In place of the anterior hyaloid artery there remains in the vitreous humor a transparent cylindric cord containing no fibers nor lamellae, as is the case in the remaining portion of the vitreous body, and consisting of a more fluid substance ; this is the hyaloid canal, or the canal of Cloquet. THE PROTECTIVE ORGANS OF THE EYE. 4.69 With regard to the posterior hyaloid vessels, the generally ac- cepted theory is that they later enter into the formation of the retinal vessels. Little is known as to the details of this process ; but the fact remains that, in the rabbit, for instance, the larger branches of the retinal vessels are internal to the inner limiting membrane, and, therefore, within the vitreous body, and that they send smaller branches into the retina (His, 80). I. INTERCHANGE OF FLUIDS IN THE EYEBALL. The anterior lymph-channels of the eye comprise (i) the lymph-canaliculi of the cornea, which communicate with similar structures in the sclera ; (2) the system of the anterior chamber, which is indirectly connected, on the one hand, with the canal of Schlemm by means of the spaces of Fontana, and with the stroma iridis, into which the ligamentum pectinatum extends ; while, on the other hand, it communicates with the posterior chamber and its recesses, and with the canal of Petit. In the posterior region of the eyeball are the lymph-channels of the retina (the perivascular spaces), those of the optic nerve, the space between the pigment layer and the remaining portion of the retina (interlaminar space, Rauber), and the lymph-spaces of the choroid and sclera. The influx and efflux of intraocular fluid occur principally by means of filtration. The influx takes place through the ciliary processes ; that the choroid has to do with this process is very improbable. The efflux takes place through the veins of the canal of Schlemm, into which the fluid filters through the cement lines of the endothelial lining of the canal of Schlemm, finally emptying into the anterior ciliary veins. A posterior efflux from the vitreous body probably does not exist, or at least occurs to a very limited extent. The anterior chamber possesses no efferent lymph-vessels (Leber, 95). J. THE PROTECTIVE ORGANS OF THE EYE. J. THE LIDS AND THE CONJUNCTIVA. At the end of the second month of embryonic life the eyelids begin to develop in the shape of two folds of skin. At the end of the third month these folds come in contact in the region of what is later the palpebral fissure, and grow together at their outer epithelial margins. Shortly before birth the two lids again separate and the definitive palpebral fissure is formed. The eyelids show three distinct layers : (i) the external cutis, which presents special structures at its free margin and continues about I mm. inward from the inner border of the free margin ; (2) the mucous membrane, or palpebral conjunctiva, beginning from 47° THE EYE. this line and covering the entire internal surface j and (3) a middle layer. 1 . The cuticular portion of the eyelid consists of a thin epider- mis and a dermis poorly supplied with papillae. Fine lanugo-like hairs with small sebaceous glands and a few sweat-glands are distributed over its entire surface. The cutaneous connective tissue is very loose, contains very few elastic fibers, and is supplied with pigment cells in the superficial layers. At the lid-margin the papillae are well developed and the epidermis is somewhat thickened. The anterior margin supports several rows of larger hairs, the cilia, the posterior row of which possesses, besides the sebaceous glands, modified sweat-glands, the ciliary glands of Moll, which also empty into or near the hair follicles. The ciliary glands are readily distin- guished from the sweat glands ; their tubules are relatively large, often showing alternating large vesicular segments and short narrow segments. A branching of the tubules has also been observed (Huber). The eyelids are further provided with numerous glands, known as the Meibomian or tarsal glands. About thirty of these glands are found in the upper, a slightly smaller number in the lower, lids. They lie within the tissue of the tarsus vertical to the palpebral margin. Each gland consists of a tubular duct, lined by stratified squamous epithelium, beset with numerous simple or branched al- veoli lined by a stratified, cubic epithelium in every respect similar to that lining the alveoli of sebaceous glands. The ducts of these glands terminate at the palpebral margin posterior to the cilia. (See Fig. 364.) 2. The conjunctival portion of the eyelids is lined by a simple pseudostratified columnar epithelium, possessing two strata of nuclei. This is continuous with the bulbar conjunctiva at the conjunctival fornix, and is characterized by the occasional presence of folds and sulci. Longitudinal folds in the upper portion of the upper lid running parallel with the lid-margin are frequently present. Goblet cells are usually found in the epithelium. According to W. Pfitz- ner (97), the epithelium of the conjunctiva consists of two or three strata of cells, of which the more superficial possess a cuticular margin. Certain structures which have always been regarded as goblet cells are in all probability similar to the cells of Ley dig — i. e., mucous cells, which do not pour their secretion out over the sur- face of the epithelium. Some lymphoid tissue is always found in the stratum proprium of the mucous membrane, and occasionally it is seen to form true lymph-nodules. It is of some interest to note that a marked production of these lymph-nodules occurs in certain diseases. • Such lymph-nodules are usually associated with epithe- lial crypts, which fact led Henle to regard them as glandular forma- tions. Small glands with a structure similar to that of the lacrimal glands are also present in the palpebral conjunctiva ; they are known as accessory lacrimal glands and are found in the upper eyelid, at the outer angle of the conjunctival fornix. Similar glands occur also at the mesial angle of the fornix. THE PROTECTIVE ORGANS OF THE EYE. 471 3. Besides the tarsus (fibrocartilage) the middle layer of the eye- lid contains : (i) The musculus orbicularis oculi, which lies beneath Fig. 364. — Vertical section of the upper eyelid of man; X *4: af> arterial arcus tar- seus ; c, cilia ; dgt, excretory duct of Meihomian gland ; glc, ciliary gland (Moll) ; McR, ciliary muscle of Riolani ; Mop, m. orbicularis palpebrarum ; Mt, nonstriated muscle-fibers of the tarsal muscle and tendon of the levator palpebrae superioris ; nlc, lymph-node of the conjunctiva palpebrse ; T, tarsus (Sobotta, "Atlas and Epitome of Histology."). the subcutaneous tissue. At the margin of the lid this structure gives off the musculus ciliaris Riolani, which is composed of two 472 THE EYE. fasciculi separated by the tarsus. (2) The connective tissue be- tween the bundles of the musculus orbicularis oculi. (3) The con- nective tissue lying behind the latter and the tarsus. In the upper lid the connective tissue mentioned under 2 and 3 is connected with the tendon of the musculus palpebralis superior. The latter is composed of smooth muscle-fibers, and is regarded as a continua- tion of the middle portion of the striated, voluntary musculus leva- tor palpebrae superioris. The middle layer of the lower lid isstruc- Fig. 365. — Meibomian or tarsal gland, reconstructed after Bern's wax-plate method; X20. turally analogous, except that here a fibrous expansion from the sheath of the inferior rectus muscle takes the place of the levator palpebrae. THE PROTECTIVE ORGANS OF THE EYE. 4/3 The blood-vessels of the eyelid lie directly in front of the tarsus, and from this region supply adjacent parts ; they reach the poste- rior portion of the lid either by penetrating the tarsus or by encir- cling it (Waldeyer, 74). The lymph-vessels form a plexus in front and one behind the tarsus. The " third eyelid," the plica semilunaris, contains, when well developed, a small plate of hyaline cartilage. At the fornix the epithelium of the palpebral conjunctiva be- comes continuous with the two- or three-layered squamous epithe- lium of the conjunctiva bulbi. Beneath this epithelium is found a loose fibre-elastic connective tissue, presenting subepithelial papillae, and quite vascular. In it are found medullated nerve-fibers, some of which terminate in free sensory nerve-endings in theconjunctival epithelium ; others terminate, especially near the corneal margin, in end-bulbs of Krause ; and still others may be traced to the cornea, to terminate in a manner previously described. 2. THE LACRIMAL APPARATUS. The lacrimal apparatus consists of the lacrimal glands, their ex- cretory ducts, the lacrimal puncta and canaliculi, the lacrimal sac, and the nasal duct. The lacrimal gland, wnich is a branched tubular gland, is sepa- rated into two portions, of which the one lies laterally against the orbit and the other close to the upper lateral portion of the superior conjunctival fornix. The structure of the gland is, on the whole, that of a serous gland (parotid), with the difference that the intralob- ular ducts are not lined by a striated epithelium such as is found in the salivary tubules, and that those cells which are wedged in between the secretory elements and functionate as sustentacular cells (basket- cells) are here much more highly developed. The excretory ducts of the orbital division generally pass by the conjunctival half of the gland, taking up a few ducts from the latter as they go, and finally empty on the surface of the conjunctiva. Aside from these, the lateral portion of the gland possesses also independent ducts. All the excretory ducts are lined by columnar epithelium and surrounded by a relatively thick connective-tissue wall having inner longitudinal and outer circular fibers. From the lateral portion of the conjunctival culdesac, into which the secre- tion is brought by the excretory ducts of the lacrimal gland, the secretion passes into the capillary space of the sac, and is then evenly distributed by means of the sulci and papillae over the con- junctival surface of the lid. In this manner the secretion reaches the mesial angle of the lid, whence it passes through the lacrimal puncta into the lacrimal canals. The nerve supply of the lacrimal glands is from the sym- pathetic nervous system. The neuraxes of sympathetic neurones accompany the gland ducts and form plexuses about the alveoli, the terminal branches of which may be traced to the gland cells. 474 THE EYE- The lacrimal canals are lined by stratified squamous epi- thelium, and possess a basement membrane as well as a con- nective-tissue layer containing circularly disposed elastic elements. Externally we find a layer of transversely striated muscle -fibers. The lacrimal sac is provided with a simple pseudostratified columnar epithelium having two strata of nuclei. In it goblet cells are also found. The nasal duct is lined by a similar epithelium. The connective-tissue wall of the latter and that of the lacrimal sac come in contact with the periosteum ; between them is a well- developed vascular plexus. Stratified squamous and ciliated epi- thelium have been described as being present in the nasal duct, as well as mucous glands in both nasal duct and lacrimal sac. (See works of M. Schultze, 72 ; Schwalbe, 87.) TECHNIC The eyes of the larger animals, after having been previously cleaned by removing the muscles and loose connective tissue, are placed in the fixing fluid and cut into two equal parts by means of an equa- torial incision. Smaller eyes with thin walls may be fixed whole. Miiller's fluid, nitric acid, and Flemming's fluid are usually employed as fixing agents. After fixing in one of these fluids, different parts of the eyeball are imbedded in celloidin or celloidin -paraffin and then sectioned. The corneal epithelium is best macerated in 33% alcohol ; the membrane of Descemet may be impregnated with silver. In order to bring the fibers of the latter into view, Nuel recommends an injection of i °jc to 2 °f0 formic acid into the anterior chamber of the eye of a dove or a rabbit, after having drawn off the aqueous humor. The cornea is then cut out, and fixed for from three to five minutes in osmic acid. The substantia propria is examined either by means of sections or by means of teased preparations from a cornea macerated in lime- water or potassium permanganate. The sections are stained with picro- carmin (Ranvier). The corneal spaces and canaliculi may be demon- strated in two ways with the aid of silver nitrate ; either the fresh cornea of a small animal is stripped of its epithelium, cauterized with a solid stick of silver nitrate, and then examined in water, in which case the corneal spaces and their canaliculi show light upon a dark ground (neg- ative impregnation) ; or the corneae of larger animals are treated in the same manner, after which tangential sections are made with a razor, and placed in water for a few days ; in this case the corneal spaces and their canaliculi show dark upon a light ground (positive impregnation, Ran- vier, 89). By means of Altmann's oil method casts of the corneal spaces and their canaliculi may be made. Treatment by the gold method often brings out not only the nerves, but also the corneal corpuscles and their processes. Ranvier (89) especially recommends a i% solution of the double chlorid of gold and potassium for the corneal nerves. The cor- nea of the frog is treated for five minutes with lemon-juice, then for a quarter of an hour with i % potassium-gold chlorid solution, and, finally, for one or two days with water weakly acidulated with acetic acid (2 TECHNIC. 475 drops to 30 c.c. of water), the whole process taking place in the light. Golgi's method may also be used, but the gold method is more certain. The sclera is treated in a similar manner. The pigmentation of the vascular layer interferes with examina- tion, and albinotic animals should therefore be selected ; or the pigment may be removed from the previously fixed eyeball with hydrogen peroxid or nascent chlorin. The latter method is applied exactly as in cases where the removal of osmic acid is desired. The adult lens is sectioned with difficulty, as it becomes very hard in all fixing fluids. The anterior capsule of the lens may be removed from previously fixed specimens and examined by itself. The lens-fibers are demonstrated by maceration in y^ alcohol (twenty-four hours) or in strong nitric acid. Before immersion the lens-capsule is opened by a puncture. The retina can rarely be kept unwrinkled in eyes that have been fixed whole. The eyeball should therefore be opened in the fixing fluid and the latter permitted to act internally ; or the external tunics are removed, thereby enabling the fixing fluid to act externally. Ranvier recommends subjecting the eyes of smaller animals (mouse, triton) for a quarter or half hour to the action of osmic acid fumes (see p. 24), after which the eyes are opened in yi alcohol with the scissors. At the end of three or four hours the posterior half of the eye is stained for some time in picrocarmin (p. 44), then carried over into \CJC osmic acid for twelve hours, washed with water, treated with alcohol, and cut. In osmic acid preparations the rod-nuclei show dark transverse bands, a condition due to the fact that the end-regions of the nuclei stain more deeply. The retina is a good object for differential staining, as, for instance, with hematoxylin-eosin, hematoxylin -orange G, etc. The latter combina- tion is particularly successful in staining the rod- and cone-ellipsoids. The examination of tangential sections should not be omitted. With the retina the best results are obtained by means of Golgi's method. Attention must be called to the fact that the supporting struc- tures of the retina are more easily impregnated than the nervous elements, and that the latter can be demonstrated to any extent only in very young eyes. Ramon y Cajal (94) recommends the following method, modi- fied after Golgi : After the removal of the vitreous humor the posterior half of the eyeball is placed for one or two days in a mixture containing 2,% potassium bichromate 20 c.c. and i% osmic acid 5 or 6 c.c. The pieces are then dried with tissue paper and placed in a 0.75% silver nitrate solution for an equal length of time. Without washing, the pieces are immersed for from twenty-four to thirty -six hours in a mixture con- taining 3% potassium bichromate 20 c.c., and i% osmic acid 2 or 3 c.c., and then again carried over into a 0.75% silver nitrate solution for twenty-four hours. In order to prevent precipitation it is advisable to roll up the retina before treating, and to cover it with a thin layer of a thin celloidin solution, which prevents it from again unrolling. The methylene-blue method (p. 184) will also bring out the nervous elements of the retina, although the results are not quite so satis- factory as those obtained by Golgi's method. 47°" THE ORGAN OF HEARING. IX. THE ORGAN OF HEARING. THE ear, the organ of hearing, consists of three parts : (i) The external ear, including the pinna or auricle and the external audi- tory canal ; (2) the middle ear, tympanum, or tympanic cavity, containing the small ear bones and separated from the external auditory canal by the tympanic membrane, but communicating with the pharynx by means of the Eustachian tube ; (3) the inner ear, or labyrinth, consisting of a bony and a membranous portion, the latter lined by epithelial cells, especially differentiated in certain regions to form a neuro-epithelium, in which the auditory nerves terminate. The first two parts serve for the collection and trans- mission of the sound-waves ; the complicated labyrinth, with its differentiated neuro-epithelium, for the perception of the same. Figure 366 presents in a schematic way the relationships of the parts here mentioned. A. THE EXTERNAL EAR. The cartilage of the ear, including that of the external auditory passage, is of the elastic variety, but differs from typical elastic carti- lage in that it contains areas entirely free from elastic fibers. The elastic reticulum is, however, never absent near the perichondrium. The skin covering the pinna is thin, and in it are found hairs with relatively large sebaceous glands ; sweat-glands are found on the outer surface. The skin lining the cartilaginous portion of the external auditory canal is somewhat mobile and possesses very few. pronounced papillae, and is characterized by the presence of so-called ceruminous glands, which represent modified and very highly differentiated sweat-glands. They are branched, tubulo-alveolar glands (Huber). They empty either into the hair follicles near the surface of the skin or on to the surface of the skin in the neighborhood of the hair fol- licles. The skin lining the osseous portion of the external auditory canal is supplied with neither hair nor glands, and possesses slender papillae, especially in the neighborhood of the tympanic membrane. The corium is closely attached to the periosteum. The tympanic membrane consists of a tense and a flaccid portion. It forms a part of both the external and the middle ear. From without inward, the following layers may be differentiated : (i) the cutaneous layer ; (2) the lamina propria ; and (3) the mucous layer. The epidermis of the cutaneous layer is identical in structure with that of the outer skin, except that the superficial layers of the stratum corneum contain nucleated cells. The corium is very thin, except along the course of the manubrium of the malleus, where it THE EXTERNAL EAR. 477 is thickened, forming the so-called cuticular ridge, which possesses papillae and is supplied with vessels and nerves. The lamina propria ends peripherally in a thickened ring of fibro- elastic tissue, the annulus fibrosus, which unites at the sulcus tym- panicus with the periosteum of the latter. The lamina propria is composed of connective-tissue fibers, in which two layers may be distinguished — externally, the radiate fibers, the stratum radiatum, and internally, the circular fibers, the stratum circulars. The exter- nal radiate layer extends from the annulus to the umbo and manu- t>rium, and is interrupted in the flaccid portion of the tympanic Pinna. BH W W Fig. 366. — Schematic representation of the complete auditory apparatus (Schwalbe). membrane by the upper fourth of the manubrium and the short process of the malleus ; it gradually thins out toward the center until it finally disappears in the vicinity of the umbo. The fibers of the inner (circular) layer are circularly disposed. This layer is thickest at the periphery of the tympanic membrane, becoming gradually thinner toward the lower end of the manubrium, where it disappears. Between the two layers of the lamina propria is a small quantity of loose connective tissue. The manubrium of the malleus is inclosed within the tympanic membrane. This is due to the union of the fibers of the radial layer with the outer strata of the manubrial perichondrium, the handle of the malleus being here covered by a thin layer of cartilage. In the posterior upper quad- rant of the tympanic membrane the two layers of the lamina propria THE ORGAN OF HEARING. intermingle, forming irregularly disposed bundles and trabeculae, the dendritic fibrous structures of Gruber. The mucous layer of the tympanic membrane consists of sim- ple squamous epithelium separated from the lamina propria by a thin connective-tissue layer containing but few cells. It likewise extends over the handle of the malleus. In the flaccid portion of the tympanic membrane the lamina propria disappears, so that in this region the cutaneous layer and the mucous membrane are in direct contact. B. THE MIDDLE EAR. The middle ear, or tympanum, is a small irregular cavity, filled with air, situated in the petrous portion of the temporal bone be- tween the bony wall of the inner ear and the tympanic membrane, and communicates with the pharynx through the Eustachian tube. It contains the small bones of the ear, their ligamentous attach- ments, and, in part, the muscular apparatus moving them. The mucous membrane lining the tympanic cavity is folded over the ossicles and ligaments of the tympanum and is joined to that of the tympanic membrane and the Eustachian tube, the line of junction with the former being marked by the presence of papilla-like eleva- tions. The epithelium of this mucous membrane is a simple pseudo- stratified ciliated epithelium, having two strata of nuclei. Cilia are, however, lacking on the surface of the auditory ossicles, on their ligaments, and on the promontory of the inner wall, as well as on the tympanic membrane. The mucosa of the mucous membrane is intimately connected with the periosteum, and may now and then contain short isolated alveolar glands, especially in the neighbor- hood of the opening of the Eustachian tube. The "auditory ossicles" are true bones with Haversian canals and lamellae ; with the exception of the stapes, they contain no marrow-cavity. Very distinct perivascular spaces are seen sur- rounding the vessels in the canals (Rauber). The malleus articu- lates with the incus, both articular surfaces being covered with hyaline cartilage. Within this articulation we find a fibrocartilagin- ous meniscus, and at the summit of the short limb of the incus another small cartilage plate. Between the lenticular process of the incus and the capitulum of the stapes is another articulation, also provided with cartilaginous articular surfaces. The basal plate of the stapes is covered both below and at its edges with cartilage, as are also the margins of the fenestra ovalis (fenestra vestibuli). The basal plate is held in place within the fenestra by an articulation, provided with tense ligamentous structures on the tympanic and vestibular sides. Between these the connective tissue is quite loose. All the cartilaginous portions of the auditory ossicles, with the ex- THE MIDDLE EAR. 479 ception of the articular cartilages, rest on the periosteum (Riidin- ger, 70). Thcfenestra rotunda (fenestra cochleae) is closed by the secon- dary or inner tympanic membrane, a connective-tissue membrane containing vessels and nerves, the outer wall of which is covered by ciliated epithelium, the inner (the surface toward the scala tympani) by flattened endothelial cells. In the antrum and mastoid cells, the mucosa of the mucous membrane is immovably fixed to the periosteum. The epithelium is of the simple squamous variety and is nonciliated. Portion of Eusta- chian tube free from glands. Cartilage. ^ Mucosa of the pharynx. — Glands. Glands. -.— r^J — Fig. 367. — Cross-section of the Eustachian tube with its surrounding parts ; X I2 (from a preparation by Professor Riidinger). The mucous membrane of the osseous portion of the Eustachian tube is very thin, and its mucosa is intimately connected with the periosteum. Its epithelium is of the simple pseudostratified ciliated variety, having two strata of nuclei. There are rio glands. The mucous membrane of the cartilaginous portion of the Eustachian tube is thicker, and its epithelium, which is of the stratified ciliated variety, is higher, and often contains goblet-cells. Lymphoid tissue may be demonstrated in the mucosa of this portion, and occasion- ally structures resembling lymph-nodules are found, especially in the vicinity of the pharyngeal opening of the tube. In the cartilaginous portion of the. tube are mucous glands, which are particularly THE ORGAN OF HEARING. numerous in the vicinity of the pharyngeal opening (Riidinger, 72, 2). The cartilage of the Eustachian tube is in part yellow elastic^ in part hyaline, and in certain portions presents the appearance of white fibre-cartilage. C THE INTERNAL EAR, The internal ear consists of an osseous and a membranous por- tion, the osseous and the membranous labyrinths ; the latter is con- tained within the former, and, although smaller, presents the same Superior semicircular canal. Horizontal semi- circular canal. Posterior semi- circular canal. Ampulla. ^^ ™^^ ^~ ™ " •••••• Bony cochlea. Vestibule. Fenestra rotunda. Fig. 368. — Right bony labyrinth, viewed from outer side : The figure represents the appearance produced by removing the petrous portion of the temporal bone down to the denser layer immediately surrounding the labyrinth (from Quain, after Sommering). general shape. The two structures are separated by a lymph-space containing the perilymph. In the bony labyrinth we recognize a central portion of ovoid shape, known as the vestibule, the outer wall of which forms the inner wall of the tympanum and presents two openings, the fenestra ovalis and the fenestra rotunda, separated by a ridge known as the promontory. This ridge becomes continuous with the lower portion of the bony cochlea, anterior and mesial to the vestibule and having the shape of a blunt cone. From the posterior portion of the ves- tibule arise three semicircular canals, known respectively as the external or horizontal semicircular canal, the anterior superior vertical, and the posterior inferior vertical semicircular canals. The canals communicate with the vestibule by means of five openings, the superior contiguous portions of the anterior and posterior canals uniting to form the canalis communis before reaching the vestibule. The three canals present near their origin from the vestibule enlarge- ments known as the osseous ampullae. The osseous labyrinth is lined throughout by a thin -layer of periosteum, covered by a layer of endothelial cells. THE INTERNAL EAR. 48l The membranous labyrinth differs in shape from the osseous labyrinth in that, in place of the single chamber (vestibule) of the latter, the membranous labyrinth presents two sacs, the utriculus and the sacculus, united by a narrow duct, the utriculosaccular duct. The utriculus is the larger, and from it arise the membran- ous semicircular canals. These present ampullae, situated within the osseous ampullae previously mentioned. The sacculus com- municates with the cochlear duct by means of the canalis reuniens (Hensen). From the utriculosaccular duct arises the ductus endolymphaticus, which passes through the aqueductus vestibuli and ends in a subdural sacciis endolymphaticus on the posterior sur- face of the petrous portion of the temporal bone. In the membranous labyrinth the nerves are distributed over certain areas known as the maculce, cristce, and papilla spiralis. ,' "i Auditory nerve with its vestihu- lar and cochlear branches. Ant. semicircular canal. Ampulla. Cochlear duct. Canalis reuniens. Ductus Ampulla. Horizontal semicir- endolymphaticus. cular canal. Fig. 369. — Membranous labyrinth of the right ear from five-month human embryo (from Schwalbe, after Retzius). There is a macula within the recess of the utriculus, the macula acustica utriculi ; and another within the sacculus, the macula acustica sacculi ; cristae are present in the ampullae of the upper, posterior, and lateral semicircular canals, the cristce ampullares sup., post., et lat. Besides these, we have the terminal arborization of the acoustic nerve in the membranous cochlea, the papilla spiralis cochlece, or the organ of Corti. 3» 482 THE ORGAN OF HEARING. J. UTRICULUS AND SACCULUS. Only the inner wall of the utriculus is connected with the peri- osteum of the vestibule. In this region lies the corresponding Membranous semicircular canal. Blood-vessel . — - — Wall of mem- branous canal. „ Epithelium of the membranous canal. Ligament of canal. Bone. Perilymphatic spaces. I Blood-vessel. Fig. 370. — Transverse section through an osseous and membranous semicircular canal of an adult human being; y<^5° (after a preparation by Dr. Scheibe): a, Connective- tissue strand representing a remnant of the embryonic gelatinous connective tissue. Such strands serve to connect the membranous canal with the osseous wall. macula cribrosa, through which the nerves penetrate to the macula of the utriculus. The utriculus and sacculus fill only a part of the inner cavity of the osseous vestibule. Between the osseous and membranous portions remains a space traversed by anastomosing connective -tissue trabeculae, and lined by endothelium, which also forms an investing membrane around the trabeculae. These trabe- culae pass on the one side into the periosteum lining the vestibule, and on the other, into the wall of the utriculus and sacculus. The cavity which they thus traverse represents a perilymphatic space. (Compare Fig. 370, which shows analogous relations in the semi- circular canals.) The wall of the utriculus, especially its inner portion, consists of dense fibrous connective tissue, most highly developed in the region of the macula acustica. In the immediate vicinity of the THE INTERNAL EAR. 483 macula utriculi the epithelium of the utriculus is high columnar in type ; in the remaining portion it consists of a single layer of low columnar cells, with a distinct basement membrane ; the epithelium of the macula itself is also high, and is composed of two kinds of elements — of sustentacular elements and of the so-called auditory hair-cells. The sustentacular cells are tall epithelial cells resting on the basement membrane by means of their single or cleft basal plates. Each possesses an oval nucleus lying at or beneath the center of the cell. The hair-cells are peculiar cylindric elements with somewhat thickened and rounded bases. One end extends to the surface of the epithelium, while the other, which contains the nucleus, extends only to the center of the epithelial layer. The free end is provided with a cuticular zone supporting a number of long, stiff hairs, which often coalesce to form single threads. On the surface of the epithelium, which must be regarded as a neuro-epithelium, are crystals of calcium carbonate, known as oto-* lit/is, each of which incloses a minute central vacuole (Schwalbe). The otoliths are inclosed in a homogeneous substance, the otolithic membrane, which coagulates in a network of filaments when sub- jected to the action of fixing agents. The nerve-fibers going to the macula penetrate the wall, and, under the epithelium, undergo dichotomous division, and, after fur- ther division, form, in the region of the basilar ends of the auditory cells, a plexus consisting of fine ramifications, and embracing the lower ends of the auditory cells. A few fibers extend still further upward, where their telodendria enter into intimate relations with the acoustic cells (v. Lenhossek, 94, i). The structure of the sacculus is in every respect like that of the utriculus, and a further description of it is therefore unnecessary. 2. THE SEMICIRCULAR CANALS. The membranous semicircular canals are attached at their con- vex surfaces to the periosteum of the bony canals, which they only partly fill, the remaining cavity being occupied by an eccentrically situated perilymphatic space traversed by connective-tissue trabeculae. The walls of the perilymphatic spaces of the semicircular canals, like those surrounding the utriculus and the sacculus, are lined by endothelium, which covers, on the one hand, the periosteal surface of the bony semicircular canals, and, on the other hand, the outer wall of the membranous canals, together with the connective-tissue trabeculae. The connective-tissue walls of the membranous canals are structurally similar to those of the utriculus and sacculus. Hensen compares their structure to that of the substantia propria of the cornea. In the adult, the inner layer of the wall of the canals supports here and there papillary elevations, which, however, 484 THE ORGAN OF HEARING. disappear along its attachment to the bony semicircular canal (Riidinger, 72, 88). The epithelium lining the membranous semicircular canals is simple squamous in character and very evenly distributed over the entire inner surface, including the papillae previously mentioned. On the concave side of each semicircu- lar canal the epithelial cells are some- what narrower and higher. This inner and higher epithelium (raphe), extending along the concave side into the ampullae, marks the region at which the semicir- cular canals were constricted off from the pocket-like anlagen. The epithe- lium of the ampullae (Fig. 371), with the exception of that in the region of the raphe, is of the squamous type. At the cristae of the ampullae, however, there is found a neuro-epithelium similar to that of the maculae. The cells adjoining both ends of the cristae are high columnar, and to these the squamous epithelium is joined. The columnar cells just men- tioned form the so-called scmilunar fold. Otoliths are also present upon the neu- ro-epithelium of the cristae. Here the structure corresponding to the otolithic membrane of the utriculus and sacculus is called the cupula. In preserved spec- imens it presents the appearance of a coagulum, showing a faint striation ; in the fresh condition, it has never been recognized as a distinct struc- ture, at least in the lower classes of vertebrates. ' — d Fig- 37L — Part of a verti- cal section through the anterior ampulla, showing the membran- ous wall, a portion of the "crista acustica," and the "planum semilunatum" (after Retzius) : a, Semilunar fold ; 6, crista acus- tica ; t, nerve-fibers ; d, blood- vessels. 3. THE COCHLEA. The cochlea consists of an osseous portion, the bony cochlea, a membranous portion, the cochlear duct, and two perilymphatic canals. The bony cochlea consists of a central bony axis of conical shape, the modiolus, around which is wound a spiral bony canal, having in man a little over two and one-half turns, the modiolus forming the inner wall of this canal. The summit of the cochlea, which has the shape of a blunt cone, is formed by the blind end of this bony canal, and is known as the cupola. The modiolus further gives support to a spiral plate of bone, the lamina spiralis ossea, which extends from the lower part of the modiolus, and, forming two and one-half spiral turns, reaches its top, where it ends in a hook-like process, the hamulus. This bony spiral lamina partly THE INTERNAL EAR. 485 divides the bony cochlear canal into two parts, the division being completed by a fibrous tissue membrane, the lamina spiralis mem- branacca, which extends from the free edge of the osseous spiral lamina to a thickened periosteal ridge, the ligamentum spirale, lining the outer wall of the bony cochlear canal. The canal above the lamina spiralis (bony and membranous) is known as the scala vestibuli, that below as the scala tympani. Both are perilymphatic canals, and communicate in the region of the last half-turn of the cochlea, by means of a narrow canal, the helicotrema, partly sur- rounded by the termination of the bony spiral lamina, the hamulus. The scala vestibuli is in free communication with the perilymphatic space of the vestibule ; while the scala tympani communicates with perivascular spaces surrounding the veins of the cochlear aqueduct, which latter empty into the jugular veins. The scala tympani ter- minates at the secondary tympanic membrane, closing the fenestra rotunda. The cochlear duct, which, as will be remembered, communicates with the sacculus by means of the canalis reuniens, is a long tube closed at both ends,' the one end representing the vestibular sac, or ccecum vestibulare, and the other the cupolar extremity, or cacum cupolare, also known as the lagena. The cochlear duct forms about two and three-fourths spiral turns, its length being about 3.5 mm. Its diameter gradually increases from its lower to its upper or distal extremity. The cochlear duct lies above the lamina spiralis, and, in a section of the cochlea parallel to the long axis of the modiolus, it is of nearly triangular shape, with the somewhat rounded apex of the triangle attached to the osseous lamina spiralis. In the cochlear duct we may distinguish the following parts : (i) the outer wall, which is intimately connected with the periosteum of the bony cochlear canal ; (2) the tympanal wall, resting on the membranous basilar membrane, with its highly differentiated neuro-epithelium, the spiral organ of Corti ; and (3) the vestibular wall, bordering on the scala vestibuli, the intervening structures forming a very delicate membrane — the vestibular or Rcissnci- ' s membrane. From the account given thus far, it may be seen that within the bony cochlear canal there are found three membranous canals, running parallel with one another and with the osseous lamina spi- ralis about which they are grouped. Two of these membranous canals, the scala vestibuli and the scala tympani, are perilymphatic spaces, and are consequently lined by endothelial cells ; between them is found the cochlear duct, from its position known also as the scala media, lined by epithelial cells. These three membranous canals retain their relative position in their spiral course about the modiolus, and, in a section through the cochlea parallel to the bony axis of the modiolus, would be met with at each turn, and at each turn present essentially the same relative position and structure. In figure 372, which is from a longitudinal section of the cochlea 486 THE ORGAN OF HEARING. of a cat, the general relations of the parts are clearly shown. Figure 373 is sketched from a longitudinal section of the cochlea of a guinea- pig, and shows the appearance presented by a section through one of the turns of the bony cochlear canal and its contents as seen under higher magnification. We may now proceed with a fuller consider- ation of the structures mentioned. msp Kn Fig. 372. — Longitudinal section of the cochlea of a cat ; X 25- This figure giresa general view of the cochlea. The cochlear duct is met with six times in the section : dct cochlear duct ; gsp, spiral ganglion ; Kn, osseous cochlear wall ; Isp, ligamentum spirale ; msp, membrana spiralis; mv, membrana vestibularis or Reissner's membrane ; «, nervus cochlearis; set, scala tympani; scv, scala vestibuli (Sobotta, "Atlas and Epitome of Histology"). The lamina spiralis ossea consists of two bony plates which in- close between them the ramifications of the cochlear nerve. The vestibular surface of the osseous lamina spiralis is covered by peri- osteum, which is continuous with a peculiar tissue, known as limbus spiralis. The latter begins at the point of attachment of Reissner's THE INTERNAL EAR. 487 membrane, extends peripherally (externally), and ends in two sharp ridges, of which the shorter, the labium vcstibulare, projects into the inner space of the cochlear duct and continues into the tectorial membrane ; while the other and longer, the labium tym- panicum, becomes attached to the wall of the scala tympani and continues into the basilar membrane. Between the two ridges is a sulcus, the sulcus spiralis interims. (Fig- 373-) The limbus spiralis Fig. 373. — Section through one of the turns of the osseous and membranous coch- lear ducts of the cochlea of a guinea-pig ; X 9° : ?> Scala vestibuli ; m, labium vestibu- lare of the limbus ; «, sulcus spiralis internus ; o, nerve-fibers lying in the lamina spi- ralis ; /, ganglion cells ; q, blood-vessels ; a, bone ; b, Reissner's membrane ; DC, ductus, cochlearis ; d, Corti's membrane;/", prominentia spiralis; g, organ of Cord; h, liga- mentum spirale ; i, crista basilaris ; k, scala tympani. is a connective-tissue formation in the region of the cochlear duct connected with the periosteum of the osseous spiral lamina and extending from the point of attachment of Reissner's membrane to the labium tympanicum. The tissue of the limbus spiralis is dense and richly cellular, and simulates in its structure the sub- stantia propria of the cornea. A casual view would seem to disclose 488 THE ORGAN OF HEARING. a high columnar epithelium, but upon closer observation, it is seen that the cellular elements are interspersed with fibers which extend to the surface. Some investigators regard this tissue as fibrocar- tilage ; others, again, as a tissue sui generis, consisting of epithelial cells mingled with connective-tissue fibers. If the labium vestibulare of the limbus spiralis be examined from the vestibular surface, a number of irregular tubercles are seen at its inner portion (near Reissner's membrane), while at its outer portion long, radially dis- posed ridges may be observed, the so-called auditory teeth of Huschke. The connective-tissue wall of the sulcus spiralis internus consists of a nonnucleated fibrillar tissue which is continued into the labium tympanicum. The latter is perforated by nerves, thus giving rise at this point to the foramina nervosa, Between the point of attachment of Reissner's membrane and the labium vestibulare, the superficial epithelium of the limbus spiralis is flat, and lines the auditory teeth and the depressions between them in a continuous layer. The epithelium of the sulcus spiralis internus is somewhat higher. The ligamentum spirale forms the thickened periosteum of the outer wall of the osseous cochlear canal. It presents two inwardly projecting ridges, the crista basilaris, to which the membranous lamina spiralis is attached, and the promincntia spiralis, which con- tains one or several blood-vessels ; between the two ridges lies the sulcus spiralis externus. The portion of the ligamentum spirale forming the periosteum of the bony cochlear canal consists of a fibrous tissue containing many nuclei, but changes internally into a looser connective tissue. The connective tissue lying external to the outer wall of the cochlear duct is veiy dense and rich in cellular elements and blood-vessels, but in the crista basilaris it changes to a hyaline, noncellular tissue, continuous with the lamina basilaris. That portion of the spiral ligament lying between the prominentia spiralis and the attachment of Reissner's membrane is known as the stria vascularis. The epithelium covering this area (a portion of the epithelium lining the cochlear duct) consists of columnar, darkly granulated cells, which now and then are arranged so as to present the appearance of a stratified epithelium, but which is more correctly interpreted as an epithelium of the pseudostratified variety. This epithelium shows no distinct demarcation from the underlying con- nective tissue. Beneath this epithelium there is found a rich capil- lary network, certain loops of which extend into the epithelium (Retzius). It is thought that the stria vascularis is concerned in the formation of the endolymph of the cochlear duct. The membranous lamina spiralis, or the basilar membrane, extends from the tympanic lip of the osseous spiral lamina to the crista basilaris of the ligamentum spirale. As already stated, the tissue composing the labium tympani- cum of the limbus extends into the basilar membrane. In this THE INTERNAL EAR. 489 membrane the surface toward the cochlear duct is known as the cochlear surface, that toward the scala tympani as the tympanic surface. Two layers are differentiated in the basilar membrane, the lamina basilaris propria and the tympanic investing layer. The lamina propria consists, in turn, of (i) radially arranged basilar fibers, or acoustic strings ; (2) two thin strata of a homogeneous substance, one above and the other below the layer of basilar fibers, the upper of which is the thicker and nucleated ; and (3) a fine cuti- cula, of epithelial origin, lying on the cochlear side. The tympanic investing layer is highly developed in youth, but later becomes thinner, and may then be differentiated into a connective-tissue layer, regarded as a periosteal continuation of the tympanic por- tion of the osseous lamina spiralis, and an endothelial cell layer belonging to the lining of the perilymphatic space or the scala tympani. In the vicinity of the labium tympanicum is a blood- vessel situated within the tympanic investing layer of the basilar membrane — the vas spirale. Reissner's membrane consists of an exceedingly thin connective- ' tissue lamella, lined on the side of the cochlear duct by a layer of flattened epithelial cells and on the vestibular side by a layer of endothelial cells. The epithelium lining the cochlear duct is occa- sionally raised into small villus-like projections. The Organ of Corti. — In the region of the labium tympan- icum of the limbus spiralis and in the greater portion of the adjoining basilar membrane, the epithelium of the cochlear duct is peculiarly modified, forming here a neuro-epithelium, which receives the terminal ramifications of the cochlear nerve and is known as the spiral organ of Corti. Passing from the labium tympanicum to the ligamentum spirale, the following three regions may be recognized in the organ of Corti : An inner region, composed of the inner sustentacular cells and the inner auditory cells ; a middle region, consisting of the arches of Corti ; and an outer region, in which are found the outer auditory cells and the outer sustentacular cells or Deiters's cells. Two cuticular membranes are in close relationship to the organ of Corti : namely, the lamina reticrdaris and the mcmbrana tcctoria, or membrane of Corti. In figure 374, a sketch of the organ of Corti and adjacent structures, it may be observed that the epithelium lining the sulcus spiralis internus (at the right of the figure) is of the pavement variety, and that the epithelium becomes gradually thicker until the organ of Corti is reached, where it becomes suddenly elevated in the form of a wall. In this, two varieties of cells are distinguished — sustentacular cells and inner auditory cells. The sustentacular cells, which follow the flattened cells, become gradually higher from within outward and occupy three or four rows. Next come the inner auditory cells, cylindric elements, somewhat rounded and 490 THE ORGAN OF HEARING. thickened at their nucleated basilar ends. The latter do not extend to the basilar membrane but end at about the level of the center of the inner pillars. At the free end of each cell is an elliptic cuti- cular zone, somewhat broader than the end-surface of the corre- sponding cell. In man about twenty rigid filaments, known as auditory hairs, are found resting on each elliptic cuticular zone. These are either arranged in a straight row or they describe a slight curve. The middle division of the organ of Corti, the arches of Corti, consists of long slender structures, known as pillar cells, or, briefly, pillars, resting firmly upon the basilar membrane and forming an arch at the vestibular side of the latter. They surround, by the \:;; Fig. 374- — Organ of Corti : At x the tectorial membrane is raised ; c, outer sus- tentacular cells ; d, outer auditory cells ; f, outer pillar cells ; g, tectorial membrane ; //, inner sustentacular cells; i,p, epithelium of the sulcus spiralis internus ; k, labium ves- tibulare ; e, tympanic investing layer ; m, outer auditory cells ; «, «, nerve-fibers which extend through the tunnel of Corti ; o, inner pillar cell ; q, nerve-fibers ; /;, b, basilar mem- brane ; a, epithelium of the sulcus spiralis externus ; r, cells of Hensen ; s, inner audi- tory cell ; /, ligamentum spirale (after Retzius). union of their free ends, a space which, as seen in figure 374> appears triangular in section. This is the tunnel of Corti. According to their position, w7e distinguish inner and outer pillars, the inner being more numerous than the outer. Including the entire extent of the lamina spiralis membranacea, we find that there are about 6000 of the inner and 4500 of the outer pillar cells. Each pillar cell originates from an epithelial cell, and is found to be composed of a protoplasmic portion containing the nucleus, which may be regarded as a remnant of the primitive cell, and of a cuticular formation derived from the primitive cell, forming the elongated body of the pillar cell — the pillar. The free adjoining ends are called the heads of the pillars. The head of the inner pillar is provided with a flattened process, the head-plate, which extends outward and forms an obtuse angle with the axis of the pillar. Under this plate, and at the outer side of the head of the THE INTERNAL EAR. 49! inner pillar, is a depression into which fits the head of the outer pillar. The latter also extends outward in the shape of a phalan- geal plate, with a thinner process, the phalangeal process, at its end. The phalangeal plate and process lie under the head-plate of the inner pillar, the process extending a little beyond this, forming an acute angle with the head of the outer pillar. At the inner side of the head of the outer pillar is a convex articular surface, with which, as a rule, two, and occasionally even three, articular sur- faces of the inner pillars come in contact. The outer and inner pillars appear to possess an indistinct longitudinal striation, and their basilar plates are continuous with the extremely fine cuticula covering the basilar membrane. The inner margins of the basilar plates belonging to the inner pillars border on the foramina ner- vosa ; while the outer margins of the basilar plates belonging to the outer pillars come in contact with the basal end of the inner- most row of the cells of Deiters in the outer region of Corti's organ. The protoplasmic portions of the pillar cells, constituting what are known as basal cells, lie against the basilar plates of the corresponding pillars, — i. e., on the basilar membrane, — and partly cover the bodies of the pillars, especially the surfaces toward the tunnel. In order to comprehend the relative position of the inner audi- tory cells to the inner pillars, it may be stated that one auditory cell rests upon every two inner pillars. The outer region of Corti's organ is joined directly to the outer pillar cells, and consists of four rows of auditory cells alternating with an equal number of sustentacular cells or Deiters's cells. Following these structures and in contact with them are the outer- most sustentacular cells, known as Hensen's cells. The outer auditory cells have a structure similar to that of the inner auditory cells, but possess a more slender body. They do not extend as far as the basilar membrane, but end at a distance from the latter equal to about double their own length. The cutic- ular zone of each outer auditory cell likewise assumes the form of an ellipse, with its long axis pointing radially. The surface of this zone also is provided with about twenty stiff auditory hairs, arranged in the form of a decidedly convex arch, the convexity of which points outward. At a short distance from the cuticular zone of each outer auditory cell is a peculiar round body, found only in these cells, the significance of which is unknown. Deiters's cells rest on the basilar membrane, and in shape resem- ble a flask with a narrow neck, known as the phalangeal process, the latter lying between the auditory cells. The nuclei of Deiters's cells lie in the upper parts of the thickened basal portions of these cells. With each Deiters's cell there is associated a cuticular structure, which extends along the surface of each cell in the form of a thin 492 THE ORGAN OF HEARING. fiber, the sustentacular fiber, and which is found partly within and partly without the cell. The sustentacular fiber begins near the center of the thicker basal portion of the cell-body and extends first into the cell itself, then passes to the surface, and, entering the phalangeal process, passes to the top of the cell and expands as a plate, to which the name phalangeal plate has been given. The latter is broader than the phalangeal process, and since, as we shall see, the phalangeal plates are joined to one another, as well as to the elliptically shaped cuticular zones of the outer auditory cells, there remains a space between the cells of Deiters and the auditory cells, as also between the outer pillars and the innermost of the outer auditory cells, known as Nuel's space. To the basal regions of the inner row of the cells of Deiters is joined the basal plate of the outer pillars of the arches of Corti. Next to the outer row of Deiters's cells are the cells of Hensen, arranged in about eight radially disposed rows. They form an eminence which is high internally, but gradually decreases in height externally. The somewhat narrowed bases of Hensen's cells prob- ably extend, without exception, to the basilar membrane. The free surfaces of these cells are likewise covered by a thin cuticular mem- brane. In man the cells of Hensen usually contain yellow pigment ; in the guinea-pig, as a rule, fat ; and in the rabbit, generally rudi- ments of sustentacular fibers. Externally the cells of Hensen grad^ ally change into elements of a more cuboid type — the cells of Claudius, of which there are about ten rows, radially disposed. The surfaces of the latter also possess a cuticular margin ; the nucleus is at the center of each cell and pigment is also present. Darker elements with more basally situated nuclei sometimes occur be- tween these cells, giving rise to the appearance of a double-layered epithelium (Bottcher's cells). Thus far we have considered in detail the cells comprising the organ of Corti, and described their relative positions and sequence from within outward. In order to give a clearer understanding of the mutual relations of these cells, from within outward and in the direction of the spiral turning of the cochlea, we shall now consider the appearance presented in a surface view of the organ of Corti. From within outward a surface view of the organ of Corti pre- sents the following characteristics : The somewhat broadened hex- agonal outlines of the inner sustentacular cells adjoin the epithelial elements of the sulcus spiralis internus and terminate externally in a spiral undulating line (if seen for only a short distance, this line appears straight). On this line border the contours of the cuticular zones belonging to the inner auditory cells. The outer margins of the cuticular zones come in contact with the head-plates of the inner pillars, the cuticular zone of one inner auditory cell coming in contact with at least two head-plates. The externally directed pro- cesses of the head-plates belonging to the inner pillars come in contact with one another and end in a spiral line which for a short THE INTERNAL EAR. 493 distance is apparently straight. The head-plates of the inner pillars cover the head-plates of the outer pillars (which also come in con- tact with each other), also their phalangeal plates, but not their phalangeal processes, which thus pro- ject beyond the line formed by the outer borders of the head-plates of the inner pillars. It should be men- tioned that about three head-plates belonging to the inner pillar cells are in apposition to every two head-plates and their phalangeal processes of the outer pillar cells. The succeeding four rows, from within outward, are made up of alternately placed cutic- ular zones of the outer hair cells and the phalangeal plates of the Deiters's cells, alternating like the squares of a chess-board. This regular arrange- ment is lost in the outer row of Deiters's cells. The cells of Hensen adjoin this row, and when viewed from the surface, present the appearance of irregular polygons. This arrangement is, however, sel- dom found to be as typical as that just described ; although the relations of the cells to one another always correspond in general to the forego- ing scheme. In the cupolar and vestibular sacs the neuro-epithelium changes into an epithelium of an indifferent type. The lamina reticularis is formed by the cementing together of the pha- langeal processes of the outer pillars and the phalangeal plates of Deiters's cells, and is continued externally by a cuticular membrane which covers the cells of Hensen and, as a much thin- ner cuticular membrane, extends over the cells of Claudius. In this mem- brane there are found three or four rows of small apertures, into which the outer hair cells project. The membrana tectoria Cortii is attached to the limbus spiralis, but becomes free at the margin of the labium vestibulare and thick- ens considerably, again becoming thinner toward its free end. Fig. 375. — Surface of the organ of Corti, with the surrounding struc- tures, from the basal turn of the cochlea of a new-born child ; the original drawing reduced one-half (after Retzius, 84): a, Epithelium of the sulcus spiralis externus ; b, Hensen' s cells; c, terminal frame; d, phalanges ; /, outer auditory cells; g, flattened processes of the outer pil- lar cells ; h, flattened processes of the inner pillar cells ; i, inner auditory cells ; k, inner sustentacular cells ; /, epithelium of the sulcus spiralis interims ; in, margin of the labium vestibulare ; «, epithelium of the limbus laminae spiralis ; o, line of attachment of the membrana Reiss- neri ; /, epithelium of the membrana Reissneri, the latter inverted. 494 THE ORGAN OF HEARING. Hence an inner attached and an outer free zone may be differentiated. This membrane has no nuclei, and shows a fine radial striation. Its free portion bridges over the sulcus spiralis internus and rests upon the organ of Corti. Its outer margin extends as far as the cells of Hensen. The development of this membrane is not thoroughly understood, although it very probably represents a dis- placed cuticular formation belonging to the cells of the limbus spiralis. This acceptation has recently been confirmed (Exner). The auditory nerve gives off, soon after entering the internal auditory meatus, vestibular branches to the maculae in the utriculus and sacculus and to the cristae in the semicircular canals, and a cochlear branch, which passes up through the modiolus in anasto- mosing bony canals. From this centrally placed column of nerve- fibers, a continuous sheet of nerve -fibers, arranged in the form of anastomosing bundles, passes radially into the osseous spiral lamina and thence to the organ of Corti. Near the base of the osseous spiral lamina, along the entire length of this sheet of nerve-fibers, there is situated in a special bony canal a ganglion, known as the spiral ganglion of the cochlea. The ganglion cells of this ganglion are bipolar, one of the processes of each cell, the dendrite, extending outward through the osseous spiral lamina to the organ of Corti, the other process, the neuraxis, passing through the bony canal in the modiolus, through the internal auditory meatus, and thence to the medulla. The dendritic processes of the nerve-cells of the spiral ganglion form bundles of medullated nerve-fibers, which pass outward within the osseous spiral lamina, forming, in the outer por- tion of the latter, a closely meshed plexus, from which small bundles of nerve-fibers proceed through the foramina nervosa of the labium tympanicum to the organ of Corti ; immediately before passing through these foramina, the medullated nerve-fibers lose their medullary sheaths and neurilemma. These nonmedullated fibers, with or without further dividing, are then arranged in small bundles, which, for a certain distance, have a spiral course : that is to say, parallel to the tunnel of Corti. One such spiral bundle is situated on the inner side of the inner pillars, under the inner row of hair cells ; another, on the outer side of the inner pillars, in the tunnel of Corti. Other fibers pass through the tunnel of Corti, so-called tunnel-fibers, to reach the outer side of the arches of Corti, where they are arranged in three or four spiral bundles, at the outer side of the outer pillars and between the rows of the cells of Deiters. From the nerve -fibers of these spirally arranged bundles, terminal branches are given off, which terminate, after further division, on the inner and outer hair cells (Retzius, Geberg). Regarding the blood-vessels of the membranous labyrinth, it should be mentioned that the internal auditory artery is a branch of the basilar artery, and divides into the rami vestibulares and rami cochleares. The branches of the former accompany those of the auditory nerve as far as the utriculus and sacculus. At the maculae THE INTERNAL EAR. 495 and cristae the capillary networks are numerous and finely meshed, but in the remaining portions of the utriculus, sacculus, and semi- circular canals, they form coarser networks. The cochlear branch accompanies the divisions of the auditory nerve as far as the first spiral turn of the cochlea ; the arteries supplying the remaining turns enter the axis of the modiolus, where they divide into numerous branches. The latter are coiled in a peculiar manner, forming the so-called glomeruli arteriosi cochlea. From these, branches are given off which penetrate the vestibular wall of the lamina spiralis ossea, where they supply the limbus spiralis and the small quantity of connective tissue in the membrana vestibularis. Other branches surround the scala vestibuli, supply the walls of the latter, and then continue to the ligamentum spirale, the stria vascu- laris, and the lamina basilaris. Fig. 376. — Scheme of distribution of blood-vessels in labyrinth (after Eichler) : g, Artery ; h, spiral ganglion ; z, vein ; v, scala vestibuli ; DC, ductus cochlearis ; c, cap- illaries in the ligamentum spirale ; d, capillaries in the limbus spiralis ; f, scala tympani. The venous trunks lie close to the arteries and receive their blood from the veins which lie at the tympanal surface of the lamina spiralis and from those which encircle the outer wall of the scala tympani. The former, in turn, receive their blood from the capil- laries of the limbus spiralis ; the latter, principally from the region of the ligamentum spirale and the basilar membrane. From this description it is seen that the arterial channels are connected with the scala vestibuli, the venous with the scala tym- pani, and that the inner blood stream circulating through the lamina spiralis and limbus spiralis is separated from the blood current of the two scalae, the ligamentum spirale, and the crista basilaris (Eichler). The entire membranous labyrinth is filled with endolympJi. The ductus endolymphaticus is, as will be remembered, a canal ending 496 THE ORGAN OF HEARING. under the dura in a saccus endolympJiaticus. In connection with the latter are epithelial tubules bordering upon lymph-channels, with which they probably communicate by means of interepithelial (intercellular) spaces (Riidinger, 88). The efferent channels for the perilymph of the vestibule extend along the nerve sheaths of those nerves supplying the maculae and cristae ; these passageways finally communicate with the subdural or subarachnoid spaces. The perilymph of the cochlea is carried off by the adventitious tissue of the vena aqueductus cochleae, the lymph-vessels of which empty into certain subperiosteal lymph-channels near the inner margin of the jugular fossa. 4. ON THE DEVELOPMENT OF THE LABYRINTH. In man the epithelium lining the membranous labyrinth origi- nates from the ectoderm as a single-layered epithelial vesicle, the auditory vesicle or the otocyst, during the fourth week of embryonic life. After being constricted off from the ectoderm, this vesicle lies in the vicinity of the epencephalon and is surrounded by mesen- chyme. The auditory vesicle then develops a dorsomesial evagina- tion, which gradually grows larger and finally becomes the ductus endolymphaticus. An evagination also occurs in the ventral wall of the vesicle, the recessus cochlea. At the same time the mesial wall is pushed inward, thus incompletely dividing the vesicle into two smaller sacs — the dorsal utriculus and the ventral sacculus. From the utricular portion there arises a horizontal evagination, flat and quite broad — the first trace of the lateral or horizontal semicircular canal ; soon after, another evagination, vertical and still broader than the first, is seen — the anlage of the other two canals. The outer portion of these pouches gradually expands, while in the middle, the two layers of each evagination come in contact with each other and coalesce, finally becoming absorbed. In the vertical evagination two such areas of adherence are found, thus forming a superior and a posterior canal, both having a com- mon crus at one end. The recessus cochleae grows both in a longitudinal and in a spiral direction, forming the cochlear duct. In the immediate vicinity of the membranous labyrinth, the mesenchyme is differentiated into a connective-tissue wall for the former. The successive layers of mesenchyme, except in those areas where the membranous labyrinth later becomes adherent to the osseous, are transformed into a mucous connective tissue. The latter is surrounded by a more compact tissue, from which are de- rived, first, cartilage ; then bone and periosteum, and thus, finally, the osseous labyrinth. By a peculiar process of regressive meta- morphosis most of the mucous connective tissue later disappears. In the adult it is replaced by the perilymphatic spaces of the laby- rinth. TECHNIC. 497 TECHNIC. In the treatment of the external and middle ear the usual methods are employed. For the study of the epithelium in conjunction with the adjacent bone the tissue is fixed and then decalcified, or sub- jected to those fixing methods which accomplish both processes at the same time. The latter method, however, can be applied only to very small objects. The manipulation of the membranous labyrinth, especially that of the adult, is a very difficult technical problem. Its isolation from the petrous portion of the temporal bone without injury can be accomplished only in well-advanced fetuses and in children, and even here a thorough knowledge of the situation of the parts in the petrous portion of the tem- poral bone is essential. Smaller animals, especially rodents, afford better specimens. In the latter, the semicircular canals and cochlea give rise to more or less distinct projections into the tympanic cavity. If the latter be opened, the situation of the parts may be ascertained from without. In the rabbit and guinea-pig, the entire cochlea projects into the tympanic cavity, and may be easily removed in toto with a strong knife, and, as the bony cochlea in these animals has very thin walls, it offers very little resistance to the decalcifying fluid (use, for instance, 3% nitric acid). According to Ranvier's method (89), the cochlea is opened with a scalpel in a 2C/C solution of osmic acid in normal salt solution. After twelve hours the cochlea is placed for decalcification in 2 % chromic acid, which is frequently changed. In guinea-pigs, for instance, decalcification is accomplished in a week. According to the method of Retzius (84), the opened cochlea is treated for half an hour with a 0.5% aqueous solution of osmic acid, and then for the same length of time with a 0.5% aqueous solution of gold chlorid. The organ of Corti is then dissected out and examined as a whole, or cut after carefully removing the bone. The labyrinth of the human adult is usually prepared as follows : The apex of the petrous portion of the temporal bone is removed and the upper semicircular canal, together with the cochlea, opened in Miiller's fluid ; in this solution the pyramid is left for three weeks ; during the first week the fluid is changed daily, and every two days during the following weeks. The specimen is then washed for twenty-four hours in running water, placed in 80% alcohol for two weeks, and finally in 96% alcohol for two days. The preparation is now ready for decalcification. This is done with 5% nitric acid, which is to be changed daily (ten days to two weeks). Then follows washing for two days in running water, carrying over into 80 % alcohol for twenty-four hours, then into 96% alcohol for from six to eight days, and, finally, infiltration and imbedding in cel- loidin (A. Scheibe). The following method may also be employed with good results : The isolated pyramid with opened semicircular canal and cochlea is treated with Miiller's fluid for two days at room-temperature, and then for three weeks in a thermostat at 23° C. During the latter period, the fluid should be changed. The specimen is then washed for forty -eight hours in running water, treated for fourteen days with 80% alcohol, then for eight days with 96% alcohol, decalcified, and further treated as in the preceding method. 32 THE ORGAN OF SMELL. Up to the present time it has been customary to cut sections in celloidin ; but the combined celloidin-paraffin method may also be em- ployed with good results, and even the paraffin method, if great care be exercised in imbedding the tissue. The nerve-fibers and nerve-endings of the cochlea may be stained with the chrome-silver method. For this purpose it is recom- mended to employ embryos or young fetuses. X. THE ORGAN OF SMELL. THE nasal cavity consists of the vestibule, the respiratory region with the accessory cavities, and the olfactory region. The vestibule is lined by stratified squamous epithelium. In the region of the anterior nares are hairs, the sebaceous glands of which are markedly developed, while at the level of the cartilage mucous glands are also present. The stratified squamous epithe- lium ceases at the anterior end of the inner turbinate bone and at the inferior nasal duct. The respiratory region possesses a simple pseudostratified, ciliated epithelium having two strata of nuclei and provided with goblet cells ; the direction of the ciliate movement is toward the posterior nares. Numerous leucocytes are usually found in the epithelium and in the underlying mucosa. Branched alveolar glands, having mucous and serous alveoli, are here present. Within the mucosa are highly developed vascular plexuses, more especially of a venous character. The accessory cavities are likewise lined by ciliated epithelium, the ciliate movement being directed exter- nally. The olfactory region is principally confined to the superior tur- binate bone and to the nasal septum lying opposite, although in the immediate vicinity of the olfactory region a few small islands of the same epithelial type are found, either entirely isolated or con- nected with the principal region by narrow bridges. In a fresh condition the olfactory region may be differentiated from the sur- rounding tissue by its color, which is distinctly yellow in man. Its pigment is contained within the sustentacular cells described on the next page. The epithelium of the olfactory region is of the columnar pseudo- stratified type, with several strata of nuclei, and consequently closely simulates a stratified columnar epithelium. Here we dis- tinguish olfactory cells and sustentacular cells. The olfactory cells occupy a peculiar position among the cells of special sense in that they represent true ganglion cells (Schultze, Golgi, Ehrlich, Ramon y Cajal). Within the epithelial layer they appear as spindle-shaped cells, with a spheric nucleus provided with a large nucleolus lying in the thickest portion of each cell. The nuclei of the different cells lie at varying levels in the middle stratum THE ORGAN OF SMELL. 499 of the epithelial layer. Toward the nasal cavity, the cells terminate in blunt cones, upon each of which are several stiff hairs, the olfac- tory hairs. The basilar ends form true centripetal nerve-processes, neuraxes, which end in the peculiar telodendria constituting the glo- meruli of the olfactory bulb. (See p. 422.) The nuclei of the sustentacular cells are more oval and are situ- ated at nearly the same level. These cells present the appearances of long columnar cells, which toward the basement membrane ter- minate in one or several processes. Between the basilar ends of these cells we find a layer of elements the broad nucleated bodies of which rest on the basement membrane, while their upper extremities terminate in short superficial processes. The mucosa contains a large number of leucocytes as well as •» » - „ "— •>•-< • - •~>^ _° • '"'', K» — _~ «=>a V >> -.'.-'.-.'•-', vs?A°,>?.:°* L'A"';7-^° »•-">;«•., -« i HI" *'"• . f -' •-" .«/**. '• v , --.'"*' '^^^| Fig. 377. — Portion of transverse section of the olfactory region of man; X I5° '• *•> zone of olfactory hairs ; ep, epithelium ; 2, zone of oval nuclei ; j, zone of round nuclei ; gl, olfactory or Bowman's glands; n, branch of olfactory nerve; tp, mucosa or tunica propria with blood-vessels (Sobotta, "Atlas and Epitome of Histology"). numerous branched tubular glands, the so-called olfactory glands or the glands of Bowman. In man these are albuminous (serous) glands, and their cells sometimes contain pigment. Jacobson's organ consists of blindly ending tubes, situated at the lower portion and at the outer side of the nasal septum. It is lined by an olfactory mucous membrane and receives a branch of the nasal nerve. This organ is rudimentary in man. The capillaries spread out immediately beneath the basement membrane of the epithelium. In the submucous connective tissue, we find a relatively well developed vascular plexus, rich in venous vessels ; this plexus is especially marked at the posterior portion of the inferior turbinate bone, forming here a tissue which resembles erectile tissue. 5OO THE ORGAN OF SMELL. A dense network of lymphatics ramifies throughout the mucous membrane, carrying the lymph to the pharynx and palate. These lymph-vessels may be injected through the subarachnoid space (Key and Retzius). The nerves (trigeminal) are widely distributed in the epithelium, ramifying through both the respiratory and olfactory regions. After repeated divisions these nerves lose their medullary sheaths, and end in telodendria which are usually provided with terminal nodules, although some are found which end in mere filaments. TECHNIC. The nasal mucous membrane is fixed in situ with osmic acid or one of its mixtures, after which small pieces are removed. It should be mentioned that the nonmedullated fibers of the olfactory nerve assume a brownish color under this treatment, while the fibers of Remak do not (Ranvier, 89). In order to isolate the epithelial elements, pieces of the mucous membrane are treated with the y$ alcohol of Ranvier. But since the prolongations of the olfactory cells (neuraxes) shrivel and curl in this fluid, Ranvier recommends that, after the epithelial cells have been macerated in ^ alcohol for one or two hours, they be treated with i 329 epithelium of, demonstration of, 343 intercalated portion of tubules, 323, 329 interlobular arteries of, 332 veins of, 334 lymphatics of, 334 medullary substance of, 323 nerves of, 334, 335 pelvis of, 336 mucosa of, 337 blood-vessels of, 338 proximal convoluted portion of tubules, 323. 325 secretory processes of, 335 straight collecting tubules of, 323 technic of, 342 tubules of, demonstration of, 342 vasa afferentia of, 332 Knife, double, 21 microtome, honing of, 37 sharpening of, 37 Kolliker's interstitial granules, 141 muscle-columns, 140 INDEX. 515 Kolliker's neuropodia, 151 Kollmann's cold carmin mass, 54 Kolossow's method of demonstrating in- tercellular bridges, 96 Kopsch's method of impregnation, 52 Krause, end-bulbs of, 170, 388 cylindric, 172 membrane of, 137 Kronecker's fluid, 22 Kuhne's method of impregnation, 48 Kupffer's stellate cells, 295 Kytoblastema, 64 LABIUM tympanicum, 487 vestibulare, 487 Laboratory microtome, 33 Labyrinth, bony, 480 development of, 496 membranous, 480, 481 blood-vessels of, 494 technic for, 497 osseous, 480 technic for, 497 Lacrimal apparatus, 473 glands, 473 . accessory, 470 nerve supply of, 473 sac, 474 Lacteals of villi of small intestine, 285 Lacunas, Howship's, 120 of bone, 112, 113 Lamellae, 105 bone, 113 compostion of, 114 method of examining, 131 circumferential, inner, 113 outer, 113 concentric, 113 fundamental, 113 periosteal, 113 Lamina basilaris propria, 489 choriocapillaris, 452, 453 cribrosa, 448, 465 elastica interna, 218 fusca, 448 propria of oral cavity, 236 of tympanic membrane, 477 reticularis, 489, 493 spiralis membranacea, 485 ossea, 484, 486 suprachoroidea, 448, 452 vasculosa Halleri of choroid, 452 Langerhans, areas of, 301 cells of, 300 Large intestine, 281. See also Intestine, large. Larynx, 309 cartilages of, 310 demonstration of, 322 mucous membrane of, 309 nerves of, 311 vascular supply of, 310 Lateral column, 408 mixed, 411 Ledges, terminal, 86 Lens, 446 apochromatic, 19 capsule, 468 collective, 19 crystalline, 467 anterior epithelium of, 468 fibers, 468 front, 19 immersion, 19 ocular, 19 suspensory ligament of, 467 technic of 475 Lenticular glands, 271 Leucocyte-nucleus, polymorphism of, 193 Leucocytes, 191 granulations of, 227 in epithelium of mucous membrane of small intestine, 275 method of counting, 232, 233 mononuclear, 192 motility of, 193 size of, 192 transitional, 192 with polymorphous nuclei, 192 Leucocytic granules, Ehrlich's, 192 Leydig's cells, 470 Lieberkiihn's glands, 88, 276 Ligament, suspensory, of lens, 467 Ligaments, 105 Ligamentum nuchae of ox, structure of, 106 pectinatum iridis, 454 spirale, 485, 488 Limbus spiralis, 486, 487 Lime-salts in bone, hematoxylin as stain for, 132 isolation of, 132 Limiting membrane, external, of retina, 459, 462 internal, of retina, 462 Lines of Retzius, 239 of Schrager, 239 Lingual mucous membrane, 247 papillae of, 247 papillae, 247 Linin, 63 Liquor folliculi, 347 Liver, 289 blood-vessels of, distribution of, dem- onstration, 306 examination of, 343 connective tissue of, 294 interlobular veins of, 293 lobules, 289 lymphatics of, 297 nerves of, 298 demonstration of, 308 reticular fibers of, demonstration of, 308 reticulum of, 294 stellate cells of, 295 technic of, 306 tissue, technic of, 307 trabeculae of, 290 Liver-cells, examination of, 306 glycogen in, demonstration of, 306 Loop, Henle's, 323 5i6 INDEX. Loop, Henle's descending limb of, 327 Lo wit's method of impregnation, 48 Lugol's solution to demonstrate glycogen in cartilage, 131 Lung alveoli, 314 blood-vessels of, 316 lobules of, 316 lymphatics of, 317 nerves of, 317 structure, units of, 316 tissue of, demonstration of, 322 Lunula, 395 Lutein, 353 cells, 353 Lymph, 186 canalicular system, 102 capillaries, 224 Lymphatic networks in endocardium, 215 system, 223 Lymph-channels, anterior, of eye, 469 injection of, 55 Lymph-follicles, germ centers of, technic for, 234 of mucosa of vermiform appendix, 281 of tongue, 251 of tonsils, 251 solitary, 197 technic for, 306 Lymph-glands, 196, 197 blood supply of, 200 capsule of, 198 hilum of, 197 lymph-sinuses of, 199 marrow, 201, 202 technic for, 233 trabeculffi of, 198 with blood-sinuses, 200 Lymph-nodules, 196 agminiited, 197 of mucosa of small intestine, 279 Lymphocytes, 191, 194 size of, 192 Lymphoid tissue, 196 Lymph-sinus, 199 Lymph-spaces, 224 injection of, 55 periaxial, 176 perichoroidal, 452 Lymph-supply of intestine, 283 Lymph-vessels, 186, 223 injection of, 55 of central nervous system, 440 of kidney, 334 of large intestine, 284 of liver, 297 of lung, 317 of mammary glands, 402 of mouth, 260 of ovary, 354 of skin, 386 of small intestine, 285 of testes, 367 of uterus, 357 MACCALLUM'S nitric acid mixture, 23 Macerating solutions, 22 alcohol, 22 caustic acid, 22 potash, 22 chromic acid, 22 hydrochloric acid, 23 MacCallum's, 23 nitric acid, 23 and chlorate of potassium, 23 sulphuric acid, 23 Maceration, methods of, 22 Macula acustica sacculi, 481 utriculi, 481 lutea, 460 region of, 460 Magenta red as stain for connective tissue, 128 Male genital organs, 361 pronucleus, 73 Mallory's differential stain for connec- tive-tissue fibrillffi and reticulum, 128 selective neuroglia fiber-staining meth- ods, 445 Malpighian corpuscles, 202, 203, 323, 324 pyramid, 323 Mammary gland, 400 alveoli of, epithelium of 401 lymphatics of, 402 nerves of, 402 Mantle fibers, 69 Marginal thread of spermatosome, 361 zone, 8 1 Marrow, bone-, 207. See also Bone- marrow. fat-, 207 lymph-glands, 102, 202 spaces, primary, 118 secondary, 120 Marrow-cells, 208 Martinotti's cells, 418, 419 Mast-cells, 104 granules, technic of, 228 Matrix of areolar connective tissue, 102 of nail, 394 sulcus of, 394 Mature ovum, 351 Mayer's solutions for staining mucin, 305 Median disc of Hensen, 137 membrane of Heidenhain, 137 Mediastinum testis, 363 Medullary cords, 199 rays, 323 sheath, 157 technic, 440 Benda's, 442 Pal's, 442 Weigert's, 440, 441 substance of cerebellar cortex, 416 of cerebral cortex, 419 of hair, 390 of kidney, 323 of ovary, 344 Meibomian gland, 472 Meissner's corpuscles, 170, 387 technic of, 405 INDEX. 517 Meissner's plexus, 287 Membrana capsulopupillaris, 468 praeformativa, 244 prima, 81 propria, 88, 92 of pancreas, 301 of uriniferous tubules, 330 pupillaris, 468 tectoria Cortii, 489, 493 Membrane, basement, 81, 88 of. small intestine, 278 basilar, 488 Bowman's, 449 cell, absence of, 62 Cord's, 489, 493 Descemet's, 450 endothelium of, 451 technic of, 474 elastic, anterior, of cornea, 449 posterior, of cornea, 450 external limiting, of retina, 459, 462 fenestrated, 107 glassy, of choroid, 452, 453 of hair, 391 Heidenhain's, 137 hyaloid, of vitreous body, 467 internal limiting, of retina, 462 Krause's, 137 median, of Heidenhain, 137 mucous. See also Mucous membrane. Nasmyth's, 238 nuclear, 63 of central nervous system, 436 oral mucous, fixation of, 303 otolithic, 483 peridental, 242 pigment, of eye, 446, 447, 457 Reissner's, 485, 489 rudder, of spermatosome, 361 tympanic, 476. See also Tympanic membrane. undulating, of spermatosome, 361 vestibular (Reissner's), 485, 489 vitreous, 452, 453 Meninges of central nervous system, 436 Menisci, tactile, 387 technic of, 405 Merkel's terminal disc, 139 Mesameboid cells, 80 Mesenchymatous tissue, 97 Mesenchyme, 80 Mesoderm, 58, 79 cells of, 80 Mesothelial cells, 80 and endothelial cells, method of studying relations, 95 Mesothelium, 80, 92 Metakinesis, 68 Metaphases, 65, 68 Methylene-blue stain, Ehrlich's, for nervous tissues, 182 for nerve-fibers, 184 Methyl-green as stain, 44 Metschnikoff's phagocytes, 60 Meyer's method of staining nerve- fibers, 184 Microcentrum, 191 Microscope and its accessories, 17 coarse adjustment of, 18 compound, 1 7 description of, 17 fine adjustment of, 18 parts of, 17 simple, 17 Microscopic preparation, 21 preparations of undecalcified bone, 131 technic, introduction to, 17 Microtome, 32 knife, honing' of, 37 sharpening of, 37 laboratory, 33 Minot automatic precision, 33, 34 rotary, 34, 35 precision, 33 rocking, 33 rotary, 35 sliding, 33 cutting celloidin sections with, 36 paraffin sections with, 35 freezing apparatus for, 36 Middle ear, 478 technic for, 497 Migratory cells, 103, 104, 193 Milk, 402 secretion, 401 Minot automatic precision microtome 33. 34 rotary microtome, 34, 35 Mitochondria, 60 Mitosis, 64 demonstration of, 75 heterotypic, 70 homeotypic, 70 Mitotic cell-division of fertilized white- fish eggs, 66, 67 ten stages of, 65 Mitral cells, 421 of olfactory bulb, 42^ Mixed gland, 258 lateral column, 411 Modified sweat-glands, 398 Modiolus, 484 Molecular layer, inner, of retina, 464 of cerebellar cortex, 413 of cerebral cortex, 417 of olfactory bulb, 421 outer, of retina, 45, 462 movement of cells, 61 Moll's glands, 398, 470 Monaster, 68 Mono nuclear leucocytes, 192 Monostratified cells of retina, 464 Montgomery's glands, 402 Morgagni, hydatids of, 360 Morula mass, 79 Mossy fibers of cerebellar cortex, 416 Mother cell, 374 nucleus, 64 skein, 67 Motor end-plate, 163 fibers, 162 nerve-endings, 162 Si8 INDEX. Motor nerve-endings, staining of, 182 neurones. 153 peripheral, 162 diagram of, 163 Mounting, 21, 52 Mouth, glands of, small, 259 lymphatics of, 260 Muchematein, 305 Mucicarmin, 305 Mucin, demonstration of, 304, 305 staining of, 305 Mucous connective tissue, ice gland, 255 layer of tympanic membrane, 478 membrane, gastric, 266 intestinal, general structure of, 264 nerves of, demonstration of, 306 nasal, technic of, 500 of Eustachian tube, 479 of Fallopian tubes, 354 of larynx, 309 of oral cavity, 236 of pelvis of kidney, 337 blood-vessels of, 338 of small intestine, 274, 277 blood-vessels of, 284 epithelium of, 274 leucocytes in, 275 lymph-nodules of, 279 villi of, 274 of stomach, fixation of, 305 of tongue, 247 of uterus, 355 of vagina, 358 epithelium of, 358 of vermiform appendix, lymph- follicles in, 281 oral, fixation of, 303 Mucus-secreting cell, 87 Miiller's fibers, 454 of retina, 462 fluid, 26 Multicellular glands, 88 classification, 91 Muscle and tendon, relation of, method of studying, 148 ciliary, 454 dilator, of pupil, 455 heart, 145 fibers of, MacCallum's nitric acid mixture for isolating, 23 motor nerve-supply of, 166 nonstriated, motor nerve-supply of, 1 66 red, 141 sphincter, of pupil, 455 striated, nerve-endings in, Sihler's method of demonstrating, 184 white, 141 Muscle-cells, cardiac, demonstration of, 148 nonstriated, 134 of fibers of Purkinje, 147 Muscle-columns of Kolliker, 140 Muscle-fibers, intrafusal, 175 nonstriated, demonstration of, 148 Muscle-fibers, striated, technic of, 147 striped, 136 voluntary, development of, 144 Muscular coat of Fallopian tubes, 355 of uterus, 356 of vagina, 359 tissue, 134 destruction of, 144 development of, 144 heart, development of, 146 nerve-fibers ending in, 162 striated, blood-vessels in, 143 technic of, 147 Muscularis mucosae of intestine 265 of small intestine, 279 of stomach, 271 Musculus ciliaris Riolani, 471 orbicularis oculi, 471 palpebralis superior, 472 Myelin sheath, 157 Myelocytes, 208 Myeloplaxes, 209 Myoblasts, 144 Myocardium, 213 NAIL, 394 bed, 394 sulcus of, 394 body of, 394 lunula of, 395 matrix, 394 sulcus of, 394 root, 394 walls, 394 Nasal artery, inferior, of retina, 466 superior, of retina, 466 cavity, 498 blood-vessels of, 499 nerves of, 500 technic of, 500 vestibule of, 498 duct, 474 mucous membrane, technic of, 500 vein, inferior, of retina, 466 superior, of retina, 466 Nasmyth's membrane, 238 Nerve end-organs, neuromuscular, 174 neurotendinous, 1 78 Nerve-cells, 149. See also Ganglion cell. Nerve-endings, annulospiral, 178 flower-like, 1 78 in striated muscle, Sihler's method of demonstrating, 184 motor, 162 staining of, 182 peripheral, 162 Ruffini's, 387 sensory, 1 66 encapsulated, 169 free, 168, 169 staining of, 182 Nerve-fibers, 157 ending in muscle tissue, 162 layer of retina, 464 INDEX. 519 Nerve-fibers, medullated, demonstration of, 1 80 of teeth, 242 methylene-blue stain for, 184 nonmedullated, 160 demonstration of, 182 of hair follicles, 393 of utriculus, 483 Nerves, auditory, 494 in taste-buds, demonstration of, 304 of bladder, 339 of bronchi, 317 of ciliary body, 456 of cornea, 451 technic, 474 of dura mater, 437 of epidermis, technic of, 405 of heart, 215 of intestine, 283 of intestinal mucous membrane, dem- onstration of, 306 of iris, 456 of kidney, 334, 335 of lacrimal gland, 473 of larynx, 311 of liver, 298 demonstration of, 308 of lung, 317 of mammary glands, 402 of nasal cavity, 500 of ovary, 354 of pancreas, 302 of penis, 372 of pia mater, 439 of prostate, 370 of salivary glands 260 of sclera, 451 of skin, 387 of suprarenal glands, 342 of tongue, 252 of sweat-glands, 397 of testes, 367 of thyroid gland, 320 of trachea, 311 of ureter, 339 of uterus, 358 of vagina, 360 olfactory, staining fibers of, 182 optic, 446, 464 pilomotor, 394 supplying blood-vessels, 223 Nerve-trunk, funiculi of, 160 compound, 162 Nervous system, central, 406 blood-vessels of, 439 fibrillar elements of, Apathy's method of demonstrating, 442 lymph vessels of, 440 membranes of, 436 technic of, 440 tissue, 148 Ehrlich's methylene-blue stain for, 182 fixation of, 183 technic of, 180 tunic of eye, 446, 457 Net-knots, 63 Networks, technic for, 235 Neura, 149 Neuraxes, 148, 151 Neurilemma, 158 Neurilemma-nuclei, 158 Neuroblasts, 148 Neurodendron, 149 Neuro-epithelial cells, 92 Neuro-epithelium, 92 Neurofibrils, Bethe's method of staining, 443 Neuroglia, 434 fibers, Benda's method of staining 445 Mallory's methods of staining, 445 staining of, 444 Neurogliar cells, 434 Neurokeratin, 157 Neuromuscular nerve end-organs, 174 Neurones, 149 cell-bodies of, 149 independence of, theory of, 156 motor, 153 peripheral, 162 diagram of, 163 relationship of, 431 sensory, peripheral, diagram of, 167 Neuroplasm, 157 Neuropodia, 151 Neurotendinous nerve end-organ, 178 Neutrophile granules, 193 technic for, 228 mixture Ehrlich's, 229 Nitric acid and chlorate of potassium as macerating solution, 23 aqueous solution, as decalcifying fluid, 133 as fixing solution, 26 as macerating solution, 23 MacCallum's, 23 Nodes of Ranvier, 158 demonstration of, 180 Nodules, 197 cortical, 198 lymph-, 196 agminated, 197 of mucosa of small intestine, 279 secondary, 197 terminal, of spermatosome, 361 Normoblasts, 208 Nuclear division, 64 layer, inner, of retina, 459, 462 membrane, 63 stains, 41 Nuclein, 63 Nucleolus, 58 true, 63 Nucleus, 58, 62 achromatic portion of, 63 chromatoid accessory, of Benda, 377 contents of, 62 daughter, 64 direct fragmentation of, 71 dorsalis, 408 gray, central, of cerebellar cortex, 416 520 INDEX. Nucleus, leucocyte-, polymorphism of, 193 mother, 64 of spermatid, 377 resting, 63 segmentation, 7 1 sole, 163 telolemma, 163 Nuel's space, 492 OCULAR lens, 19 Odontoblasts, 240, 241, 244 Odontoclasts, 247 Olfactory bulb, 421 glomerular layer of, 421 granular layer of, 421 mitral cells of, 421 molecular layer of, 421 peripheral fibers of, 421 cells, 498 hairs, 499 region, 498 epithelium of, 498 nerve, fibers of, staining of, 182 Oocytes, 350 Optic cup, 447 nerve, 446, 464 blood-vessels of, 465 papilla, 460 region of, 460 stalks, 446 vesicles, primary, 446 secondary, 447 Ora serrata, 461 Oral cavity, 235 glands of, 253 mucous membrane of, 236 fixation of, 303 submucosa of, 236 Orbiculus ciliaris, 453 Orcein as stain for connective tissue, 128 Osmic acid as fixative for cartilage, 130 as fixing solution, 24 as stain for adipose tissue, 130 Osseous labyrinth, 480 Ossicles, auditory, 478 Ossification, centers of, 116 groove, 121 of cartilage, in ridge, 121 Osteoblasts, 118 Osteoclasts, 120 Otolithic membrane, 483 Otoliths, 483 Outer fiber layer of retina, 461 molecular layer of retina, 459, 462 Ovarian tissue, fixation of, 378 Ovary, 344 antrum of, 347 blood-vessels of, 354 cortex of, 344 germinal epithelium of, 345 lymphatics of, 354 medullary substance of, 344 nerves of, 354 Ovary, stratum granulosum of, 345 stroma of, 344 technic of, 378 Ovula Nabothi, 356 Ovum, 71, ^44 changes in, during development, 350 mature, 351 primitive, '345, 350 ripe, 350 technic for, 378 vacuole of, 344 Oxychromatin granules, 63 PACCHIONIAN bodies, 438 Pacinian corpuscles, 388 technic of, 405 Pal's method for demonstration of medullary sheath, 442 Pancreas, 298 blood-vessels of, 302 cells of, inner and outer zones, methods of differentiating, 308 intermediate tubule of, 300 intertubular cell-masses of, 301 intralobular duct of, 300 membrana propria of, 301 nerves of, 302 technic of, 308 zymogen granules in, demonstration of, 308 Pancreatic duct, 298 Panniculus adiposus, 384 Papilla, 84 circumvallate, 249 dentinal, 243 filiform, 248 foliate, 249 fungiform, 248 hair, 389 lingual, 247 optic, 460 region of, 460 spiralis cochlea, 481 tactile, 383 vascular, 383 Papillary artery, inferior, of retina, 466 superior, of retina, 466 vein, inferior, of retina, 466 superior, of retina, 466 Paracarmin as stain, 42 in bulk, 46 Paradidymis, 367 Paraffin imbedding, 27 diagram for, 30 infiltration, 27 diagram for, 30 removal of, 40 sections, cutting of, with sliding microtome, 35 dextrin method of fixing, 40 distilled water for fixing of, to slide, 39 fixing of large numbers to cover-slips, 39 INDEX. 521 Paraffin sections, glycerin-albumen for fixing of, to slide, 38 Japanese method of fixing to slide, 39 Paralinin, 63 Paranuclein, 63 Paraplasm, 60 Parareticular cells, 464 Parathyroid glands, 321 Paroophoron, 360 Parotid gland, 255 Pars ciliaris retinae, 453, 461 iridica retinas, 461 papillaris, 382 reticularis, 382 Partsch's cochineal solution, 42 Pellicula, 62 Pelvis of kidney, 336 mucosa of, 337 blood-vessels of, 338 renal, 336 Penis, 370 erectile tissue of, 371 nerves of, 372 Pepsin, effect of, on connective tissue, 128 Peptic glands, 268 Perforating fibers of cornea, 450 Periaxial lymph-space, 176 Pericardium, 214 Pericellular plexuses, 428 Perichondrium, 109 Perichoroidal lymph-spaces, 452 Peridental membrane, 242 Perilymph of cochlea, 496 Perilymphatic spaces, 224 Perimysium, 143 Perineurium, 161 Periosteal lamellae, 113 Periosteum, 112 alveolar, 242 future, 116 Peripheral fibers of olfactory bulb, 421 motor neurones, 162 diagram of, 163 nerve terminations, 162 sensory neurone, diagram of, 167 Peritendineum, 105 Perivascular spaces, 224 Petit's canal, 467 Petit and Ripart's solution, 22 Peyer's patches, 265 technic for, 306 Pfliiger's egg tubes, 345 Phagocytes, 193, 194 Metschnikoff's, 60 Phalangeal plate, 491, 492 process, 491 Pharyngeal tonsils, 252 Pharynx, 262 Physiologic excavation of retina, 460 Pia intima, 438 mater, 438 nerves of, 439 Pial funnels, 439 Picric acid as fixing solution, 25 as stain, 45 Picric acid for fixing cells, 75 Picric-nitric acid as a fixing solution, 25 Picric-osmic-acetic acid solution as fixing fluid, 25 Picric-sublimate-osmic solution as fixing fluid, 25 Picrocarmin as stain for connective tissue in cartilage, 131 for elastic fibers in cartilage, 131 of Ranvier, 44 of Weigert, 45 Picrosulphuric acid as fixing solution, 25 Pigment, 97 cells, 77, 104 membrane of eye, 446, 447, 457 of cells, 6 1 of skin, 384 technic of, 404 Pillar cells, 490 heads of, 490 inner, 490 outer, 490 Pilomotor nerves, 394 Pineal gland, 422 Pituitary body, 423. See also Hypophy- sis. Plasma, blood, 187 cells, 104 Plate, phalangeal, 491, 492 Platelets, blood, fixation of, 227 Plates, technic for, 235 Pleura, visceral and parietal layer of, 319 Plexus, choroid, 439 epilamellar, 261, 397 ground, of cornea, 451 Heller's, 283 hypolamellar, 261 intracapsular, 429 myentericus, 286 of Auerbach, 286 of Meissner, 287 pericellular, 428 subepithelial, of cornea, 451 superficial, of cornea, 451 Plica? palmataj, 356 semilunares, 282, 473 sigmoideae, 266 transversales recti, 282 Plural staining, 44 Plurifunicular cells, 408 Polar body, 72 field, 70 rays, 68 Polarity of cells, 81 Polygonal cells of cerebral cortex, 417 Polykaryocyte, 193 Polymorphism of leucocyte-nucleus, 193 Polymorphous cells of cerebral cortex, 418 Polynuclear cells, 70 Polystratified cells of retina, 464 Portal vein, 292 Posterior hyaloid arteries, 468 vertical semicircular canal, 480 522 INDEX. Potash, caustic, as macerating solution, 22 Potassium bichromate and formalin as fixing fluid, 27 chlorate of, nitric acid and, as macerat- ing solution, 23 hydrate, action of, on connective tissue, 128 Precapillary arteries, 218 veins, 220 Precision microtome, 33 Prepuce, 372 Primary blastodermic layers, 79 egg tubes of Pfliiger, 345 germ layers, 79 marrow spaces, 118 optic vesicles, 446 tendon bundles, 105 Primitive ova, 345, 350 seminal cells, 372 Primordial ova, 345 Prisms, enamel, 238 Projection fibers of cerebral cortex, 419 Prominentia spiralis, 488 Pronucleus, female, 74 male, 73 Prophases, 64, 66 Prostate, 368 blood-vessels of, 370 concretions of, 370 corpora amylacea of, 370 nerves of, 370 secretion of, 370 Prostatic bodies, 370 Protoplasm, 58, 59 of spermatids, chromatoid accessory nucleus of, 377 sphere substance of, 376 Protoplasmic currents, 75 stains, 41 Protozoa, 58 Pseudopodia, 60 Pulp cords, 204 splenic, structure of, 205 tooth-, 241 Pupil, dilator muscle of, 455 sphincter muscle of, 455 Purkinje's cells, 153 of cerebellar cortex, 415 fibers, 213 isolated, demonstration of, 148 muscle-cells of, 147 vesicle, 344 Purpurin, alkaline, as stain for calcium carbonate in bone, 132 Pyloric glands, 269 Pyramidal cells of cerebral cortex, 153 large, of cerebral cortex, 417 small, of cerebral cortex, 417 columns, crossed, 411 tract, direct, 411 Pyramids of Ferrein, 324 of Malpighian, 323 QUINTUPLE hydroquinon developer, 51 RABL'S hematoxylin-safranin, 46 solutions, 25 Rami cochleares, 494 vestibulares, 494 Ramon y Cajal's technic for retina, 475 Ranvier's crosses, demonstration of, 180 method for examination of connective tissue, 126 of demonstrating glycogen in liver- cells, 306 spaces in bone, 132 of impregnation, 48 nodes, 158 demonstration of, 180 picrocarmin, 44 solution of iodin and potassium iodid, 22 Recessus camerae posterioris, 467 cochleae, 496 Rectum, 281 Red bone-marrow, 207 muscles, 141 Red-blood corpuscles, 187. See also Erythrocytes . Reissner's membrane, 485, 489 Remak's fibers, 160 demonstration of, 182 Renal artery, 332 lobes, 323 pelvis, 336 Renflement biconique, 158 Respiration, organs of, 309 technic of, 322 Respiratory bronchioles, 313 elastic fibers, demonstration of, 322 epithelium, 315 examination of, 322 region, 498 Resting nucleus, 63 Rete testis, 363 canals of, 365 Retia mirabilia, 222 _ arterial, 333 _ Reticular connective tissue, 100 cells of, 100 fibers of liver, demonstration of, 308 tissue, demonstration of, 234 Reticulum of liver, 294 Retina, 446, 447, 457 arteries of, 466 blood-vessels of, 465 central artery of, 465 vein of, 465 cone-fibers of, 459 external limiting membrane of, 459, 462 fiber-baskets of, 462 ganglion-cell layer of, 459, 464 inferior nasal artery of, 466 vein of, 466 papillary vein of, 466 inner molecular layer of, 464 nuclear layer of, 459, 462 internal limiting membrane of, 462 macula lutea of, 460 Muller's fibers of, 462 INDEX. 523 Retina, nerve-fiber layer of, 464 optic papilla of, 460 or a serrata of, 461 outer fiber layer of, 461 molecular layer of, 459, 462 pars ciliaris retinae, 461 iridica retinae, 461 physiologic excavation of, 460 relation of elements of, to one another, 462 rod-fibers of, 458 superior nasal artery of, 466 vein of, 466 papillary artery of, 466 vein of, 466 technic of, 475 Retinaculae cutis, 384 Retzius, end-piece of, 361 lines of, 239 Ring, contraction-, 158 Ripart and Petit' s solution, 22 Ripe ovum, 350 Rocking microtome, 33 Rod-fibers of retina, 458 Rod-visual cells, 458 bipolar cells of, 463 Rolando's gelatinous substance, 408 Root-sheaths of hair, 389. See also Hair, root-sheaths of. Rose's carmin-bleu de Lyon, 45 Rouleaux, 187 Rudder membrane of spermatosome, 361 Ruffini end-organ, 388 SABIN'S modification of Mallory's differ- ential stain for connective-tissue fi- brillse and reticulum, 129 Sacculus, 481, 482 ventral, 496 Saccus endolymphaticus, 481, 496 Safranin as stain, 44 Salivary glands, 253, 255 blood supply of, 259 nerve supply of, 260 Salts, lime-, in bone, hematoxylin as stain for, 132 isolation of, 132 Sarcolemma, 135, 137 Sarcolytes, 144 Sarcomeres, 138 Sarcoplasm, 135, 137 Sarcous elements, 141 Scala media, 485 tympani, 485 vestibuli, 485 Schachowa's spiral segment, 327 Schlemm's canal, 448 Schmidt-Lantermann-Kuhnt's segments, 157 Schmorl's method of staining bone cor- puscles, 133 Schrager's lines, 239 Schron's granule, 344 Schultze's iodized serum, 22 Schwann, sheath of, 158 Sclera, 446, 448 blood-vessels of 449 nerve supply of, 451 technic of, 475 Scleral conjunctiva, 448 sulcus, inner, 449 Sebaceous glands, 398 Secondary marrow spaces, 120 optic vesicle, 447 tendon bundles, 105 Secretion, milk, 401 of intestine, 288 of prostate, 370 process of, 92 vacuoles, 291 Secretory processes of kidney, 335 Sectioning, 32 Sections, 2 1 staining of, 41 Sectionwork, appropriate stains for, 235 Segmentation nucleus, 71 Segments, Schmidt-Lantermann-Kuhnt's, 157 spiral, of Schachowa, 327 Selective stains, 41 Semicircular canal, 483 anterior superior vertical, 480 external, 480 horizontal, 480 posterior inferior vertical, 480 Semilunar fold, 484 Seminal cells, primitive, 372 fluid, examination of, 378 vesicles, 368 Sense cells, 81 Sensory nerve-endings, 166 encapsulated, 169 free, 168, 169 staining of, 182 neurone, peripheral, diagram of, 167 Septa renis, 324 Septum posticum, 438 Serous cavities, 224 gland, 255 Sertoli's cells, 364 Sexual cells, fertilization of, 71 male, development of, 72 matured, 7 1 Sharpening microtome knife, 37 Sharpey, fibers of, 115 method of isolating, 134 Sheath, axial, 176 Henle's, 162 medullary, 157 technic, 440 Benda's, 442 Pal's, 442 Weigert's, 440, 441 myelin, 157 of axial thread of spermatosome, 361 of Schwann, 158 Shedding hair, 393 Sihler's method of demonstrating nerve- endings in striated muscle, 184 Silver nitrate as injection fluid, 55 method of impregnation, 47 524 INDEX. Simple epithelium, 82. See also Epithe- lium, simple. microscopes, 17 Sinus, blood, 222 lactiferus, 400 lymph-, 199 pocularis, 370 Sinuses, 222 , Sinusoids, 221 Skein, mother, 67 Skin, 379 and appendages, 379 technic of, 403 glands of, 396 lymph-vessels of, 386 nerves of, 387 pigment of, 384 technic for, 404 structure of, technic for, 404 true, 379 vascular system of, 386 Slide digestion for connective tissue, 129 Slides, 20 Sliding microtome, 33. See also Micro- tome, sliding. Small intestine, 274. See also Intestine, small. Smell, organ of, 498 Sole nuclei, 163 plate, granular, 163 Somatic cell, 71 Specimens, permanent, preparation of, 52 Spermatids, 72, 374 develoment of, into spermatosomes, 374, 37° nucleus of, 377 protoplasm of, chromatoid accessory nucleus of, 377 sphere substance of, 376 Spermatoblast, 376 Spermatocytes, 70, 72 of first order, 374 of second degree, 374 of third degree, 374 Spermatogenesis, 372 schematic diagrams of, 373 technic of, 378 Spermatogones, 72 Spermatogonia, 372 Spermatosome, 361 accessory thread of, 361 axial thread of, 361 sheath of, 361 development of, from spermatids 374 376 flagellum of, 361 head of, 361 marginal thread of, 361 middle piece of, 361 rudder membrane of, 361 tail of, 361 terminal nodule of, 361 undulating membrane of, 361 Spermatozoa, 60, 71, 73 Spermatozoon, 361. See also Sperma- tosome. Sphere substance of protoplasm of spermatids, 376 Sphincter muscle of pupil, 455 Spider cells, 435 Spinal cord, 406 anterior median fissure of, 406 commissures of, 412 gray substance of, 406, 409 horns of, 408 posterior median septum of, 406 white substance of, 406, 409 ganglia, 424 ganglion cell of Dogiel, 426 Spindle, achromatic, 68 central, 68 Spindle-shaped cells of cerebral cortex, 417 Spiral ganglion of cochlea, 494 organ of Corti, 489 segment of Schachowa, 327 Spirem, 67 Spleen, 202 blood supply of, 203 lobules, 204 diagram of, 205 trabeculae of, 203 Splenic pulp, structure of, 206 tissue, demonstration of, 234 Splenolymph glands, 201 Spongioblasts, 434 diffuse, 464 stratum, 464 Spongioplasm, 60, 274 Spot, Wagner's, 344 Staining, 41 blood-cells, 227 blood films, Wright's method, 229 bone corpuscles, Schmorl's method, 133 double, 44 of cells, 76 fibers of olfactory nerve, 182 in bulk, 46 diagram for, 47 in sections, diagram for, 47 motor nerve-endings, 182 neurofibrils and Golgi-nets, Bethe's method, 443 neuroglia, 444 fibers, Benda's method, 445 Mallory's methods, 445 plural, 44 section, 41 sensory nerve-endings, 182 Stains, 41 acid, 41 fuchsin-picric acid solution, van Gieson's, 45 hemalum, 43 alkaline purpurin, for calcium carbon- ate in bone, 132 alum-carmin, 42 for bulk, 46 anilin, 44 basic, 41 Biondi-Heidenhain triple, 46 INDEX. 525 Stains, Bismarck brown, 44 borax-carmin, alcoholic, 41 for bulk, 46 aqueous, 41 carmin, 41 carmin-bleu de Lyon, 45 coal-tar, 44 Czocor's cochineal solution, 42 differential, for connective-tissue fi- brillae and reticulum, 128 Ehrlich's methylene-blue, for nervous tissues, 182 eosin, for blood-cells, 227 for adipose tissue, 130 for canalicular system in cartilage, 131 for mucin, 305 for sectionwork, 235 fuchsin-resorcin elastic fibers, 128 gold chlorid, for capsules of cartilage, J31 Heidenhain's iron, for bulk, 46 hemalum, 43 acid, 43 for bulk, 46 hematoxylin, Bohmer's, 42 for bulk, 46 Delafield's, 43 Ehrlich's, 43 for nuclei and granules, 228 for lime-salts in bone, 132 Friedlander's glycerin-, 43 hematoxylin-eosin, 45 hematoxylin-safranin, 46 hematoxylon, 42 Heidenhain's iron, 43 iodo-iodid of potassium, to demonstrate glycogen in cartilage, 131 magenta red, for connective tissue, 128 methylene-blue, for nerve-fibers, 184 methyl-green, 44 nuclear, 41 orcein, for connective tissue, 128 paracarmin, 42 for bulk, 46 Partsch's cochineal solution, 42 picric acid, 45 picrocarmin, Ranvier's, 44 Weigert's, 45 protoplasmic, 4 1 safranin, 44 selective, 41 Sudan III, for fat, 130 triple, 46 Stars, daughter, 374 Stellate cells of cerebellar cortex, 415 large, of cerebellar cortex, 416 of cerebral cortex, 417 of liver, 295 Stellular vasculosae, 453 Steno's ducts, 253 Stomach, 264, 266 blood-vessels of, 284 crypts of, 266 epithelium and secretory cells of, changes in, during secretion, 271 foveolae of, 266 Stomach, glands of, 267 cardiac, 267 fundus, 268 pyloric, 269 mucous membrane of, 266 fixation of, 305 muscularis mucosae of, 271 Stomach-pits, 266 Straight tubules of testes, 363 Stratified epithelium, 83. See also Epithe- lium, stratified. Stratum circulare, 477 of intestine, 266 corneum, 379, 381 fibrosum of intestine, 265 germinativum, 379 granulosum, 379 of ovary, 347 longitudinale of intestine, 266 lucidum, 381 technic for, 403 Malpighii, 379 technic of, 403 proprium of oral cavity, 236 radiatum, 477 spongioblasts, 464 submucosum of oral cavity, 236 Stria vascularis, 488 Striated muscle, nerve-endings in, Sih- ler's method of demonstrating, 184 muscle-fibers, technic of, 147 muscular tissue, blood-vessels in, 143 Striation of Baillarger, 421 of Bechtereff and Kaes, 421 of iris, 455 of ovary, 344 of red blood-corpuscles, 187 Subarachnoid space, 437 Subdural space, 437 Subepithelial plexus of cornea, 451 Sublingual gland, 255 Submaxillary gland, 258 Submucosa of intestine, 265 of oral cavity, 236 of urethra, 372 Substantia propria of cornea, 449 technic for, 474 Succus prostaticus, 370 Sudan III as stain for fat, 130 Sudoriparous glands, 396. See also Sweat- glands. Sulcus of matrix of nail, 394 spiralis internus, 487 Sulphuric acid as macerating solution, 23 Superficial plexus of cornea, 45 1 Superior nasal artery of retina, 466 vein of retina, 466 papillary artery of retina, 466 vein of retina, 466 Suprarenal capsule, demonstration of, 343 glands, 339 blood-vessels of, 341 nerves of, 342 Suspensory ligament of lens, 467 Sustentacular cells, 92, 250, 372, 483 526 INDEX. Sustentacular fiber 492 Sweat-glands, 396 capillaries of, 397 coiled portion of, 396 modified, 398 nerves of, 397 Sympathetic ganglia, 427 Syncytium, 97 development and differentiation, 98 TACTILE corpuscles, Meissner's, 387 technic of, 405 menisci, 387 technic of, 405 papilla?, 383 Tsenise coli, 266 semilunares, 282 Tannic acid, effect on red blood-cor- puscles, 189 Tapetum cellulosum, 453 fibrosum 453 Tarsal gland, 472 Taste-buds, 249 nerves in, demonstration of, 304 technic for, 303 Taste-pore, 250 Teasing, 2 1 Teeth, 238 adult, structure of. 238 auditory, 488 blood-vessels of, 242 development of, 243 method of studying, 303 medullated nerve-fibers of, 242 pulp of, 241 technic for, 303 Teichmann's crystals, 188 method of obtaining, 230 Tela submucosa, 236 Tellyesnicky's fluid, 26 Telodendria, 150, 162 Telolemma nuclei, 163 Telophases, 65, 70 Temperature, high, effect on tissues, 29 Temporal artery, inferior, of retina, 466 superior, of retina, 466 vein, inferior, of retina, 466 superior of retina, 466 Tendon, 105 and muscle, relation of, method of studying, 148 bundles, primary, 105 secondary, 105 cells from tail of rat, 107 fasciculi, 105 Tenon's capsule, 448 Terminal bronchioles, 314, 315 fibers of cerebral cotex, 420 ledges, 86 nodule of spermatosome, 361 Testes, 362 blood-vessels of, 367 convoluted tubules of, 363 examination of, 378 lymph-vessels of, 367 Tests, nerves of, 367 straight tubules of, 363 vasa efferentia of, 363, 365 Theca folliculi, 347 Third eyelid, 473 Thoma's ampullae, 204 Zwischenstiick, 204 Thoma-Zeiss hemocytometer, 232 Thread-granules, 60 Thrombocytes, 194 Thymus gland, 210 blood supply of, 212 Thyroid gland, 319 acini of, chief cells of, 320 colloid cells of, 320 blood supply of, 320 demonstration of, 322 nerves of, 320 granules, 149 Tissue, 79 adipose, 107 stain for, 130 connective, 96. See also Connective tissue. effect of high temperature on, 29 elastic, effect of trypsin digestion on, 127 method of obtaining, 127 epithelial, 80 erectile, 371 fibrous, elastic, 106 liver, technic of, 307 lymphoid, 196 mesenchymatous, 97 muscular, 134 destruction of, 144 development of, 144 heart, development of, 146 nerve-fibers ending in, 162 striated, blood-vessels in, 143 technic of, 147 nervous, 148 Ehrlich's methylene-blue stain for, 182 fixation of, 183 technic of, 180 ovarian, fixation of, 378 pulmonary, demonstration of, 322 reticular, demonstration of, 234 splenic, demonstration of, 234 Toison's fluid for diluting blood, 232 Tomes' granular layer, 246 processes, 244 Tongue, 247 lymph -follicles of, 251 mucous membrane of, 247 nerve supply of, 252 papillae of, 247 Tonsils, lymph-follicles of, 251 pharyngeal, 251 Trabeculae of liver, 290 of lymph-glands, 198 of spleen, 203 Trachea, 310 demonstration of, 322 nerves of, 311 INDEX. 527 Transitional epithelium, 85 leucocytes, 192 Triple stains, 46 Trophoplasts, 385 Trypsin digestion, effect on connective and elastic tissues, 127 Tubular glands, 89 coiled, 90 compound branched, 90 reticulated, 90 simple branched, 90 Tubules, convoluted, of testes, 363 dentinal, 240 intermediate, of pancreas, 300 of kidney, demonstration of, 342 straight collecting, of kidney, 323 of testes, 363 uriniferous, 323 membrana propria of, 330 Tubuli recti, 363 Tubulo-alveolar gland, 90 Tunica albuginea, 92, 344, 362 dartos, 384 externa of eye, 446 fibrosa of eye, 446, 448 interna of eye, 446, 457 mucosa of intestine, 265 propria of oral cavity, 236 sclerotica, 446, 448. See also Sclera. vaginalis, 362 vasculosa, 362 of eye, 446, 452 Tunics of eye, 446 Tunnel-fibers, 494 Tympanic investing layer, 489 membrane, 476 cutaneous layer of, 476 epidermis of, 476 lamina propria of, 477 mucous layer of, 478 Tympanum, 478 Tyson, glands of, 372 UNDECALCIFIED bone, microscopic prep- arations of, 131 Undulating membrane of spermatosome, 361 Unicellular glands, 87 Unna's orcein stain for connective tissue, 128 Ureter, 336 nerves of, 339 technic of, 343 Urethra, epithelium of, 371 submucosa of, 372 Urinary organs, 323 Uriniferous tubules, 323 membrana propria of, 330 Uterus, 355 blood supply of, 357 lymphatics of, 357 mucous membrane of, 355 muscular coat of, 356 nerves of, 358 Utriculosaccular duct, 481 Utriculus, 481, 482 dorsal, 496 nerve-fibers of, 483 wall of, 482 VACUOLE of ovum, 344 Vacuoles, 61 secretion, 291 Vagina, 358 mucous membrane of, 358 epithelium of, 358 muscular coat of, 359 nerves of, 360 vestibule of, epithelium of, 360 Valves, auriculoventricular, of heart, 213 of veins, 220 Valvulas conniventes, 265, 274 Van Gieson's acid fuchsin-picric acid solution, 45 Vas aberrans Halleri, 366 deferens, 367 epididymidis, 364, 366 spirale, 489 Vasa afferentia, 197 of kidney, 332 cfferentia, 197 of testes, 363, 365 recta spuria, 334 Vascular canals, 112 papillae, 383 supply of larynx, 310 system, 213 of skin, 386 tunic of eye, 446, 452 Vater-Pacinian corpuscles, 1 73 distribution of, 174 Veins, 219 central, of retina, 465 interlobular, of kidney, 334 of liver, 293 nasal, inferior, of retina, 466 superior, of retina, 466 papillary, inferior, of retina, 466 superior, of retina, 466 portal, 292 precapillary, 220 small, 220 temporal, inferior, of retina, 466 superior, of retina, 466 valves of, 220 Venae arciformes, 334 stellatae, 334 vorticosae, 452 Ventral sacculus, 496 Ventrolateral column, 408 Ventromesial column, 408 Venulae rectae, 334 Vermiform appendix, mucosa of, lymph- follicles of, 281 Vesicles, germinal, 71 optic, primary, 446 secondary, 447 Purkinje's, 344 seminal, 368 (Reissner's), 485, 489 INDEX. Vestibule of ear, 480 of nasal cavity, 498 of vagina, epithelium of, 360 Villi of mucous membrane of small in- testine, 274 of small intestine, lacteals of, 285 Virchow's bone corpuscles, method of isolating, 134 Visual cells, 458 Vitreous body, 446, 467 hyaloid membrane of, 467 membrane, 452, 453 Volkmann's canals, 115 vom Rath's solutions, 25 von Ebner's method of decalcification, 133 von Koch's technic for bone, 132 WAGNER'S spot, 344 Wandering cells, 60, 103, 104 Water, distilled, for fixing paraffin sections to slide, 39 effect on red blood-corpuscles, 188 Wax plates, 56 apparatus for making, 56 reconstruction by, 55 Bern's method, 56 cutting out parts to be recon- structed, and completing model, J 57. drawing apparatus, 56 serial sections, 56 Weigert's fuchsin-resorcin elastic fibers stain, 128 Weigert's methods for demonstration of medullary sheath, 440, 441 picrocarmin, 45 Wharton's ducts, 253, 254 jelly, 100 White blood-corpuscles, 191. See also Leucocytes. fibers, 99 fibrocartilage, no muscles, 141 rami communicantes, 429 fibers, 429, 456 substance of spinal cord, 406, 409 Wirsungian duct, 298 Wolffian duct, 360 Wright's method of staining blood films, 229 Wrisberg, cartilages of, 310 YELLOW bone-marrow, 207, 210 gelatin mass as injection fluid, 54 ZENKER'S fluid, 26 Zinn's arterial circle, 465 zonule, 467 Zona pellucida, origin of, 350 Zone, boundary, of choroid, 453 marginal, 8 1 Zonula ciliaris, 446, 467 Zonule of Zinn, 467 Zymogen, 255 granules in pancreas, demonstrating, 308 SAUNDERS' BOOKS on Pathology, Physiology Histology, Embryology and Bacteriology W. B. 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D., Professor of Pathology in Rush Medical College, Chicago. 172 colored figures on 77 lithographic plates, 36 text-cuts, many in colors, and 35 3 pages. Cloth, $5.00 net. In Saunders* Hand- Atlas Series. This new Atlas will be found even more valuable than the two preceding volumes on Special Pathologic Histology, to which, in a manner, it is a com- panion work. The text gives the generally accepted views in regard to the signifi- cance of pathologic processes, explained in clear and easily understood language. The lithographs in some cases required as many as twenty-six colors to reproduce the original painting. Dr. Hektoen has made many additions of great value. W. T. Councilman, M. D., Professor of Pathologic Anatomy, Harvard Univenity. " I have seen no plates which impress me as so truly representing histologic appearances as do these. The book is a valuable one." Howell's Physiology A Text=Book of Physiology. By WILLIAM H. HOWELL, PH.D., M. D., Professor of Physiology in the Johns Hopkins University, Balti- more, Md. Octavo of 1018 pages, 306 illustrations. Cloth, $4.00 net. THE NEW (4th) EDITION Dr. Howell has had many years of experience as a teacher of physiology in several of the leading medical schools, and is therefore exceedingly well fitted to write a text-book on this subject. Main emphasis has been laid upon those facts and views which will be directly helpful in the practical branches of medicine. At the same time, however, sufficient consideration has been given to the experimen- tal side of the science. The entire literature of physiology has been thoroughly digested by Dr. Howell, and the important views and conclusions introduced into his work. Illustrations have been most freely used. The Lancet, London " This is one of the best recent text-books on physiology, and we warmly commend it to the attention of students who desire to obtain by reading a general, all-round, yet concise survey of the scope, facts, theories, and speculations that make up its subject matter." SAUNDERS' BOOKS ON McFar land's Pathology A Text-Book of Pathology. By JOSEPH McFARLAND, M. D., Pro- fessor of Pathology and Bacteriology in the Medico-Chirurgical College of Philadelphia. Octavo of 856 pages, with 437 illustrations, many in colors. Cloth, $5.00 net; Half Morocco, $6.50 net. THE NEW (2d) EDITION You cannot successfully treat disease unless you have a practical, clinical knowledge of the pathologic changes produced by disease. For this purpose Dr. McFarland's work is well fitted. It was written with just such an end in view — to furnish a ready means of acquiring a thorough training in the subject, a training such as would be of daily help in your practice. For this edition every page has been gone over most carefully, correcting, omitting the obsolete, and adding the new. Some sections have been entirely rewritten. You will find it a book well worth consulting, for it is the work of an authority. St. Paul Medical Journal " It is safe to say that there are few who are better qualified to give a resume1 of the modern views on this subject than McFarland. The subject-matter is thoroughly up to date." Boston Medical and Surgical Journal " It contains a great mass of well-classified facts. One of the best sections is that on the special pathology of the blood." McFarland's Biology: Medical and General Biology: Medical and General — By JOSEPH MCFARLAND, M. D., Professor of Pathology and Bacteriology in the Medico-Chirurgical Col- lege of Phila. 1 2mo, 440 pages, 1 60 illustrations. Cloth, $1.75 net. ILLUSTRATED This work is both a general and medical biology. The former because it dis- cusses the peculiar nature and reactions of living substance generally; the latter because particular emphasis is laid on those subjects of special interest and value in the study and practice of medicine. The illustrations will be found of great assistance. Frederic P. Gorh&m, A. M., Brown University. " I am greatly pleased with it. Perhaps the highest praise which I can give the book is to say that it more nearly approaches the course I am now giving in general biology than any other work." BA CTERIOL OGY AND HISTOL OGY. McFarland's Pathogenic Bacteria The New (6th) Edition, Revised A Text-Book Upon the Pathogenic Bacteria. By JOSEPH McFAR- LAND, M. D., Professor of Pathology and Bacteriology in the Medico- Chirurgical College of Philadelphia, Pathologist to the Medico-Chirur- gical Hospital, Philadelphia, etc. Octavo volume of 709 pages, finely illustrated. Cloth, $3.50 net FULLY ILLUSTRATED This book gives a concise account of the technical procedures necessary in the study of bacteriology, a brief description of the life-history of the important patho- genic bacteria, and sufficient description of the pathologic lesions accompanying the micro-organismal invasions to give an idea of the origin of symptoms and the causes of death. The illustrations are mainly reproductions of the best the world affords, and are beautifully executed. In this edition the entire work has been practically rewritten, old matter eliminated, and much new matter inserted. H. B. Anderson, M. D., Professor of Pathology and Bacteriology, Trinity Medical College, Toronto. " The book is a satisfactory one, and I shall take pleasure in recommending it to the students of Trinity College." e The Lancet, London " It is excellently adapted for the medical students and practitioners for whom it is avowedly written. . . . The descriptions given are accurate and readable." Hill's Histology and Org'anog'raphy A Manual of Histology and Organography. By CHARLES HILL, M. D., formerly Assistant Professor of Histology and Embryology, Northwestern University, Chicago. I2mo of 468 pages, 337 illustra- tions. Flexible leather, $2.00 net. THE NEW (2dJ EDITION Dr. Hill's work is characterized by a completeness of discussion rarely met in a book of this size. Particular consideration is given the mouth and teeth. Pennsylvania Medical Journal " It is arranged in such a manner as to be easy of access and comprehension. To any contemplating the study of histology and organography we would commend this work." SAUNDERS' BOOKS ON GET A • THE NEW THE BEST /V HI C f 1 C <& II STANDARD Illustrated Dictionary New (6th) Edition, Entirely Reset The American Illustrated Medical Dictionary. A new and com- plete dictionary of the terms used in Medicine, Surgery, Dentistry, Pharmacy, Chemistry, Veterinary Science, Nursing, and kindred branches ; with over 100 new and elaborate tables and many handsome illustrations. By W. A. NEWMAN BORLAND, M.D., Editor of " The American Pocket Medical Dictionary." Large octavo, 986 pages, bound in full flexible leather. Price, $4.50 net ; with thumb index, $5.00 net IT DEFINES ALL THE NEW WORDS— IT IS UP TO DATE Dorland's Dictionary defines hundreds of the newest terms not defined in any other dictionary — bar none. These new terms are live, active words, taken right from modern medical literature. It gives the capitalization and pronunciation of all words. It makes a feature of the derivation or etymology of the words. In some dictionaries the etymology occupies only a secondary place, in many cases no derivation being given at all. In ' ' Dorland, ' ' practically every word is given its derivation. In "Dorland" every word has a separate paragraph, thus making it easy to find a word quickly. The tables of arteries, muscles, nerves, veins etc., are of the greatest help in assembling anatomic facts. In them are classified for quick study all the necessary information about the various structures. In "Dorland" every word is given its definition — a definition that defines in the fewest possible words. In some dictionaries hundreds of words are not defined at all, referring the reader to some other source for the information he wants at once. Howard A. Kelly, M. D., Johns Hopkins University, Baltimore " Dr. Dorland's dictionary is admirable. It is so well gotten up and of such convenient size. No errors have been found in my use of it." J. Collins Warren. M. D., LL.D., F.R.C.S. (Hon.), Harvard Medical School " I regard it as a valuable aid to my medical literary work. It is very complete and of convenient size to handle comfortably. I use it in preference to any other." PATHOLOGY. Stengel's Text-Book of Pathology The New (5th) Edition A Text-Book of Pathology. By ALFRED STENGEL, M. D., Professor of Medicine in the University of Pennsylvania. Octavo volume of 979 pages, with 400 text-illustrations, many in colors, and 7 full-page colored plates. Cloth, $5.00 net; Sheep or Half Morocco, $6.50 net. WITH 400 TEXT-CUTS. MANY IN COLORS, AND 7 COLORED PLATES In this work the practical application of pathologic facts to clinical medicine is considered more fully than is customary in works on pathology. While the subject of pathology is treated in the broadest way consistent with the size of the book, an effort has been made to present the subject from the point of view of the clinician. In the second part of the work the pathology of individual organs and tissues is treated systematically and quite fully under subheadings that clearly indicate the subject-matter to be found on each page. In this edition the section dealing with General Pathology has been most extensively revised, several of the important chapters having been practically rewritten. A very useful addition is an Appendix treating of th- technic of pathologic methods, giving briefly the most important methods at present in use for the study of pathology, including, however, only those methods capable of giving satisfactory results. The book will be found to maintain fully its popularity. PERSONAL AND PRESS OPINIONS William H. Welch. M. D.. Professor of Pathology, Johns Hopkins University, Baltimore, Md. " I consider the work abreast of modern pathology, and useful to both students and practi- tioners. It presents in a concise and well-considered form the essential facts of general and special pathologic anatomy, with more than usual emphasis upon pathologic physiology." Ludvig Hektoen, M. D.. Professor of Pathology, Rush Medical College, Chicago. " I regard it as the most serviceable text-book for students on this subject yet written by an American author." The Lancet, London " This volume is intended to present the subject of pathology in as practical a form as pos- sible, and more especially from the point of view of the 'clinical pathologist.' These subjects have been faithfully carried out, and a valuable text-book is the result. We can most favorably recommend it to our readers as a thoroughly practical work on clinical pathology." SAUNDERS' BOOKS ON Mallory and Wright's Pathologic Technique New (5th) Edition, Revised Pathologic Technique. A Practical Manual for Workers in Patho- logic Histology, including Directions for the Performance of Autopsies and for Clinical Diagnosis by Laboratory Methods. By FRANK B. MALLORY, M. D., Associate Professor of Pathology, Harvard Univer- sity ; and JAMES H. WRIGHT, M. D., Director of the Pathologic Labora- tory, Massachusetts General Hospital. Octavo of 500 pages, with 152 illustrations. Cloth, $3.00 net. WITH CHAPTERS ON POST-MORTEM TECHNIQUE AND AUTOPSIES In revising the book for the new edition the authors have kept in view the needs of the laboratory worker, whether student, practitioner, or pathologist, for a practical manual of histologic and bacteriologic methods in the study of patho- logic material. Many parts have been rewritten, many new methods have been added, and the number of illustrations has been considerably increased. Among the new matter are the following : Smith's staining method for encapsulated bacteria ; the antiformin method for detection and cultivation of tubercle bacilli ; Musgrave's and Clegg's method for the cultivation of amebas ; Wright's method for staining myelin sheaths in frozen sections ; Ghoreyeb's method for spirochetes ; Alzheimer's method for cytologic examination of cerebrospinal fluid ; Giemsa's new method for protozoa and bacteria in sections, and the Wassermann-Noguchi tests for syphilis. PERSONAL AND PRESS OPINIONS Wm. H. Welch, M. D., Professor of Pathology, Johns Hopkins University, Baltimore. " I have been looking forward to the publication of this book, and I am glad to say that I find it a most useful laboratory and post-mortem guide, full of practical information and well up to date." Boston Medical and Surgical Journal " This manual, since its first appearance, has been recognized as the standard guide in patho- logical technique, and has become well-nigh indispensable to the laboratory worker." Journal of the American Medical Association " One of the most complete works on the subject, and one which should be in the library «rf every physician who hopes to keep pace with the great advances made in pathology." EMBRYOLOGY. Heisler's Text-Book qf Embryology Third Edition A Text-Book of Embryology. By JOHN C. HEISLER, M.D., Pro- fessor of Anatomy in the Medico-Chirurgical College, Philadelphia. Octavo volume of 435 pages, with 212 illustrations, 32 of them in colors. Cloth, $3.00 net. WITH 212 ILLUSTRATIONS, 32 IN COLORS The fact of embryology having acquired in recent years such great interest in connection with the teaching and with the proper comprehension of human anatomy, it is of first importance to the student of medicine that a concise and yet sufficiently full text-book upon the subject be available. This new edition represents all the latest advances recently made in the science of embryology. Many portions have been entirely rewritten, and a great deal of new and impor- tant matter added. A number of new illustrations have also been introduced and these will prove very valuable. The previous editions of this work filled a gap most admirably, and this new edition will undoubtedly maintain the reputation already won. Heisler's Embryology has become a standard work. PERSONAL AND PRESS OPINIONS G. Carl Huber, M.D., Professor of Embryology at the Wistar Institute, University of Pennsylvania. " I find the second edition of ' A Text-Book of Embryology' by Dr. Heisler an improve- ment on the first. The figures added increase greatly the value of the work. I am again recommending it to our students." William Wathen, M. D., Professor of Obstetrics, Abdominal Surgery, and Gynecology, and Dean, Kentucky School of Medicine, Louisville, Ky, " It is systematic, scientific, full of simplicity, and just such a work as a medical student will be able to comprehend." Birmingham Medical Review, England " We can most confidently recommend Dr. Heisler's book to the student of biology or medicine for his careful study, if his aim be to acquire a sound and practical acquaintance with the subject of embryology." io SAUNDERS' BOOKS ON Wells* Chemical Pathology Chemical Pathology. — Being a Discussion of General Pathology from the Standpoint of the Chemical Processes Involved. By H. GIDEON WELLS, PH. D., M. D., Assistant Professor of Pathology in the University of Chicago. Octavo of 549 pages. Cloth, $3.25 net. A PRACTICAL BOOK Dr. Wells' work is written for the physician, for those engaged in research in pathology and physiologic chemistry, and for the medical student. In the intro- ductory chapter are discussed the chemistry and physics of the animal cell, giving the essential facts of ionization, diffusion, osmotic pressure, etc., and the relation of these facts to cellular activities. Special chapters are devoted to Diabetes and to Uric-acid Metabolism and Gout. Wm. H. Welch. M. D. Professor of Pathology, Johns Hopkins University. " The work fills a real need in the English literature of a very important subject, and I shall be glad to recommend it to my students." Lusk's Elements of Nutrition Elements of the Science of Nutrition. By GRAHAM LUSK, PH. D., Professor of Physiology at Cornell Medical School. Octavo volume of 302 pages. Cloth, $3.00 net. THE NEW (2d) EDITION— TRANSLATED INTO GERMAN Prof. Lusk presents the scientific foundations upon which rests our knowledge of nutrition and metabolism, both in health and in disease. There are special chapters on the metabolism of diabetes and fever, and on purin metabolism. The work will also prove valuable to students of animal dietetics at agricultural stations. Lewellys F. Barker, M. D. Professor of the Principles and Practice of Medicine, Johns Hopkins University. " I shall recommend it highly to my students. It is a comfort to have such a discussion of the subject in English." HISTOLOGY. ii Bohm, Davidoff, and Huber's Histology A Text=Book of Human Histology. Including Microscopic Tech- nic. By DR. A. A. BOHM and DR. M. VON DAVIDOFF, of Munich, and G. GARL HUBER, M.D., Professor of Embryology at the Wistar Insti- tute, University of Pennsylvania. Handsome octavo of 528 pages, with 361 beautiful original illustrations. Flexible cloth, $3.50 net. THE NEW (2d) EDITION, ENLARGED The work of Drs. Bohm and Davidoff is well known in the German edition, and has been considered one of the most practically useful books on the subject of Human Histology. This second edition has been in great part rewritten and very much enlarged by Dr. Huber, who has also added over one hundred origi- nal illustrations. Dr. Huber's extensive additions have rendered the work the most complete students' text-book on Histology in existence. Boston Medical and Surgical Journal " Is unquestionably a text-book of the first rank, having been carefully written by thorough masters of the subject, and in certain directions it is much superior to any other histological manual." DrewV Invertebrate Zoology A Laboratory Manual of Invertebrate Zoology. By OILMAN A. DREW, PH.D., Professor of Biology at the University of Maine. With the aid of Members of the Zoological Staff of Instructors of the Marine Biolog- ical Laboratory, Woods Holl, Mass, tamo of 200 pages. Cloth, #1.25 net. A LABORATORY WORK The subject is presented in a logical way, and the type method of study has been followed, as this method has been the prevailing one for many years. Prof. Allison A. Smyth, Jr., Virginia Polytechnic Institute " I think it is the best laboratory manual of zoology I have yet seen. The large number of forms dealt with makes the work applicable to almost any locality." » 12 SAUNDERS BOOKS ON Norris' Cardiac Pathology Studies in Cardiac Pathology. By GEORGE W. NORRIS, M.D., Associate in Medicine at the University of Pennsylvania. Large octavo of 235 pages, with 85 superb illustrations. Cloth, $5.00 net. SUPERB ILLUSTRATIONS The wide interest being manifested in heart lesions makes this book particu- larly opportune. The illustrations are superb and are faithful reproductions of the specimens photographed. Each illustration is accompanied by a detailed description ; besides, there is ample letter press supplementing the pictures. Considerable matter of a diagnostic and therapeutic nature has been interwoven. Boston Medical and Surgical Journal "The illustrations are arranged in such a way as to illustrate all the common and many of the rare cardiac lesions, and the accompanying descriptive text constitutes a fairly continuous didactic treatise." McConnell's Pathology A Manual of Pathology. By GUTHRIE McCoNNELL,M.D., Professor of Bacteriology and Pathology at Temple University, Philadelphia. I2mo of 523 pages, with 170 illustrations. Flexible leather, $2.50 net. NEW (2d) EDITION Dr. McConnell has discussed his subject with a clearness and precision of style that make the work of great assistance to both student and practitioner. The illustrations have been introduced for their practical value. New York State Journal of Medicine " The book treats the subject of pathology with a thoroughness lacking in many works of greater pretension. The illustrations — many ef them original — are profuse and of exceptional excellence." Hektoen and Riesman's Pathology AMERICAN TEXT-BOOK OF PATHOLOGY. Edited by LUDVIG HEK- TOEN, M.D., Professor of Pathology, Rush Medical College, Chi- cago; and DAVID RIESMAN, M.D., Professor of Clinical Medicine, Philadelphia Polyclinic. Octavo of 1245 pages, ^43 illustra- tions, 66 in colors. Cloth, $7.50 net ; Half Morocco, $9.00 net. HISTOLOGY. 13 Diirck anc Hektoen's Special Pathologic Histology Atlas and Epitome of Special Pathologic Histology. By DR. H. DURCK, of Munich. Edited, with additions, by LUDVIG HEKTOEN, M. D., Professor of Pathology, Rush Medical College, Chicago. In two parts. Part I. — Circulatory, Respiratory, and Gastro-intestinal Tracts. 120 colored figures on 62 plates, and 158 pages of text. Part II. — Liver, Urinary and Sexual Organs, Nervous System, Skin, Muscles, and Bones. 123 colored figures on 60 plates, and 192 pages of text. Per part : Cloth, $3.00 net. In Saunders' Hand- Atlas Series. The great value of these plates is that they represent in the exact colors the effect of the stains, which is of such great importance for the differentiation of tissue. The text portion of the book is admirable, and, while brief, it is entirely satisfac- tory in that the leading facts are stated, and so stated that the reader feels he has grasped the subject extensively. William H. Welch, M. D.. Professor of Pathology, Johns Hopkins University, Baltimore. "I consider Diirck's 'Atlas of Special Pathologic Histology,' edited by Hektoen, a very useful book for students and others. The plates are admirable." Sobotta and Htiber's Human Histology Atlas and Epitome of Human Histology. By PRIVATDOCENT DR. J. SOBOTTA, of Wiirzburg. Edited, with additions, by G. CARL HUBER, M. D., Professor of Histology and Embryology in the University of Michigan, Ann Arbor. With 214 colored figures on 80 plates, 68 text-illustrations, and 248 pages of text. Cloth, $4.50 net. In Saunders' Hand-Atlas Series. INCLUDING MICROSCOPIC ANATOMY The work combines an abundance of well-chosen and most accurate illustra- tions, with a concise text, and in such a manner as to make it both atlas and text- book. The great majority of the illustrations were made from sections prepared from human tissues, and always from fresh and in every respect normal specimens. The colored lithographic plates have been produced with the aid of over thirty colors. Boston Medical and Surgical Journal " In color and proportion they are characterized by gratifying accuracy and lithographic beauty." 14 SAUNDERS1 BOOKS ON Bosanquet on Spirochaetes Spi rochretes : A Review of Recent Work, with Some Original Ob- servations. By W. CECIL BOSANQUET, M.D., Fellow of the Royal Col- lege of Physicians, London. Octavo of 1 52 pages, illustrated. $2.50 net. ILLUSTRATED This is a complete and authoritative monograph on the spirochaetes, giving morphology, pathogenesis, classification, staining, etc. Pseudospirochaetes are also considered, and the entire text well illustrated. The high standing of Dr. Bosanquet in this field of study makes this new work particularly valuable. Levy and Klemperer's Clinical Bacteriology The Elements of Clinical Bacteriology. By DRS. ERNST LEVY and FELIX KLEMPERER, of the University of Strasburg. Translated and edited by AUGUSTUS A. ESHNER, M. D., Professor of Clinical Medicine, Philadelphia Polyclinic. Octavo volume of 440 pages, fully illustrated. Cloth, $2.50 net. S. Solis-Cohen, M. D., Professor of Clinical Medicine, Jefferson Medical College, Philadelphia. " I consider it an excellent book. I have recommended it in speaking to my students." Lehmann, Neumann, and Weaver's Bacteriology Atlas and Epitome of Bacteriology : INCLUDING A TEXT-BOOK OF SPECIAL BACTERIOLOGIC DIAGNOSIS. By PROF. DR. K. B. LEHMANN and DR. R. O. NEUMANN, of Wurzburg. From the Second Revised and Enlarged German Edition. Edited, with additions, by G. H. WEAVER, M. D., Assistant Professor of Pathology and Bacteriology, Rush Medical College, Chicago. In two parts. Part I. — 632 colored figures on 69 lithographic plates. Part II. — 5 1 1 pages of text, illustrated. Per part: Cloth, $2.50 net. In Saunders1 Hand-Atlas Series. PATHOLOGY, BACTERIOLOGY, AND PATHOLOGY. 15 Eyre's Bacteriologic Technique THE ELEMENTS OF BACTERIOLOGIC TECHNIQUE. A Laboratory Guide for the Medical, Dental, and Technical Student. By J. W. H. EYRE, M. D., F. R. S. Edin., Lecturer on Bacteriology at the Medical and Dental Schools, London. Octavo of 375 pages, with 170 illustrations. Cloth, $2.50 net. American Text-Book of Physiology second Edition AMERICAN TEXT-BOOK OF PHYSIOLOGY. In two volumes. Edited by WILLIAM H. HOWELL, PH. D., M.D., Professor of Physiology in the Johns Hopkins University, Baltimore, Md. Two royal octavos of about 600 pages each, illustrated. Per volume: Cloth, $3.00 net; Half Morocco, $4.25 net. " The work will stand as a work of reference on physiology. To him who desires to know the status of modern physiology, who expects to obtain suggestions as to further physio- logic inquiry, we know of none in English which so eminently meets such a demand." — The Medical News. Warren's Pathology and Therapeutics second Edition SURGICAL PATHOLOGY AND THERAPEUTICS. By JOHN COLLINS WARREN, M. D., LL.D., F. R. C. S. (Hon.), Professor of Surgery, Harvard Med- ical School. Octavo, 873 pages, 136 relief and lithographic illustrations, 33 in colors. With an Appendix on Scientific Aids to Surgical Diagnosis and a series of articles on Regional Bacteriology. Cloth, $5.00 net; Half Morocco, $6.50 net. Gorham's Bacteriology A LABORATORY COURSE IN BACTERIOLOGY. For the Use of Medical, Agricultural, and Industrial Students. By FREDERIC P. GORHAM, A. M., Associate Professor of Biology in Brown University, Providence, R. I., etc. lamo of 192 pages, with 97 illustrations. Cloth, $1.25 net. " One of the best students' laboratory guides to the study of bacteriology on the mar- ket. . . . The technic is thoroughly modern and amply sufficient for all practical pur- poses."— American Journal of the Medical Sciences. Raymond's Physiology New (3d) ^^^ HUMAN PHYSIOLOGY. By JOSEPH H. RAYMOND, A. M., M. D., Pro- fessor of Physiology and Hygiene, Long Island College Hospital, New York. Octavo of 685 pages, with 444 illustrations. Cloth, $3.50 net. "The book is well gotten up and well printed, and may be regarded as a trustworthy guide for the student and a useful work of reference for the genera! practitioner. The illustrations are numerous and are well executed." — The Lancet, London. 16 BACTERIOLOGY, PHYSIOLOGY, AND HISTOLOGY. Ball's Bacteriology Sixth Edition, Revised ESSENTIALS OF BACTERIOLOGY : being a concise and systematic intro- duction to the Study of Micro-organisms. By M. V. BALL, M. D., Late Bacteriologist to St. Agnes' Hospital, Philadelphia. i2mo of 289 pages, with 135 illustrations, some in colors. Cloth, $1.00 net. In Saunders* Question- Compend Series. " The technic with regard to media, staining, mounting, and the like is culled from the latest authoritative works." — The Medical Times, New York. Budgett's Physiology New od) Edition ESSENTIALS OF PHYSIOLOGY. Prepared especially for Students of Medi- cine, and arranged with questions following each chapter. By SIDNEY P. BUDGETT, M. D., formerly Professor of Physiology, Washington Uni- versity, St. Louis. Revised by HAVAN EMERSON, M. D., Demonstrator of Physiology, Columbia University. i2mo volume of 250 pages, illus- trated. Cloth, $ i . oo net. Saundcrs1 Question- Compend Series. "He has an excellent conception of his subject. . . It is one of the most satisfactory books of this class" — University of Pennsylvania Medical Bulletin. Leroy's Histology New (4th) Edition ESSENTIALS OF HISTOLOGY. By Louis LEROY, M. D., Professor of Histology and Pathology, Vanderbilt University, Nashville, Tennessee. i2mo, 263 pages, with 92 original illustrations. Cloth, $1.00 net. In Saunders1 Question- Compend Series. " The work in its present form stands as a model of what a student's aid should be ; and we unhesitatingly say that the practitioner as well would find a glance through the book of lasting benefit." — The Medical World, Philadelphia. Barton and Wells' Medical Thesaurus A THESAURUS OF MEDICAL WORDS AND PHRASES. By WILFRED M. BARTON, M. D., Assistant Professor of Materia Medica and Therapeutics, and WALTER A. WELLS, M.D., Demonstrator of Laryngology, Georgetown University, Washington, D. C. i2ino, 534 pages. Flexible leather, $2.50 net; thumb indexed, $3.00 net. American Pocket Dictionary New (7th) Edition DORLAND'S POCKET MEDICAL DICTIONARY. Edited by W. A. NEW- MAN DORLAND, M. D., Editor "American Illustrated Medical Dic- tionary." Containing the pronunciation and definition of the principal words used in medicine and kindred sciences, with 64 extensive tables. 610 pages. Flexible leather, with gold edges, #1.00 net; with patent thumb index, $1.25 net. " I can recommend it to our students without reserve." — T. H. HOLLAND M D of the Jefferson Medical College, Philadelphia.