^ k Ppk^ 1SK8 * K »?, m m - W> C.V 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 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, NEW YORK, LONDON . B. SAUNDERS & COMPANY 1904 Set Up, Printed, and Copyrighted October, 1900. Reprinted July, 1901. Revised, Reprinted, and Recopyrighted, August, 1904. Copyright, 1904, by W. B. Saunders & Company. Registered at Stationers' Hall, London, England. PRESS OF W. B. SAUNDER8 & COMPANY TO THEIR TEACHER PROFESSOR C VON KUPFFER THIS BOOK IS DEDICATED BY THE GRATEFUL AUTHORS 258251 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, August, i go 4. EDITOR'S PREFACE TO THE FIRST EDITION. THE " Text-book of Histology " by Bohm and v. Davidoff, as stated by 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 IL 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 O 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 100 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 186 Formation of Blood 186 Red Blood-corpuscles 187 White Blood-corpuscles I91 Blood Platelets— Thrombocytes 194 Behavior of Blood-cells in the Blood Current I96 Lymphoid Tissue, Lymph-nodules, and Lymph-glands - 196 Spleen 202 Bone-marrow 2O7 Thymus Gland 2I° IL CIRCULATORY SYSTEM. Vascular System 2I3 Heart 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. IIL 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) 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 VL THE SKIN AND ITS APPENDAGES. Skin (Cutis) 370, Hair _ ^Q Nails g* 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 VIIL 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 Retinae, and Pars Iridica Retinae 461 Muller'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 4°7 Fetal Blood-vessels of the Eye 4°8 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 4?8 Internal Ear 480 Utriculus and Sacculus 4^2 Semicircular Canals 4^3 Cochlea 4^4 Organ of Corti 4§9 Development of the Labyrinth 49^ 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 11. Cylindric ciliated cells from the primitive kidney of Petromyzon 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. Process of 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 v 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 nuchse 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 IO2 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 111 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 no. 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 1 18. 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) .... 161 123. Cross-section through a peripheral nerve IDI 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 X39- 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 ... .188 153. 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 206 166. 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 225 179. Thoma-Zeiss hemocytometer 232 1 80. 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 rnolar from (he 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 266 208. 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 2 1 6. 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) 3°2 245. 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 3X3 250. Internal surface of human respiratory bronchiole (Kolliker) 3I4 251. Inner surface of human alveolus, showing respiratory epithelium (Kolliker) . 315 252. Respiratory epithelium in amphibia 3*6 253. 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 3*8 257. Cross-section of thyroid gland of man (Huber) 3*9 258. Section from parathyroid of man (Huber) 321 259. Kidney of new-born infant 323 260. Isolated uriniferous tubules 324 ILLUSTRATIONS. I 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 32^ 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 (Ruckert) 351 283. Scheme of the development and maturation of an ascaris ovum (Boveri) . . 352 284. Section of oviduct of young woman 355 285. Section from uterus of young woman 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. vSection 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 Born'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 Born's wax-plate method 398 l6 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 (Nagelj 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 Purkinje 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 43 1 347. 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 47 J 365. Meibomian or tarsal gland, reconstructed after Born' 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) 49° 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. I. 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 o way that by revolving the disc the apertures may be brought one 2 17 i8 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 a1 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. 1 9 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 two- 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 and 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 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 (a2 b2), and the projection of the retinal image into the field of vision as the virtual image (bz a3).— (Fig 21, Gage, The Microscope, eighth edition. ) eye. 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 rrficroscopist 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, 0.3 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 (95%) witn 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 \o/0 to i % osmic acid. Useful for macerating epithelia. 2. Dilute solutions of chromic acid, \c/c 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 2oc/c 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. Carnoy's Acetic-alcohol 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. Carnoy'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 ic/c 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 % aqueous solution . . lo parts. Chromic acid, I cfo aqueous solution ... 25 " Glacial acetic acid, \cfo 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 cfo aqueous solution . . .15 " Glacial acetic acid I part. FoPs Solution. — Fol has recommended the following modification of Flemming 's solution : Osmic acid, I $ aqueous solution .... 2 parts. Chromic acid, \c/0 aqueous solution ... 25 " Glacial acetic acid, 2.c/0 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 80^ for the same length of time, and then preserved in 90% 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 70%, 80% 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 io^ 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 75% 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^ 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 "fee-fa 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 90% 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 Muller's fluid. Potassium bichromate 3 Sm- 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. 2J 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 °/c 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 ^ 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. Fltered 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. S.-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. 2Q 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 wrhich 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. I 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 t Alcohol -xylol mixture t —Xylol -*r— t Xylol -paraffin (cold) t Xylol -paraffin (in paraffin oven) t Soft paraffin •«£ t 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 IO 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- AMETER. Absolute alcohol . . . 2 6 24 For a longer or Xylol K 3 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. CELLOID1N. 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. 3! The hardened tissues are cut into small pieces, which should not be much more than y% 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 80% alcohol, where it remains until the celloidin is hard, or until it is desired to cut sections. 3 2 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 t Thick celloidin solution t Imbedding t 80% alcohol 3. CELLOIDIN-PARAFF1N. 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 to 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 (a), and a slide (£) which supports a block (V), to which is fastened a knife by means of a thumb- screw (X). 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 (Wro °f an inch) ; 4 notches, 20 microns (yjVir °f an inch), 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 (or 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 y1^ 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 Soc/G 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 80 yc 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. \. 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. 3Q 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 (yid. 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 40 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 10° 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 logm. 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 gendy 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 (/c 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 i% 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% 100 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 overstaming (P. Mayer, 92). Czocor's Cochineal Solution. — 7 gm. of powdered cochineal and 7 gm. of roasted alum are kept suspended in 100 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 €/c 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). — 100 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. Hematoxylin. — 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 100 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 Absolute alcohol 25 c.c. Ammonia alum, saturated aqueous solution 400 " Alcohol, 95^ loo " Glycerin loo " 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 100 c.c. Distilled water 100 " Glycerin 100 " 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 \ saturated with .... 60 " Distilled water / 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 Carnoy's fluid. Very thin sections (in case of amniota not over 4^) are fixed to the slide with water and put, into a 2.5% 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 */$ 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% 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 % to 2 % aqueous solution of eosin or in a i% 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 % 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. Hematoxylirusafranin 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. Biondi-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 modificaton 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 y2 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% 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% aclueous solution of hematoxylin ; transfer for an equal length of time to a 0.5% 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. In Sections. 90% alcohol Celloidin sections Paraffin sections in 90 % alcohol 1 X' Remove paraffin Water 1 Absolute alcohol Distilled 4- ;i r water 90 % alcohol x Stain,. f 4* ,^ > \ /Distilled water Wash in water Wash in acid alcohol N^ / 4- 1 X'-Jnh/ •&• jt Y Ti£ 70^ alcohol 70^ alcohol Stain Absolute alcohol Wash in water 4- Alcohol s Absolute alcohol Wash in acid alcohol 4- 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 0.5% 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 : Cohnheim1 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 °/c 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 ic/c 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 r/c 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 Lowit' 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 c/c 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 \c/c arseni- ous acid in the sunlight. METHODS OF IMPREGNATION. 49 Golgi's Chromsilver or Chromsublimate Method. — This method depends on the formation of a very fine precipitate, which forms in cer- tain tissue elements or in preexisting space, 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 MUller's fluid, and increased the percentage of bichromate on changing the fluid (up to 4 %}. 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 \% 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 MUller'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 MUller'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 (if0 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 O.66f0 and the quantity about 200 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 12.5% 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 alum 20 gm., ammonium sulpho- cyanid IO gm., sodium chlorid 40 gm., and water IOOO gm. (the mixture must stand for eight days and then be filtered) ; (b) a \(fc gold chlorid solution. The specimen is placed for a few minutes in a solution composed of 60 c.c. of a and 7 c.c. of b, washed again in distilled water, dehydrated with alcohol, and mounted in Canada balsam. After toning and washing, the sections may still be stained. Golgi's methods are extremely inconstant in their results. When successful, how- ever, only a few elements are blackened each time, an advantage not to be underesti- mated ; for if all nerves should stain equally well, discrimination between the various elements in the preparation would be very difficult. Neither are the same structures always impregnated ; sometimes it is the ganglion cells and fibers, at other times the neu- rogliar cells, and occasionally only the vessels. After the foregoing explanation of Golgi's methods as applied by himself, we shall append a description of these methods as modified and employed at the present time (Ramon y Cajal, Kolliker, von Lenhossek and others). Golgi's methods are classified as the slow, the mixed, and the rapid. The slow method requires a preliminary treatment. Pieces of tis- sue from i to 2 cm. in diameter are placed for from three to five weeks in a 2 c/c potassium bichromate solution ; they are then transferred for from twenty-four to forty -eight hours to a 0.75% silver nitrate solution, or for a much longer time to a 0.5% solution of corrosive sublimate. In the mixed method the specimens are allowed to remain for four or five days in a 2 % aqueous potassium bichromate solution ; then for from twenty -four to thirty hours in a mixture consisting of i% osmic acid i vol., and 2% potassium bichromate solution 4 vols. They are then treated with a 0.75% silver nitrate solution for one or two days. When the rapid method is employed, the specimens are immedi- ately placed in a mixture consisting of i vol. of i % osmic acid and 4 vols. of a 3.5% potassium bichromate solution, and, finally, for one or two days in a 0.75% silver nitrate solution, to every 200 c.c. of which one drop of formic acid has been added. When employing these methods, and more particularly the one last described (which seems to be the most efficient), the following conditions must be carefully observed : If possible, the material should be absolutely METHODS OF IMPREGNATION. 51 fresh, the specimens must not exceed 3 or 4 mm. in thickness, and for every piece of tissue treated about 10 c.c. of the osmium-potassium bichromate mixture should be employed, the specimens remaining in the latter (in the dark) at a temperature of 25° C. for a length of time vary- ing according to the result desired (two or three days for the neurogliar cells, from three to five days for the ganglion cells, and from five to seven days for the nerve-fibers of the spinal cord). The objects are now dried with blotting-paper or washed quickly in distilled water and then placed for two or three days in a 0.75% silver nitrate solution at room-tempera- ture. In this they may remain for four or five days without damage, but not longer, as otherwise the precipitate becomes markedly granular (vid. v. Lenhossek, 92). If Golgi's method be unsuccessful (this applies to all its modifica- tions), the preparations may be transferred from the silver nitrate solu- tion back into a potassium bichromate-osmic acid mixture containing less osmic acid, in which they remain several days, and are then again placed in the silver nitrate solution for from twenty-four to forty-eight hours. This procedure may even be repeated. Cox obtains a precipitate in both cells and fibers by treating small pieces of the central nervous organs with a mixture composed of potassium bichromate 20 parts, 5% corrosive sublimate 20 parts, distilled water 30 to 40 parts, and 5% potassium chromate of strong alkaline reaction 16 parts. The specimens remain in this mixture from one to three months, according to the temperature, and are then further treated according to Golgi's method. As the chrome-silver preparations are not permanent, and can not, therefore, be subsequently stained, Kallius has suggested that the chrome- silver precipitate be reduced to metallic silver by treatment with the " quintuple hydroquinon developer" (hydroquinon 5 gm., sodium sulphite 40 gm., potassium carbonate 75 gm., and distilled water 250 gm. ). For this purpose 20 c.c. of the solution are diluted with 230 c.c. of distilled water ; this mixture may be preserved in the dark for some time if desired. Before using this latter solution, it should be mixed with y^t or at the most ^, of its volume of absolute alcohol. The sections are placed in a watch-crystal containing some of the latter mixture until they turn black (a few minutes). As soon as the silver salt is completely reduced, the sections are placed for from ten to fifteen minutes in 70% alcohol, then for five minutes in a 20% solution of sodium hyposulphite and, finally, washed for some time in distilled water, after which they may be stained, and even treated with acid alcohol and potassium hydrate. The following simple method for permanently mounting Golgi prepar- ations under a cover-glass has been recommended by Huber. After impregnation with chrome-silver the tissues are hastily dehy- drated, imbedded in celloidin, and cut in sections varying from 25 jj. to 100 fi in thickness. The sections are then dehydrated and placed for from ten to fifteen minutes in creosote, from which they are carried into xylol, where they remain another ten minutes. The sections are then removed to the slide. The xylol is then removed by pressing sev- eral layers of filter-paper over the section. On removing the filter-paper the sections are quickly covered by a large drop of xylol balsam and the slide is carefully heated over a flame for from three to five minutes. Be- 52 THE MICROSCOPIC PREPARATION. fore the balsam cools the preparation is covered with a large cover-glass, warmed by passing several times through the flame. Kopsch (96) places specimens in a solution composed of 10 c.c. of formalin (40% formaldehyd) and 40 c.c. of a 3.5% solution of potas- sium bichromate. For objects 2 c.c. in size 50 c.c. of the fluid are em- ployed ; but if the specimens be large, the mixture must be changed in twelve hours. At the end of twenty -four hours this fluid is replaced by a fresh 3.5% potassium bichromate solution, and the specimens are then transferred to a 0.75% solution of silver nitrate (after two days, if the tissue be the liver or stomach ; and after from three to six days, if retina or central nervous system). After this treatment the objects are car- ried over into 40% alcohol and, finally, into absolute alcohol, imbedded as rapidly as possible, and cut. The sections are mounted in balsam without a cover-glass. PREPARATION OF PERMANENT SPECIMENS, The resinous media used in the final mounting of preparations are Canada balsam and damar. Canada Balsam. — Commercial Canada balsam is usually dissolved in turpentine ; it should be slowly evaporated in a casserole and then dissolved in xylol, toluol, or chloroform, etc. The proper concentration of the solu- tion is found with a little experience. A thick solution penetrates the interstices of the section with difficulty, and usually contains air-bubbles which often hide the best areas of the preparation, and can only be re- moved with difficulty by heating over a flame. Thin solutions, on the other hand, have also their disadvantages ; they evaporate very quickly, and the empty space thus created between the cover-slip and slide must again be filled with Canada balsam. This is best done by dipping a glass rod into the solution and placing one drop at the edge of the cover-slip, whereupon the fluid spreads out between the cover-slip and slide as a result of capillary attraction. Canada balsam dries rather slowly, the rapidity of the process depending upon the temperature of the room. To dry quickly, the slides may be held for a few moments over a gas or alcohol flame, or they may be placed in a warm oven, where the prepara- tions become so dry in twenty-four hours that they can be examined with an oil -immersion lens. The oil used for this purpose should be wiped away from the cover-slip after examination. This can only be done, with- out moving the cover-slip, when the balsam is thoroughly dry and holds the cover-slip firmly in place. Damar. — Damar is dissolved preferably in equal parts of oil of tur- pentine and benzin. It has the advantage of not rendering the prepara- tion as translucent as Canada balsam. Otherwise it is used as the latter. Clearing Fluids. — Since alcohol does not mix with Canada balsam or damar, an intermediate or clearing fluid is used in transferring objects from the former into the latter. Xylol, toluol, carbol-xylol (xylol, 3 parts; carbolic acid, i part), oil of bergamot, oil of cloves, and oil of origanum are ordinarily used. The process is somewhat simpler where sections are fixed to the slide. Xylol is dropped onto the surface of the slide, or better, the whole preparation is placed for a few minutes in a vessel containing xylol until the diffusion currents have ceased (which may be seen with the naked eye). The slide is then taken out, tilted to allow the xylol to run off, wiped dry around the object with a cloth, and placed upon the METHODS OF INJECTION. 53 table with the specimen upward. A drop of Canada balsam is now placed on the section (usually on its left side), and a clean cover-slip grasped with a small forceps. It is then gently lowered in such a way that the Canada balsam spreads out evenly and no air-bubbles are im- prisoned under the glass. When this is done the preparation is finished. If one is dealing with loose sections, a spatula or section-lifter is very useful in transferring them from absolute alcohol into the clearing fluid — carbol-xylol or bergamot oil (xylol evaporates very rapidly) — and from this onto the slide. In doing this it is necessary that the sec- tion should lie well spread out on the section -lifter, wrinkles being re- moved with a needle or small camel' s-hair brush. In sliding the section off the spatula (with a needle or brush) a small quantity of the clearing fluid is also brought onto the slide. This must be removed as far as possible by tilting or with blotting-paper. The section can now be mounted in Canada balsam as before. For esthetic and practical reasons the student should see that during the spreading of the drop of Canada balsam the section remains under the middle of the cover-slip. Should it float to the edge, it is best to raise the cover-slip and lower it into place again. The cover-slip should never be slid over the specimen. Glycerin. — To mount in glycerin the sections are transferred from water to the slide, covered with a drop of glycerin and the cover-slip ap- plied. This method is employed in mounting sections colored with a stain that would be injured by contact with alcohol, and where clearing is not especially necessary. Farrant's Gum Glycerin. In place of pure glycerin the following mixture may be used : Glycerin 50 c.c. Water 50 " Gum-arabic (powder) 50 gm. Arsenious acid i " Dissolve the arsenious acid in water. Place the gum-arabic in a glass mortar and mix it with the water; then add the glycerin. Filter through a wet filter-paper or through fine muslin. To preserve such preparations for any length of time the cover- glasses must be so fixed as to shut off the glycerin or acetate of potash from the air. For this purpose cements or varnishes are employed which are painted over the edges of the cover-slip. These masses adhere to the glass, harden, and fasten the cover-slip firmly to the slide, hermetically sealing the object. The best of these is probably Kronig's varnish, pre- pared as follows : 2 parts of wax are melted and 7 to 9 parts of colophonium stirred in, and the mass filtered hot. Before employing an oil-immersion lens it is advisable to paint the edge with an alcoholic solution of shellac. METHODS OF INJECTION. The process of injection consists in filling the blood- and lymph-ves- sels with colored masses in order to bring out clearly their relation to the neighboring tissue elements. The instruments required are a syringe of suitable size or a constant pressure apparatus and cannulas of various sizes. Serviceable and instructive injections of blood-vessels are readily made ; good injections require skill, experience, and patience. Injection masses may be classed under two heads — cold injection masses and warm injection 54 THE MICROSCOPIC PREPARATION. masses. The vehicle of the latter is most generally gelatin. For inject- ing the blood-vessels either the cold or the warm masses may be employed, although the latter gives better results. The cold masses are to be used for injecting the lymphatic vessels. In injecting the blood-vessels it is well to wash out the vessels with warm normal salt solution before the in- jection mass is forced into the vessels. The following masses may be recommended : Gelatin=carmin. — The first is a gelatin -carmin mass, and is prepared as follows: (i) 4 gm. of carmin are stirred into 8 c.c. of water and thoroughly ground. Into this a sufficient quantity of ammonia is poured to produce a dark cherry color and render the whole transpar- ent. (2) 50 gm. of finest quality gelatin is placed in distilled water for twelve hours until well soaked. It is then pressed out by hand and melted at a temperature of 70° C. in a porcelain evaporating dish. The two solutions are now slowly mixed, the whole being constantly stirred until a complete and homogeneous mixture is obtained. To this mass is added, drop by drop, a 25% acetic acid solution until the color begins to change to a brick red and the mass becomes slightly opaque. This should be very carefully done, as a single drop too much may spoil the whole. During this procedure the substance should be kept at 70° C. and constantly stirred. The change in color indicates that the reaction of the mass has become neutral or even slightly acid (an ammoniac solu- tion should not be used, since the stain diffuses through the wall of the vessel and colors the surrounding tissues); the whole is filtered through flannel while still warm. As this mass hardens on cooling it is injected warm. The instruments used are also warmed before the injection is begun. Gelatin-Berlin Blue. — One part of oxalic acid is powdered in a mortar; to this is added one part of Berlin blue and 12 parts of water. Stir and rub until a solution is obtained. Prepare a gelatin vehicle as directed in the preceding paragraph ; to 1 2 parts of the gelatin mass add slowly while stirring 12 parts of the Berlin blue solution. The whole is filtered through flannel while still warm. Yellow Gelatin Mass (Hoyer). — Prepare a gelatin vehicle consist- ing of i part of gelatin and 4 parts of distilled water ; a cold, saturated solution of bichromate of potassium and a cold, saturated solution of lead acetate. Take equal volumes of each. Add the bichromate of potassium solution to the gelatin and heat almost to boiling; then add slowly, while stirring, the lead acetate solution. Carmin Mass, Cold (Kollmann). — One gm. of carmin is dissolved in a small quantity of ammonium hydrate and 20 c.c. of glycerin added. To another 20 c.c. of glycerin there is added 20 drops of hydrochloric acid and this added to the glycerin -carmin mixture while stirring. Saturated aqueous solutions of Berlin blue or a Prussian blue may also be used for cold injections. Injection masses already prepared are to be had in commerce. Besides those already mentioned, still others colored with China ink, etc., are in general use. Small animals are injected as a whole by passing the cannula of a syringe into the left ventricle or aorta. In the case of large animals, or where very delicate injections are to be made, the cannula is inserted into one of the vessels of the respective organs. The proper ligation of the remaining vessels should not be omitted. RECONSTRUCTION BY MEANS OF WAX PLATES. 55 Organs injected with carmin are fixed in alcohol and should not be brought in contact with acids or alkalies. Such parts as are injected with Berlin blue are less sensitive in their after-treatment. Pieces or sec- tions that have become pale regain their blue color in oil of cloves. If objects or sections injected with Berlin blue be treated with a solution of palladium chlorid, the bluish color changes to a dark brown which afterward remains unchanged (Kupffer). By means of the above injection methods other lumina can be filled, as, for instance, those of the glands. As a rule, these are only par- tially filled, since they end blindly, and their walls are less resistant and may be damaged by the pressure produced by the injection. Silver Nitrate. — In thin membranes and sections the vessel-walls can be rendered distinct by silver-impregnation, which brings out the out- lines of their endothelial cells. This may be done either by injecting the vessel with a i % solution of silver nitrate, or, according to the process of Chrzonszczewsky, with a 0.25% solution of silver nitrate in gelatin. This method is of advantage, since, after hardening, the capillaries of the injected tissue appear slightly distended. Organs thus treated can be sectioned, but the endothelial mosaic of the vessels does not appear defi- nitely until the sections have been exposed to sunlight. The injecting of lymph -channels, lymph -vessels, and lymph- spaces is usually done by puncture. A pointed cannula is thrust into the tissue and the syringe emptied by a slight but constant pressure. The injected fluid spreads by means of the channels offering the least resist- ance. For this purpose it is best to employ aqueous solutions of Berlin blue or silver nitrate, as the thicker gelatin solutions cause tearing of the tissues. Altman's Process. — To bring out the blood capillaries and the lymphatic channels, Altman's process (79), in which the vessels are in- jected with olive oil, is useful. The objects are then treated with osmic acid, sectioned by means of a freezing microtome and finally treated with eau de Javelle (a concentrated solution of hypochlorite of potassium). By this process all the tissues are eaten away, the casts of the blood-vessels remaining as a dark framework (corrosion) . The manipulation of these pre- parations is extremely difficult on account of the brittleness of the oil casts. For lymph-channels Altman (ibid,} used the so-called oil-impregnation. Fresh pieces of tissues, thin lamellae of organs, cornea, etc., are placed for five to eight days in a mixture containing olive oil i part, absolute alcohol yz part, sulphuric ether ^ part (or castor oil 2, absolute alcohol i, etc.). The pieces are then laid for several hours in water, where the externally adherent globules of oil are mechanically removed and those in the lymph-canalicular system are precipitated. The objects are now treated with osmic acid, cut by means of a freezing microtome, and corroded. In this case, the corrosive fluid (eau de Javelle) should be diluted two or three times. RECONSTRUCTION BY MEANS OF WAX PLATES. It is often impossible to obtain a clear conception of the form of minute anatomic structures, nor of their relations, by means of sections or by the methods of maceration and teasing. To obviate such difficulties methods of reconstruction have been devised, by means of which such structures may be reproduced in an enlarged form without losing their inherent morpho- 56 THE MICROSCOPIC PREPARATION. logic features. Of these methods, we shall here describe that suggested by Born (1876) and known as Bern's method of reconstruction by wax plates. . This method has found wide application in embryologic investi- gations, and has proved very valuable in ascertaining the form, relation, and metamorphosis of embryonic structures and organs. It has not been so extensively used in the study of the form of fully developed anatomic structures ; it deserves, however, a fuller appreciation of its value as an aid in microscopic study. Necessary are serial sections, wax plates of desired thickness, and a drawing apparatus. Serial Sections. — One of the requisites of wax plate reconstruction is a perfect series of sections of uniform thickness. The thickness of the sections should depend on the character and size of the object to be reconstructed and on the magnification necessary to give the model ob- tained such a size as to enable it to be readily manipulated. In the reconstruction of fully developed anatomic structures, such as parts of glands or entire glands, it is generally not possible to make an outline drawing of the parts to be reproduced. When this is possible, it forms the first step of the method. Wax Plates. — Several methods have been suggested for obtaining wax plates of uniform and desired thickness. The instrument devised by Fig- 9- — Apparatus for making wax plates, used in reconstruction by Born's method. Huber and figured in Fig. 9 may be recommended for this purpose. It consists of a heavy cast-iron plate, supported by three adjustable legs. On two sides of the plate are found movable side- pieces which may be raised or lowered by micrometer screws to a desired height and then tightly clamped. There is, further, a heavy iron roller which runs on the adjustable side pieces. This roller needs to be heated in boiling water before use, and is kept in boiling water when not in use during the process of making wax plates. The method of making plates is as fol- lows : The side plates are adjusted so that their upper surface projects above the main plate for a distance representing the thickness of the wax plate desired. Melted wax is then poured on the main plate, in an even layer somewhat thicker than the wax plate desired, and then rolled out with the hot roller until the roller runs evenly on the side pieces. The wax plate is now allowed to cool, when it is removed from the apparatus and placed in a pan of cold water, where it remains for a few minutes or until thoroughly cooled. Drawing of the Portions of the Sections to be Reconstructed. — The drawings of the portions of the sections representing the portion to be reconstructed, at the magnification selected, may be made with the RECONSTRUCTION BY MEANS OF WAX PLATES. 57 aid of a camera lucida, or by means of a projection apparatus. Bardeen has devised a drawing table which is placed horizontally, over which is placed a mirror at an angle of 45 degrees. The table may be made to move by means of a windlass toward or away from the microscope so that any magnification may be quickly obtained. An ordinary micro- scope with the tube placed horizontally may be used, the illumination being obtained from an arc light. (For further details see Bardeen, "Johns Hopkins Bulletin," vol. xii, p. 148.) Sharp outlines of the parts to be reconstructed should be made and the drawing for each sec- tion labelled with reference to the series of drawings and with reference to the number of the section, as it is often necessary to refer to the sec- tions while reconstructing. After the drawings have been completed they are transferred to the wax plates, which is conveniently done by placing the drawing over the wax plate and tracing the outline with a blunt-pointed instrument, using some pressure while doing so. The wax plates are numbered with reference to the drawings. It is necessary to maintain an equal ratio between the diameter of the magnification of the drawing of the sections, the thickness of the plates used and the thickness of the sections. Thus, if it is desired to reconstruct portions of a series of sections 5 /j. in thickness and to use wax plates 2 mm. thick, the draw- ings need to be made at a magnification of 400 diameters. Cutting Out the Parts to be Reconstructed and Completing the Model. — Those portions of the wax plates representing the parts to be reconstructed as outlined by the tracings are cut out with a sharp knife with narrow blade, the wax plate being placed on a glass plate during this procedure. If the parts of the sections to be reconstructed consist of a number of disjointed pieces, these are retained in their rela- tive positions by means of remaining bridges of wax, which should be firm enough to keep all parts in their proper relation. The parts of each wax plate representing the portions of the section to be reconstructed are piled up in their proper sequence as they are cut out. The completion of the model consists in accurately adjusting the portions obtained from each wax plate to those which precede and follow them. This process is facilitated by building up the model in blocks representing five sections, as has been suggested by Bardeen. Those parts representing the portions of the sections to be reconstructed are united together by pins or small nails ; other parts, such as wax bridges, are removed by means of a hot knife. The successive blocks are then similarly united and the model is Completed by smoothing over the surfaces by means of a hot iron. GENERAL HISTOLOGY. I. THE CELL. DURING the latter part of the seventeenth century, Hooke, Mal- pighi, and Grew, making observations with the simple and imperfect microscopes of their day, saw in plants small compartment-like spaces, surrounded by a distinct wall and filled with air or a liquid ; to these the name cell was applied. These earlier observations were extended in various directions during the latter part of the seven- teenth and the eighteenth century. Little advance was made, however, until Robert Brown (1831) directed attention to a small body found in the cell, previously mentioned by Fontana, and known as the nucleus. In the nucleus Valentin observed (1836) a small body known as the nucleolus. In 1838 Schleiden brought forward proof to show that plants were made up wholly of cells, and especially emphasized the importance of the nuclei of cells. In 1839 Schwann originated the theory that the animal body was built up of cells resembling those described for plants. Both Schleiden and Schwann defined a cell as a small vesicle, surrounded by a firm membrane inclosing a fluid in which floats a nucleus. This conception of the structure of the cell was destined, however, to undergo important modification. In 1846 v. Mohl recognized in the cell a semifluid, granular substance which he named protoplasm. Other investigators (Kolliker and Bischoff) observed animal cells devoid of a distinct cell membrane. Max Schultze (1861) attacked vigorously the older conception of the structure of cells, proclaim- ing the identity of the protoplasm in all forms of life, both plant and animal, and the cell was defined as a nucleated mass of protoplasm endowed with the attributes of life. In this sense the term cell is now used. The simplest forms of animal life are organisms consisting of only one cell {protozoa). Even in the development of the higher animals, the first stage of development, the fertilized egg, is a single cell. This by repeated division gives rise to a mass of similar cells, which, owing to their likeness in shape and structure, are said to be undifferentiated. As development proceeds, the cells of this mass arrange themselves into three layers, the germ layers, the outer one of which is the ectoderm, the middle one the mesoderm, and the inner one the entoderm*. In the further development, the cells of the germ layers change their form, assume new qualities, adapting themselves to perform certain definite functions ; a division of labor ensues, — the cells become differentiated. Cells having similar shape 58 THE CELL-BODY. 59 and similar function are grouped to form tissues, and tissues are grouped to form organs. We shall now consider the structure of the cell. Every cell consists of a cell-body and a nucleus. A* THE CELL-BODY. The body of the cell consists of a substance known as proto- plasm or cytoplasm. This is not a substance having uniform Vacuoles. Chromatin network. Linin network. Nuclear fluid. Nuclear membrane. Cell-membrane. Exoplasm. g &. Spongioplasm. Hyaloplasm. Nucleolus. Chromatin net-knot. Centrosome. Centrosphere. Foreign inclosures. Metaplasm. Fig. 10. — Diagram of a cell. physical and chemical qualities, but a mixture of various organic compounds concerning which knowledge is not as yet conclusive, but which in general are proteid bodies or albumins in the widest sense. In spite of the manifold differences in its composition, proto- plasm exhibits certain general fundamental properties which are always present wherever it is found. Ordinarily, protoplasm ex- hibits certain structural characteristics. In it are observed two con- stituents,— threads or plates, which are straight or winding, which branch, anastomose, or interlace, and which are generally arranged in a regular framework, network, or reticulum. These threads probably consist of or contain small particles arranged in rows, called cell- microsomes (vid. van Beneden, 83 ; M. Heidenhain, 94; and others). Benda, who has devoted much time to the study of certain proto- plasmic structures, has found in these threads small granules or 6O THE CELL. A rod-shaped structures to which he has given the name "thread- granules" or mitochondria. The mitochondria can be differentially stained and are not distributed irregularly through the cell proto- plasm, but in certain definite regions. They are regarded as in part identical with the microsomes. This thread-like substance is known as protoplasm in the stricter sense (Kupffer, 75); also as spongio- plasin, or the fibrillar mass of Flemming (82). The other constit- uent of the cytoplasm is a more fluid substance lying between the threads in the meshes of the spongioplastic network, and is known as paraplasm (Kupffer), hyaloplasm, cytolymph, or the interfibrillar substance of Flemming. According to most investigators, the more important vital processes of the cell are to be identified with the spongioplasm, and are controlled by the nucleus, while the para- plasm assumes an inferior or passive role. With special methods Altman (94) was able to demonstrate granules in the protoplasm, associated with, but not in the spongioplastic threads. To these he gave the name bioblasts, and referred the vital qualities of the proto- plasm to them. Butschli believes the protoplasm to consist of _ Cilia. Fig. II. — Cylindric ciliated cells from the primitive kidney of Petromyzon planeri ; X 1200. separate, honeycomb-like spaces, which give it a foam-like structure — foam-structure of protoplasm. Protoplasm displays phenomena of motion, shown on the one hand by contraction, and on the other by the formation of processes that take the form either of blunt projections or lobes, or of long, pointed, and even branched threads or processes known as pseudo- podia. The extension and withdrawal of the pseudopodia enable the cell to change its position. The point of such a process fastens to some object and the rest of the cell is drawn forward, thus giving the cell a creeping motion — wandering cells. Certain cells take up and surround foreign bodies by means of theirpseudopodia. If these bodies are suitable for nutrition, they ar£**aslimilated ; if not, they can, under certain circumstances, be deposited by the cell in cer- tain localities (Metschnikoff 's phagocytes). Similar thread-like processes which, however, can not be drawn into the cell, occur in some cells in the shape of cilia, which are in constant and energetic motion — ciliated cells. Certain cells possess only a single long pro- cess, by means of which unattached cells are capable of direct or rotating motion — -flagellate cells, spermatozoa. THE CELL-BODY. 6 1 Inside of the cell-body the protoplasm also shows phenomena of motion, the streaming of the protoplasm. In plant cells there is often a noticeable regularity in the direction of the current. Men- tion should not be omitted of the so-called molecular or Brownian movement in the cells, which consists in a rapid whirling motion of particles or granules suspended in the protoplasm (Brown). Living protoplasm is irritable in the highest degree, and reacts very strongly to chemic and physical agents. It is very sensitive to changes in temperature. All the phenomena of life occur in greater intensity and more rapidly in a warm than in a cold temperature, this fact being very strikingly shown by the phenomena of motion in the cell, as also in its propagation. By subjecting protoplasm to different temperatures, its various movements can be slowed or quickened. It dies in too high or too low a temperature. Certain substances coming in contact with the cell from a given direc- tion have on it an attracting or repelling action. These phenomena are known as positive and negative chemotropism (chemotaxis) . The action of chemic agents on the different wandering cells of the body and on cer- tain free-swimming unicellular organisms naturally varies to a great degree. Among these phenomena must be included those produced by water (hydrotropism) and light (heliotropism). It is very probable that all these phenomena are of importance to the proper appreciation of some of the processes going on in the vertebrate body (as, for instance, in the origin of diseases caused by micro-organisms). Protoplasm may contain various structures. Of these, the vacuoles deserve special mention. They are more or less sharply defined cavities filled with fluid, and vary considerably in number and size. The fluids that they contain differ somewhat, but are always secreted by the protoplasm, and are, as a rule, finally emp- tied out of the cell. As a consequence, vacuoles are best studied where the function of the cell is a secretory one. Here they are often large, and sometimes fill up the whole cell, the contents of which are then emptied out (glandular cells). Contents of a solid nature, such as fat, pigment, glycogen, and crystals, are peculiar to certain cells. By these deposits the cell is more or less changed, the greatest variation in form taking place in the production of fat. The latter, as a rule, takes the shape of a globule, and greatly modifies the position of the normal con- stituents of the cell. Deposits of pigment alter the cells to a less degree. This substance occurs in the protoplasm either in solution or in the form of fine crystalline bodies. Glycogen is more gener- ally diffused, occurring very generally in embryonal cells and in the liver- and cartilage-cells of the adult. Occasionally we find larger crystals in animal cells, as, for instance, in the red blood-corpuscles of the teleosts. So-called margarin crystals sometimes occur in large numbers as stellate figures in dead fatty tissues kept at low temperatures. 62 THE CELL. Many cells are without a distinct cell membrane, another con- stituent of the protoplasm. In such cells the outer layer of the protoplasm is often more homogeneous and less dense than that lying more centrally, which has often a more granular appearance; the outer layer of the protoplasm is in such cells known as the exoplasm, in contradistinction to the more granular endoplasm. In other cells, however, the outer layer of the cell-protoplasm shows differentiation, leading to the formation of a distinct cell-mem- brane (as in fat-cells, cartilage-cells, goblet-cells, etc.). F. E. Schulze has given i£ the name pellicula in cases where the entire cell is sur- rounded by a homogeneous layer, and cuticida or cuticle where only one side of the cell is supplied with the membrane (as in the intestinal epithelium). It is assumed that both spongioplasm and paraplasm are concerned in the formation of this membrane. In the protoplasm of many cells there is found a small body known as the centrosome. This is usually situated near the nucleus of the cell, occasionally in the nucleus. Generally, it has the appear- ance of a minute granule, sometimes scarcely larger than a micro- some. It is often surrounded by a small area of a granular or finely reticular or radially striated cytoplasm, known as the attraction- sphere or centrosphere. B. THE NUCLEUS. The second constant element of the cell is the nucleus. As a rule, it is sharply defined, and in its simplest form consists of a round vesicle of a complicated structure composed of several sub- stances. The form of the nucleus corresponds in general to the shape of the cell ; in an elongated cell, it is correspondingly long, and flattened where the cell is plate-like in shape. The nucleus of a wandering cell that is in the act of passing through a narrow inter- cellular cleft adapts itself to the changes of form in the cell without being permanently altered in shape. In other words, the nucleus is soft, and can be easily distorted by any solid substances within or without the protoplasm, only to resume its original form when the pressure is removed. It possesses, then, a certain amount of elas- ticity. Movements of certain nuclei, entirely independent of the sur- rounding protoplasm, have often been observed. It is only rarely that the general form of the nucleus differs materially from the general form of the cell. This, however, occurs in the nuclei of leucocytes and many of the giant cells of bone-marrow, which are often irregular, and may even be ring-shaped. In certain arth- rozoa, branching forms of nuclei occur, as also in the skin glands of turtles. The proportionate size of nucleus to cell-body varies greatly in different cells. Especially large nuclei are found in im- mature ova, in certain epithelial cells, etc. The contents of the nucleus consist of a framework or reticu- lum, in the meshes of which there is found a semifluid substance. THE NUCLEUS. 63 In treating the nuclei with certain stains, the nuclear reticulum will be seen to consist of two constituents, a substance appearing in the form of variously shaped, minute granules, which stains deeply, and is, therefore, known as chromatin. This is imbedded in and deposited on a less stainable network, the linin. The meshes of this network are occupied by a transparent, semifluid substance, which does not stain easily, and is known as the achromatic portion of the nucleus. It is also known as paralinin, nuclear sap, karyolymph, or nucleoplasm. Chemically, chromatin belongs to those albuminous substances known as nucleins. In well-stained nuclei of considerable size the chromatin gran- ules are seen closely placed in a continuous row throughout the net- work of linin, which penetrates the nuclei in all directions. In every resting nucleus one or more small round bodies are found imbedded in the nucleoplasm. These are known as true nucleoli, and do not stain quite so deeply as the chromatin. The fact that certain reagents dissolve the chromatin, but not the true nucleoli, proves that the substance of which the latter are composed is not identical with chromatin, — and is, therefore, known as paranuclein (F. Schwartz). In many cases we find in the linin, granules of a substance known as lanthanin, which displays a marked affinity for the so- called acid anilin stains, in contradistinction to chromatin, which stains principally with the basic anilin colors. These are known as oxychromatin granules in contradistinction to the basichromatin granules of the chromatin (M. Heidenhain, 94). The true nucleoli should not be confused with the slight swell- ings of the chromatin network found at the junction of the threads, and known as net-knots, or karyosomes. Surrounding the resting nucleus is usually a nuclear membrane (amphipyrenin) resembling in many respects chromatin. As a rule, it does not form a continuous layer, but is perforated, having open- ings that contain nuclear fluid. We have, then, both substances, chromatin and nucleoplasm, as elements of the nuclear membrane. Besides this, the nuclear membrane receives an outer layer, differ- entiated from the protoplasm. Later investigations have shown that even during a period of rest the relationship of the nucleus to the protoplasm of the cell is much more intimate than was hereto- fore believed (vid. Reinke, 94). A resting nucleus — i.e., one not in process of division — usually consists, therefore, of a sharply defined membrane (amphipyrenin), which has in its interior a chromatic (nuclein) and an achromatic (linin) network, a nuclear fluid (paralinin), and nucleoli (paranuclein). The chromatin of the nucleus is not always in the form of a net- work. In some cases — as, for instance, in the premature ova of certain animals (O. Hertwig, 93. II) and in spermatozoa — it is col- lected in compact bodies. In the ova it may often be mistaken for a true nucleolus (germinal spot). In this case, however, it consists of nuclein, and not of paranuclein. 64 THE CELL. C NUCLEAR AND CELL-DIVISION. The founders of the cell theory believed in what may be known as a modification of the theory of spontaneous generation, stating that cells might originate from a structureless substance known as kyto- blastema or blastema, in which a nucleus was formed by precipita- tion. Henle (1841) drew attention to the fact that cells might mul- tiply by the separation of small portions of the cell-body, a process known as budding ; and Barry (1841) stated that during the multi- plication of cells the nuclei divided. The same year Remak observed division of cells in the blood of embryos. Goodsir (1845) originated the theory that all cells were developed from preexisting cells. This was first clearly stated as a general law by Virchow (1855), and his saying, " Omnis celLida a-cettula" is constantly being verified. Our more accurate knowledge of cell-division dates, how- ever, from more recent times (1873—80), when Schneider, Fol, Stras- burger, Flemming, and many others demonstrated that during the division of the cell the nucleus passed through a series of compli- cated changes which resulted in an exact division of the chromatin. The phenomena which usher in cell-division are especially /noticeable -in the nucleus, the elements of which are arranged and transformed in a typic manner. During the division of the nucleus the nuclear membrane is lost, and the relationship of the substances of the nucleus to the protoplasm of the cell is a very intimate one. As a consequence, during the middle phases of division there is no well-defined demarcation between the nucleus and the cell-body. As a rule, the mother cell and nucleus divide into two daughter cells, each having a nucleus, alike in every particular. It was early observed, however, that occasionally cells divided by a much sim- pler process, in which case the nucleus did not pass through such complicated changes. Accordingly, two distinct types of cell- division are recognized, which are distinguished as mitosis, karyoki- nesis, or indirect cell-division, and amitosis, or direct cell-division. Both lead to the formation of two nuclei, which are known as daughter nuclei as distinguished from the original mother nucleus. J. MITOSIS OR KARYOKINESIS (INDIRECT CELL-DIVISION). Th-e description of the process of mitotic cell-division is compli- cated by the fact that structural changes are observed which occur simultaneously in the nucleus, centrosome, and cytoplasm. This fact should be borne in mind, as, for the sake of clearness, a sepa- rate description of the changes involving each of these structures seems demanded. The process of mitotic cell-division may be divided into four periods or phases, which follow one another with- out clearly defined limits : The prophascs, in which the nuclear membrane disappears, the chromatin is transformed into definite threads, and the centrosome NUCLEAR AND CELL-DIVISION. Figs. 12-21. — Ten stages of mitotic nuclear division from the oral epithelium of the larva of a salamander. (Plate I, Sobotta and Huber's "Atlas and Epitome of Human Histology," 1903) : Fig. 12, Cell with resting nucleus ; Fig. 13,' cell with nucleus at the beginning of mitosis; Fig. 14, nuclear membrane has disappeared, chromosomes in a loose skein, pole field at the left; Fig. 15, monaster viewed from above ; Fig, 16, mo- naster viewed from the side, achromatic spindle is also shown ; Fig. 17, monaster viewed from the side, with chromosomes crowded closely about the equator of the spindle ; Fig. 18, stage of metakinesis ; Fig. 19, diaster with beginning constriction of the cell-body; Fig. 20, dispirem with completion of the cell division ; Fig. 21, telophase. and centrosphere undergo important changes. This is the prepar- atory stage. The metaphases, in which the division and the separation of the chromatin take place. The anaphases, in which the daughter nuclei are formed and the cell-protoplasm begins tojiivide. The telopkases, in which the division of the cell is completed. 5 66 THE CELL. Fig. 22 Fig. 23. Fig. 24. Fig. 25. Fig. 26. Figs. 22-26. — Mitotic cell-division of fertilized whitefish eggs — Coregonus albtis. Fig. 22, Cell with resting nucleus, centrosome, and centrosphere to the right of the nucleus ; Fig. 23, cell with two centrospheres, with polar rays at opposite poles of nucleus; Fig. 24, spirem ; Fig. 25, monaster ; Fig. 26, metakinesis stage. To give a better understanding of the process we have inserted a series of figures in which several phases of mitotic division are portrayed. In figures 12-21 are shown ten stages of mitotic nu- clear division from the oral epithelium of the larva of a salamander, in which changes undergone by the nucleus and centrosome are clearly brought out. And, further, a series of figures (22—29) show- ing the different phases of mitotic cell-division of the fertilized eggs of the whitefish {Coregonus albns) ; the changes involving the centro- some, centrosphere, and cytoplasm are illustrated. Figure 30, show- ing a small portion of a section through the testis of the salamander, the object in which Flemming first observed this complicated series of changes, presents the appearance more generally seen during mitotic cell-division of the tissue cells of the higher vertebrates. (a) Prophases. — The changes occurring in the nucleus will be considered first. At the beginning of the process of mitosis, the chromatin network, consisting of chromatin granules, is transformed into a twisted skein of threads, beginning at the periphery of the NUCLEAR AND CELL-DIVISION. Fig. 29- Figs 27-29. — Mitotic cell-division of fertilized whitefish eggs — Coregonus albus. Fig. 27, Metakinesis stage; Fig. 28, diaster; Fig. 29, late stage of dispirem, the cell -protoplasm almost divided. nucleus. This skein of threads is known as the spirem or mother skein, and may appear as a single thread, which breaks up into a definite number of segments, or the segments may appear as such when the skein is forming. At first the threads are coarse and often somewhat irregular, staining much more deeply than the limn network. The separate segments of chromatin are known as chromosomes (Waldeyer, 88). They appear, as a rule, in the form of rods varying in length and thickness, and staining very deeply, and often bent into characteristic U-shaped loops. The bent portion of each loop is called its crown. " Every species of plant or ani- mal has a fixed and characteristic number of chromosomes, which regularly recurs in the division of all its cells ; and in all forms arising by sexual reproduction the number is even" (Wilson, 96). In man the number of chromosomes is given as sixteen by Barde- leben (92) and Wilson (96), and as twenty-four by Flemming (98). During the formation of the spirem the nuclear membrane, as a rule, disappears. The nucleolus is also lost sight of, although the manner of its disappearance can not be definitely stated. The net- knots are no doubt taken up by the chromosomes. The chromo- 68 THE CELL. somes are now free in the protoplasm ; gradually the crown of each chromosome approaches the center of the space occupied by the nucleus, and the chromosomes form a characteristic, radially arranged stellate figure, known as the monaster, in the equatorial plane of the cell. During the progress of the changes affecting the chromatin of the nucleus and resulting in the formation of the chromosomes, important phenomena are observed, connected partly with the achromatic substance of the nucleus, more especially with the centrosome, centrosphere, and cytoplasm of the cell. These phenomena result in the formation of a complicated structure known as the achromatic spindle or amphiaster. Its development is as fol- lows : The centrosome and centrosphere, as has been stated, usu- ally lie in the protoplasm to one side of the nucleus. If, at the be- ginning of the division, the centrosome be single, it divides, and the two centrosomes begin to separate, causing a division of the centro- sphere. Between the centrosomes are usually seen finely drawn-out connecting threads. The centrosomes, each of which is surrounded by a centrosphere, now move apart, and a structure known as the central spindle, and consisting of fine threads arranged in the form of a spindle, develops between them. At each end of the central spindle is found a centrosome surrounded by a centrospherefrorn which radiate into the cytoplasm fine fibers known as pottirrdy^. During the formation of the achromatic spindle the nuclear mem- brane disappears and the chromosomes develop, as above described. Some fibers, which seem to have their origin from the centrosphere, grow into the spirem formed of chromosomes, which they appear to pull into the equatorial plane of the cell, which is also the equator of the central spindle. Thus, the nuclear figure above described as the monaster is formed. In other cases the centrosomes and centrospheres continue moving apart until opposite each other and separated by the nucleus (Figs. 23, 24). As the nuclear membrane disappears and the spirems and chromosomes are form- ing, the central spindle develops, its fibers running from centro- sphere to centrosphere. The polar rays also develop in the cyto- plasm at the same time. As the central spindle develops, the chromosomes arrange themselves or are arranged about its equator — monaster. (U) Metaphases. — Usually, during the formation of the monaster, or immediately after its formation (sometimes in the spirem stage or even earlier), the most important process of cell -division takes place. Each chromosome divides longitudinally into two daughter chromosomes. The loops first divide at the crown, the cleft extend- ing up either limb until the free ends are reached. The smallest particle of chromatin divides, retaining the exact relative position in the twin chromosomes that it possessed in the mother chromo- some. The daughter chromosomes now wander over the central spindle, their crowns presenting, in opposite directions toward the poles of the cell. This process is known as metakinesis. Two stel- NUCLEAR AND CELL-DIVISION. 69 late figures are developed about the respective poles of the central spindle. The appearance presented is known -as a diaster. Our knowledge of the part taken by the amphiaster- or achromatic spindle in metakinesis is not above controversy. It would appear, however, that certain cytoplastic fibers, which arise from the cen- trosphere and hang over the central spindle and chromosomes, designated as mantle fibers, assist in drawing the daughter chromo- somes toward the poles of the central spindle. (c) Anaphases. — After the formation of the diaster, the loops be- longing to each stellate figure are joined together to form a skein, thus forming the dispirem. The chromatin threads of the two skeins gradually assume the disposition found in the resting nucleus. This process takes place in such a way that the threads of the Resting nucleus. MY &i * JW/.'^i^ltW^'-rf^SSx Diaster. ^ mBM __ M-M_ vii -- Metakinesis. ^W^K^B ^7^1 -- Daughter cells. XfcHEiras&fiffii Monaster. Spirem. 30. — Mitotic division of cells in testis of salamander (Benda and Guenther). skeins (or the single thread) send out lateral processes. These interlace, and little by little reproduce the network of the resting nucleus ; at the same time the nuclear membrane and the nucleolus reappear. In this stage the changes that lead to the division of the cell-body are observed. In some cases the division of the cell-body is ushered in by an equatorial differentiation of the connecting threads of the central spindle. Chains of granules, arranged in double rows, are seen to appear in this region. The cell now begins to contract at its equator, the contraction extending between the two chains of granules until the cell is completely divided. At this time, also, the threads of the amphiaster disappear or are drawn into the nucleus. The centrosomes, with centrospheres, again lie by the side of the daughter nuclei. 70 THE CELL. According to the opinion of C. Rabl (85), there remains in the nucleus, even after it has fully returned to a state of rest, a polar arrangement of the chromatin loops — that is, an arrangement of the axis of the loops in the direction of the centrosphere. The area toward which the crowns of the loops point is known as the polar field. The equatorial differentiation of the connecting threads of the central spindle, above mentioned, was first observed in vegetable tissue, and is known as the cell-plate. (Fig. 29.) In animal cells such a plate is relatively rare, and, when seen, is found developed in a rudimentary form (v. Kostanecki 92, I). (d] Telophases (M. Heidenhain 94). — In these phases of mitosis the cell divides completely. The daughter nuclei and centrospheres, which do not yet occupy their normal position in the daughter cells, show movements that result in their assuming their normal positions. From our description it is seen that the anaphases represent the same stages as the prophases, only in an inverted sequence. In the latter case, the result is the resting nucleus, while the prophases lead to the metaphases. The fertilized ovum also divides by indirect nuclear division. (Figs. 22—29.) From it are derived, by this process, the seg- mentation cells, or blastomeres, from which the whole embryo is developed. (e) The Heterotypic Form of Mitosis. — The above-described type of indirect or mitotic nuclear division (homeotypic mitosis) is the usual one. Variations, however, occur, as, for instance, in the so-called heterotypic form of division (Flemming 87), which occurs in certain cells of the testes (spermatocytes). In this form the first stages are lacking, the nucleus possessing from the beginning a skein-like structure. The longitudinal splitting and division of the chromatin threads take place during the first spirem stage, after which there is a phase in \vhich the figure may be compared with an aster of ordinary mitosis, although the free ends of the threads in this case are seldom observed. The latter is due to the fact that after the longitudinal splitting, the ends of the chromosomes remain united, or, if entire separation occurs, they are again joined. In this way closed loops are formed extending from pole to pole. Later the threads break at the equator and move toward the poles, again dividing to form the daughter stars. 2. AMITOSIS, Very different from the indirect form of nuclear division is the direct or amitotic. It appears to occur seldom as a normal process, and is only exceptionally followed by a subsequent cell-division (vid. Flemming, 91, III). As% consequence, this process, in most cases, results in the formation of polynuclear cells (polynu clear leu- cocytes, giant-cells, etc.). The complicated nuclear figures of PROCESS OF FERTILIZATION. ?I indirect division are here entirely absent. The nucleus merely con- tracts at a certain point and separates into two or more fragments (direct fragmentation, Arnold) ; often the nucleus first assumes an annular form and then breaks up into several fragments, which remain loosely connected (polynuclear cells). Centrospheres are also present, and appear to take a prominent part in the whole pro- cess, although the exact relationship between the achromatin and chromatin has not as yet been determined. Nemiloff has recently called attention to two locations where amitotic divisions may readil>^fje~5bserved — namely, in the large surface cells of transitional epithelium of the bladder of mammals and in the lymphoid tissue layer of the liver of amphibia. In the cells of the former type the nuclear division is initiated by a division of the nucleolus which is followed by a division of nucleus and later the protoplasm. Centrosomes and attraction spheres were not noticed in these cells. The division of the lymphoid cells of the amphibian liver is initiated by a depression found in one side of their spherical nuclei. This depression deepens until the nuclei be- come perforated and assume an annular shape. These ring-shaped nuclei then break through in two or more places and two or more daughter nuclei are formed. During the process of division a cen- trosome with attraction sphere may often be observed, generally situated in the depression which initiates the division and later in the center of the perforated nucleus-. Its rolejn the division of the nucleus and the cell-body is, however, not fully understood. D. PROCESS OF FERTILIZATION. The sexual cells form a special group among cells in general. Before the division of the egg-cell leading to the development of the embryo can take place, the ovum must .-be impregnated (the so- called parthenogenetic ova are an exception to this rule). Fertili- zation is produced by the male sexual cell, the spermatozoon. J"he process of fertilization consists in a conjugation of two sex- ual cells, vand in this process certain peculiarities in the behavior of both cells must be mentioned. The cell forming the ovum and the one forming the spermato- zoon must pass through certain stages before fertilization can be accomplished. These consist in the loss of half their chromosomes by the nuclei of both sexual cells. In this way are produced the matured sexual cells (ova and spermatozoa), which retain only half of the number of chromosomes of a somatic (body-) cell. In the conjugation of the male and female sexual cells their nuclei unite to form a single nucleus, known as the segmentation nucleus. Consequently, this nucleus contains the same number of chromo- somes as does that of a somatic cell. In its earlier developmental stages the ovum is an indifferent cell, the nucleus of which is known as the germinal vesicle. As the THE CELL. Membrane of ovum. Nucleus of ovum. Spermatozoon entering. Protoplasm of ovum with deutoplastic granules. Fig. 31. Female pronu- cleus. - Male pronu- cleus. Fig- 32. Fig. 33. Figs. 31-33. — Diagrams of the process of fertilization, after Boveri. Figure 31, the ovum is surrounded by spermatozoa, one of which is in the act of penetration. Toward it the yolk is pushed forward in a short, rounded process. Figure 32, the tail of the spermatozoon has disappeared. Beside the head is a centrosome with polar radiation. Figure 33, the pronuclei approach each other. ovum matures the germinal vesicle approaches the periphery, and a peculiar metamorphosis, which may be regarded as a double, un- equal division of the egg-cell, takes place. One portion, in the case of both divisions, is much smaller than the other, and is known as a polar body. At the close of these divisions, during which the chromosomes have been reduced to half the original number, there are, therefore, two polar bodies and the matured ovum, which is now ready for impregnation. The development of the male sexual cell in its earlier stages is sim- ilar to that of the ovum. They are derived from cells known as sper- matogones. These divide into equal parts, forming the cells of a second generation, the spermatocytes. From a further division of the spermatocytes, during which division the chromosomes are reduced to half the number, the spermatids are produced. These latter are then changed directly into spermatozoa. The reduction division of the egg-cell and that of the spermatocytes is in principle the same, except that in spermatogenesis all cells become matured sexual cells PROCESS OF FERTILIZATION. 73 — Centrosome. Female pro- nucleus. Chromo- somes of male pro- nucleus. Centrosome. Fig. 34- Fig. 35- Chromosomes from egg-nu- cleus. __ Chromosomes from sperm- nucleus (male pronucleus). — Centrosome. Fig. 36. Figs. 34-36. — Diagrams of the process of fertilization, after Boveri. Figure 34, from the spirems in the pronuclei, chromosomes have been formed. The cemrosphere has divided. Figure 35, the double chromosomes of the two pronuclei lie in the equatorial plane of the ovum. Figure 36, the ovum has divided. Chromosomes from the male and female elements are seen in equal numbers in both daughter nuclei. (spermatozoa). In short, there is here an absence of structures analogous to the polar bodies, which degenerate after maturation of the ovum. The spermatozoa are flagellate cells. The head consists prin- cipally of nuclear substance, to which is added a smaller middle- piece containing, according to the investigations of Pick, the centro- some. These two portions of the male sexual cell, the head- and middle-piece, are the most important, and are exclusively con- cerned in fertilization, the flagellum or tail playing no part in this process. The spermatozoon usually penetrates the ovum after the first polar body has been extruded. The tail disappears during this process, being either left at the periphery of the egg or dissolved in the protoplasm. From this time the head represents the so-called male pronucleus, and the middle-piece the Centrosome. From this stage the male pronucleus undergoes changes, the first of which consists of a loosening of the chromatin. Chromatin granules are 74 THE CELL. formed, which later arrange themselves in the form of chromo- somes. After the second polar body has been extruded, the chro- matin remaining in the ovum is transformed into the female pro- nucleus. The latter then approaches the male pronucleus, the membranes of both nuclei disappearing. The chromosomes of the two nuclei thus formed are of equal number, and now come to lie together. After a longitudinal division of the chromosomes, the daughter chromosomes glide along the filaments of the achro- matic spindle, developed from the centrosome of the male pronu- cleus, toward its two poles, as in ordinary mitosis. This they do in such a manner that an equal distribution of the male and female daughter chromosomes results. Then follow the stages of the ana- phase. From the above description of the process of fertilization it is seen that it consists, in the end, of a union of the nuclei of both sexual cells. If paternal qualities are inherited by the offspring, this can only take place through the nucleus, or through the centrosome of the male sexual cell. In other words, it can be safely said that these structures, or the nucleus alone, are the principal means of trans- mitting inherited qualities. The same may also be said of the female pronucleus. There is no doubt that the first two seg- mentation cells of the ovum are equally provided with male and female nuclear elements. Since all future cells are derivatives of these two, it is possible that the nucleus of every somatic cell (body-cell) is hermaphroditic. , E. CHROMATOLYSIS. In the living organism many cells are destroyed during the various physiologic processes and replaced by new ones. On the death of a cell, changes take place in its nucleus which result in its gradual disappearance. These processes, which seem to follow certain definite but as yet unfamiliar laws, have been known since their study by Flemming (85, I) by the name of chromatolysis (karyolysis). The nuclei during the course of these changes show many varied pictures. TECHNIC In a fresh condition, cells do not show much of their internal structure. Epithelial cells of the oral cavity, which can easily be ob- tained and examined in the saliva, show really nothing except the cell outlines and the nuclei. More, however, can be seen in young ova iso- lated from the Graafian follicles of mammalia ; or the examination may be facilitated by using the ovary of a young frog. Tissues that are especially adapted for the observation of cells in a fresh condition are small ova, blood-corpuscles, and epithelia of certain invertebrate animals (shellfish, CHROMATOLYSIS. 75 etc.). Unicellular organisms such as amebse, infusoria, and many low forms of vegetable life make also good material for this purpose. Protoplasmic currents are best seen in the tactile hairs of the net- tle. Should fresh animal cells be desired, amebae can occasionally be found in muddy or marshy water. The same phenomena may be ob- served in the leucocytes of the frog or, better still, in the blood of the crab. In order to make a detailed study of the minute relationship of the different cellular structures, it is necessary to fix the cells ; the same is true of nuclear division and cell proliferation. Although this process has been observed in living cells, it was not until it had been thoroughly worked out in preserved preparations. The best results in the study of the cell are obtained by methods that will be subsequently described. Fresh tissues are absolutely essential. According to Hammer, mitosis in man does not cease immediately after death. The nuclei suffer chromatolytic destruction, and the achro- matic spindle is the last element to disappear. Flemm ing's solution here deserves first mention as a fixative. The tissues are imbedded, sectioned, and stained with safranin. An equally good fixative is Hermann's solution, which may be combined with a sub- sequent treatment with pyroligneous acid. Rabl fixes with a 0.1-0.12 c/( solution of chlorid of platinum, washes with water, passes into gradually stronger alcohols, then stains with Delafield's hematoxylin, and finally examines the preparation in methyl alcohol. Mitoses can also be seen by fixing in corrosive sublimate, picric acid, chromic acid, etc., and staining in bulk with hematoxylin or carmin, although perhaps not so well as by the preceding method. The objects to be examined are best when obtained from young and grow- ing animals, especially those possessing large cells. Above all are to be recommended the larvae of amphibia, like the frog, triton, and sala- mander. If examination by means of sections be undesirable, thin structures should be procured, such as the mesentery, alveoli of the lungs, epithelium of the pharynx, urinary bladder, etc. These have the advan- tage of enabling one to observe the whole cell instead of parts or frag- ments of cellular structures. In sections of a larva that has been fixed in toto, mitotic figures can be seen in almost all the organs, and are particu- larly numerous in the epithelium of the epidermis, gills, central canal of the brain and spinal cord, etc. Other organs, such as the blood, liver, and muscle, also show mitoses. Certain vegetable cells are peculiarly adapted to the study of mitosis, as, for instance, those in the ends of young roots of the onion. The onion should be placed in a hyacinth glass filled with water and kept in a warm place. After two or three days numbers of small roots will be found to have developed. Beginning at the points, pieces 5 milli- meters in length are cut, which are treated in the same manner as animal tissues. These are then cut, either transversely or longitudinally, into very thin sections (not over 5 ;j. in thickness). In one plane, polar views of the mitoses are obtained ; in the other, lateral views. The methods used for demonstrating the remaining parts of the cell and its nucleus (except the chromatin) are, as a rule, more compli- cated, and consequently less reliable. In order to see the centrosome, the spindle fibrils, the linin threads, and the polar rays, one of the 76 THE CELL. methods already described may be used ; viz., the treatment with pyro- ligneous acid of objects previously fixed in osmic acid mixtures. According to Hermann (93, II), sections from such preparations can be double=stained as well as those that have not been treated with pyroligneous acid. They are accordingly stained with safranin in the usual manner, and afterward treated from three to five minutes with the following solution of gentian violet : 5 c.c. of a saturated alco- holic solution of the stain is dissolved in 100 c.c. of anilin water. The latter is composed of 4 c.c. of anilin oil in 100 c.c. of distilled water. This is shaken in a test-tube and then filtered through a wet filter. The sections are then placed in a solution of iodin and iodid of potassium (iodin i gm., iodid of potassium 2 gm., water 300 c.c.) until they have become entirely black, after which they are immersed in alcohol until they receive a violet tinge with a slight dash of brown. By this means the chromatin network, the resting nuclei, and the chromosomes in both of the spirem stages appear bluish -violet, while the true nucleoli are pink. The chromosomes of the aster and diaster are colored red. Flemming (91, III) recommends the following method: Fixation by his mixture ; the specimens or thin sections are then placed in safranin from two to six days, washed for a short time in distilled water, and then immersed in absolute alcohol weakly acidulated with hydrochloric acid (i : 1000), until no more color is given off. They are then washed again with distilled water and placed in a concentrated solution of anilin-water- gentian -violet from one to three hours. After a third rinsing in distilled water, they come into a concentrated aqueous solution of orange G, until they begin to assume a violet color. Then wash with absolute alcohol, clear in clove or bergamot oil, and mount in Canada balsam. A comparatively simple method showing the different structures of the cell and its nucleus with great clearness consists in staining with Heidenhain's hematoxylin. Solger (89, I and 91) has discovered that both chromosomes and polar rays are shown in an exquisite manner in the pigment cells of the skin (corium) of the frontal and ethmoidal regions of the common pike (vid. Fig. 37). The preliminary treatment is optional, Flemming's solution or corrosive sublimate being the best. These cells illustrate the stability of the radiate structures of protoplasm, the polar rays showing as parallel rows of pigment granules. The various structures of resting and dividing nuclei and cells are of such a complicated nature that they can be observed only with great difficulty in ordinary objects, because of the crowding of so many elements into a comparatively small space. For example, salamandra maculosa, which has become a classic histologic object through the researches of Flemming, possesses somatic cells whose nuclei have no less than twenty-four chromosomes. (It may here be remarked that, curiously enough, salamandra atra has only half this number. ) Consequently, van Beneden's discovery (83), that the somatic cells of ascaris megalocephala have only four primary chromosomes, is a fact of considerable import- ance. Boveri (87, II and 88) has even found an ascaris showing only two chromosomes. As these animals also show distinct achromatic fig- ures in the protoplasm of their ova and sperm cells, they are certainly worthy of being regarded as typic specimens for laboratory purposes. The processes of cell -proliferation are almost diagrammatic in their dis- tinctness. CHROMATOLYSIS. 77 After opening the abdominal wall of the animal, the ovisacs are removed, their numerous convolutions separated as much as possible, and then fixed for twenty-four hours in a picric-acetic acid solution (a concentrated aqueous solution of picric acid diluted with 2 vols. of water to which i per cent, glacial acetic acid is added). Then fol- lows washing for twenty-four hours with water, after which the specimen is transferred to increasing strengths of alcohol (Boveri, ibid.). Differ- ent regions of the ovisacs contain ova in various stages of development, those nearest the head containing cells ripe and ready for fecundation, while in the more posterior regions are ova in varying stages of segmen- tation showing mitoses. Specimens fixed in the manner above described can be stained with a borax-carmin solution. After staining, the ova are gently pressed out with needles upon a slide, separated, covered with a cover- glass, and cleared by gradual irrigation with glycerin. The ova, especially the segmentation spheres, are very small, and can be examined only under high magnification. In spite of the minuteness of the ob- ject and the fact that the yolk does not take the stain, and, on account of Centrosphere. •Nucleus. Fig- 37- — Pigment cell from the skin of the head of a pike ; ><^ 650. its high refractive index, distorts the picture to a considerable extent, the mitotic figures are beautifully distinct. Certain methods of treatment bring out in both cells and nuclei the presence of peculiar granules. The latter have been especially studied and described by v. Altmann (94, 2ded. ). The methods that he applies are as follows : The specimens of organs of recently killed animals are fixed in a mixture consisting of equal volumes of a 5% aqueous solution of potassium bichromate and a 2% solution of osmic acid, remaining in the mixture for twenty-four hours. They are then washed for several hours in water and treated with ascending strengths of alcohol ; viz., 70, 90, and 100%. The specimens are now placed in a solution of 3 parts of xylol and i part of absolute alcohol, then in 78 THE CELL. pure xylol, and finally in paraffin. The tissues imbedded in paraffin must not be cut thicker than i to 2 p.. Altmann mounts according to the following method : A rather thick solution of caoutchouc in chloroform (the so-called traumaticin of the Pharmacopeia — i vol. guttapercha dissolved in 6 vols. chloroform) is diluted before use with 25 vols. of chloroform and the resulting mixture poured upon a slide. The latter is tilted, and after evaporation of the chloroform, heated over a gas flame. The paraffin sections are mounted upon the slides so prepared and then painted with a solution of guncotton in aceton and alcohol (2 gm. guncotton dissolved in 50 c.c. of aceton, 5 c.c. of which is diluted with 20 c.c. of absolute alcohol). After painting with this solution, the sections are firmly pressed upon the slide with tissue paper, and after drying are made to adhere more closely by slight warming. Fixation to the slide with water is equally good. The sections can now be treated with various staining solutions without becoming detached from the slides. The paraffin is gotten rid of by immersing in xylol, after which the specimens are placed in absolute alcohol. Fuchsin S. can be used as a stain (20 gm. fuchsin S. dissolved in 100 c.c. anilin water). A small quantity of this solution is placed upon the section, and the slide warmed over a flame until its lower surface becomes quite perceptibly warm and the staining solution begins to evaporate. The slide is then allowed to cool, washed with picric acid (concentrated alcoholic solution of picric acid diluted with 2 vols. of water), after which it is covered with a fresh quantity of picric acid, and again, but this time vigorously, heated (one-half to one minute). Occasionally the same results can be obtained by covering the section for five minutes with a cold solution of picric acid of the above strength. This last procedure has a decided influence upon the granula, and gives rise to a distinct differentiation between them and the remaining portions of the cell, the latter appearing grayish -yellow, while the granula themselves appear bright red. In some cases where the granula can not be sharply differentiated from the remaining structures, it may be necessary to repeat the staining process. Xylol-Canada balsam should not be used for mounting, as it has a bleaching effect upon the osmic acid in the specimen. Mount either in liquid paraffin (Altmann) or in undiluted Canada balsam, which is easily reduced to a fluid state, whenever needed, by heating. There is another method used by Altmann which deserves mention, but practical application of which must be improved upon in the future ; this consists in freezing the specimens and drying them for a few days in the frozen condition in a vacuum over sulphuric acid at a temperature of about — 30° C. According to Fischer, dilute solutions of pepton when treated with various reagents (especially with a potassium bichromate-osmium mix- ture) form precipitates and granules which are remarkable in that they react to stains exactly as do Altmann 's granula. It is, therefore, doubt- ful whether Altmann 's granules should be regarded as vital structures. Altmann (92) has also devised a simpler negative method for demonstrating the granula. Fresh specimens are placed for twenty-four hours in a solution consisting of molybdate of ammonium 2.5 gm., chromic acid 0.35 gm., and water 100 c.c. ; then treated for several days with absolute alcohol, sectioned in paraffin, and colored with a nuclear stain such as hematoxylin or gentian. The intergranular network THE TISSUES. 79 is colored, while the granula remain colorless. The amount of chromic acid used (0.25 to i%) varies according to the object treated ; if molyb- date of ammonium alone be used, the nuclei will appear homogeneous, while if an excess of chromic acid be employed, the nuclei will appear coarsely reticulated. This method leads to the formation of granula in the cells as well as in the nucleus. Biitschli's Foam-structure. — Fixing is done either in picric acid solution or in weakly iodized alcohol. The specimens are then stained with iron-hematoxylin — /'. e., first treated with acetate of iron, rinsed in water, and transferred to a 0.5% aqueous solution of hematoxylin (simi- lar to the method of R. Heidenhain). Very thin sections are required (^2 to i /;.). Mounting is done, when the lighting is good, in media having low refractive indices, which emphasize the alveolar or foam-like structure of the protoplasm. Of various animal objects, Butschli espe- cially recommends young ovarian eggs of teleosts, and blood-cells and intestinal epithelium of the frog, etc. It is still a matter of uncertainty whether or not the structures are actually present in living protoplasm. II. THE TISSUES. The first few generations of cells which result from the segmen- tation of the fertilized ovum have no pronounced characteristics. They are embryonic cells of rounded form, and are known as blas- tomeres. As they increase in number they become smaller and of polygonal shape, owing to the pressure to which they are subjected. From the mass of blastomeres, known as the morula mass, there are formed, under various processes described under the name of gastndation, two layers of cells, the so-called primary germ layers, of which the outer is the ectoderm, the inner the entoderm. To the primary germ Bayers is added still a third layer, called the meso- derm ; it is derived from both the ectoderm and entoderm, but principally from the latter. From these three layers of cells, known as the primary blastodermic layers, are developed all the tissues, each layer developing into certain tissues that are distinct for this layer. In their further development and differentiation the cells of the blas- todermic layers undergo a change in shape and structure character- istic for each tissue, and there is developed an intercellular substance varying greatly in amount and character in the several tissues. In the tissues developed from the ectoderm and entoderm the cellular elements give character to the tissue, while the intercellular sub- stance is present in small quantity ; in the majority of the tissues developed from the mesoderm, the intercellular substance is abun- * dant, while the cellular elements form a less conspicuous portion. The tissues derived from the ectoderm are : The epidermis of the skin, with the epidermal appendages and glands ; the epithelium lining the mouth, with the salivary glands a.nd the enamel of the teeth ; the epithelium and glands of the nasal tract and the cavities opening into it ; the lens of the eye and retina, 8O THE TISSUES. and the epithelium of the membranous labyrinth of the ear ; and finally, the entire nervous system, central and peripheral. From the entoderm : The epithelium lining the digestive tract, and all glands in con- nection with it, including the liver and pancreas ; the epithelium of the respiratory tract and its glands ; the epithelium of the bladder and urethra (in the male, only the prostatic portion, the remainder being of ectodermal origin). The cells of the mesoderm are early differentiated into three groups (Minot, 99) : (a) Mesothelium. — The mesothelial cells retain the character of epithelial cells. They form the lining of the pleural, pericardial, and peritoneal cavities, and give origin to the epithelium of the uro- genital organs (with the exception of the bladder and urethra), and striated and heart muscle tissue. (b) Mesenchyme, from which are derived all the fibrous connective tissues, cartilage, and bone, involuntary muscle tissue, the^spleen, lymph-glands, and bone -marrow ; and cells of an epithelioid charac- ter, lining the blood and lymph-vessels and lymph-spaces, known as endothelial cells. (c) Mesameboid cells, comprising all red and white blood-cells. It would be extremely difficult to attempt a classification of tis- sues according to their histogenesis, as identical tissue elements owe their origin to different germinal layers. The classification adopted by us is based rather on the structure of the tissues in their adult stage. We distinguish : yA. Epithelial tissues with their derivatives. B. Connective tissues ; adipose tissue ; supporting tissues (car- tilage, bone). C. Muscular tissue. D. Nervous tissue. E. Blood and lymph. A. EPITHELIAL TISSUES. Epithelial tissues are nonvascular, and composed almost wholly of epithelial cells, united into continuous membranes by a substance known as intercellular cement. They serve to protect exposed surfaces, and perform the functions of absorption, secretion, and excretion. The epithelia are developed from all of the three layers of the blastoderm. They secrete the cement-substance found between their contigu- ous surfaces. This takes the form of thin lamellae between the cells, gluing them firmly together. In certain regions the epithelial cells develop short lateral processes (prickles), which meet like structures EPITHELIAL TISSUES. 8 I from neighboring cells, thus forming intercellular bridges. Between these bridges are intercellular spaces filled with lymph-plasma for the nourishment of the cells. Epithelia do not, as a rule, possess processes of any length. However, it would appear that the base- ment membranes, situated beneath the epithelia, consist chiefly of processes from the basal portion of the cells. Some authors ascribe to them a connective-tissue origin, a theory which conflicts with the fact that such membranes are present in the embryo before connective tissue, as such, has been developed (membrana prinia, Hensen, 76). //The free surfaces of epithelia often support cuticular structures which are to be regarded as the products of the cells. The cutic- ulae of neighboring cells fuse to form a cuticular membrane or mar- ginal zone which can be detached in pieces of considerable size (cuticula). In longitudinal sections the cuticula show, in many cases, a striation which would seem to indicate that they are com- posed of a large number of rod-like processes cemented together by a substance possessing a different refractive index. The cell-body is also striated for more than half its length, corresponding to the rods of the marginal zone. In the region of the nucleus at the basal por- tion the striation disappears, the cell here consisting of granular pro- toplasm of a more indifferent character. Since one surface of each epithelial layer lies free, and Is conse- quently exposed to other conditions than the inner surface, certain differences are noticed between the two ends of e£ch cell. The cells may develop cuticular structures as above stated. In other cases motile processes (ciliaj) are developed on their exposed surface, which move in a definite direction in the medium surrounding them, and by meansvof this motion sweep away foreign bodies. It is not strange that the free surface of ttye epithelia, exposed- as it is to stimulation from without, should develop special structures for the reception of sensations (sense cells). On the other hand, the inner or basal surfaces of the cells usually retain a more indifferent character, and serve for -the attachment ef the cells and the conveyance of their nourishment../ For this reason the nuclei of such cells are usually situated^near th£ basal surface. From the above it is seen that the two ends'of the epithelial cell undergo varying processes of differentiation, the outer being adapted more to the animal, the inner more to the vegetative functions. This differentiation has recently been known as the polarity of the cell. This polarity appears to be retained even when the cell loses its epithelial character and assumes other functions (Rabl, 90). With few exceptions, blood- and lymph-vessels do not penetrate into the epithelia, but the latter are richly supplied with nerves. The finer morphology of the epithelia will be described in the chap- ters on the different organs in Part II. Epithelia are classified according to the shape and relation of the epithelial cells. 6 THE TISSUES. We give the following classification : 1. Simple epithelia (with or without cilia). (a) Squamous epithelium. (^) Cubic epithelium. (<:) Columnar epithelium. (d] Pseudostratified columnar epithelium. 2. Stratified epithelia (with or without cilia). (a) Stratified squamous epithelium, with superficial flattened cells (without cilia). (£) Transitional epithelium. ( in a solution of silver nitrate (l %) until the tissue becomes opaque. If then the tissue is exposed to sunlight, the silver is reduced in the ground-substance, giving it a brown color, while the cell-spaces remain unstained. The ground- substance of areolar connective tissue contains mucin. THE CONNECTIVE TISSUES. 103 The cellular elements of areolar connective tissue, which, as above stated, are found in the cell -spaces, are either fixed con- 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 vitam). 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. Protoplas Nucleus. Bacteria in a vacuole. 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 A* to 10 /*. 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 cells, while others regard them as derived from the white blood-cells (lympho- cytes). They are found in various mucous membranes and in lymph- oid 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 are often found in such numbers that they cover up the nucleus. The granules are stained by a 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 skins, 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 Fig. 69. — Leucocyte of a frog with pseudopodia. The cell has included a bacterium which is in process of diges- tion. (After Metschnikoff, from O. Hertwig, 93, II.) 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. -—»-- 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 fas- 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, interfascicu- 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 >c Tendon cell. „ Tendon fibers. Tendon cell. 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 o " 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 (infra vitam}. 74- — Cross-section of ligamentum nuchse 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 (ind. 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.'.v. Nucleus. Protoplasm. • Fat drop. Cell-membrane. Fig. 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. - 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 lacunae are surrounded by a narrow zone of ground-substance, which does not stain as does the ground- THE CONNECTIVE TISSUES. 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- afttr— 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 &r perichondrium, Fig- 77- — From a section through the cranial cartilage of a squid (after M. Fiirbinger, 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 ".: .' fc, ' .'' t- ' 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 °5°' 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. I I I 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°°- network in the immediate neighborhood of a capsule. Fine elastic 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 man 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 I 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. Figs. 8 1 and 82. — Lamellae seen from the surface ; X4°° (after v. Ebner 75). ay Primitive fibrils and fibril-bundles; r, 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 /J. 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 mar- 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. - I > Outer circum- ferential lamellae. •~^' Haversian or concentric lamellae. > ->/-^//:<\V;< -Haversian ' ' v *-r-\" *» ' ^ ' H$ canal. *j&t , ?> ^ J ' , ' — - - -'' / , ^ : - , f » ' ' O>» . 'y r •^^'-•^~ * 1 - j' ..- V/ /•- '• ."L^i-i— ^,-*4_V ^--* Interstitial - t r~s j * ^ lamellae. ,V.-" 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 ; y^ 56. The lamellae of bone are composed 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 /j. to 31 p. long, 6 /JL to I 5 /* wide, and 4 JJL to 9 p. 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 «-*•-- Canaliculi. - 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 Sharp ey ; in the adult they contain elastk: 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 Volkmanris canals. The structure of bone-marrow will be discussed with the blood- forming organs. I 1 6 THE TISSUES. (b) 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, 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 intramembranous 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-development. — 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 layei THE CONNECTIVE TISSUES. II/ 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 into 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. Periosteum. Unaltered hyaline cartilage. 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- n8 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 — Groove of ossification. Periosteum. Periosteal bone lamella. Primary marrow spaces. Fig. 86. — Longitudinal section of the proximal end of a long bone (sheep embryo) ; X 30- 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- beculae 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 bone-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. I2O 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 // to 91 p. long and 30 jn 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 Howship's lacuna. 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 — /'. e., it goes through the stages of vesicular cartilage and the formation 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 the longitudinal growth of the bone is made possible ; at the same time, 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 columns, 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 Epiphyseul-- ^J^^^-T^sL ; - •••' vessel. bone. (£'•••' »- V* v-^v> ';•• * Ossification ridge. Epiphyseal cartilage." Fig. 88 — Longitudinal section through epiphysis of arm bone of sheep embryo ; X I2> a, /;, Primary marrow spaces and bone lamellae of the diaphysis. cranial vault and the greater number of the bones of the face, and also in bone developed from the periosteum (perichondrium) sur- rounding the cartilage fundaments of endochondral bone. All fibrous-tissue bone is developed in the same way. The intramembranous bone-development begins by an approxi- mation and more regular arrangement of the osteoblasts of the osteogenetic layer of the periosteum about small fibrous-tissue 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-vessels de- veloped from the tissue elements of the periosteum not engaged in the formation of bone. THE CONNECTIVE TISSUES. I23 Intrarnembranous bone first appears in the form of a thin lamella of bone, which increases in size and thickness by the formation of trabeculse 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- Osteo !_:x- blast. ...~ > ' I c ' • c ioR 3 '<•'«£ -:- -:.i ,"/* ••Vr Fig. 89. — Section through the lower jaw of an embryo sheep (decalcified with picric acid) ; X 3°°' At # 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 trabeculae 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, i • Fig. 90. — 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- tissue. Outer fibrous - layer of periosteum. Osteogenetic layer of periosteum Osteoblasts space. Blood-ves-_ sel. Osteoblasts.- I fJ"A: matrix. ' Osteobiasts.^ Fig. 91. — From a cross-section of a shaft (tibia of a sheep) ; X 55°- In the 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 % 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. I2/ fibrils. Such preparations are also useful in studying the action of reagents (see below). The substance resembling mucin which cements the fibrillse 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 — /. e., 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 in 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 hours. They also swell in hydrochloric acid in strengths of o. i % to 6% . In strengths of 6% to 25 , 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. r, Cross- sections of the connective-tissue membranes separating involuntary muscle-cells ; of, an area showing so-called intercellular bridges; they are attached to the connective tissue membranes surrounding the cells (Mallory's differential connective- tissue stain). 1 36 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. "— Free ending. --» Nucleus. Sarcolemma. 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*75- 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 /M. 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- 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, Plensen'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. P'ig. 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) ; £, 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. Fibrils. 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 9CO- 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 muscie-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 the fibril bundles or muscle-columns 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 in closing the mus- cle-columns. Thus, we have in a cross-section slightly darker areas, the cross-sections of the muscle-colurnns, known as Cohn- heiiris fields or areas, separated by the network of sarcoplasm. (Fig. 98.) Fig. 99- — From a striated muscle of man ; obtained by teasing ; X 1200. hy A median disc lying in the transverse disc Q; 2, 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) : from Lacerta viridis ; X 160. Figs. 128 and 129, from a guinea-pig ; X 700. Fig. 130, from a hedge-hog; X I2O°- 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. 1 66 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 (infra mtani) (Huber, DeWitt) : A, Surface view ; J3, cross- section ; s, s, sarcolemma ; «/, neurilemma. * * If -^ mustttctll j Fig. 132. — Motor nerve-ending on Fig. 133. — Motor nerve-ending on heart muscle-cells of cat ; methylene-blue involuntary nonstriated muscle-cell from stain (Huber, De Witt). 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. 1 67 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 Cell-body. Process of cell. Neuraxis, ends 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 tela- 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 general 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. T-, J ^ i i 11 r -tr 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'" vo1' XXXVII)« all been investigated recently by I/O THE TISSUES. Dogiel, and the account here given follows closely his descrip- tion. End-bulbs of Krausc. — 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 // to 50 /2 broad and from 1 10 p. to 1 8o// long. They possess a thin connective-tissue capsule, in which are found round or oval nuclei, some 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. Phys.," vol. IX). SENSORY NERVE-ENDINGS. \*J \ 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 (Dogiel, "Arch. f. mik. Anat.," vol. XLI). 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.io 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 ; inn, 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 interlarnellar 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 branches on central nerve-fiber ; £, 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 /J. (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 intraf used fibers, 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 sheatb of Henle. Fig. 142. — Corpuscle of Herbst from bill of duck; X 600. 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 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 trabeculae 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 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. 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. VII). organ. As these nerves enter the capsule, the sheath of Henle blends with the capsule. The medullated nerve-fibers now and 12 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 or 3 mm. long. A capsule consisting of from 2 to 6 fibrous tissue lamellae, and broadest at the equatorial part of the Jour. Comp. Neural.," vol. x). end-organ, surrounds a number of in- Fig. 146. - Neurotendinous nerve end-organ from rabbit; teased 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. Fig- 147- — Cross-section of neurotendinous nerve end-organ of rabbit ; from tissue stained in methylene-blue : ;//, Muscle-fibers ; /, tendon ; c, 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 l8O 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 (sensory 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 % osmic acid. A nerve (not too thick) is placed in a i 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 O 9 Fig. 156.— Red blood-corpuscles from various vertebrate animals; XIQOO (Welker'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 ; z, from goat ; /£, 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 llarna 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- 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 y. (7.2 p. to 7.8 //), in their long diameter, and 1.6 fj. to 1.9/1 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 : s •PECIES. SIZE. No. IN CUBIC MILLI- METER. Man . . {Homo) . . 7.2-7. 8/z . . . 5,000,000 Monkey . . 7 ft . . . . . 6,355,000 Hare Guinea-pig . . (Lepus cuniculus} . . (Cavia cob.} . . . . .7-16 . . . . 7.48 . . . . , 6,410,000 . . . 5,859,500 Dog . . (Canis fam.} . . . . . 7.2 . . . . . . . . 6,650,000 Cat . . (Felis dom.} . . . . . 6.2 . . . . . 9,900,000 Horse . . (Equus cab . ) . . . . .5.58 . . . . . 7,403,500 Musk-deer Spanish goat .... . . (Moschus jav.} . . . . (Capra his.} . . . . 2.5 . . 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 . . ( Testudo grceca} . . L. 21.2 . . . . 629,OOO B. 12-45 Lizard . . (Lacerta agil.} . . . . L. 15-75 • . . . 1,292,000 B. 9-1 Snake . . L. 22.0 . . . . 829,400 B. 13.0 Frog . . (Rana temp. ) . . . . L. 22.3 . . . . 393,200 B. 15-7 Toad . . L. 21.8 . . . . 389,000 B. 15-9 Triton . . (Triton crist.} . . . . L. 29-3 • . . . 103,000 B. 19.5 BLOOD AND LYMPH. No. IN " SPECIES. SIZE. CUBIC MILLI- METER. Salamander (Salamandra mac.} . . Length, 37.8 80,000 Breadth, 23.8 {Proteus angu.} . . Sturgeon (Acipenser St.] . . . Carp (Cyprinus Gobio] . . L. L. B. L B. 5« 35 13-4 10.4 17.7 10. 1 35,000 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 p. 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 ; e, 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. 192 BLOOD AND BLOOD-FORMING ORGANS. They vary in size from 5 // to 7. 5 jj. 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 p. to 10 fjt. 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 nuclei, 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 polynuclear 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, Fig. 158. — Ehrlich's leucocytic granules; X 1800 (from preparations of H. F. Miiller) : #, Acidophile or eosinophile granules, relatively large and regularly distributed ; £ , neutrophile granules ; j3, amphophile granules, few in number and irregularly dis- tributed ; 7, mast cells with granules of various sizes ; (5, basophile granules, (a, ff, 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-, ft-, 8-, Y-, 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 eosinophilc granules. In normal blood from I Jj 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 ?- 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 0.5% to I Jo of the white blood-cells. It cannot at this time be definitely stated whether the different varieties of granules are to be looked upon as specific products of the protoplasm of the leucocytes, possibly of the nature of granules which may be likened to the secretory granules of glandular cells, or whether they are to be regarded as cell inclusions. It has also not been clearly shown whether one variety of granules may develop into another variety, — neutrophile into eosinophile, — although this has been suggested. According to Weidenreich, eosinophile gran- ules are thought to represent fragments of erythrocytes, enclosed within the protoplasm of leucocytes. The polymorphism of the leucocyte-nucleus has induced many investigators to advance the theory that a direct division takes place (fragmentation — Arnold, Lowit). Flemming (91, III), however, succeeded in demonstrating that true rnitotic processes actually take place, so that in this respect there really exists no difference between leucocytes and other cells (compare also H. F. Miiller, 89, 91). It is only in the formation of polynuclear leucocytes that the poly- morphous nucleus sometimes undergoes a fragmentation process which results in several parts. But even in this case pluripolar mitoses have been observed. A division of the cell-body subse- quent to that of the nucleus is lacking in the processes just described. As a result a single cell with several nuclei is formed (polykaryocyte). The fate of such cells is still in doubt. The extraordinary motility which most leucocytes possess, is in great part responsible for their wide distribution, even outside of the vascular system. They have the power of creeping through the walls of the capillaries (diapedesis, Cohnheim 67, I), and of penetrating into the smallest connective-tissue clefts, between the cells of epithelia, etc., whence they either pass on (migratory cells) or remain stationary for a time. An important function falls to the lot of the leucocytes in the absorption of superfluous tissue particles or in the removal of foreign bodies from certain regions of the body. In the first case they take part in a process of tissue-disintegration ; in the second, they take up the particles by means of their pseudo- podia for the purpose either of assimilation or of removal (phago- '3 194 BLOOD AND BLOOD-FORMING ORGANS. cytes). It piay be readily understood that the latter function of the leucocytes is of the greatest importance in certain pathologic pro- cesses. It is somewhat venturesome at the present state of our knowl- edge to make definite statements as to the origin in postembryonic life of the various forms of white blood-cells "above described. The following statement, however, seems warranted from the evidence at hand. The lymphocytes would seem to be developed in the meshes of adenoid tissue, especially in the so-called germ centers of Flemming, m the adenoid tissue of lymph-glands and lymph-follicles (see under these). Here the cells undergo active karyokinetic division, but where the cells which pass through the process originate is a matter concerning which there is a difference of Opinion. Some investi- gators believe that they penetrate the germ centers with the lymph, and find there a suitable place for division. Again, others see in Flemming's germ centers permanent organs whose elements remain stationary and supply the blood with a continuous quota of lympho- cytes. Be this as it may, the fact remains that the germ centers are the most important regions for the formation of lymphocytes. From these they pass out with the lymph current into the blood circulation, or directly into the blood-vessels, there to enter upon the functions which they are called upon to perform. The leucocytes with neutrophile granules are probably developed in the blood and lymph from mononuclear leucocytes which have their origin in the spleen pulp, possibly also in the bone-marrow. The leucocytes of circulating blood with eosinophile granules in all probability come from mononuclear cells with such granules found in bone-marrow. Under certain conditions it would seem that they also develop in connective tissue. The leucocytes with the basophile granules prob- ably enter the circulation from the connective tissue of certain re- gions. The lymphocytes and leucocytes found in the blood are also found in the lymph-vessels and lymph-spaces. 4. BLOOD PLATELETS-THROMBOCYTES. The third element of the blood is the blood platelets (Bizzozero) (blood-placques, Laker ; hematoblasts , Hayem ; thrombocytes, Deck- huysen). They are extremely delicate and transitory structures, whose existence in the living blood was denied for a long time by many in- vestigators, but whose presence in the wing vessels of the living bat was conclusively demonstrated by Laker (84). They are free from hemoglobin, are of round or oval shape, and in mammals measure about 3 fJL in diameter. Owing to the fact that they readily clump together when blood leaves the vessels, and undergo change, it is somewhat difficult to give an estimate of their number. They are said to be present in human blood to the extent of 200,000 to 300,000 in every cubic millimeter. By the exercise of great care BLOOD AND LYMPH. and the employment of special methods on the part of a number of recent observers (Detjen, Deckhuysen, Kopsch and Argutinsky), they have been able to show that these structures present a more complicated structure than was formerly thought. When exam- ined in an isotonic salt solution (for mammals 0.9 to 0.95 sodium chlorid solution), they present an oval or short spindle-shaped form, and in them there can be made out a relatively large structure, which stains in certain basic aniline stains and is interpreted as a nucleus (Deckhuysen). When examined after a method suggested by Detjen (with a I per cent, agar solution there is mixed 0.6 per cent, sodium chlorid, 0.3 per cent, of sodium metaphos- phate and dipotassium phosphate; a thin layer of this agar mixture is spread on the slide and a drop of blood mounted between it and the cover), the blood platelets or throm- bocytes may be observed on the warm stage for several hours, and it may be seen that they present ameboid move- ment, in that short, thread-like pro- cesses pass out from the cell, which may alter their shape and position and which may be again withdrawn. When the blood leaves the blood- <_J . ^ , vessels, the blood platelets or throm- bocytes break down very quickly, Fig. 159. — Fibrin from laryngeal unless the above-mentioned methods vessel of child ; x about 300. are made use of, so that in ordinary fresh preparations or generally in dried films they are not to be observed in an unaltered state. The nuclei disappear and the pro- toplasm becomes granular or vacuolated. The breaking down of the blood platelets or thrombocytes is accompanied by the forma- tion of fibrin (coagulation of the blood), the fibrin threads beginning at the borders or processes of the platelets, and radiating in all directions (Kopsch). Hemokonia. — H. F. Muller (96) found in the blood of healthy and diseased individuals highly refractive, colorless, and round (seldom rod- like) bodies, which he terms ' ' hemokonia. ' ' Their numbers vary, although they are normal constituents of the blood. Their nature and origin are obscure. They do not dissolve in acetic acid, nor are they blackened by osmic acid. The latter would seem to indicate that they do not consist of ordinary fat substance, although they are probably com- posed of a substance closely allied to fat. They are usually i fj. in diam- eter. BLOOD AND BLOOD-FORMING ORGANS. 5. BEHAVIOR OF BLOOD-CELLS IN THE BLOOD CURRENT. In the circulating blood the behavior of the formed elements varies. The more rapid axial current contains very nearly all the erythrocytes, and as a consequence very few are found adjacent to the walls of the vessels. In the peripheral current, on the other hand, are found most of the leucocytes, and in a retarded circulation they are seen to glide along the walls of the vessels. At the bifurcations of the vessels, especially of the capillaries, the erythrocytes are sometimes caught and elongated by the division of the current, the one-half of the cell extending into the one and the other half into the other branch of the vessel, while the corpuscle oscillates back and forth. When again free the cell immediately resumes its original shape. From this it is seen that erythrocytes are very elastic structures. In the smaller vessels and capillaries, especially when the latter are altered by pathologic conditions, the leucocytes may be seen passing out of the vessels, and it would seem that they are able to penetrate through the walls and even through the endo- thelial cells lining the blood-vessels (compare also Kolossow, 93). First, they' send out a fine process, which is probably endowed with a solvent action. This penetrates the wall of the vessel, after which the remainder of the cell pushes its way through slowly. B. LYMPHOID TISSUE, LYMPH-NODULES, AND LYMPH- GLANDS. As to the origin of lymphoid tissue, the lymph-glands, and the spleen, there is still considerable difference of opinion. Most authors believe that these structures are developed from the middle germinal layer (Stohr, 89 ; Paneth ; J. Schaffer, 91 ; Tomarkin). Others believe in an entodermic origin (Kupffer, 92; Retterer; Klaatsch ; C. K. Hoffmann, 93, II). The framework of lymphoid tissue is a reticular connective tis- sue (adenoid connective tissue — His, 61). This consists of a net- work of fine fibrils of reticular and white fibrous connective tissue and of cells (endoplasm and nuclei) which are situated on the reticulum, often at nodal points. Within its meshes the lymph-cells lie in such numbers and so densely arranged that on microscopic examination the network is almost entirely covered unless very thin sections are used. The cells may be removed from the meshes of the reticulum by stippling and brushing section with a fine brush or by placing sections in a test-tube partly filled with water and sub- jecting them to vigorous shaking, or, still better, by subjecting sections or pieces of lymphoid tissue to digestion with pancreatin. Lymph tissue may be diffuse, as in the mucous membrane of the air-passages and as in that of the intestinal tract, uterus, etc. (vid. Sauer, 96). Lymphoid tissue may be also sharply defined in the LYMPHOID TISSUE, LYMPH -NODULES, AND LYMPH-GLANDS. form of round nodules, simple follicles or nodules. These are either single, solitary lymph-follicles, or gathered into groups, agminated lymph -nodules. They are found scattered in the mucous mem- brane of the mouth, pharynx, and intestine. In lymph-nodules also we find the characteristic lymph-cells and the adenoid reticulum. As a rule, the former are arranged concentrically at the periphery ; and in the center of the nodule the reticular tissue usually has wider meshes, and the lymph-cells are less densely placed. (Fig. 160.) In the center of the nodule the cells often show numerous mitoses, and it is here that an active proliferation of the cells takes place. The cells may either remain in the lymph-follicle or the newly formed cells are pushed to the periphery of the nodule, and are then swept into the circulation by the slow lymph current which circu- lates between the wide meshes of the reticular connective tissue. Flemming (85, II) has called that central part of the nodule con- taining the proliferating cells the germ center or secondary nodule (compare p. 194). The germ centers are transitory structures, and are consequently found in different stages of development. They may even be absent for a time. — -———-—- —-Epithelium < of intes- \ tine. A Gland. Fig. 160. — A solitary lymph-nodule from the human colon. At a is seen the pronounced concentric arrangement of the lymph-cells. The lymph-glands are organs of a more complicated structure, but also consist of lymphoid tissue. They are situated here and there in the course of the lymph-vessel and are widely distributed. Their size varies greatly. In shape they are much like a bean or kidney, and the indentation on one side is known as the hilum. The affer- ent lymph-vessels, the vasa afferentia, enter at the convex surface of the organ, while the efferent vessels, the vasa efferentia, pass out 198 BLOOD AND BLOOD-FORMING ORGANS. at the hilum. The whole gland is surrounded by a capsule consist- ing of two layers : the outer is made up of a loose, and the inner of a more compact, connective tissue in which elastic fibers and a few smooth muscle-fibers are imbedded. Portions of the inner layer pass into the substance of the gland to form septa, or trabeculce, by means of which the organ is divided into a number of imperfectly separated compartments. These trabeculae may be very well developed, as in the lymph-glands of the domestic cattle, or only If Fig. 161. — Transverse section of human cervical lymph-gland, showing the general structure of a lymph-gland ; X l%- bg. Blood-vessels ; cf, fibrous capsule ; /£, hilum ; kz, germ-center ; «/, lymph-nodule ; sc, cortical substance ; gm, medullary substance ; try trabeculae ; 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 trabeculse 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- LVMPHOID 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. 1 6 1.) 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) Germ center. Mitosis. — Lymph-sinus. 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 loose 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 (Blutlymphcfrusen, 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 trabeculae 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 mononuclear 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 trabeculse 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-formine function. This has also o 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 (trabeculae), 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 -z'r Fig. 163. — Portion of section of human spleen ; X '5- 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 (Tkomrf s Zwischcn- stiick), 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. 205 "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 Intralobular trabecula. Artery to one of the ten -- compartments. - Capsule. -- Intralobular venous spaces. Intralobular vein. Ampulla of Thoma. - - Spleen pulp cord. — Interlobular vein. — Intralobular vein. Intralobular artery. - ^ Interlobular trabecula. Intralobular trabecula. ^—"—5 Malpighian corpuscle. 1-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. A Fig. 165. — Cells containing pigment, blood-corpuscles, and hemic masses from the spleen of dog; X 1800 (from cover-glass of H. F. Miiller). _ b Fig. 1 66. — From the human spleen ; X 80 (chrome- silver method) : a, Larger fibers of a Malpighian body ; £, 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 vertebrae. 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. BLOOD AND BLOOD-FORMING ORGANS. The typical cellular elements of red bone-marrow are : i. The Marrow-cells, or Myehcytes.—ltesz 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- • 1 Fig. 167. — Cover-glass preparation from the bone-marrow of dog ; X 1200 (from preparation of H. F. Miiller) : a, Mast-cell ; 6, lymphocyte ; c, eosinophile cell ; d, red blood-cell ; e, erythroblast in process of division ; fy f, normoblast ; £, 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 ery throb lasts, 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 Granules. — 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. rs* 'n/a /&- - . i*^%. &} w Fig. 168. — From a section through human red bone-marrow; X 680. Technic No. 216 : a, f, Normoblasts ; l>, reticulum ; c, mitosis in giant cell ; d, giant cell ; f, h, myelocytes ; gt mitosis ; z, space containing fat-cells. 4. Cells with basophilic granules. In the bone-marrow are found mononuclear cells in which basophile granules may be differentiated with special reagents. 5. The various forms of leucocytes and the lymphocytes found in blood and lymph. 6. The giant cells (myeloplaxes), which are situated in the center of the marrow, and contain simple or polymorphous nuclei, or lie adjacent to the bone in the form of osteoclasts, which are, as a rule, polynuclear (compare p. 120). The physiologic significance of the giant cells is still obscure. They probably originate from single leucocytes by an increase in size of the latter, and not, as many assume, from a fusing of several leucocytes. The giant cells are endowed with ameboid movement, and often act as phagocytes (the latter quality is denied them by M. Heidenhain, 94). 14 2IO BLOOD AND BLOOD-FORMING ORGANS. M. Heidenhain (94) has made a particular study of the giant cells. According to him the nuclei of these cells take the form of per- forated hollow spheres whose thick walls contain "endoplasm." The latter is continuous with the remaining protoplasm of the cell, the " exo- plasm " through the "perforating canals" of the nuclear wall. The exoplasm is arranged in three concentric layers, separated from each other by membranes, the external membrane of the outer zone being the membrane of the cell. The outer layer or marginal zone is of a transient nature, but is always renewed by the cell. Thus, the cell -membrane is replaced by the secondary membrane situated between the second and third zone. According to the same author the functions of the giant cells appear to consist in " the selection and elaboration of certain albu- minoid substances of the lymph and blood currents, which are later returned to the circulation. ' ' The number of centrosomes occurring in the mononuclear giant cells of the bone-marrow is very large, and in some cases, as in a pluripolar mitosis, may even exceed one hundred in number. The distribution of the blood-vessels in the bone-marrow is as follows : On entering the bone the nutrient arteries divide into a large number of small branches, which then break up into small arterial capillaries. The latter pass over into relatively large venous capillaries with relatively thin walls, which appear perforated in certain places, so that the venous blood pours into the spaces ii of the red bone-marrow where the current is very slow. The blood passes out by means of smaller veins formed by the conflu- ence of the capillaries which collect the blood from the marrow. It is worth mentioning that the venous vessels, while inside of the bone-marrow, possess no valves ; but, on the other hand, they have an unusually large number of valves immediately after leaving the bone. Yellow bone-marrow is derived from red bone-marrow by a change of the marrow-cells into fat-cells. The gelatinous marrow, on the contrary, is characterized by the small quantity of fat which it contains. Neither the yellow nor the gelatinous bone-marrow is a blood-forming organ (compare Neumann, 90; Bizzozero, 91 ; H. F. Miiller, 91 ; van der Stricht, 92). E. THE THYMUS GLAND. The thymus gland is usually considered as belonging to the lymphoid organs, although in its earliest development it resembles an epithelial, glandular structure. In the epithelial stage, this gland develops from the entoderm of the second and third gill clefts. Mesodermic cells grow into this epithelial structure, proliferate and then differentiate into a tissue resembling adenoid tissue. It retains this structure until about the end of the second year after birth, when it slowly begins to retrograde into a mass of fibrous tissue, adipose tissue, and cellular debris, which structure it presents in adult life. THE THYMUS GLAND. 2 I I By means of connective-tissue septa, the thymus is divided into larger lobes, and these again into smaller lobes, until finally a number of small, irregularly spheric structures are formed — the lobules of the gland. These are, however, connected by cords of lymphoid tissue, the so-called medullary cords. The lobules of the Fig. 169. — A small lobule from the thymus of child, with well-developed cortex, presenting a structure similar to that of the cortex of a lymph-gland ; X °° : ay Hilus ; b, medullary substance ; c, cortical substance ; d, trabecula. thymus gland consist of a reticular connective tissue much more deli- cate at the periphery than at the center of the lobule. The reticulum supports branched connective-tissue cells, with relatively large nuclei. In the meshes of the reticular tissue are cellular elements, in structure similar to the lymphocytes, which are more numerous at the periph- ery of the lobule than at its center, so that we may here speak of Fig. 170. — Hassal's corpuscle and a small portion of medullary substance, showing reticulum and cells, from thymus of a child ten days old. the lobule as divided into a cortical and a medullary portion. Leucocytes with polymorphous nuclei, also leucocytes with eosino- phile granules, are also found. The medullary portion is usually entirely surrounded by the cortical substance, but may penetrate to the periphery of the lobule, allowing the blood-vessels to enter and 212 BLOOD AND BLOOD-FORMING ORGANS. leave at this point. In the cortical substance occur changes which result in the formation of structures closely resembling the cortical nodules of lymph-glands. Until recently, little was known of the significance of this organ. A careful study revealed a similarity between certain cellular ele- ments of the thymus and the constituents of the blood-forming organs, — a similarity still more striking from the presence of nucleated red blood-cells in the thymus. Logically, then, the embryonal thymus is to be regarded as one of the blood-forming organs (Schaffer, 93, I). During embryonic life from the fourth month on and for some time after birth, there are found in the thymus peculiar epithelial bodies, known as the corpuscles of Hassal. They are spheric struc- tures, about o. i mm. in diameter, whose periphery shows a con- centric arrangement of the epithelial cells. In their central portions are found a few nuclear and cellular fragments. These bodies occur only in the thymus gland. They are remnants of the primary epithelial, glandular structure of the thymus, and are formed by an ingrowth of mesoderm which breaks down the epithelium into small irregular masses, mechanically compressed by the proliferating mesoderm. The thymus gland has a relatively rich blood supply. Arterial branches enter the lobules usually near the medullary cords and form capillary networks at the boundaries of the medullary and cortical portions ; from this anastomosing capillaries radiate to the periphery of the lobules, joining to form a relatively dense capillary network under the connective-tissue covering. The veins arise from this cap- illary network and are situated mostly in the interlobular connec- tive tissue. Certain of the veins are in the medullary portions of the lobules, where they accompany the arteries (Kolliker, v. Ebner). The lymph-vessels are in the interlobular connective tissue in close apposition with the adenoid tissue. Nerve-fibers accompanying the blood-vessels have been ob- served. II. THE CIRCULATORY SYSTEM. THE walls of the blood-vessels vary in structure in the different divisions of the vascular system. All the vessels, including the heart, possess an inner endothelial lining. In addition to this, the larger vessels are provided with other layers, which consist, on the one hand, of connective and elastic tissue and, on the other, of non- striated muscle-fibers. The vessels are also richly supplied with nerves, that form plexuses in which ganglion cells are sometimes found, and in the larger vessels the outer layer is honeycombed by nutrient blood-vessels, called vasa vasorum. In the heart, the mus- cular tissue is especially well developed. According to the structure THE VASCULAR SYSTEM. 213 •of the vessels, we distinguish, in both arteries and veins, large, medium-sized, small, and precapillary vessels, and finally, the capil- laries themselves. The latter connect the arterial and venous pre- capillary vessels. In the lymphatic system we must further dis- tinguish between the larger lymph-vessels, the sinuses, and the capillaries. A. THE VASCULAR SYSTEM. J. THE HEART, In the heart there are recognized three main coats — the endo- cardium, the myocardium, and the pericardium or epicardium. The endocardium consists of plate-like endothelial cells, with very irregular outlines. Beneath this endothelial layer is a thin membrane composed of unstriped muscle-cells, together with a small number of connective-tissue and elastic fibers. Below this is a somewhat thicker and looser layer of elastic tissue connected ex- ternally with the myocardium. Between the two layers are found, here and there, traces of a layer of Purkinje 's fibers (compare p. 147). Purkinje's fibers are found in the heart of many mammalia, although absent in the heart of the human adult. The auriculoventricular valves of the heart represent, in general, a duplication of the endocardium. The layer of smooth muscle- fibers found in the latter is better developed on the auricular surface. At the points of insertion of the chordae tendineae the connective- tissue layer is strongly developed and assumes a tendon-like con- sistency. The semilunar valves of the aorta and pulmonary artery have a similar structure. In the nodules of these valves the elastic fibers are especially dense in their arrangement. The myocardium is made up of the heart muscle-fibers already described (yid. p. 145). Between the heart muscle-fibers and bundles of such fibers are thin layers of fibrous connective tissue containing a network of capillaries. The myocardium of the auricles may be divided into two layers, of which the outer is common to both auricles, the fibers of which have a nearly circular arrangement, while the deeper layer is separate for each chamber. The arrange- ment of the heart muscle-fibers of the ventricles is complicated. With special methods of maceration J. B. MacCallum was able to show that "the superficial fibers are found to have origin in the auriculoventricular ring, to wind about the heart spirally, and to end in tendons of the papillary muscle of the opposite ventricle. The deep layers also begin in the tendon of one auriculoventricular ring, pass around to the interventricular septum, cross over backward or forward in this septum, and end in the papillary muscle of the other ventricle. In the light of this, the heart consists of several bands of muscles with tendons at each end, rolled up like a scroll or like the letter S." The musculature of the auricles is almost completely 214 THE CIRCULATORY SYSTEM. separated from that of the ventricles by means of the annulus fibro- sus atrioventricularis , or the auriculoventricular ring, which consists in the adult of connective tissue containing numerous delicate and densely interwoven elastic fibers. The pericardium consists of a visceral layer, the epicardium, ad- hering closely to the myocardium, and a parietal layer (pericardium), loosely surrounding the heart and continuous at the upper portion of the heart with the visceral layer. Between the two layers is the pericardial cavity, containing a small quantity of a serous fluid — the pericardial fluid. In the visceral layer (the epicardium) we find a connective-tissue stroma covered by flattened mesothelial cells. A similar structure occurs also in the parietal layer, although here the connective -tissue stroma is considerably reinforced. De- posits of fat, in most cases in the neighborhood of the blood-vessels, are sometimes seen between the myocardium and the visceral layer of the pericardium. According to Seipp, the distribution of the elastic tissue in the heart is as follows : The endocardium of the ventricles contains far more elastic tissue than that of the auricles, especially in the left ventricle, where even fenestrated membranes may be present. In the myocardium of the ventricles there are no elastic fibers aside from those which are found in the adventitia of the contained blood- vessels. In the myocardium of the auricles, on the contrary, such fibers are very numerous and are continuous with the elastic elements in the walls of the great veins. The epicardium also pre- sents elastic fibers in the auricles continuous with those of the great veins emptying into the heart, and in the ventricles continuous with those in the adventitia of the conus arteriosus. In those portions of the heart-wall containing no muscular tissue the elastic elements of the epicardium are continuous with those of the endocardium. In the new-born the cardiac valves possess no elastic fibers, although they are present in the adult. They are developed on that side of each valve, which, on closing, is the more stretched — for instance, on the auricular side of the auriculoventricular valves. The heart has a rich blood supply. The capillaries of the myo- cardium are very numerous, and so closely placed around the muscle bundles that each muscular fiber comes in contact with one or more capillaries. In the endocardium the vessels are confined to the connective tissue. The auriculoventricular valves con- tain blood-vessels, in contradistinction to the semilunar valves, which are non-vascular, while the chordae tendineae are at best very poorly supplied with capillaries. The coronary arteries, which terminate in the capillaries above mentioned, are terminal arteries in the sense that " the resistance in the anastomosing branches is greater than the blood pressure in the arteries leading to those branches (Pratt, 98). This observer has further shown that the vessels of Thebesius (small veins which open on the endocardial surfaces of the ventricles and auricles and THE VASCULAR SYSTEM. communicate directly with all the chambers of the heart) "open from the ventricles and auricles into a system of fine branches that communicate with the coronary arteries and veins by means of capillaries, and with the veins, but not with the arteries, by passages of somewhat larger size"; so that, although the blood supply through the coronary arteries for a given area of the myocardium is cut off, the heart muscle of this area may receive blood through the vessels of Thebesius. Lymphatic netivorks have been shown to exist in the endocar- dium, and their presence in the pericardium is not difficult to demon- strate. Little is known with regard to the lymph -channels of the myocardium. The nerve supply of the heart includes numerous medullated nerve-fibers, the dendrites of sensory neurones, and numerous non- medullated fibers, the neuraxes of sympathetic neurones. Smirnow (95) described sensory nerve -endings in the endocardium of amphibia and mammalia, which he suggests may be the terminations of the depressor nerve. Dogiel (98) has corroborated and extended these observations, and has described complicated sensoiy telodendria situated both in the endo- and pericardium. The latter states that, after forming plexuses and undergoing repeated division, the medul- lated sensory nerves lose their medullary sheaths, the neuraxes further dividing in numerous varicose fibers, variously interwoven and terminating in telodendria, which vary greatly in shape and configuration. These tek>dendria are surrounded by a granular substance containing branched cells, probably connective-tissue cells, the interlacing branches of which form a framework for the telodendria. Similar sensory nerve-endings occur in the adventitia of the arteries and veins of the pericardium (Dogiel, 98) ; and Schemetkin has shown that sensory nerve-endings occur in the adven- titia and intima, especially in the latter, of the arch of the aorta and pulmonary arteries. In the heart, under the pericardium on the posterior wall of the auricles and in the sulcus coronarius, are found numerous sympathetic neurones whose cell-bodies are grouped to" form sympathetic ganglia. The neuraxes of these sympathetic neurones — varicose, nonmedullated nerve-fibers — form intricate plexuses situated under the pericardium and, penetrating the myo- cardium, surround the bundles of heart muscle-fibers. From the varicose nerve-fibers constituting these plexuses, fine branches are given off, which terminate on the heart muscle-cells in a manner previously described (see p. 166 and Fig. 132). The cell-bodies of the sympathetic neurones, the neuraxes of which thus terminate on the heart muscle-fibers, are surrounded by end-baskets, the telodendria of small medullated nerve-fibers which reach the heart through the vagi. The slowed and otherwise altered action of the heart-muscle, produced on stimulating directly or indirectly the vagus nerves is therefore due not to a direct action of these nerve- fibers on the heart muscle-cells, but to an altered functional activity 2l6 THE CIRCULATORY SYSTEM. produced by vagus stimuli in at least some of the sympathetic neu- rones situated in the heart, the neuraxes of which convey the im- pulse to the heart muscle. The heart receives further nerve supply through sympathetic neurones, the cell-bodies of which are situated in the inferior cervical and stellate ganglia, the neuraxes of which enter the heart as the augmentor or accelerator nerves of the heart. The mode of ending of these nerve-fibers has not as yet been fully determined. It may be suggested as quite probable that they ter- minate on the dendrites of sympathetic neurones, the cell-bodies of which are not inclosed by end-baskets of nerves reaching the heart through the vagi, as above described. It is also possible that they end directly on the heart muscle-cells. The cell-bodies of the sympathetic neurones, the neuraxes of which form the augmentor nerves, are surrounded by the telodendria of small medullated fibers, forming end-baskets, which leave the spinal cord through the anterior roots of the upper dorsal nerves. Besides the nerves here described, nonmedullated nerves (whether the neuraxes of sympa- thetic neurones, the cell-bodies of which are situated inside or out- side of the heart has not been fully determined), form plexuses in the walls of the coronary vessels, terminating, it would seem, on the muscle-cells of the media (vasomotor nerves). 2. THE BLOOD-VESSELS. A cross-section of a blood-vessel shows several coats. The inner consists of flattened endothelial cells, and is common to all vessels. The second varies greatly in thickness, contains most of the contractile elements of the arterial wall, and is known as the media, or tunica media. Its elastic fibers have in general a circular arrangement and are fused at the inner and outer surfaces to form fenestrated membranes, the lamina elastica interna and externa. Outside of the media lies the adventitia or tunica externa, consist- ing in the arteries almost entirely of connective tissue and in the veins principally of contractile elements, smooth muscle-fibers. Between the internal elastic membrane and the endothelial layer is a fibrous stratum which varies in structure in the different -vessels of larger caliber. This is the subendothelial layer, or the inner fibrous layer, and forms, together with the endothelium, the intima or tunica intima. Bonnet (96), as a result of his own investigations, suggests a somewhat different classification of the layers composing the arterial wall. According to him, the endothelium alone con- stitutes the intima. The elastic membranes, both internal and external, together with the tissue lying between them, and that between the internal elastic membrane and the intima, constitute the media. The tissue layers outside the external elastic membrane form the tunica externa (adventitia). (a) Arteries. — In the great arterial trunks, such as the pulmo- nalis, carotis, iliaca, etc., the tunica media possesses a very typical THE VASCULAR SYSTEM. structure. It is divided by means of elastic fibers and membranes Intima. - Elastica in- terna. Endothelium of the intima. *~ > Media. Fenestrated elastic mem- brane. Elastica ex- terna. Inner layer of adventitia. Outer layer of adventitia. .1 Vasa vasorum. Fig. 171. — Cross-section of the human carotid artery ; X 15°' (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 2l8 THE CIRCULATORY SYSTEM. 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 fibro-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 in 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 brae hi al, 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 Fig. 173.— Precapillary vessels from longitudinal fibers, the subendo- mesenteryofcat: a, Precapillary artery ; thelial layer, which is thin and is b, precapillary vein possessing no muscu- i • • , j 11 t_ L\ c lar tissue. 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 muscuiar 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. 2I9 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. (b) 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. Elastica interna. Media. Fenestrated elastic membrane. Inner layer of the adventitia with longitudinally ar- ranged muscle- cells. Connective tissue of the adventitia. 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 220 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. Intima. - Media. — Adventitia xvith nonstriated muscle-cells in cross-sec- tions. Fig. 175. — Section of small vein (human); X 64O- 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). (c) 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. 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 very 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. 1 66 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 are found not only in the adventitia, but also in the intima, as has been shown by Schemetkin. (See p. 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. Timofeew 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 perilymphatic 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 J6o. (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 i«ito 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, hematoxylin. 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 «-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 hematoxylin 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 /^-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 ^-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 overstaining 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 1 20 Methyl-green, " " " . . 125 Distilled water, 300 Absolute alcohol, 200 Glycerin, 100 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-green, 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 ( Griibler' 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, hemoglobin 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 c/c 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 BIood=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: «, Slide used in counting ; l>, sectional view ; cy a portion of ruled bottom of the well ; d, pipette. the ampulla). The pipette is then carefully shaken to mix thoroughly the blood and the diluting fluid. Either of the following two solutions may be used for diluting the blood : Hay em' 's Solution : Bichlorid of mercury 0.5 gm. Sodium chlorid i.o gm. Sodium sulphate 5.0 gm. Distilled water 2CO.O c.c. Toisorf s Fluid {as given by V. Kahlderi) : Methyl violet 5 B 0.025 gm. Neutral glycerin 30.0 c.c. Distilled water 80.0 c.c. Mix the methyl violet with the glycerin and distilled water ; to this solution is added — Sodium chlorid (C. P.) i.o gm. Sodium sulphate (C. P.) 8.0 gm. Distilled water . 80.0 c.c. TECHNIC (BLOOD AND BLOOD-FORMING ORGANS). 233 Filter, and the solution will be ready for use. The white blood-cells are stained violet, and may thus be counted with the red. The diluting fluid contained in the capillary tube is then blown out, and a small drop of the diluted blood is placed on the center of the small glass disc. The small disc is surrounded by a ring of glass, cemented to the slide. This glass ring is o. i mm. thicker than the glass disc. When this small chamber is covered with a thick cover-glass, we have a layer of blood o. i mm. deep between the disc and the cover-glass. On the upper surface of the small glass disc (on which the drop of diluted blood was placed) there are marked off 400 small squares. The sides of the small squares are ^ mm. long. It will be seen that the layer of blood over each of the squares would have a cubical contents of — ?oVo of a cubic millimeter (fo X ^ X TV == ?in>cf)- The hemocytometer slide is now placed on the stage of the micro- scope, where it should remain undisturbed for several minutes before counting. The red blood-cells in 25 to 50 squares are then counted. To ascertain the number of red cells' in a cubic millimeter the following formula may be useful : {each mass of) f ,., . ^ fin') f blood counted, L yd J fllutlon' I X" \ ^ C6"S [ j here ico [ / } counted f , r , TftW c.mm. ) i. j. J_ _ J number of red n (number of squares counted) | blood-cells in I c.mm. Or, ascertain the average of the red blood-cells in the squares counted, and multiply this number by 400,000. In case it is desired to count only the white blood- corpuscles, a ft per cent, solution of glacial acetic acid is used for diluting the blood. This solution bleaches the red cells, and brings out clearly the white corpuscles. The blood is diluted only ten times, using for this purpose the Thoma- Zeiss pipette for counting white corpuscles. The formula then reads as follows : ( the number of white ^j 4000X^(10) Xni blood -corpuscles I ( 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 /Jt). 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 rilled 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 o* 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 gat 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 corneum 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 glandulce labiates 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 r— mem- i brane \ with \ igh \ ^ tpil- \ Duct of 1 gland. Epithe- ) 1 1 i u m of mu- cous mem- brane. Gland. ; fiedep- / iderm- / is. '- Striated I muscle. Hairfol- licles. Epider - mis. 180. — Section through the lower lip of man ; X 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.) \. 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 // 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 -^%m / W ft--- Pulp cavity. Cementum. Fig. 181. — Scheme of a longitudinal section through a human tooth. In the enamel are seen the ''lines of Retzius." 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 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 ju. to 4.5 fjL 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 — 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 I7°- 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 Q * B c -• 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 /}, 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 ' - ^ v ^\ ^ r f- +-M>^ '3-* - ^ A 1 -^-'^ ~> ^ •* -,V tVVr ^- C> ^ 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 cementum. or enamel germs. In their first stage of development the enamel germs are knob- like, but later' their bases spread, and they become a\ 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 J 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 vitani) : 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 prcefonnativa. 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 prseforma- Fig. 188. Fig. 189. Figs. 186-189. — ^our 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. 1 88, 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. 1 86, 187, and 188, X IIQ 5 Fig- l89> X 4°- <*» a> a> a-> Epithelium of the oral cavity ; ^, 3, b, b, its basal layer; c, c, c, the superficial cells of the enamel organ ; d, d, d, d, enamel pulp; /, />, /, dentinal papilla ; .r, j, enamel-forming elements (enamel cells) ; o, odontoblasts ; S, enamel germ of the permanent tooth ; v, part of the enamel ledge of a temporary tooth ; u, surrounding connective tissue. 245 246 THE DIGESTIVE ORGANS. 44- pulp. — Enamel cells. r~ — Odontoblasts. tiva), the axial portion of the cells remaining intact as the dentinal fibers ; the basal portions of the cells containing the nuclei persist, later constituting the odontoblasts of the adult pulp. By the fusion of the segments of the ground-substance formed by each cell, it becomes a homogeneous mass, but soon displays connective-tissue fibrils which gradually undergo a process of calcification. The mem- . __ . -tr brana praeformativa has no fibers and calcifies .« lat( X 720 : The dentin is formed, but has become homo- that part of it in the 1'C- geneous from calcification. Bleu de Lyon differen- gion of the fang is ab- tiates it into zones (a and b\. At c is seen the in- •, • 11 timate relationship of the odontoblasts to the tissue of SOrbed in order to allow the dental pulp. the cementum to reach the surface of the dentin. Remains of this regressive portion persist as the epithelial nests of the dental root (compare p. 242). The contents of the dentinal papillae change into the tissue of the dental pulp. As early as the third month outgrowths appear on the inner side of the enamel ledge next to the partly developed milk-teeth, which represent the anlagen of the enamel organs of the permanent teeth. Their further development is similar to that of the milk teeth. The enamel organs of the molars are also developed from an enamel ledge which is practically a backward continuation of the embryonic enamel ledge. With their crowns presenting, the temporary teeth at last break through the epithelium of the gums. When the de- velopment of the permanent teeth is so far advanced that they are ready to perforate, regressive processes begin at the roots of the THE ORAL CAVITY. 247 milk-teeth, which are due, as in like conditions of the bone, to the action of certain cells, which are here known as " odontoclasts." The crowns of the milk-teeth are then thrown off, one by one, by the growing permanent teeth. For further information as to the teeth and their development, see the articles by v. Ebner (Scheff's " Handbuch der Zahnheil- kunde" and in Kolliker's "Handbuch der Gewebelehre," Bd. iii), whose studies we have to a great extent followed on this subject. 2. THE TONGUE. The Lingual Mucous Membrane and its Papillae. — The mucous membrane of the tongue differs in general very little from Fig. 191. — Fungiform papilla from human tongue. that lining the rest of the oral cavity. It must, however, be borne in mind that in the greater part of the tongue the submucosa is poorly developed, and as a consequence the mucous membrane on the upper surface and base of the tongue is scarcely movable. Other peculiarities of the lingual mucous membrane are the absence of glands in the mucosa on the upper surface of the tongue, — although glands are found in the musculature of the tongue, their ducts passing through the mucosa, — the presence of epithelial papillae, and of lymph-follicles at the base of the tongue. The upper surface of the tongue is roughened by the presence of epithelial projections, the lingual papilla. The latter are almost entirely epithelial structures, and should not be confused writh those papillae which are composed exclusively of connective tissue. There 248 THE DIGESTIVE ORGANS. are several classes of lingual papillae — the filiform, the fungiform, and the circumvallate papillae. The most numerous are the thread- like or filiform papilla (from 0.7 to 3 mm. long). These are scat- tered over the entire upper surface of the tongue, and consist of conic projections of the epithelium and of the mucosa. The con- nective-tissue portions of these papillae are very thin and long. The basal layers of the epithelium can not be distinguished from the same layers covering the surrounding mucosa, but the more super- "Papilla filiformis. m $ '• ' K Tongue epithe- yX-vJ Hum Hum. 'T%tjkr "Connective-tissue papilla. V — Mucosa. '" ', '•' , •' / 4&- - — Basal epithelial layer. Fig. 192. — From a cross-section of the human tongue, showing short, thread-like papillae (filiform) ; X I4°- ficial layers are differentiated, in that their cells are arranged parallel to the long axes of the papillae and overlap each other like tiles (Fig. 192). Their free ends are often continued into several spine- like processes. Less numerous than the filiform are the fungiform papilla (from 0.7 to 1.8 mm. in height) scattered here and there between the former. They are nearly hemispheric in shape, and are joined to the surface of the tongue by a slightly constricted base. At times they are even partly sunk into the mucous membrane. The mucosa is raised under the epithelium to form connective-tissue papillae (Fig. 191). On the free surface of the fungiform papillae THE ORAL CAVITY. 249 are sometimes found taste-buds, or taste-goblets, which lie im- bedded in the epithelium and extend through its entire thickness. The circumvallate papillce occupy a definite region on the upper surface of the tongue, and are arranged in two rows, forming almost a right angle, with the apex directed backward and situated just in front of the foramen caecum (Morgagni). These papillae are few in number, about eight to fifteen in all. In shape they are similar to those of the fungiform type, but are much larger (about I or 2 mm. in diameter), and sunk so deeply into the mucous membrane that the latter forms a wall around their sides. Here also the mucosa passes up into the papillae and forms con- nective-tissue papillae of its own at the upper surface, while at the sides it merely adheres to the smooth inner surface of the epithelial layer. Taste-buds are found in the epithelium at the sides of the papillae, and also in that of the ridges surrounding the papillae. At the sides of the human tongue and near its base are the so-called fimbrice linguce. These are irregular folds of mucous membrane, ,.:;-.• >> - ujr, • Fig. 193. — Longitudinal section of foliate papilla of rabbit, showing taste-buds. the sides of which also contain taste-buds. In the rabbit they are more regular in structure and consist of parallel folds of mucous membrane thickly dotted with taste-buds, and are termed the foliate papillce. In place of the circumvallate papillae, the guinea-pig pos- sesses structures similar to the foliate papillae of the rabbit. Into the depressions in which the circumvallate papillae lie and into those between the folds of the fimbriae linguae open the ducts of numerous serous glands, the glands of v. Ebner (see below). The Taste-buds. — The gustatory organs in the form of taste- buds are found on the surface of the tongue, principally on the lateral surfaces of the circumvallate papillae and the fimbriae linguae (foliate papillae). They, are also occasionally met with in the epithelium of the fungiform papillae and the soft palate, and on the posterior surface of the epiglottis. They always lie imbedded in the epithelium and extend through its entire thickness ; they are ovoid in form, with base downward and the smaller pole at the 250 THE DIGESTIVE ORGANS. surface. The whole structure is surrounded by the epithelium of the mucous membrane of the regions in which they occur, except at the attenuated outer end of the taste -bud, where, by means of a small opening, the taste-pore, it communicates with the oral cav- ity. Most of the cells constituting the taste-buds are elongated, spindle-shaped structures, extending from one end of the organ to the other, with spaces between them. There are four varieties of these cells : (i) The outer sustentacular or tegmental cells, lying at the periphery of the organ with a nucleus in their center, and having a short, cone-shaped cuticular projection ; (2) the inner sustentacular or rod-shaped cells, which are more slender structures with basally situated nuclei and without a cuticular projection ; between the latter are (3) elongated, spindle-shaped, neuro-epithe- Epithelium. Taste-buds. " Groove sur- rounding papilla. Ebner's gland. Fig. 194. — Longitudinal section of a human circumvallate papilla ; X 2O- lial cells, with the nucleus of each in the thickest portion of the cell, and with slender, stiff processes projecting into the taste -pore ; (4) a few broad basal cells, communicating with each other as well as with the sustentacular cells by numerous processes. We have, therefore, in the cells of the first, second, and probably fourth varieties, elements which belong exclusively to the sustentacular apparatus of the organ (Hermann, 85, 88). Von Ebner found in the taste-buds of the circumvallate papillae of man, monkey, and cat, as well as of the papillae foliatae of the rabbit, an open space situated between the taste-pore and the tip of the taste-bud (Fig. 195). These spaces vary according to the species, and are bounded above by the summits of the tegmental cells and laterally and below by the more centrally situated sus- THE ORAL CAVITY. 251 by the development of a solid encircling Epithe- lium. tentacular cells. The cavities are often 10 p in depth, and are filled with a fluid apparently in communication with the fluid of the depression into which the circumvallate papillae are sunk. The processes of the neuro-epithelial cells project into the cavity from its floor and lateral walls, but do not extend as far as the taste- pore. The circumvallate papillae are differentiated from the adjacent surface of the tongue epithelial ridge. Nu- merous taste-buds ap- pear on the surface quite early in the his- tory of the embryo. These, however, dis- appear completely when the permanent taste - buds develop from the basal cells of the epithelial ridge. Similar phenomena occur in the fungiform papillae (Hermann, 88). The neural epith- elia of the taste-gob- lets were formerly re- garded as directly connected with the nerve-fibers by means of long processes, but the latest researches have shown that dendrites of sensory neurones (sensory nerves) enter the taste-buds and end free in telodendria. The latter sur- round the neuro-epithelial and, to some extent, the sustentacular cells, their relations depending upon contact. The Lymph-follicles of the Tongue (Folliculi linguales) and the Tonsils. — At the root of the tongue, and especially at its sides, are numerous elevations due to the increased quantity of lymphoid tissue found in the mucosa of these regions, the lingual tonsils, or lingual follicles. In the center of each follicle is a cavity communicating with the exterior and caused by an invagination of the epithelium. The lymphoid tissue contains a number of -more or less distinctly defined lymph-nodules, some even showing germ centers (vid. p. 197). The whole structure is surrounded by a connective-tissue capsule. The epithelial walls of the follicular cavities often show extensive degenerative changes, which are accompanied by increased migration of leucocytes into the oral cavity. These leucocytes change (according to Stohr, 84) into the so-called mucous or salivary corpuscles of the saliva. Pharyngeal Tonsil. — The pharyngeal tonsils may be regarded Tegmental cell. Neuro-epithe- lial cell. Sustentacular cell. Terminal branches of nerves. Fig. 195. — Schematic representation of a taste-goblet (partly after Hermann, 88). 252 THE DIGESTIVE ORGANS. as clusters of small lymph-follicles, similar to those found in the tongue. The pharyngeal tonsil presents numerous irregularly formed crypts, lined by stratified pavement epithelium. These crypts are often widened at the base and are provided with irregular sac- cular enlargements. The crypts are all surrounded with lymphoid tissue, which may be regarded as diffuse lymphoid tissue in which are found numerous lymphoid follicles, often showing germ-centers. The lymphoid tissue is bounded externally by fibrous tissue, septa of which pass into the lymphoid tissue surrounding the crypts. Fig. I96. — Section through the pharyngeal tonsil of man; X 6K : a^A Arcus glosso-palatinus ; .f/>, epithelium;//, crypt; M, striated muscle; »/, lymphoid nodules; S, connective tissue septa ; st, remains of tonsillar sinus. The epithelium lining the crypts or cavities of the tonsils shows, as in the lingual follicles, extensive degenerative changes, resulting mainly in the formation of variously shaped communicating spaces filled with lymphocytes and leucocytes. (See Fig. 197.) Besides the nerves terminating in the taste-buds, the tongue is richly supplied with sensory nerves which terminate in free sen- sory endings, which may be traced into the epithelium, and which are especially numerous in the fungiform and cjrcumvallate papillae ; or in smaller or larger end-bulbs of Krause found in the mucosa of THE ORAL CAVITY. 253 the fungiform papillae. The motor nerves of the tongue terminate in motor endings. GLANDS OF THE ORAL CAVITY. The glands of the oral cavity comprise numerous branched tubulo-alveolar glands situated in the mucosa and submucosa of the lips, cheek, and tongue; branched tubular glands in the region of the circumvallate papillae; of a pair of compound branched alveolar glands, the parotid ; and of two pairs of compound branched tubulo-alveolar glands, the submaxillary and sublingual. These are classified according to their secretions into those secret- ing principally mucus (human sublingual and many of the smaller oral glands), and known as mucous glands ; those secreting a fluid albuminoid substance containing no mucus, the serous glands (parotid glands and the small glands near the circumvallate papillae) ; Fig. 197. — The area designated by a in the previous illustration, shown by a higher magnification; X about 150: a, Leucocytes in the epithelium; b, one of the spaces in the epithelium filled with leucocytes and more or less changed epithelial cells ; cy blood-vessel ; < I4°- The demilunes of Heidenhain are more deeply shaded (Maziarski, " Anatomische Hefte," 1901). preparations teased in serum or in 2 % to 5 % sodium chlorid solu- tion (Langley), or when fixed and stained after special methods, it may be seen that the secretion is first formed in the form of large granules, consisting of a substance known as mucigen, which breaks down to form the mucus, much as described for mucous or goblet cells (see page 87). In preparations the cells of which are stored with mucigen the nuclei are situated at the periphery of the tubules and alveoli, near the basement membranes. The cells with serous secretion are situated in close apposition to the basement membrane; they resemble in structure serous cells, and are found either singly or in groups of crescentic shape. These groups are known as the crescents of Giannzzi or the demilunes of Heidenhain. The margins of the individual cells composing the crescents are SALIVARY GLANDS. 257 often so faintly outlined that the whole structure has the appearance of a large polynuclear giant cell. The demilunar cells have been variously interpreted by different observers. They have been regarded as permanent cells with a special secretion, as transitional structures, and again as cells des- tined to replace the degenerated mucous cells. Stohr (87) be- lieves that the cells of the acini are never destroyed in the process Crescents of Gianuzzi. 200. — From section of human submaxillary gland. of mucous secretion, and that the crescents of Gianuzzi are there- fore merely a complex of cells containing no secretion, which have been crowded to the wall by the adjacent enlarged and distended cells. Solger (96), on the other hand, does not regard the demi- lunes as transitional structures whose function is to replace the destroyed cells, but considers them to be permanent secreting cells — an opinion which he bases on the results of special methods of Connective — - tissue. Gland cell of acinus. — — Intralobu- lar duct. Intermedi- ate duct. Fig. 201. — Section from parotid gland of man. investigation. According to him, then, the mucous salivary glands are mixed glands, in that the demilunes consist of cells of a serous type, while the remaining elements are mucous in character. The destruction of mucous cells during secretion is not admitted by him (compare also R. Krause). This latter view seems more in accord with recent observations. 17 258 THE DIGESTIVE ORGANS. The Submaxillary Gland (Mixed Gland).—- The submaxillary gland of man is a gland composed of tubules similar in shape and structure to those found in the parotid gland, having a serous secre- tion, and of tubules with alveolar enlargements, lined with cells Fig. 202. — Portion of a model of a salivary gland with mucous secretion : a, intralo- bular duct ; b, intermediate duct ; c, tubules and alveoli lined by mucous cells ; d, demi- lunes of Heidenhain (from Bohm and DavidofT, third German edition). which form mucus. These mucus-secreting tubules are joined to intermediary ducts which are branches of intralobular ducts with striated epithelium. The mucus-forming tubules show the demi- lunes of Heidenhain. The submaxillary glands of man are there- ^-*»" Fig. 203. — A number of alveoli from the submaxillary gland of dog, stained in chrome- silver, showing some of the fine intercellular tubules. fore mixed glands, with both serous and mucous secretion, the re- spective tubules or groups of tubules showing the characteristics of mucous and serous glands. In Fig. 202 is shown a portion of a model of a salivary gland, with mucous secretion. SALIVARY GLANDS. 259 By means of various methods the existence of a network of tubules surrounding the glandular cells may be demonstrated both in the serous and mucous glands. The same arrangement may be observed in the case of the cells forming the demilunes. The course of these tubules may be followed to their junction with the lumen of the secreting portion of the gland tubule, and the whole structure would seem to indicate that the entire surface of the cells is concerned in the act of secretion (Erik Miiller, 95 ; Stohr 96, II). As to the part that the intermediate tubules and the intralobular tubes play in the process of secretion, Merkel's (83) theory is of interest. He believes that the former yield a part of the water in the saliva, while the salts are furnished by the rod-shaped epithe- lium of the intralobular tubes. These views of Merkel have been questioned, as it has been shown by chemic analysis that the relative quantity of water and salts in the secretion of the salivary glands is not at all proportionate to the number of the intermediate tubules and intralobular tubes. For example, Werther finds that although a great many intermediate tubules are present in the par- otid gland of the rabbit and none at all in the submaxillary gland of the dog, nevertheless the secretions of these glands contain equal quantities of water. Furthermore, the secretions of the parotid of the rabbit and of the sublingual of the dog show equal quantities of salts, in spite of the fact that in the former there are large numbers of intralobular tubes with rod-shaped epithelium and in the latter none at all. THE SMALL GLANDS OF THE MOUTH. Besides the larger glands, there are in the oral cavity numerous small lobular, tubulo-alveolar and simple branched tubulo-alveolar glands. They are mostly glands with mucous secretion. In many of them demilunes of Heidenhain may be made out, most clearly in those of the lips (J. Nadler). They are known, accord- ing to their location, as glandulae labiales, palatinae, and linguales. The absence of intralobular tubes and well-defined intermediate tubules is characteristic of all the smaller glands of the oral cavity. As a consequence the secreting tubules are composed almost entirely of those parts corresponding to the acini of the larger glands. Branched tubular glands, with serous secretion, known as v. Ebner's glands, occur in the tongue, their ducts opening into the depressions of the circumvallate and foliate papillae, while the secreting tubules extend into the muscular portion of the tongue. The general character of v. Ebner's glands is shown in Fig. 204. The salivary glands and smaller glands of the mouth have a rich blood supply. In the salivary glands the arteries follow the ducts through their repeated branching, ultimately ending in capil- laries which form a network inclosing the acini and the terminal 260 THE DIGESTIVE ORGANS. portions of the system of ducts. The blood-vessels for each lobule are quite distinct, forming only few anastomoses with those of neighboring lobules. The Lymphatics. — In the connective tissue surrounding and separating the acini there are found clefts, which contain lymph. These clefts are in part between the blood-capillaries and the base- ment membranes. Lymph-vessels are found in the connective tissue separating the lobules and lobes of the gland, in which they follow the duct system. Lymph-vessels have not been found in the lobules. Fig. 204. — Model of a gland of v. Ebner, from a boy .fourteen years old; X J7°- (Maziarski, " Anatomische Hefte," 1901.) The nerve supply of the salivary glands, may, owing to the im- portance of these structures, receive somewhat fuller consideration. Their nerve supply is from several sources. That of the sublin- gual and submaxillary glands will be considered first. Sensory nerve-fibers (no doubt the dendrites of sensory neurones, the cell- bodies of which are situated in the geniculate ganglion) terminate in free sensory endings in the large excretory ducts and their branches. These medullated fibers accompany the ducts in the form of small bundles. From place to place one or several fibers leave these bundles and, after dividing a number of times, lose their medullary sheaths. After further division the nonmedullated branches form plexuses under the epithelial lining of the ducts. From the fibers of these plexuses terminal fibrils are given off, which enter the epithelium, to end, often near the free surface, on the epithelial cells (Arnstein, 95; Huber, 96). The secretory cells of the acini receive SALIVARY GLANDS. 26 1 their innervation from sympathetic neurones. The cell-bodies of those supplying the sublingual glands are grouped in a number of small, sympathetic ganglia situated in a small triangle formed by the lingual nerve, the chorda tympani and Wharton's duct, the chorda- lingual triangle. These ganglia may be known as the sublingual ganglia (Langley). The cell-bodies of the sympathetic neurones supplying the secretory cells of the subrnaxillary glands are grouped in small ganglia situated on Wharton's duct just before it enters the gland, in the hilum of the gland, and on the interlobar and inter- lobular ducts ; they may be spoken of collectively as the submax- illary ganglia. In the glands under discussion, the neuraxes of the sympathetic neurones are grouped to form small bundles, which divide repeatedly, the resulting divisions joining to form plexuses situated in the outer portion of the walls of the ducts, and as such may be followed along the ducts, the bundles of nerve-fibers be- coming smaller and the division of the bundles of fibers and the individual fibers occurring oftener as the smaller divisions of the system of ducts are reached. On reaching the acini, the terminal branches of the nerve-fibers form a plexus outside of the basement membrane, epilamellar plexus ; from this branches are given off which penetrate the basement membrane, some forming zhypolam- ellar plexus, others ending on the gland-cells in small granules or clusters of granules (Arnstein). Throughout their entire course the neuraxes of the sympathetic neurones are varicose, nonmedullated nerve-fibers. The nerve-fibers of the chorda tympani end in ter- minal end-baskets, inclosing the cell-bodies of the sympathetic neurones found in the sublingual and subrnaxillary ganglia, and not in the glands, as generally stated by writers. The increase of secre- tion from the subrnaxillary and sublingual glands on direct or indi- rect stimulation of the chorda tympani is due, therefore, not to a direct stimulation of the gland-cells by the fibers of this nerve, but to a stimulation of the sympathetic neurones of the sublingual and submaxillary ganglia, the neuraxes of which convey the impulse to the gland-cells. These glands have a further nerve supply from the superior cervical ganglia of the cervical sympathetic. The neuraxes of sympathetic neurones, the cell-bodies of which are situated in the superior cervical ganglia, accompany the blood-vessels to the sub- lingual and submaxillary glands ; their mode of termination is, however, not as yet determined. The cell-bodies of the sympathetic neurones here in question are surrounded by end-baskets of nerves which leave the spinal cord through the second, third, and fourth dorsal spinal roots. The blood-vessels of the salivary glands are also richly supplied with vasomotor nerves, the neuraxes of sympa- thetic neurones, which terminate on the muscle-cells of their walls. The nerve supply of the parotid glands is, in the main, like that of the other salivary glands here described, although it has not been worked out with the same detail. The cell-bodies of the sympathetic neurones, the neuraxes of which innervate the gland-cells, are, it 262 THE DIGESTIVE ORGANS. would appear, situated in the otic ganglia. The nerve-ending in the smaller glands of the mouth is similar to that given for the salivary glands, as has been shown by Retzius and other observers. It is very probable that the cell-bodies of the sympathetic neu- rones, the neuraxes of which innervate the glands of the tongue, are situated in the small sympathetic ganglia found on the lingual branches of the glossopharyngeal and lingual nerves. B. THE PHARYNX AND ESOPHAGUS. Pharynx. — The epithelium of the pharynx is of the stratified squamous variety, and also contains prickle cells and keratohyalin. (See Skin.) A stratified ciliated epithelium is present only in the fornix in the region of the posterior nares. The area covered by this type of epithelium is more extensive in the fetus and new-born, and extends over the whole nasopharyngeal vault. In the human embryo the superficial epithelial cells of the esophagus possess cilia up to the thirty-second week (Neumann, 76). The papillae of the mucosa are loosely arranged and are in the form of slender cones. The mucosa of the pharynx contains diffuse adenoid tissue rich in cells which in some places forms accessory tonsils (vid. p. 251); it is bounded externally by a well-developed layer of elastic fibers which occupies the same relative position as does the muscularis mucosae in the esophagus. External to this elastic layer, there is found a muscular coat consisting of striated muscle-fibers. Esophagus. — The esophagus is lined by a stratified pavement epithelium, which rests on a papillated mucosa, consisting of fibrous tissue which contains few elastic fibers and is bounded externally by a muscularis mucosae, the majority of the cells of which show a longitudinal arrangement. External to the muscularis mucosae there is found a well-developed submucosa, consisting of loosely woven fibro-elastic connective tissue. Outside of the submucosa there is found a muscular layer, consisting of an inner circular and an outer longitudinal layer. These muscular layers consist in the upper half of the esophagus mainly of striated muscle-fibers, while in the lower half they consist almost wholly of nonstriated muscular tissue. There is, however, no sharply defined line of demarcation between the two types of muscular tissue, as the fibers of the unstriped variety penetrate for some distance upward into the substance of the striated muscle, giving the tissue here a mixed character. The esophagus contains two varieties of glands: (i) Mucous glands of the type of branched tubulo-alveolar glands. The secret- ing portions of these glands are situated in the submucosa, while the ducts pass through the muscularis mucosae to the surface. The secreting tubules and alveoli are lined by mucous cells ; demi- lunes are absent. The ducts, which often show cystic dilations, THE PHARYNX AND ESOPHAGUS. are lined for the greater part by a single layer of columnar cells ; at their termination they often possess a lining of stratified pave- ment epithelium. (2) The other variety of glands are found in two zones, the one situated at the upper end of the esophagus, in a region opposite the cricoid cartilage to the fifth tracheal cartilage (superficial glands of esophagus, Hewlett; upper cardiac gland, Schaffer), the other at the end of the esophagus, just before it enters the stomach — the esophageal cardiac glands. These glands are situated above the muscularis mucosae, and are of the branched £•• Epithelium. ~— Mucosa. — • Muscularis mucosae. -• Submucosa. •-• Circular layer of muscle. s /•-- Longitudinal muscle layer. — Outer connec- tive-tissue coat. Fig. 205. — Section of esophagus of dog ; X l tubular variety. The ducts of these glands, \fhich reach the surface through the apices of the connective tissue papillae, are lined by a single layer of columnar epithelial cells. The secreting portions of the tubules are lined by shorter columnar cells. Here and there cells like the parietal cells of the fundus glands of the stomach, to be described later, are also found, as also cells showing a mucous secretion. The cardiac glands of the esophagus are similar to the glands of the same name found at the cardiac end of the stomach, with which they may be said to be continuous, and which will receive further consideration. 264 THE DIGESTIVE ORGANS. C THE STOMACH AND INTESTINE. J. GENERAL STRUCTURE OF THE INTESTINAL MUCOUS MEMBRANE. The mucous membrane of the stomach and intestine, unlike that of the esophagus and oral cavity, possesses an epithelium of the simple columnar variety with elongated cells (about 22 fj. in Alveolus - of gland. Mucosa. Basal epi- thelial cells. ,-- Gland- cells. - Lumen. Branched papilla of mu- cosa. Fig. 206. — Part of section of human esophagus, showing a cardiac gland with a • dilated duct; X I2°- height). At the cardia the stratified squamous epithelium of the esophagus terminates abruptly, the basilar layer of the esophageal epithelium being continued as the simple columnar epithelium of the stomach. In the intestine the epithelium shows a well-marked striated cuticular border, striated protoplasm in the outer ends of the cells, extending to the immediate vicinity of the nuclei, which THE STOMACH AND INTESTINE. 265 are situated in the basal portions of the cells. The basal portion of each cell consists of nonstriated protoplasm, ending in a longer or shorter process which extends to the basement membrane, or possibly even penetrates it. The epithelial cells have the power of produc- ing mucus, a phenomenon which, in the normal condition, rarely embraces whole areas of epithelium ; these cells (goblet cells) are usually surrounded by others which are unchanged (for details about goblet cells see General Histology, p. 87). Throughout the entire intestinal tract the epithelium forms simple, branched, and compound tubular and alveolar glands. These are depressions lying in the mucosa, and only in the duodenum extend beyond it into the sub- mucosa. The mucosa consists of adenoid tissue, consisting of reticular fibers and a fine network of elastic fibers, containing relatively few cells. It fills the interstices between the glands, and often forms a thin but continuous layer (granular layer of F. P. Mall) below the glands. It is therefore obvious that the development of the mucosa is inversely proportionate to the number and the density of arrangement of the glands ; when the latter are present in large numbers, as, for instance, in the stomach, the mucosa is reduced to a minimum. In the small intestine it forms not only the perma- nent folds, but also certain leaf-like and finger-like elevations kno\vn as villi, which are covered with epithelium and project into the lumen of the intestine, thus increasing to a considerable extent the surface area of the mucous membrane. In the mucosa are found small nodules of adenoid tissue. These are spoken of as lenticular glands when occurring in the stomach, as solitary glands when found in the upper portion of the small intestine and in the large intestine. In the lower portion of the small intestine they are grouped to form the agminated glands, or Peyer's patches, which, when large, extend into the submucosa. In the external portion of the mucosa there is found a thin, somewhat -denser layer, known as the stratum fibrosum (F. P. Mall), consisting mainly of white fibrous tissue (Spalteholz) ; and external to this is a layer consisting of two or three strata of unstriped muscle-fibers, the muscularis miicoscz. As a rule, it is composed of an inner circular and an outer longitudinal layer. This arrangement is interrupted only where the larger glands and follicles penetrate into the submucosa. The epithelium with the glands, the mucosa with its lymph-nodules, and the muscularis mucosae form together the mucous membrane, or tunica mucosa. Below the mucous membrane is the connective-tissue submucosa. This is characterized by its loose structure, and consequently affords considerable mobility to the mucous membrane. In the small intes- tine it forms a large number of permanent transverse folds known as valvulce conniventcs (Kerkring). In the submucosa of the duodenum occur the secreting portions of Brunner* s glands (gland- ule duodenales), and in the small intestine the larger lymph-nodes and Peyer's patches. 266 THE DIGESTIVE ORGANS. External to the submucosa is the muscular coat, which generally consists of two layers of unstriped muscle-tissue. The inner layer is composed of circular fibers (stratum circulare) ; the outer layer, of longitudinal fibers (stratum longitudinale). In the colon the longi- tudinal layer forms definite bands, the t Bodies of gas- tric glands. Fundus. -- Mucosa. Fig. 208. — From vertical section through fundus of human stomach ; X 60 : a and b, Inter- lacing fibers of the muscularis mucosae ; from a and b muscular fibers enter the mucosa. The fibers of the layer b penetrate those of layer a. Fig. 209. — A number of fundus glands from the fundus of the stom- ach of young dog, stained after the chrome-silver method, showing the system of fine canals surrounding the parietal cells and communicat- ing with the lumen of the glands. I. The cardiac glands have recently been subjected to careful investigation by Bensley; his account is here followed. They occur in the region of the junction of the esophagus and stomach, occupying a zone varying somewhat in width, but may be as wide as 4.3 cm. The glands are of the type of branched tubulo-alveolar glands. The tubules and alveoli are not of uniform structure. The majority of the lining cells are mucus secreting cells, and 268 THE DIGESTIVE ORGANS. may be recognized as such in suitably stained preparations, cells with zymogen granules, similar to the chief cells of the body of the fundus glands (see these), are also found, as also the parietal cells, as found in the latter glands. The cardiac glands may be regarded as decadent structures. 2. The fundus glands (peptic glands) consist of a crypt or foveola, into which empty three to five, or even more, unbranched and branched tubules, which often show irregular terminal enlarge- --T. Epithelium of esophagus. - Cardiac gland. Junction of esophagus arid stomach. Epithelium of stomach. I Gastric crypt. Fig. 210. — From a section through the junction of the human esophagus and cardia ; X5°- ments. The tubules vary in length, measuring from 0.4 to 2.2 mm. The upper end of a fundus tubule is slightly narrower and presents structural peculiarities, and is known as the neck of the gland. The main portion of the gland is called its body, and the region at its distal blind end the fundus. The fundus glands, as their name suggests, are found in the fundus or cardiac end of the stomach, and are lined by two kinds of cells : parietal (border cells, acid, oxyntic, or delomorphous cells — R. Heidenhain, 69; Roller., 70) and chief, central, peptic, or adelomor- THE STOMACH AND INTESTINE. 269 phous cells. The parietal cells lie against the walls of the gland — that is, they rest on its basement membrane — and are particularly numer- ous in the neck and body of the gland, but not so numerous in its fun- ' dus. Their bodies often extend more or less beyond the even line of the remaining cells, thus forming, together with the membrana •propria, a protuberance (particularly noticeable in the pig, where almost the entire cell may be enveloped by the basement membrane, giving it an appearance of being entirely extraglandular). Toward the lumen of the gland the contour of these cells is modified by pressure on the part of the adjacent cells belonging to the other variety, and they are indented according to the number of the latter. Occasionally, a process is seen extending from a parietal cell to the lumen of the gland. The parietal cells are larger than the cells of the other variety and richer in protoplasm ; they are of an irregular oval or triangular shape and possess, as a rule, a single nucleus, although in man numerous parietal cells with two nuclei are found. The parietal cells are clearer in fresh preparations than are the chief cells, while in fixed preparations the reverse is generally the case. They stain deeply in Heidenhain's iron-lac-hematoxylin, are dark- ened by osmic acid, and show an affinity for acid stains, especially for eosin, also for congo-red and for neutral carmine solutions. According to Erik Muller and Golgi (93), there exists in the peripheral protoplasm of each parietal cell a system of canals in the form of a network communicating with the lumen of the gland and varying in structure according to the physiologic condition of the cell — wide-meshed in a state of hunger and fine-meshed during digestion. A peripheral zone differing from the rest of the cell- body may occasionally be demonstrated in the parietal cells (mouse) by using the method of von Altmann. The chief cells are short, irregular, columnar structures whose narrower portions point toward the lumen of the gland. They are situated either directly between the lumen and the basement mem- brane of the gland, or their basilar surfaces border on a delomor- phous cell. They are found throughout the tubule of the gland and occupy the spaces between the delomorphous cells. The chief cells of the fund us glands are of two varieties, as has been shown by Bensley. The chief cells of the body of the gland are charac- terized by the possession of relatively large zymogen granules,! which are found in the inner portion of the cells. These granules are used up during secretion. The outer or basal portion of the cells contains a pro zymogen, not in granular form but recognized by its staining reaction. The chief cells of the neck are slightly smaller than those of the body, and differ from these in that they do not possess zymogen granules and prozymogen only in small amounts, but show by their reaction to certain stains that they are mucus-secreting cells. The structure of the pyloric region of the stomach differs in some respects from that of the cardiac end and fundus. There 270 THE DIGESTIVE ORGANS. is, however, no sharply defined boundary between fundus and pylorus, but a transitional zone in which changes gradually take place. Toward the pylorus the gastric crypts gradually become deeper and the parietal cells decrease in number. Here also the glands branch more freely. In the pylorus itself the crypts fre- quently extend half-way through the thickness of the mucous' membrane, often even penetrating nearly to the muscularis mucosae, in which case the corresponding tubules become tortuous and arch over the muscularis mucosae. The glands of the pyloric region are therefore to be classified as branched tubular glands (De Witt). Epithelium - of fold be- tween gas- tric crypts. Gastric crypt. Pyloric gland. Mucosa. . Muscularis ^*. mucosse. Fig. 211. — From vertical section through human pylorus ; X about 60. Among the branched pyloric glands are found glands which show no distinct branching. The most important feature is that in the great majority of the tubules only a single variety of cell is pres- ent in the pyloric gland. (Only here and there are found parietal cells in the pyloric glands of the human stomach.) These cells may be compared with the chief cells of the neck regions of the fundus glands, in that they show no zymogen granules, and prozy- mogen only in small quantity, and on staining with special stains, it can be shown that their secretion is mucus. They are of colum- nar shape, and more uniformly so than the chief cells of the fundus THE STOMACH AND INTESTINE. 271 glands — a condition probably due to the general absence of delo- morphous cells. In the immediate vicinity of the gastroduodenal valve the pyloric glands become shorter, and other glands, which extend into the submucosa, and which are identical in structure with the glands of Brunner in the duodenum, make their appear- ance. In this portion of the pylorus are also a few scattered villi, which from their structure may be considered as belonging to the duodenum (yid. Fig. 218). In the normal condition the mucosa of the stomach contains solitary lymph-nodules (lenticular glands) in the fundus region; they are, however, more frequent in the pyloric region ; well-defined lymph-nodules are constantly present in the immediate vicinity of the pylorus. The muscularis mucosae is usually composed of three layers, the fibers of the individual layers forming distinct interlacing bun- dles. Individual muscle-fibers very frequently branch off from the inner layer, assume a vertical position and disappear among the glands. This arrangement is especially well seen in the muscularis mucosae of the fundus of the stomach (Fig. 208). Only the inner and middle layers of the muscular coat of the stomach enter into the formation of the sphinc- ter pylori (Fig. 2 1 8). The fibers of the outer layer, however, pene- trate through the sphinc- ter pylori and may even be traced into the sub- mucosa. When these alone contract, the mus- cular bundles of the sphincter act somewhat as pulleys, and a mod- erate dilatation of the lumen of the pylorus is the result ( dilatator pylori, Riidinger, 97 ). (For further particulars about the stomach, compare Oppel, 96.) The changes which the epithelium and the secretory cells of the stomach undergo during secretion are of special importance. These relations have been carefully studied in animals by R. Heidenhain (83). As far as our present knowledge goes, it would seem that the same processes also occur in man. In a state of hunger the chief cells of the fundus are large and contain numerous zymogen gran- ules, while the parietal cells are small ; in certain cases the parietal cells abandon their mural position and, like the chief cells, border upon the lumen of the gland. During the first few hours of diges- tion the chief cells remain large, while the parietal cells increase in Mucosa. Fig. 212. — From section through human pylorus; X6oo. 272 THE DIGESTIVE ORGANS. size. In the dog, from the sixth to the ninth hour of digestion, the chief cells diminish in size and contain fewer zymogen granules, while the parietal cells remain large and even increase in size. From the fifteenth hour on, the process becomes reversed; the Fig. 213. — Section through fundus of human stomach in a condition of hunger ; X 5°°' Lumen. _- _ Mucosa. Chief cell. Parietal cell. Fig. 214. — Section through fundus of human stomach during digestion ; X 5°°- chief cells enlarge and the parietal cells diminish in size. In a con- dition of hunger the cells of the pylorus are clear, of medium size, and do not begin to enlarge until six hours after feeding. From the fifteenth hour on, the cells become smaller and more turbid, THE STOMACH AND INTESTINE. 273 Flg- 215. — Illustrations of models, made after Corn's wax-plate reconstruction method, of glandular structures and duodenal villi of the human intestine ; X loo : a, Funclus 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 ; c, leaf-shaped villi and crypts of Lieberkuhn of the duodenum ; dt crypts of Lieberkuhn 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 muc'osa 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 muc.ous 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 cell 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. 2/5 The epithelial cells undergo a special metamorphosis, after which, 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 =-— Mucosa. Muscularis mucosae. Fig. 2 1 6. — Section through mucous membrane of human small intestine ; X 88. At a is a col- lapsed chyle-vessel in the axis of the villus. 2/6 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 Lieb er kukri s glands, or crypts. Their length varies from 320 p. to 450 fj.. 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- i a b Fig. 217. — Longitudinal section through sum- mit of villus from human small intestine ; X 9ot) ( Flamming' 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 Lieberkiihn 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 Lieberkiihn, and in the pylorus with the pyloric glands. These glands, Brunner'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, and 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. Lymph- nodule. ----%-- — Muscularis mucosae. Pyloric glands. Brunner's glands. Villus. 1&~ J£JI-?'~'~ Muscularis mucosae. m "- Submucosa. Villus. Brunner's glands. ' ptP> \Blood-vessel. ''-•Glands of Lieberkuhn. Fig. 218. — 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 closely 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 mucosa 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 in epithe- lium. Epithelium. Crypt. . ^KJX. Intermedi- ary zone. Submucosa. - Fig. 219. — Section of solitary lymph-nodule from vermiform appendix of guinea- pig, showing crypt ; X about 40x5 (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 2°°' 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- ofif) ; 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. 281 THE STOMACH AND INTESTINE. 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 Lieberkiihn. 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 Lieberkiihn, 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 subtnucosa. The glands of Lieberkiihn are displaced in the regions of the lymph- follicles. Gland ^^~/ Submu- IV 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 semilunares 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. 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 ofif 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 Heller's plexus (F. P. Mall). From this, radiating branches are Epithelium of stomach. -- Region of the bodies of the gastric glands. i=^s=£- -• Muscularis mucosae. 223- — Section through fundus of cat's stomach. The blood-vessels are injected ; X DO- 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- cosae, 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. 235 down between the glands, and are arranged in the form of a net- work just above the muscularis mucosse, 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. H Central chyle- vessel of vil- lus. ~~f"f~ Chyle-vessel. Vein. Mucosa. Muscularis mucosse. Submucosa. Plexus of lymph -ves- sels. Circular mus- cular layer. Plexus of lymph-ves- sels. Long. muse. layerwiththe serous coat. 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 the 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 \helacteals 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 (yid. 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 (intra 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 t\\e 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 the prin- cipal physiologic function of the glands of Lieberkiihn 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. Altmann (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- in or 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. 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 fibre-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 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 intralobular 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 trabeculcz, 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 Anastomos- es between vessels of s eve ral lobules. 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 iS fj. 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 IIQ (chrome- silver method). Vacuole of secretion. Tubule of same. Bile capillary. Fig. 230. — Human bile capillaries as seen in section ; X 48° (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 292 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 (exo- 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. e., 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 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- Bile capillaries. 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- ™E LIVER- lobular connective tissue the portal vein and its branches are accom- panied by divisions of the hepatic actej^ 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 ; \ 100. 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 294 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. 295 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 Gitterfa'sern of KupfTer 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 KupfTer (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 Kupfifer (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 (KupfTer) 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 L/ 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, fibro-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 mucosae. The mucosa presents folds, covering ridges of connective tissue of the fibro-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. • ftr Interlobular con- nective tissue. Intralobularvei,,... > ," ' *,.;{£ . "• * «' "t *• ^~W Stellate cells. V* 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 neu raxes 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-duets 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 300 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-aqinal cell. Intermediary duct. Intralobular _ duct. Alveolus. upp 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 s>een, 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 membrana 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 intertubular 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. .— In .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 s mallei- branches in the lobules, and finally break up into capillaries which 243« Scheme showing relation of three adjoining alveoli to excretory duct, illustrating origin of centro-acinal cells. »> Vi Blood capillary. Alveolus of 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 osmicacid. 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 fi.) and stained with safranin- gentian-violet. 3°4 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 TECHNIC. 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 100 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. (£) 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 3O6 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 peri vascular 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 o/0 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 of 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 0.75% 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 {Gitterfasern) , 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 0.5% 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 100 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. \ Stratified ciliated col- umnar epithelium. Glands. — — Muscle. Muscle. Fig. 245. — Vertical section through the mucous membrane of the human larynx ; X5- 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 strarrds, 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. 311 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 ; b, 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 312 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 columnar epithelium. Elastic fibers, cut trans- versely. — Gland. 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. 3 I 3 D. TERMINAL DIVISIONS OF BRONCHI AND ULTIMATE AIR-SPACES, The bronchioles are continued as the respiratory bronchioles. - - Artery. — 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 fibre-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. 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 atrium 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 p. 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 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 p. 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 e^ich 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 respiratory 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 : b, Basilar portion ; a, the thin process. The lower figure is a sec- tion : «, Respiratory epithelial cell ; b, blood- vessel ; c, connective tissue around the al- veoli. TERMINAL DIVISIONS OF BRONCHI AND ULTIMATE AIR-SPACES. 3 I 7 to the structure they supply — finer and more closely arranged in the mucous membrane, and coarser in the connective-tissue walls. In the neighborhood of the terminal bronchial tubes the capillary nets anastomose freely with those of the respiratory capillary system. From the capillaries of the bronchial arteries, veins are formed which empty either into the bronchial veins or into the branches of the pulmonary veins. 253- — Scheme of lung lobule after Miller: b. r., Respiratory bronchiole ; d. a/., alveolar duct (terminal bronchus); <0^ K|;:£ •7 ^^&>--S~~\ L^ ®**A*m •-...fi | $>^vNj •dS^::;^- Fig. 257. — Portion of a cross-section of thyroid gland of a man ; X 3°- fafir, Interstitial connective tissue ; bg, blood-vessel ; c, colloid substance ; ts, gland alveoli. is later the foramen caecum of the tongue; a part of both lateral portions, the right and left lobes, are formed from a complicated metamorphosis of the epithelium of the fourth visceral pouch. These various parts unite in man into one, so that in the adult the struc- ture of the organ is.continuous. The thyroid gland consists of numerous noncommunicating acini or follicles of various sizes lined 32O ORGANS OF RESPIRATION. by a nearly cubic epithelium ; the lobules are separated from each other by a highly vascularized connective tissue, continuous with the firm connective-tissue sheath surrounding the whole gland. The connective-tissue framework of the thyroid has been studied by Flint by means of the destructive digestion method. Relatively greater amounts of connective tissue are found in connection with the blood- vessels, while the follicular membranes are delicate. The follicles are either round, polyhedral, or tubular, and are occasionally branched (Streiff). At an early stage the acini are found to con- tain a substance known .as "colloid" material. Langendorff has shown that two varieties of cells exist in the acini of the thyroid body — the chief cells and colloid cells. Those of the first variety apparently change into colloid cells, while the latter secrete the colloid substance. During the formation of this material the colloid cells become lower, and their entire contents, including the nuclei, change into the colloid mass. Hiirthle distinguished two processes of colloid secretion ; in the one the cells remain intact, in the other they are destroyed. He claims that the colloid cells of Langendorff participate in the former pro- cess, while in the latter they are first modified (flattened) and then changed into the colloid substance. The secretion is formed in the cells in the form of secretory granules. The colloid material may enter the lymph-channels, either directly by a rupture of the acini, or indirectly by a percolation of the substance into the intercellular clefts, whence it is carried into the larger lymphatics. The thyroid gland has a very rich blood supply. The vessels, which enter through the capsule, break up into smaller branches which form a very rich capillary network surrounding the follicles. The veins, which are thin-walled, arise from this capillary network. The gland is provided with a rich network of lymphatic vessels. Anderson (91) and Berkley (94) have studied the distribution of the nerve-fibers of the thyroid gland with the chrome-silver method ; the account given by the latter is the more complete and will be followed here. The nonmedullated nerves entering the gland form plexuses about the larger arteries, which are less dense around the smaller arterial branches. Some of these nerve-fibers are vascular nerves and end on the vessels ; others form perifollicular meshes surrounding the follicles of the gland. From the network of nerve- fibers about the follicles, Berkley was able to trace fine nerve fila- ments which seemed to terminate in end-knobs on or between the epithelial cells lining the follicles. Even in the best stained prepa- rations, however, not nearly all the follicular cells possess such a nerve termination. In methylene-blue preparations of the thyroid gland (Dr. De Witt) some few medullated fibers were found in the nerve plexus surrounding the vessels. In a number of preparations these were traced to telodendria situated in the adventitia of the vessels, showing that at least a portion of these medullated nerves are sensory nerves ending in the walls of the vessels. THE THYROID GLAND. 32I PARATHYROID GLANDS. Small glandular structures found on the posterior surfaces of the lateral lobes of the thyroid were discovered by Sandstrom in 1880. They are surrounded by a thin connective-tissue capsule and divided into small imperfectly developed lobules by a few thin fibrous-tissue septa or trabeculae, which support the larger vessels. The epithelial portions of these structures consist of relatively large cells and capil- lary spaces. According to Schaper (95), who has recently subjected these structures to a careful investigation, the epithelial cells have a diameter which varies from 10 //to 12 //, possessing nuclei 4 // in diameter. These cells are of polygonal shape and have a thin cell-membrane, a slightly granular protoplasm, and a nucleus pre- senting a delicate chromatic network. The cells are arranged either in larger or smaller clusters or, in some instances, in anastomosing trabeculae or columns, consisting either of a single row or of several rows of cells. Between the clusters or columns of cells are found rela- Fig. 258. — From parathyroid of man. tively large capillaries, the endothelial lining of which rests directly on the epithelial cells. Connective-tissue fibrils do not, as a rule, follow the capillaries between the cell-masses. These vessels may therefore be regarded as sinusoids (Minot). The structure of the parathyroid resembles in many respects that of certain embryonic stages of the thyroid, and it has been suggested that these bodies represent small masses of thyroid gland tissue, retaining their em- bryonic structure. Schaper has observed parathyroid tissue, the cells of which were here and there arranged in the form of small follicles, some of which contained colloid substance. Such obser- vations lend credence to the view regarding the parathyroid as an embryonic structure. Whether in this stage they form a special secretion has not been fully determined. (See Schaper, 95.) 21 322 ORGANS OF RESPIRATION. TECHNIC For the demonstration of the larynx and trachea, young and healthy subjects should be selected. Pieces of the mucous membrane or the whole organ should be immersed in a fresh condition. Sections through the entire organ present only a general structural view • but if a close examination of accurately fixed mucous membrane be desired, the latter should be removed with a razor before sectioning and treated separately. Chromic-osmic acid mixtures are recommended as fixing agents, and safranin as a stain. Besides the nuclear differentiation, the goblet cells stain brown, and the elastic network of the stratum proprium and the submucosa a reddish -brown. For examining the epithelium, isolation methods are employed, such as the y$ alcohol of Ranvier. The examination of the respiratory epithelium is attended with peculiar difficulty ; it is, perhaps, best accomplished by injecting a 0.5% solution of silver nitrate into the bronchus until the lumen is completely filled, and then placing the whole in a 0.5% solution of the same salt. After a few hours, wash with distilled water and transfer to 70% alcohol. Thick sections are now cut and portions of the respiratory passages examined ; the silver lines represent the margins of the epithe- lial cells. Such sections should not be fastened to the slide with albumen, as the latter soon darkens and blurs the picture. These specimens may also be stained. For the elastic fibers, especially those of the alveoli, fixation in Mutter's fluid or in alcohol and staining with orcein is a good method, as also Weigert's differential elastic tissue stain. Fresh pieces of lung tissue treated with potassium hydrate show numerous isolated elastic fibers. Pulmonary tissue may be treated by Golgi's method, which brings out a reticular connective-tissue structure in the vessels and alveoli. The pulmonary vessels may be injected with comparative ease. The thyroid gland is best fixed in Flemming's solution ; it is then stained with M. Heidenhain's hematoxylin solution or, better still, with the Ehrlich-Biondi mixture which differentiates the chief from the colloid cells ; the former do not stain at all, while the latter appear red with a green nucleus (Langendorff ). The colloid substance of the thy- roid gland does not cloud in alcohol or chromic acid, nor does it coagu- late in acetic acid, but swells in the latter; 33% potassium hydrate hardly causes the colloid material to swell at all, though in weaker solu- tions it dissolves after a long time. THE URINARY ORGANS. 323 V. THE GENITO-URINARY ORGANS. A. THE URINARY ORGANS. J. THE KIDNEY. THE kidney is a branched tubular lobular gland, which in man consists of from ten to fifteen nearly equal divisions of pyramidal shape known as the renal lobes. The apex of each pyramid (the Malpighian pyramid) projects into the pelvis of the kidney. The kidney is surrounded by a thin but firm cap- sule consisting of fib- rous connective tissue a --T^P^t^S^P^IlByi^lP^ Artery< containing a few elas- tic fibers and, in its Vein. _. deeper portion, a thin layer of nonstriated muscle-cells Fig* 259-— Kidney of new-born infant, showing a '* . distinct separation into reniculi ; natural size. At a is The secreting por- seen the consolidation of two adjacent reniculi. tion is composed of a large number of tubules twisted and bent in a definite and typical manner, the uriniferous tubules. In each one of these tubules we distinguish the following segments : (i) Bowman's capsule, or the ampulla, surrounding a spheric plexus of capillaries, the glomerulus, which, with the capsule of Bowman, forms a Malpighian corpuscle ; (2) a proximal convoluted portion ; (3) a U-shaped portion, con- sisting of straight descending and ascending limbs and the loop of Henle ; (4) a distal convoluted portion or intercalated portion ; and (5) an arched collecting portion ; from the confluence of a num- ber of these are formed the larger straight collecting tubules, which, in turn, finally unite to form the papillary ducts or tubules of Bellini, which pass through the renal papillae and empty into the renal pelvis. Besides the uriniferous tubules the kidney con- tains a complicated vascular system, a small amount of connective tissue, etc. In a longitudinal median section the kidney is seen to be com- posed of two substances, — the one, the medullary substance, pos- sessing relatively few blood capillaries and containing straight collecting tubules and the loops of Henle ; the other, the cortical substance, richer in blood-vessels, and containing principally the Malpighian corpuscles and the proximal and distal convoluted tu- bules. In each renal lobe we find these two substances distributed as follows : The Malpighian pyramid consists entirely of medullary substance, which sends out a large number of processes, the medul- 324 THE GENITO-URINARY ORGANS. lary rays, or pyramids of Ferrein, toward the surface of the kidney. The latter do not, however, quite reach the surface, but terminate at a certain distance below it ; they are formed by collecting tubules which extend beyond the medullary substance. The entire remain- ing portion of the kidney is composed of cortical substance ; be- tween the medullary rays it forms the cortical processes, and at the periphery of the kidney, where the medullary rays are absent, the cortical labyrinth. Those portions of the cortical substance sep- arating the Malpighian pyramids are known as the columns of Bertini, or septa renis. B c Fig. 260. — Isolated uriniferous tubules : A and B, from mouse ; C, from turtle. In all three figures a represents the Malpighian corpuscle ; b> the proximal convoluted tubule; c, the descending limb of Henle's loop; d, Henle's loop; e, the straight col- lecting tubule ; f> the arched collecting tubule. The various segments of the uriniferous tubule are characterized by their shape and size and by their epithelial lining. The Malpighian corpuscle has a diameter of from 1 20 fj. to 220 //. The capsule surrounding the glomerulus consists of two layers, which are to be distinguished from each other when its relation to the glomerulus is taken into consideration. The capsule forms a double-walled membrane around the glomerulus ; a condition which is easily understood by imagining an invagination of the THE URINARY ORGANS. 325 glomerulus into the hollow capsule. Between the inner wall cov- ering the surface of the glomerulus (glomerular epithelium) and the outer wall (Bowman's capsule) there remains a cleft-like space which communicates with the lumen of the corresponding urinifer- ous tubule. In the adult the glomerular epithelium is very flat, with nuclei projecting slightly into the open space of the Malpig- hian corpuscle. The epithelium of the outer wall is somewhat higher, but still of the squamous type. The capsule of Bowman communicates with the proximal convoluted tubule by means of a short and narrow neck. Its epithelium passes over gradually into Column of Ber- „. tini. MeduHary ,- rays. vs Malpighian — K pyramid. Lobule of adi- pose tissue. Blood-vessel. i, --c Fig. 261.. — Median longitudinal section of adult human kidney ; nine-tenths natural size. In the peripheral portion the limits between its renal lobes are no longer recogniz- able. the cubical epithelium of the neck, which, in turn, merges into that of the proximal convoluted tubule. The proximal convoluted portion is from 40 fj. to 70 // in diameter and is lined by a single layer of irregular columnar cells, the boundaries of which are made out with difficulty. The structure of these cells has been studied in recent years by a number of in- vestigators, among whom may be mentioned Disse, whose account 326 THE GENITO-URINARY ORGANS. is here followed. In the epithelial cells of the proximal convoluted portion there may be recognized an outer or basal portion of the cells, in which there is found a spongioplastic network with rectan- gular meshes, with cytoreticular fibrils- running parallel and at right angles to the basement membrane. In the meshes of this network there is found hyaloplasm. The cytoreticular fibrils which are at right angles to the basement membrane contain numerous granules, giving the basal portions of the cells a striated appear- ance. The inner portions of the cells contain a cytoreticulum and hyaloplasm; the reticular fibrils do not, however, contain granules, Fig. 262. — From section of cortical substance of human kidney ; X 24° : at Epi- thelium of Bowman's capsule; b and d, membrana propria ; a> The irregular interlacing pro- jections. 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 [i. to 28 fj. 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 fj. to 45 p. 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. Fig. 266. — Section of medulla of human kidney ; X about 300 : a, a, a, Ascending limb of Henle's loop ; b, l>, b, blood vessels ; to 300 fi. 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. I 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 what 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. •J*-*'-- 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 afferentia. 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 rectse spuriae) and form there capillaries which ramify throughout the papillae with oblong meshes. Artery of ^.-~ capsule. Arched collecting tubule. Straight collect- ing tubule. Distal convoluted tubule. Malpighian cor puscle. Proximal convo- / luted tubule. " v- 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 vena stellata. 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 B Fig. 270. — A, Direct anastomosis between an artery and vein in a column of Bertin of child ; £t 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 Wolffian 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 Wolffian 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. — Mucosa. 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- 22 33^ 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 aim, rrn Fig. 272. — Transverse section of the wall of the human bladder, giving a general view of its structure. X I5- eP> Epithelium ; tp, tunica propria or mucosa ; sm, sub- mucosa ; ilm, inner longitudinal layer of muscle ; rni, circular layer of muscle ; al//i, ex- ternal longitudinal layer of muscle ; tay 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 ofT, 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 sheaths 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 contai-ning 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 - Capsule. . Zona glomerulosa. . Zona fasciculata. Zona reticularis. Fig. 273. — Section of suprarenal cortex of dog ; X I2°* salts are therefore known as chromafrin 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 plexus 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 GENITO-URINARY 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 YZ 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. J. 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 // to 1 1 p. 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 p. to 4.0 p. in diameter, tet/med 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 j p to \op. 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 jrregular, 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 — Germinal epi- thelium. Stroma of-^-;!*!*' ovary. V\M Ovum with fol- licular epithe- lium. gfl «K- , / /\ v^___ 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 GENITOURINARY 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 Granular layer of large Graafian follicle. 276. — From ovary of young girl ; X of Pfliiger ; (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 Pfluger. 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 invaginations 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 theca 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 •' a> a-> a> ai Germinal spots ; b, b, b, b, germinal vesicles ; c, c, c, c, ova ; , ovum in the discus proligerus ; t/ie, thecaexternafolliculi ; thi, theca interna folliculi with blood-vessels (Sobotta, "Atlas and Epitome of Human Histology"). observed in the ovaries of the aged. Changes in the size and structure of the ova accompany the proliferation of the follicular cells in the growing follicles. As soon as the follicular cells of a primitive follicle proliferate, as above described, the ovum of the follicle increases in size until it has attained the size of a fully developed ovum. The zona pellucida now makes its appearance, and after this has reached a certain thickness, yolk granules (deuto- plastic granules) develop in the protoplasm of the ovum. In a fully developed Graafian follicle the ovum presents an outer clearer protoplasmic zone and an inner fine granular zone containing yolk 35O THE GENITOURINARY ORGANS. granules ; in the former lies the germinal vessel. Between the protoplasm of the ovum and the zona pellucida is found a narrow space known as the perivitelline space. The germinal vesicle (nucleus), which is usually of spheric shape, possesses a doubly contoured membrane and a large germinal spot (nucleolus), which shows ameboid movements. The origin of the zona pellucida has not as yet been fully de- termined. It probably represents a product of the egg epithelium, and may be regarded in general as a cuticular formation of these cells. At all events it contains numerous small canals or pores into which the processes of the cells composing the corona radiata ex- tend. These processes are to be regarded as intercellular bridges (Retzius, 90) ; and, according to Palladino, they occur not only between the ovum and the corona radiata, but also between the follicular cells themselves. In the ripe human ovum the pores are apparently absent (Nagel), and it is very probable that they have to do with the passage of nourishment to the growing egg. Retzius believes that the zona pellucida is derived from the processes of the cells composing the corona radiata, which at first interlace and form a network around the ovum. Later, the matrix of the membrane is deposited in the meshes of the network, very probably by the egg itself. Further developmental changes are, however, necessary before a fully developed ovum (ripe ovum) may be fertilized. These are grouped under the head of maturation of the ovum. They have in part been described in a former section (p. 71), but may receive further consideration at this time. During maturation the chromo- somes are reduced in number, so that the matured ovum presents only half the number found in a somatic cell of the same animal. The manner in which this reduction takes place has been described for many invertebrates and vertebrates, and in all ova studied with reference to this point essentially the same phenomena have been observed. In this account we shall follow the process as it occurs in the Copepoda (Riickert, 94). During the period of growth the cells composing the last gen- eration of oogonia (primitive ova) increase in size, and are then known as " oocytes " (the ripe ova). These then undergo mitotic division, and in each a spirem is formed which divides into 12 chromosomes, and not into 24 as in the case of the somatic cells. These 12 chromosomes split longitudinally, so that the germinal vesicle is seen to contain 12 pairs of chromosomes, or daughter loops. By this process the oogonia have become egg mother cells (O. Hertwig, 90) or oocytes of the first order. The loops now begin to shorten and each soon divides crosswise into two equal rods, thus giving rise to 12 groups of 4 chromosomes, or 12 tetrads. The mother cell now divides into 2 unequal parts, the process con- sisting in a distribution of the rods composing the tetrads in such a THE FEMALE GENITAL ORGANS. 351 way that the pairs of rods derived from one set of daughter loops pass to the one daughter cell, and those derived from the other set to the second daughter cell. In this manner are formed the large egg daughter cells (O. Hertwig) or oocytes of the second order, and a smaller cell, the first polar body. From this it is seen that the daughter cell still retains 1 2 pairs of rods. A second unequal division immediately follows without a period of rest, but in this case the corn- Fig. 282.— Schematic representation of the behavior of the chromatin during the maturation of the ovum (from Riickert, 94). Instead of 12 chromosomes we have drawn, for the sake of simplicity, only four : a, a, a, First, and (b) second polar body. ponent parts of the pairs of rods are so divided that each separate rod moves away from its fellow, although they both originated from the same daughter loop. In this manner a cell of the third gen- eration is formed, the oocyte of the third order, or mature ovum, as well as a second polar body. The second division in the period of maturation is peculiar in that here daughter chromosomes are 352 THE GENITOURINARY ORGANS. formed, not by a longitudinal splitting of the chromosomes, but by a transverse division. In the process of development of the ova, three periods are therefore distinguishable. The first, or period of proliferation, rep- resents a stage of repeated mitotic division in the oogonia, during which the latter become gradually reduced in size. In the second, or period of growth, the oogonia increase in size and are then ready for the third, or period of maturation. In the latter, by means of a modified double mitotic division, uninterrupted by any resting stage, the matured ovum and the polar bodies are formed. These several periods are represented in figure 283. The manner in which the fully developed Graafian follicle Primordial egg-cell. Germinal zone. /Zone of mitotic division. \ / (The number of genera- tions is much larger than here represented.) Zone of growth. ^ Oocyte 1. order. Oocyte II. order. ^HB *v *• p- B- /Zone of maturation. Matured ovum. T^* P.B. Fig. 283. — Scheme of the development and maturation of an ascaris ovum (after Boveri) : P. B., Polar bodies. (From " Ergebn. d. Anat. u. Entw.," Bd. I.) bursts and its ovum is freed is still 'a subject of controversy ; the following may be said regarding it : By a softening of the cells forming the pedicle of the discus proligerus, the latter, together with the ovum, are separated from the remaining granulosa, and lie free in the liquor folliculi. At the point where the follicle comes in contact with the tunica albuginea of the ovary, the latter, with the theca folliculi, becomes thin, and in this region, known as the stigma, the blood-vessels are obliterated and the entire tissue grad- ually atrophies ; thus a point of least resistance is formed which gives way at the slightest increase in pressure within the follicle, or in its neighborhood. THE FEMALE GENITAL ORGANS. 353 The part of the Graafian follicle which remains after the ovum has been released forms a structure known as the corpus luteum, a struc- ture which passes through certain developmental stages and then undergoes degeneration. The regressive metamorphosis is much slower in a corpus luteum whose ovum has been fertilized and is in process of further development than in those whose ova have not been impregnated ; the former is known as the corpus luteum verum, the latter as the corpora lutea spuria. There is as yet difference of opinion as to the mode of development of the corpora lutea, certain observers maintaihing that the cells ofthezona granulosa contribute largely to the development of these structures, while others trace their origin to the cells of the theca interna. In this account we shall follow Sobotta, whose careful observations on the development of the corpora lutea of the mouse and rabbit support strongly the former view. According to this observer, the walls of the Graafian follicle collapse after its rup- ture. The cells of the follicular epithelium, which remains within the collapsed follicle, hypertrophy, the cells attaining many times their original size. As the epithelial cells enlarge, a yellowish pigment known as lutein makes its appearance. The cells are now designated as lutein cells. At the same time the vascular connective tissue of the inner thecal layer penetrates between the hypertrophied epithelial cells in the shape of processes accompanied by leucocytes. The structure which thus develops is known as the corpus luteum. On the rupture of the follicle hemorrhages often take place on account of the laceration of the blood-vessels. The re- mains of such hemorrhages are found in the form of hematoidin crystals. After a variable time the corpora lutea degenerate ; in this regres- sive metamorphosis the epithelial cells (lutein cells) undergo fatty degeneration, and the connective tissue trabeculae become atrophied. Each corpus luteum is thus changed into a corpus albicans, which in turn is absorbed, and in its place there remains only a connective tissue containing very few fibers. Not all of the eggs and follicles reach maturity ; very many are destroyed by a regressive process known as atresia of the fol- licles. This process may begin at any stage, even affecting the primitive ova while still imbedded in the germinal epithelium — first attacking the egg itself and later the surrounding follicular epithe- lium, although in both the degenerative process is identical. The germinal vesicle and the nuclei of the follicular cells usually undergo a chromatolytic degeneration, although they sometimes disappear without apparent chromatolysis (direct atrophy), while the cell-bodies are generally subjected to a fatty degeneration or may even undergo what is known among pathologists as an albu- minous degeneration — i. e., one characterized by granulation and showing no fat reaction but numerous reactions such as are ob- served where albumin is present. These two forms of metamor- 23 354 THE GENITOURINARY ORGANS. phosis result in a liquefaction of the cell-body, and finally lead to a hyaline swelling, which renders the substance of the cell homo- geneous. The zona pellucida softens, increases in volume, becomes wrinkled, and after some time is absorbed. A further stage in the regressive process consists in the formation of scar tissue, as in the case of the corpus luteum. Here leucocytes accompany the proliferation from the tunica interna of the theca folliculi, and assist in absorbing the products of degeneration, the result being a connective-tissue scar (vid. G. Ruge, and Schottlander, 91, 93). The blood-vessels of the ovary enter at the hilum and branch in the medullary substance of the ovary. From these medullary vessels branches are given off which penetrate the follicular zone, giving off branches to the follicles and terminating in a capillary network in the tunica albuginea (Clark, 1900). The relations of the branches to the follicles are such that in the outer layer of the theca folliculi the vessels form a network with wide meshes while the inner layer contains a fine capillary network. The veins are of large caliber and form a plexus at the hilum of the ovary. The lymphatics of the ovary are numerous. They begin in clefts in the follicular zone, which unite to form vessels lined by endothelial cells in the medulla. They leave the ovary at the hilum. The nerves accompany and surround the blood-vessels, while very few nerve-fibers penetrate into the theca folliculi ; those doing so form a network around the follicle and end often in small nodules without penetrating beyond the theca itself. Ganglion cells of the sympathetic type also occur in the medulla of the ovary near the hilum (Retzius, 93; Riese, Gawronsky). 3. THE FALLOPIAN TUBES, UTERUS, AND VAGINA. The Fallopian tubes or ova ducts consist of a mucous mem- brane, muscular coat, and peritoneal covering. The mucous membrane presents a large number of longitudinal folds which present numerous secondary folds which frequently communicate with one another. Very early in the development four of these folds are particularly noticeable in the isthmus ; these may also be recognized at times in the adult. These are the chief folds, in contradistinction to the rest, which are known as the accessory folds (Frommel). The accessory folds are well developed in the isthmus, and are here so closely arranged that no lumen can be seen with the naked eye. The epithelium lining the tubes is composed of a single layer of ciliated columnar cells which entirely cover the folds as well as the tissue between them. Glands do not occur in the oviducts, unless the crypts between the folds may be considered as such. The mucosa beneath the epithelium contains relatively few connective-tissue THE FEMALE GENITAL ORGANS. 355 fibers, but numerous cellular elements. In the isthmus it is com- pact, but in the ampulla and infundibulum its structure is looser. The mucosa contains a few nonstriated muscle-fibers, which have a longitudinal direction and extend into the chief folds, but not into the accessory folds. External to the mucosa is found the muscular coat, consisting of an inner circular and an outer and thinner longitudinal layer consisting of bundles of nonstriated muscular tissue separated by connective tissue and blood-vessels. The longitudinal layer is im- perfectly developed in the ampulla and may be entirely absent in the infundibulum. The peritoneal layer consists of a loose connec- tive tissue covered by mesothelium. Mucosa. Crypt -3 - Crypt. Fig. 284. — Section of oviduct of young woman. To the left and above are two enlarged ciliated epithelial cells from the same tube ; X I7°- The ova ducts have a rich blood-supply. The terminal branches of the arteries pass into the primary and secondary folds of the mucosa, where they form capillary plexuses under the epithelium. The blood is returned by means of a well-developed venous plexus. The lymphatic vessels have their origins in the folds of the mucosa. Nerve-fibers have been traced to the musculature and to the lining epithelial cells. The uterus is composed of a mucous, a muscular, and a peri- toneal coat. The mucosa of the body of the uterus and cervix is lined by a single layer of columnar ciliated epithelial cells ; these are some- 35^ THE GEN7ITO-URINARY ORGANS. what higher in the cervix than in the corpus. Barfurth (96) has found intercellular bridges between the cells of the uterine epithelium in the guinea-pig and rabbit. In the cervix of the virgin the ciliated columnar epithelium extends as far as the external os, at which point this usually changes to a stratified squamous epithelium. In multiparae the squamous epithelium extends into the cervical canal and may be found, with occasional exceptions (islands of ciliated epithelium), throughout its entire lower third. This arrangement is subject to considerable variation, so that even in children the lower portion of the cervical canal may sometimes be lined by stratified epithelium. Recent investigations have established the fact that in both the uterus and oviducts the general direction of the wave-like ciliary motion is toward the vagina (Hofmeier). In the body of the uterus the mucosa is composed of a reticular connective tissue consisting of relatively few connective-tissue fibers and branched connective-tissue cells arranged in the form, of a network, in the meshes of which are found lymphocytes and leucocytes. Under low magnification the mucosa presents more the appearance of adenoid tissue than of areolar connective tissue. The mucosa of the cervix is somewhat denser, containing more fibrous tissue. In the cervical canal the mucosa of the anterior and posterior walls is elevated to form numerous folds, extending laterally from larger median folds. These folds are known as the pliccz palmatce. The mucosa of the body of the uterus and of the cervix contains numerous glands, the uterine and cervical glands. The uterine glands are branched tubular in type, and extend through the mucosa and certain ones may even extend for a short distance into the muscular layer. They are lined by ciliated columnar epithelium, resting on a basement membrane. The cervical glands are larger and more branched than those of the body of the uterus, and belong to the type of tubulo-alveolar glands ; they have a mucous secretion. The glands and crypts extend as far as the external os. In the mucous membrane of the cervical region we find peculiar closed sacs of varying size lined by simple cylindric epithelium, the so- called ovula Nabothi, which probably represent cystic formations (vid. A. Martin). Three layers of muscular tissue are to be seen both in the corpus and cervix uteri — an inner longitudinal, a middle nearly cir- cular, in which the principal blood-vessels are found, and an outer longitudinal. The inner and outer layers are known respectively from their position as the stratum mucosum and stratum serosum, the middle and more vascular as the stratum vasculosum. As com- pared with the middle, the inner and outer muscle layers are poorly developed. The complicated conditions found in the uterine musculature can be better understood if some attention be paid to its origin. The circular layer should be regarded as the original musculature of the Mullerian ducts. The outer longitudinal layer develops later, and is derived from the musculature of the broad THE FEMALE GENITAL ORGANS. 357 ligament. Between these two are the large vessels accompanied by a certain amount of muscular tissue — a condition which persists throughout life in the carnivora. In man the blood-vessels pene- trate into the circular musculature and only appear later in the inner muscular layer. A true muscularis mucosse is not present in the human uterus (Sobotta, 91). The serous or peritoneal layer consists of a layer of mesothelial cells and submesothelial connective tissue. The uterus derives its blood supply from the uterine and ovarian arteries, which enter from the broad ligament through its lateral portion. These vessels pass to the stratum vasculosum of the muscular layer, where they branch repeatedly, some of the branches Uterine epithelium. £; Gland. - Mucosa. Fig. 285. — From uterus of young woman ; X 34- J. Amann.) (From a preparation by Dr. 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 GENITO- URINARY 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 GENITOURINARY ORGANS. The epithelium of the vestibulum 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 (Wolffian 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 flagellum. 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 P'ig. 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 ; ft middle piece ; g, marginal thread ; h, axial thread ; iy undulating membrane ; k, fibrils of the axial thread ; /, fibrils of the marginal thread ; ;//, end piece of Retzius ; n, rudder-membrane. is continued into the tail of the spermatozoon, and is here sur- rounded by a transparent substance — the sheath 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. — d 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 // to 5 fj. long, and from 2 fji to 3 ft in breadth ; the middle piece is 6 IJL long and I p in breadth ; the tail is from 40 fj. to 60 p. long, and the end-piece 6 p long. The spermatozoa are actively motile, a phe- nomenon due to the flagella, which give them a spiral, boring motion. They are character- ized by great longevity and are very resistant to the action of low temperatures (vid. Pier- In some species of bat the sper- 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.) Fig. 289. — Human spermatozoa. The two at the left after Retzius (8 1) ; the one at the extreme left is seen in profile ; the other in surface view ; the one at the right is drawn as described by Jensen : a, Head ; b, terminal nod- ule ; c, middle piece; SQj o~\ d, tail ; e, end-piece of Retzius. 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. 363 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- dymiais. ' Corpus Highmori ~~~ and rete testis. — — Blood-vessel . Tubuli recti. — — — - Vas epididymidis. Fig. 290. — Longitudinal section through human testis and epididymis. The light areas between the lobules are the fibrous- tissue septa of the testis ; X 2> sections may be observed in sections of the testis. When isolated, these tubules are seen to begin in the testis as closed canals, which are closely coiled upon each other (convoluted tubules) and describe a tortuous course, until they finally reach the corpus Highmori. Immediately before they reach the latter, the convoluted tubules change into short, straight and narrow segments — the straight tubules, or tubuli recti. Within the corpus Highmori, all the straight tubules of the testis unite to form a tubular network — the rete testis (Haller). From this network about fifteen tubules — the vasa efferentia — THE GENITOURINARY ORGANS. arise. The latter, at first straight, soon begin to wind in such a man- ner that the various convolutions of each canal form an independent system, invested by a fibrous sheath of its own — coni vasculosi Halleri. These lobules constitute the elements of the globus major of the epididymis. In cross-section the vasa erferentia are seen to be stellate in shape. The vasa erTerentia gradually unite to form one canal — the vas epididymidis. This is markedly convoluted and is situated in the body and tail of the epididymis itself. The epithelium of the convoluted seminiferous tubules consists of sustentacular cells (cells or columns of Sertoli) and of sperma- togenic elements. The former are high, cylindric structures (see below), the basilar surfaces of which are in contact. They do not form a continuous layer, but their basal processes are interwoven to form a superficial network surrounding the epithelium of the Fig. 291. Fig. 292. Sustentacular cells (cells of Sertoli) of the guinea-pig (chrome-silver method). Figure 291, surface view of the seminiferous tubules ; figure 292, profile view ; X 22° : a, Basilar surface of a cylindric sustentacular cell ; b, flattened sustentacular cell ; c, c, depressions in the sustentacular cells due to pressure from the spermatogenic cells ; J, basilar portion of sustentacular cells. seminiferous tubules. (Fig. 292.) In the meshes of the reticulum are deposited numbers of plate-like cells, which lie in contact with the basement membrane and also represent sustentacular elements (vid. Merkel, 71). Between the sustentacular cells are found from four to six rows of cells, possessing relatively large nuclei, rich in chromatin, and derived from cells of the deeper strata by mitotic cell division. The epithelium of the convoluted portion of the seminiferous tubules is, therefore, a stratified epithelium. The cells of this epithelium present various peculiarities according to their stage of development, and will be considered more fully in discussing spermatogenesis. Externally, the walls of the convoluted tubules are limited by a single layer or several layers of spindle-shaped, epithelioid cells. A basement membrane is present, but very thin, and in some cases THE MALE GENITAL ORGANS. hardly capable of demonstration. The convoluted tubules are separated from each other by a small amount of connective tissue, in which, in addition to the vessels, nerves, etc., are found peculiar groups of large cells containing large nuclei, and known as interstitial cells. Nothing definite is known regarding the significance of these cells ; but they are probably remains of the Wolffian body. Reinke (96) found repeatedly crystalloids of problematic significance in the interstitial cells of the normal testis. The stratified epithelium of the convoluted tubules changes in 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 efiferentia 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 ; c, 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 HallerL Num- THE MALE GENITAL ORGANS. 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 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. Fig. 296. — Section of dog's testis with in- jected blood-vessels (low power) : a, Seminifer- ous tubule ; &, connective-tissue septum ; c, blood- vessel. 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. Mucosa. k /TV Inner longi- tudinal muscular layer. Middle cir- cular mus- cular layer. 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. The 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 trabeculae. 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 37° 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 y2 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 trabeculae 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. 3/1 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 trabeculae 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. mu'cosa 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 gland-s (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 — tMtfollicularoi sustentacu- SPERMATOGENESIS. 3/3 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. ,;: "/\ I \ ">r K ft Spermatocyte I order. • '^ " - Spermatocytes II order.- —• • [ Zone of maturation. 5. • • • Spermatids. 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 cell ; 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 Fi^. 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 : «, 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 of 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 SPERM ATOGENESIS. 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 portr®n 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 fornfced 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 inter -cellular 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 ; ^, 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 fibrillse, 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. 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.t 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 Stratum corneum. Stratum Malpighii. Duct of sweat- gland. Corium. Subcutis. , ~~ 'Blood-vessel. Sweat-gland. ' Fig. 304. — 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 lucidum. 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 382 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 direc- 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 48°- THE SKIN. 383 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 no // to 220 />« 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 « corneutn. 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 p. to 50 p. 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 retinacula cutis, and inclose in their meshes masses of fatty tissue which form the patiniaihis 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 >k I Stratum corneum. Pigment •- cell with two pro- cesses. PK.'~ '"=- 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. tive-tissue cells are found immediately beneath the epidermis, 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 ; c, subcutis ; of, subpapillary arterial network ; Cervical region in the plane of the sixth spinal nerve-root ; B, lumbar region ; C, thoracic region ; Dt sacral region (compare with Fig. 328). (From preparations of H. Schmaus.) 4O8 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 nucleus 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 gelatinosa (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 terminate. 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 cellsr 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. xDendrite. Blood-vessel.- V- —Nerve-fiber layer. Fig- 33°. — Section through the human cerebellar cortex vertical to the surface of the con- volution. Treatment with Miiller's fluid ; X IJ5- The molecular layer contains three varieties of nerve-cells, those of Purkinje, which border upon the granular layer, the stel- ^fe •1 - rt ft V 1 w be V ^ 0 w 3,13 s ba . ft; b§ §a rt 'S "^ ** -J 2 0 .0 rt u » &^ M 1) : *o* 'Sf'rt c "S i U x^ ii -2 i'g3 "1 c« "1. ^ ^^ 'O m 'o o 2 '£) -i to'3 If \ u .•) •n.^ ^5 1 2-=- o ^ H — Id » ••>. = ' . **Si ! 4>^ ' Y< '- \* g V 1 CO 1 p c sJS -s bfl £ 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°- - — Nauraxis. — 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, cla^v-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. 41 J 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 — i. 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 p in diameter) and large pyramidal cells (from 20 //to 30 p. 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, in1 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 cells 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 pjcr 774 —Portions of vertical section of human cerebral cortex, treated by the Golgi method • V 70. The figure shows the arrangement of the different cells of the cerebral cortex • gP Layer of large pyramidal cells ; kP, layer of small pyramidal cells ; /Z, 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. - - Serondarv detidrite Basal dendrite. — Neuraxis with collaterals. Fig. 335. — Large pyramidal cell from the human cerebral cortex. method; X '5°. Chrome-silver 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. •> . £*- *:*. > It during their passage through the basal nuclei ; (2) the commissural fibers, which, according to the original definition, pass through the corpus callo- H~ a sum and anterior commis- ^^i^^S^j§^^M^ 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; 6, striation of Bechtereff-Kaes ; <:, layer of small pyramidal cells ; d, stripe of Bail- larger; e, radial bundles of the medullary sub- stance ; /, 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, with 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, fifth, 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 // to 300 />« 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. Molecu- lar layer. Layer of olfactory glomeruli. Peripheral nerve fibers. Fig. 337. — The olfactory bulb, after Golgi and Ram6n y Cajal. not shown. The 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 infundibulum ; 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. 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 (infra vitant). 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 £, small spinal ganglion cells with T-shaped division of process ; <:, 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 // to 45 /JL 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 — 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 fji. 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 rami communic antes. 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. 431 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 M Fig. 346. — Schematic diagram of a sensorimotor reflex arc according to the modern neurone theory ; transverse section of spinal cord : mN, Motor neurone ; sAT, sensory neurone ; C l , nerve-cell of the motor neurone ; C'2, nerve-cell of the sensory neurone ; Cerebral cortex ; mNl, motor neurone of the first, sN2, sensory neurone of the second, degree ; C 1, motor cell of the spinal cord ; C2, sensory cell of a spinal ganglion; C3, pyramidal cell of the brain cortex (pyschic cell) ; C4, nerve-cell of a sensory neurone of the second degree ; n, n, n, n, neuraxes ; d, d, dendrites ; c, c, c, c, collaterals ; /, t, telodendria ; sNl, sensory neurone first degree ; mN2, motor neurone second degree. 28 434 THE CENTRAL NERVOUS SYSTEM. H. THE NEUROGLIA* The neuroglia tissue is an especially differentiated supporting tissue found in the central nervous system, the optic chiasm, optic nerve and retina and for some distance, at least, in the olfactory nerve. Its relation to other tissues has long been a matter of con- troversy, but modern observers have shown quite conclusively that neuroglia tissue is of ectodermal origin. It should not be under- stood, however, that the neuroglia tissue forms the only supporting tissue of the central nervous system. In all parts of the central nervous system, more especially, however, in the spinal cord, there is found true connective tissue of mesoblastic origin, more especially in connection with the blood-vessels. At an early stage of embryonic development there are seen in the spinal cord, and also in the brain, elements radially disposed around the neural canal, which upon closer observation appear to be processes emanating from the epithelial cells lining the neural canal. These processes may undergo repeated dichotomous divi- sion, ending finally in a swelling near the periphery of the cord. These cells are known as epen- dymal cells, and are differenti- ated from ectodermal cells, called Spongioblasts. In later stages the radial arrangement is still preserved, but the cell-bodies no longer all border upon the cen- tral canal, many being found at varying distances from the latter. At this stage in the development of the spinal cord, the elements retaining their original charac- teristics are situated only in the region of the ventral and dorsal fissures of the spinal cord, and during further development in- crease in number. These observations would seem to 'indicate that at least a portion of the neurogliar cells, which develop from the ependymal cells previously mentioned, originate from the epithelium of the central canal, and that from here they are gradually pushed toward the periphery of the cord. This assumption is still further strength- ened by the fact that later the epithelial cells of the central canal still continue to divide. Later observations (Schaper, 97) show, how- ever, that neurogliar cells develop also from certain undifferentiated germinal cells of the neural canal, of ectodermal origin, which Fig. 349. — Neurogliar cells : <7, From spinal cord of embryo cat ; b, from brain of adult cat ; stained in chrome-silver. THE NEUROGLIA. 435 wander from their position near the neural canal toward the per- iphery of the medullary tube, where they develop into neuroglia cells. Owing to the fact that of the several methods now at hand for studying neuroglia tissue no two give identical results, the views concerning this tissue are still at variance. The Golgi or chrome- silver method was for many years the only method by means of which the elements of neuroglia tissue were brought to light with any degree of clearness. In preparations of the central nervous system treated with this method all the neuroglia elements appear as cells with processes. The cell bodies of these cells as also the processes being stained black or nearly black (as seen with trans- mitted light) so that the relations of the processes to the cellular constituents can not be ascertained, investigators who have made use of this method in their study of neuroglia distinguish two essentially different cellular elements of the neuroglia: ependymal cells, pre- viously mentioned, and neuroglia cells, so-called spider cells or astrocytes. The astrocytes are grouped under two main heads : short-rayed astrocytes, possessing a few short processes, found in the gray matter, and long-rayed astrocytes with many fine and long pro- cesses, which do not appear to branch, found both in the gray and white matter. The two types of astrocytes are not clearly defined, as intermediate types are also found. In figure 349 are shown two astrocytes (long-rayed) as seen in chrome-silver preparations. A number of investigators have in recent years perfected methods by means of which neuroglia tissue could be stained differentially — Weigert, Mallory, Benda. In tissues treated after any one of these rather complicated differential staining methods the processes of the neuroglia cells as seen in chrome-silver preparations appear in the form of well-contoured fibrils, which are not interrupted by the cell- bodies of the neuroglia cells, from which they are either entirely separated or are seen to pass through the protoplasm of the cells without losing their identity. In preparations of the central nervous system stained after Benda' s differential neuroglia tissue staining method, numerous neuroglia cells may be observed both in the gray and white matter. Certain of these cells possess very little proto- plasm, others — and these are in the majority — present it to an ap- preciable extent. The shape of such cells varies. When situated in the main mass of the white matter of the spinal cord, and seen in cross-sections of the cord, they present an irregular triangular and quadrangular form, with protoplasmic branches which arise from the angles and which extend for a variable distance between the nerve- fibers. In such preparations it may be seen that the neuroglia fibers pass in close proximity to the neuroglia cells, apparently em- bedded in the outermost part of their protoplasm, and often follow- ing the protoplasmic processes. This view of the structure of neu- rogliar tissue is more in accord with recent investigations on this 43^ THE CENTRAL NERVOUS SYSTEM. subject (Weigert, Mallory, Benda, Krause, Hardesty, Huber). In figure 350 are shown two neuroglia cells from a cross-section of a human spinal cord, in which the relation of neuroglia fibers to neu- roglia cells is shown. Fig. 350. — Typical neuroglia cells, from cross-section of the white matter of the human spinal cord, stained after Benda' s selective neuroglia tissue staining method; X 1 200 (Huber, "Studies on Neuroglia Tissue," Vaughan Festschrift, 1903). L THE MEMBRANES OF THE CENTRAL NERVOUS SYSTEM. The membranes of the central nervous system (meninges) are three in number: the outer, or dura mater ; the middle, or arach- noid; and the inner, or pia mater. Around the brain the dura mater is very intimately connected with the periosteum and presents a smooth inner surface. It con- sists of an inner and an outer layer, the two being separated from each other only in certain regions. At such points either the inner layer is pushed inward to form a duplicature, as in the falx cerebri and falx cerebelli, tentorium, and diaphragma sellae, or the outer layer is pushed outward to form small, blindly ending sacs. The venous and lymphatic sinuses lie between the two layers. The outer THE MEMBRANES OF THE CENTRAL NERVOUS SYSTEM. 437 layer of the dura is continued some distance along the cerebrospinal nerves. The dura mater of the spinal cord does not form the periosteum for the bones forming the vertebral canal ; these possess their own periosteum. The spinal dura mater is covered on its outer surface by a layer of endothelial cells and is separated from the wall of the vertebral canal by the epidural space, containing a venous plexus imbedded in loose areolar connective tissue and adipose tissue. The dura consists chiefly of connective-tissue bundles having a longitudinal direction along the spinal cord. Within the cranium, however, the bundles of the inner and outer layers cross each other ; those of the outer having a lateral direction anteriorly and a mesial posteriorly ; those of the inner, a mesial direction anteriorly and a lateral posteriorly. In the falx cerebri, tentorium, etc., the fibers are arranged radially, extending from their origin toward their borders. The shape and size of the connective-tissue cells vary greatly, and their processes form a network around the bundles of connective tissue. Few elastic fibers are present, and, according to K. Schultz, these are entirely absent in the new-born ; they are somewhat more numerous in the dura of the spinal cord. The dura is very rich in blood-capillaries, and the presence of lymphatic channels in com- munication with the subdural space may be demonstrated by means of puncture-injections. The inner surface of the dura mater is cov- ered by a layer of endothelial cells. The dura mater is quite richly supplied with nerves, especially in certain regions. These are of two varieties : ( i) Vasomotor fibers, which form plexuses in the adventitial coat of the arteries, and would seem to terminate in the muscular coat of the arteries ; (2) medullated nerve-fibers, which either accompany the blood-vessels in the form of larger or smaller bundles or have a course inde- pendent of the vessels. After repeated division these medullated nerve-fibers lose their medullary sheaths and terminate between the connective-tissue bundles in fine varicose fibrils, which may often be traced for long distances (Huber, 99). The arachnoid is separated from the dura by a space which is regarded as belonging to the lymphatic system — the subdural space. The outer boundary of the arachnoid consists, as does the inner lin- ing of the dura, of a layer of flattened endothelial cells. The arach- noid is made up of a feltwork of loosely arranged connective-tissue trabeculse, which also penetrate into the lymph-space between it and the pia — the subarachnoid space. For a short distance from their points of origin the cerebrospinal nerves are accompanied by arachnoid tissue. In the brain the arachnoid covers the convolu- tions and penetrates with its processes into the sulci. These pro- cesses are especially well developed in the so-called cisterns ; in the cisterna cerebellomedullaris, fossae Sylvii, etc. In the spinal cord the subarachnoid space is separated by the ligamenta den- ticulata into two large communicating spaces — a dorsal and a ven- 438 THE CENTRAL NERVOUS SYSTEM. Gray matter. tral. The dorsal space is further divided by the septum posticum, best developed in the cervical region. At certain points, usually along the superior longitudinal sinus, the outer surface of the arachnoid is raised into villi, which are covered by the inner layer of the dura, and form with the latter the Pacchionian bodies or granulations. These villi are connected with the arachnoid by pedicles so delicate that they often seem to be suspended free in the venous current of the sinus. The subarachnoid space contains numerous blood-vessels, some of which are free and others attached to the arachnoid. Their adventitia is covered by endothelium ; hence the subarachnoid space would seem to assume here the character of a perivascular space. The trabeculae and membranes composing the arachnoid tissue show a great similarity to those of the mesentery, and es- pecially to those of the omentum. The whole constitutes a typical are- olar connective tissue, interrupted at numerous points and covered by a continuous layer of en- dothelial cells. Large numbers of spiral fibers are found here twining around single or groups of connective-tissue fi- bers. The arachnoid possesses neither blood- vessels nor nerves. The pia mater cov- ers the entire surface of the brain and spinal cord, dipping down into every fissure and crevice. In the spinal cord it consists of an outer and an inner lamella, the former being com- posed of bundles of connective tissue containing elastic fibers. As a rule, the course of the fibers is longitudinal. Externally this layer is covered by a layer of endothelium. The blood- vessels lie between the outer and inner layers of the pia. The inner layer (pia intima) is made up of much finer elements, and is covered on both sides by endothelium. It is this layer which accompanies the blood-vessels penetrating into the spinal cord, surrounding their adventitia and forming with the latter the limits of their perivascular spaces. These are in communication with the White matter. Fig- 351. — Section through the cerebral cortex of a rabbit. The blood-vessels are injected ; X 4°* BLOOD-VESSELS OF THE CENTRAL NERVOUS SYSTEM. 439 interpial spaces, and, by means of the adventitia of the blood-vessels, with the subarachnoid space. Aside from those just described, numerous fine, nonvascular, connective-tissue septa penetrate from the pia mater into the substance of the spinal cord. Wherever the pia mater penetrates the spinal cord, the latter is hollowed out, forming the so-called pied funnels. Just beneath the pia there is found in the spinal cord of man a well-developed layer of neuroglia fibers. The posterior longitudinal septum of the spinal cord consists (in the thoracic region) exclusively of neurogliar elements, but in the cervical and lumbar regions the pia also enters into its peripheral formation. In the brain, however, the conditions are somewhat different. Here the external layer of the pia disappears, leaving only a single layer analogous to the pia intima of the spinal cord. The pia mater enters into the formation of the choroid plexus. This structure consists of numerous freely anastomosing blood- vessels, which form villus-like processes, the surfaces of which are covered by squamous or cubic epithelial cells. This epithelium is regarded as a continuation of the ventricular epithelium, and is cili- ated, at least in embryonic life and in the lower classes of verte- brates. From an embryologic point of view the whole structure represents the brain-wall reduced to a single layer of epithelium (internal epithelial investment) pushed forward into the ventricle by the vessels and pia mater. Since the dura and arachnoid accompany the cerebrospinal nerves for some distance, it is obvious that the lymph-vessels of the nasal mucous membrane (see these) may also be injected from the subarachnoid space (compare also Key and Retzius). The pia mater, like the dura mater, receives two varieties of nerve-fibers : (i) Vasomotor fibers, which form plexuses in the ad- ventitial coat of the arteries and terminate in the muscular layer of the arteries. These may be traced to the small precapillary branches of the vessels. (2) Larger and smaller bundles of rela- tively large, medullated nerve-fibers, which accompany the larger pial vessels, forming loose plexuses in or on the adventitial coat of the vessels. After repeated divisions these medullated nerves lose their medullary sheaths and terminate in the adventitia of the ves- sels, in long, varicose fibrils or in groups of such fibrils (Huber, 99)- J. BLOOD-VESSELS OF THE CENTRAL NERVOUS SYSTEM. The blood-vessels of the central nervous system present certain peculiarities which deserve special consideration. The spinal cord receives its arterial blood mainly through vessels which accompany the spinal nerve roots and through numerous anastomoses from a plexus in the pia mater in which there may be 44° THE CENTRAL NERVOUS SYSTEM. recognized a median ventral unpaired line of anastomosis and along each half of the spinal cord four other lines of anastomosis. From the median unpaired line of anastomosis some 200 to 2 50 branches pass into the anterior fissure, each of which generally divides into a right and left branch just in front of the commissure, each branch being distributed to the gray matter in its immediate vicinity. The white matter receives its blood-supply from vessels of the plexus in the pia mater, from which numerous fine branches are given off which terminate in capillary networks and extend as far as the gray matter. The veins return the blood to the veins of the pia mater, following in the main the course of the arteries. The central and peripheral arteries do not anastomose except through capillaries and now and then precapillaries (Adamkiewicz and Kadyi). In the cerebral cortex the capillaries are particularly numerous, and are closely meshed wherever groups of ganglion cells occur. In the medullary substance they are somewhat less closely arranged, their meshes being oblong. In the cerebellum the arrangement is analogous. Of all the layers composing the cerebellum the granu- lar is the most vascular; within it the capillaries are also densely arranged and form a close network. Lymphatic vessels with definitive walls have thus far not been discovered in the central nervous system. The blood-vessels through the central nervous system are, however, surrounded by perivascular spaces, which may be regarded as performing the function of lymph- atic vessels. TECHNIC The organs of the central nervous system are best fixed in Miiller's fluid, washed with water, cut in celloidin, and stained with carmin. Such preparations are suitable for general topographic work. Special structures — as, for instance, the medullary sheaths of the nerve- fibers, the ganglion cells, the relations of the different neurones and den- drites to one another, etc. — require different treatment. The medullary sheath may be demonstrated as follows (Weigert): Pieces of tissue (spinal cord, for instance), fixed as usual in Miiller's or Erlicki's fluid, are transferred without washing to alcohol, imbedded in celloidin, and cut. Before staining the sections it is necessary to subject them to the mordant action of a neutral copper acetate solution (a saturated solution of the salt diluted with an equal volume of water). The sections may be subjected to the mordant action of this solution, but the following procedure is more convenient : The specimens, imbedded in celloidin and fastened to a cork or a block of wood, are placed for one or two days in the copper solution just described. At the expiration of this time the pieces of tissue will have become dark, and the surrounding celloidin light green. They are then placed in 80% alcohol, in which they may be preserved for any length of time. The sections are then stained in the following solution : i gm. of hematoxylin is dissolved in 10 c.c. absolute alcohol, and 90 c.c. of distilled water are then added (the fluid must remain exposed to the air for a few days) ; the addition of an alkali — as; for instance, a cold satu- TECHNIC. 441 rated solution of lithium bicarbonate (i. c.c. to 100 c.c. of hematoxylin solution) — brings out the staining power of the solution at once. In this stain the sections are placed (at room -temperature) for a day, and then in a thermostat (40° C.) for a few hours. The sections, now quite dark, are washed in distilled water and then placed in the so-called dif- ferentiating fluid. The latter consists of borax 2 gm., ferrocyanid of potassium 2.5 gm., and distilled water 100 gm. In this fluid the color of the sections is differentiated by virtue of the circumstance that the medullary sheath retains the dark stain, while the remaining structures, such as the ganglion cells, etc. , are bleached to a pale yellow. The time required for this differentiation varies, but it is usually complete at the end of a few minutes. The sections are then washed in distilled water, dehy- drated in alcohol, cleared in carbol-xylol (carbolic acid i part, xylol 3 parts) and mounted in balsam. Weigert's new method is more complicated, but fruitful of cor- respondingly better results. The preliminary treatment remains the same. After the tissues have been imbedded in celloidin and this hardened in 80% alcohol, they are transferred to a mixture composed of equal parts of a cold saturated aqueous solution of neutral copper acetate and 10% aqueous solution of sodium and potassium tartrate, and the whole is placed in the thermostat. Larger pieces — as, for instance, the pons Varolii of man — may remain in the solution longer than twenty-four hours, after which time, however, the mixture must be changed ; but in no case should the specimens be permitted to remain longer than forty-eight hours in this solution. The temperature in the thermostat should not be high, otherwise the specimens will become brittle. The objects are now placed in a simple aqueous solution of neutral copper acetate, either saturated or half diluted with water, and again put in the oven. They are then rinsed in distilled water and placed in 80% alcohol; after remaining in this for one hour, they are in a condition to cut, but may be preserved still longer if desired. Cut and stain in the customajy manner. The staining solution is prepared as follows : (#) lithium carbonate 7 c.c. and distilled water 93 c.c. (saturated aqueous solution) ; (^) hematoxylin r gm., absolute alcohol 10 c.c. ; both a and b keep for some time, and may be kept on hand as stock solutions. Shortly before using, 9 parts of a and i part of b are mixed. After remaining in this solution for from four to five hours at room -temperature the sections are well stained, but do not overstain even if allowed to remain in the solution for twenty-four hours. In the case of loose celloidin sections the use of the differentiat- ing fluid is superfluous. Hence this method is particularly advantageous when the gray and the white matter can not be distinguished macro- scopically. Finally, the sections are washed in water, placed in 95% alcohol, cleared with carbol-xylol or anilin-xylol (in the latter case carefully washed with xylol), and mounted in xylol -balsam. The medul- lated fibers appear dark blue to black, the background pale or light pink, and the celloidin occasionally bluish. In order to remove the latter color, it is only necessary to wash the sections in 0.5% acetic acid in- stead of ordinary water ; a process, however, not to be recommended in the case of very delicate preparations — as, for instance, the cerebral cortex. In applying Weigert's methods a certain thickness of section (not exceeding 25 /JL) is essential, since in thicker sections the medullary sheaths are not sharply differentiated from the surrounding tissue. 442 THE CENTRAL NERVOUS SYSTEM. For thick sections the modified Weigert method, or Pal's method, is employed. After the specimens have been treated according to Wei- gert's method up to the point of staining with hematoxylin, they are placed for from twenty to thirty minutes in a 0.25% solution of potassium per- manganate. As differentiating fluid a solution of oxalic acid i gm., potassium sulphite i gm., and water 200 c.c. is used, care being taken, as in the case of Weigert' s differentiating fluid, that the gray matter is thoroughly bleached (here entirely colorless) and the white matter dark. By this method the medullary sheaths are stained blue, while the rest of the structure remains colorless. The staining is very precise, but not so intense as by Weigert' s method. Hence its adaptability for thicker sections. Benda's method is a modification of the Weigert-Pal methods. The tissues are hardened in Miiller's or Erlicki's fluid, imbedded in celloidin, and cut. The sections are then subjected to the action of the following mordant for from twelve to twenty-four hours : liquor ferri ter sulphatis i part, distilled water 2 parts. They are then thoroughly rinsed in two tap-waters and one distilled water and then stained in the following hem- atoxylin solution: hematoxylin i gm., absolute alcohol 10 c.c., distilled water 90 c.c.; in which they remain for twenty-four hours. They are next washed in tap-water for from ten to fifteen minutes and treated with a 0.25% aqueous solution of permanganate of potassium until the gray and the white matter are differentiated, after which they are rinsed in distilled water and bleached in the following solution until the gray mat- ter has a light yellow color : hydric sulphite 5 to 10 parts, distilled water 100 parts. The sections are then washed in tap -water for from one to two hours, rinsed in distilled water, dehydrated, cleared in carbol-xylol, and mounted in balsam. Medullary sheaths will be stained a bluish-black ; other structures, a light yellow. Sections stained after the Weigert, Pal, or Benda method may be counterstained in Van Gieson's picric-acid- fuchsin stain (i% aqueous solution of acid fuchsin, 15 parts; saturated aqueous solution of picric acid, 50 parts; distilled water, 50 parts). The fibrous connective tissue in the sections and degenerated areas stains a light red. Apathy (97) demonstrates the fibrillar elements of the nervous system in invertebrates and vertebrates by means of his gold method. Fresh tissue may be used, or tissue already fixed. In the first case thin membranes are placed for at least two hours in a i % solution of yellow chlorid of gold in the dark, then carried over without washing into a i °/o solution of formic acid (sp. gr. 1.223), an(^ finally exposed for from six to eight hours to the light (the formic acid may have to be changed). These specimens are best mounted directly in syrup of acacia or in con- centrated glycerin. In his second method Apathy fixes vertebrate tissues for twenty-four hours in sublimate-osmic acid (i vol. saturated solution of corrosive sublimate in 0.5% sodium chlorid solution combined with i vol. i °/c osmic acid solution), washes repeatedly in water, and places for twelve hours in an aqueous iodo-iodid of potassium solution (potassium iodid i % and iodin 0.5%). The further treatment is the same as after or- dinary corrosive sublimate fixation. Finally, the specimens are imbedded in paraffin with the aid of chloroform, cut, and mounted by the water method. The whole process, up to the point of imbedding in paraffin, is carried out in the dark. The sections are then passed through chloro- TECH NIC. 443 form and alcohol into water, where they are allowed to remain for at least six hours ; or they may be washed in water, placed for one minute in i % formic acid, again washed in water, immersed for twenty-four hours in a i % solution of gold chlorid, rinsed in water, and finally placed in a i % formic acid solution and exposed to the light. For the latter purpose glass tubes are employed in which the slides are placed obliquely, with the sections downward. A uniform illumination of the section with "as intense a light and low a temperature " as possible are conditions indis- pensable to the attainment of successful results. The sections are then again washed in water and mounted in glycerin or syrup of acacia, or in Canada balsam after being dehydrated. Thin membranes are stretched upon small frames of linden wood especially prepared for this purpose. Their further treatment is then the same as that of sections fixed to the slide. If successful, the nerve -fibrils appear dark violet to black. The large ganglia in the spinal cord of lophius, the calf, etc., are especially recom- mended as appropriate vertebrate material. Bethe (1900) has recommended the following method for staining neurof ibrils and Golgi-nets in the central nervous system of vertebrates : The perfectly fresh tissue is cut in thin lamellae, varying in thickness from 4 to 10 mm. These are placed on pieces of filter-paper and then in 3 to 7-5% nitric acid, in which they remain twenty-four hours. From the hardening fluid the pieces of tissue are transferred into 96% alcohol, where they remain for from twelve to twenty-four hours. They are then placed in a solution of ammonium -alcohol, — ammonium (sp. gr. 0.95 to 0.96), i part; distilled water, 3 parts; 96% alcohol, 8 parts, — in which they remain for from twelve to twenty-four hours. The temper- ature of this solution should not exceed 20° C. The tissues are then placed for from six to twelve hours in 96% alcohol, and next in a hydro- chloric acid-alcohol solution, — concentrated hydrochloric acid (sp. gr. 1. 1 8 — 37%), i part; distilled water, 3 parts; and 96% alcohol, 8 to 12 parts, — in which they remain for several hours. The temperature of this solution should not exceed 20° C. The tissues are then again placed in 96% alcohol for from ten to twenty-four hours, and next in distilled water for from two to six hours. The tissues are now placed for twenty -four hours in a 4% aqueous solution of ammonium molybdate. (This solution should be kept at a temperature varying from 10° to 15° C., if it is de- sired to stain the neurofibrils ; or at a temperature varying from 10° to 30° C., if it is desired to bring out the Golgi-nets. ) After the ammo- nium molybdate treatment, the tissues are rinsed in distilled water, placed in 96% alcohol for from ten to twenty- four hours, then in absolute alco- hol for a like period, cleared in xylol or toluol, and imbedded in par- affin. Sections having a thickness of 10 // are now cut and fixed to slides with Mayer's albumin -glycerin. Since the various solutions used in the fixation and further treatment of the tissues do not act with the same in- tensity on all parts of the piece of tissue to be studied, and since the differ- entiation and staining depend on a relative proportion (not yet fully de- termined) of the mordant (ammonium molybdate) and the stain in a given section, it is advised by Bethe to cut large numbers of sections and fix to each slide about three sections from different parts of the series. After fixation of the sections to the slide the paraffin is removed with xylol ; and they are then carried through absolute alcohol into distilled water, in which, however, the sections remain only long enough to re- 444 THE CENTRAL NERVOUS SYSTEM. move the alcohol. The slides (with the sections fixed to them) are then taken from the water and rinsed with distilled water from a water-bottle. The slide is then wiped dry on its under surface and edges with a clean cloth, and about i c.c. to 1.5 c.c. of distilled water placed on the slide over the sections. The slides are now placed in a warm oven with a tem- perature of 55° C. to 60° C. for a period of time varying from two to ten minutes. No definite time can here be given ; sections from each block of tissue need to be tested until the right stay in the warm oven is ascertained. The slides are then taken from the warm oven and rinsed two or three times in distilled water and again dried as previously directed. They are then covered with the following staining solution and again placed in the warm oven for about ten minutes : toluidin-blue, i part ; distilled water, 3000 parts. The stain is washed off with dis- tilled water and the sections are placed in 96% alcohol until no more stain is given off — usually for from three-fourths to two minutes. They are then dehydrated in absolute alcohol, passed through xylol twice, and mounted in xylol balsam. For a fuller discussion of this method the reader is referred to Bethe's account in " Zeitsch. f. Wissensch. Mikrosk.," vol. xvii, 1900. For staining neuroglia Weigert (95) has recommended a method, from which we give the following : A solution is made consisting of 5% neutral acetate of copper, 5% ordinary acetic acid, and 2.5% chrome-alum in water. The chrome-alum and water are first boiled together, the acetic acid then added, and finally the finely pulverized neutral copper acetate, after which the mixture is thoroughly stirred and allowed to cool. To this solution 10% formalin may be added. Objects not over 0.5 cm. in diameter are placed in this fluid for eight days, the mixture being changed at the end of a few days. By this means the pieces of tissue are at the same time fixed and prepared for subsequent staining by the action of the mordant. If separation of the two processes be desired, the specimens are fixed for about four days in a 10% formalin solution (which is changed in a few days), and then placed in the chrome-alum mixture without the addition of formalin. Specimens thus fixed may be preserved for years without disadvantage, and may then be subjected to further treatment by other methods, Golgi's for instance. Washing with water, dehydration in alcohol, and imbedding in celloidin are the next steps. The sections are then placed for about ten minutes in a 0.33% solution of potassium permanganate, washed by pouring water over them, and placed in the reducing fluid (5% chromogen and 5% formic acid of a specific gravity of 1.20; then filter carefully, and add 10 c.c. of a 10% solution of sodium sulphite to 90 c.c. of the fluid). The sections, rendered brown by the potassium permanganate, readily decolorize in a few minutes, but it is better to leave them for from two to four hours in the solution. If it be desirable to decolorize entirely the connective tissue, no further steps need be taken preliminary to staining ; if not, the reducing fluid is poured off and the sections are rinsed twice in water and then placed in an ordinary saturated solution of chromogen (5% chromogen in distilled water, carefully filtered). The sections are left in this solution overnight, and the longer they remain in it, the more marked will be the contrast, as far as stain is concerned, between the con- nective and nervous tissues ; then water is again twice poured upon the sections and they are ready for staining. This process consists in a TECHNIC. 445 modified fibrin stain (vid. Technic). The iodo-iodid of potassium solu- tion is the same (saturated solution of iodin in a 5% iodid of potassium solution). Instead of the customary gentian -violet solution, a hot satu- rated alcoholic (70% to 80% alcohol) solution of methyl -violet is made, and, after cooling, the clear portion decanted off; to every 100 c.c. of this fluid 5 c.c. of a 5% aqueous solution of oxalic acid is added. The staining takes place in a very short time. The sections are then rinsed and normal salt solution and the iodo-iodid of potassium solution poured over them (5% iodid of potassium solution saturated with iodin), and washed off with water and dried with filter-paper and decolorized in the anilin oil-xylol solution in the proportion of 1:1. The reactions are rapid, and the thickness of the section should not exceed 20 p.. This method is best adapted to the central nervous system of the human adult ; it has as yet not been sufficiently tested for other vertebrates. Mallory' s Selective Neuroglia Fiber -Staining Methods. — Fix tissues in 10% formalin four days ; place in saturated aqueous solution of picric acid four days ; place in 5 % aqueous solution of ammonium bichromate four to six days in warm oven at 38° C.; dehydrate and imbed in cell- oidin ; sections may be stained in Weigert's fibrin stain and differenti- ated with equal parts of anilin oil and xylol, or they may be treated as follows: Place sections in 0.5% aqueous solution of permanganate of potassium twenty minutes ; wash in distilled water one to three minutes ; place in i % aqueous solution of oxalic acid thirty minutes ; wash in dis- tilled water ; stain in phosphotungstic-acid-hematoxylin solution (hemat- oxylin i g., distilled water 8oc.c.,io% aqueous solution of phosphotung- stic acid [Merk], 20 c.c., peroxid of hydrogen [U.S. P.], 2 c.c.) for twelve to twenty-four hours ; rinse in distilled water and place for five to twenty minutes in an alcoholic solution of ferric chlorid (ferric chlorid 30 g-> 3°% alcohol 100 c.c.) ; rinse in distilled water and dehydrate quickly, clear in oil of bergamot, and mount in xylol-balsam. Benda1 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, may be 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 44-6 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 % 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 zonula 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. Ciliary pro- cesses. Post, con- junctival __ vessels. ~~ Anterior JJ ciliary // vessels. Chorio- capillaris. Vena vor- ticosa. Hyaloid canal Post, cili- ary arte-. ries. A. cen- tral is ret- inae. — Pigment I 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 Descernet'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. 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, while the two or three most 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-canalicufi. 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 // 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 Fig. 353. — Section through the anterior portion of human cornea ; X 5°°- 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 64°- 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 sub epithelial 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 perichoroidal lymph-spaces. Sclera Lamina supra- ._ choroidea. Lamina vascu- losa Halleri. Lamina chorio- Fig. 355. — Section through the human choroid ; X I3(>- The lamina vasculosa of the choroid fs 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 vena vorticosce. 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 tapetum fibrosum. 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 cellulosum. 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 p) 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 orbicnlus 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 fj. 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 retinae). 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 evaluations 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 ^^^^ nT Corneal epithe- lium. I Substantia pro- pria. Loose connec- _ . live tissue of ^^~^r".: \ Iris. the conjunc- J^ ^~*^ m 't^ Pigment layer. 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 interrupted by circular bundles. The third or inner division {fibra circulares y 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 the 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 Fohtana. 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 t/te 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 circuits 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 Margin of pupil. Orbiculus ciliaris. Choroid. the cavernous plexuses, to ter- minate, it is thought, — in part, at least, — in the dilator of the since stimulation of these nerves causes a dilatation of the Fig. 357. — Injected blood-vessels of the human choroid and iris ; X 7- ins, THE INTERNAL OR NERVOUS TUNIC OF THE EYE. 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. K 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. J. 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 fi 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 Muller'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 [J. 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 Muller's fibers. In the lower classes of vertebrates a rod-ellipsoid Layer of nerve- fibers. Ganglion-cell layer. -- Inner molecular layer. Inner nuclear layer. -- 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. 459 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 p to 25 fj. 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 Muller'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. Blood-vessels. r* — "~" Layer of nerve-fibers. ... I Inner molecular layer.,_ V— _ Inner nuclear layer.. I-_5j* Outer molecular layer ____ - 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, \hzfimdus, 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 layer, 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 ~^MBHHHM^^ i nerve-fibers. ©'•©, Ganglion-cell .J 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. 481 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 saccus 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 macula, cristcz, and papilla spiralis. 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 crista ampullares sup., post., et lat. Besides these, we have the terminal arborization of the acoustic nerve in the membranous cochlea, the papilla spiralis cochlea, or the organ of Corti. 482 THE ORGAN OF HEARING. 1. 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. Blood-vessel. Fig. 370. — Transverse section through an osseous and membranous semicircular canal of an adult human being; X 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- liths, 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 neur-o-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- Pig ^71. Part of a verti- tioned form the so-called semilunar fold. cal section through the anterior Otoliths are also present upon the neu- \ — d ampulla, showing the membran ous wall, a portion of the "crista acustica," and the "planum semilunatum" (after Retzius) : a, Semilunar fold ; b, crista acus- tica ; c, nerve-fibers ; d, blood- vessels. 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. 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- branacea, 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 ccecum 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 veiy delicate membrane — the vestibular or Rcissner's membrane. Fr,om 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. •gsp Kn Fig. 372. — Longitudinal section of the cochlea of a cat ; X 25- Tnis figure gives a general view of the cochlea. The cochlear duct is met with six times in the section : dc, cochlear duct ; gsp, spiral ganglion ; Kn, osseous cochlear wall ; hp, 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 vestibulare, 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 inter mis. (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° : A Scala vestibuli ; m, labium vestibu- lare of the limbus ; n, sulcus spiralis internus ; 0, nerve-fibers lying in the lamina spi- ralis ; /, ganglion cells ; g, blood-vessels ; a, bone ; £, Reissner's membrane ; DC, ductus cochlearis ; d, Corti' s membrane ; f, prominentia spiralis ; gy organ of Corti ; h, liga- mentum spirale ; z, crista basilaris ; /£, 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 prominentia 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 very 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 reticidaris and the membrana tectoria, 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 49° 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 x c d f ^j-V\A ! \ ;" a b b K "V i i *r'G('-: ' ',.4 o . - ; t>,<-o,:r. q / i^ °^ im i J \ Fig. 374. — Organ of Corti : At x the tectorial membrane is raised ; — '^^-^^^f^BI^BHte^V " '^.»°.v^r** Fig. 377. — Portion of transverse section of the olfactory region of man ; X I5° • A zone of olfactory hairs ; , epithelium ; 2, zone of oval nuclei ; j, zone of round nuclei ; ,<,-•/, olfactory or Bowman's glands; «, branch of olfactory nerve; //, 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 y$ alcohol for one or two hours, they be treated with i % osmic acid for a quarter of an hour. If shreds be now placed in water and teased, the cells, together with their prolongations, may be isolated without the curling of the latter. The chrome-silver method applied to the nasal mucous membrane of young animals and fetuses has been the means of establishing the important fact that the olfactory cells of the olfactory region are in reality peripherally situated ganglion cells. NDEX. ABBE'S apparatus, 19 Absorption of fat by intestine, 288 method of studying, 306 Accessory disc of Engelman, 139 lacrimal glands, 470 thread of spermatosome, 361 Acervulus, 423 Acetic acid, effect on connective tissue, 128 on red blood-corpuscles, 188 sublimate solution as fixing fluid, 25 Achromatic portion of nucleus, 63 spindle, 68 Acidophile granules, technic for, 227 Adenoid connective tissue, 196 Adipose tissue, 107 stain for, 130 Agminated lymph-nodules, 197 Air-cells, 314 Air-spaces, ultimate, 313 Akrosome, 377 Alcohol as fixing solution, 23 as macerating solution, 22 Alcoholic borax-carmin solution as stain, 4i in bulk, 46 Alkalies, effect on red blood-corpuscles, 189 Altmann's method of demonstrating granules in cells, 77 of mounting, 78 process, 55 Alum-carmin as stain, 42 in bulk, 46 Alveolar ducts, 314, 315 glands, 91 compound branched, 91 simple, 91 branched, 91 periosteum, 242 Alveoli, 88 lung, 314 of mammary gland, epithelium of, 401 Amacrine cells, 464 Amitosis, 64, 70 Amitotic cell-division, 70 Amphiaster, 68 Amphipyrenin, 63 Amphophile granules, technic for, 228 Ampullae of Thoma, 204 Anaphases, 65, 69 Anastomoses, 222 Anilin stains, 44 Animals, injection of, 54 Anisotropic transverse disc, 138 Annulospiral nerve-ending, 178 Annulus fibrosus, 477 atrioventricularis, 214 Anterior epithelium of crystalline lens 468 ground bundle, 411 hyaloid artery, 468 lymph-channels of eye, 469 superior vertical canal, 480 Anterolateral columns, ascending, 411 descending, 411 Antrum of ovary, 347 Anus, 281 Apathy's method for demonstration of fibrillar elements of nervous system, 442 Apochromatic lens, 19 Aponeuroses, 105 Aqueous borax carmin solutions as stain, 4i humor, 446 Arachnoid, 437 Arches of Corti, 490 Arcuate fibers of cornea, 450 Area cribrosa, 332 vasculosa, 186 Areas of Langerhans, 301 Arrectores pilorum, 393 Arteriae arciformes, 332 capsulares glomeruliferae, 334 Arterial circle of Zinn, 465 retia mirabilia, 333 Arteries, 216 coronary, 214 hyaloid, anterior, 468 posterior, 468 interlobular, of kidney, 332 medium-sized, 218 of choroid, 455 of retina, 466 precapillary, 218 Arteriolae rectae spuriae, 333 verae, 334 Artery, auditory, internal, 494 central, of retina, 465 hepatic, 293 nasal, inferior, of retina, 466 superior, of retina, 466 papillary, inferior, of retina, 466 superior, of retina, 466 renal, 332 501 502 INDEX. Artery, temporal, inferior, of retina, 466 superior, of retina, 466 Ascending anterolateral columns, 411 Association fibers of cerebral cortex, 420 Astrocytes, 435 Atresia of ovarian follicles, 353 Atria, 314 Attraction-sphere, 62 Auditory artery, internal, 494 cells, outer, 491 hairs, 490 nerve, 494 ossicles, 478 teeth, 488 Auerbach's plexus, 286 Auriculoventricular valves of heart, 213 Axial canals of small intestine, 285 cords, 157, 1 60 fibrils of, demonstration of, 181 sheath, 176 thread of spermatosome, 361 sheath of, 361 Axillary glands, 398 Axis-cylinder, 159 naked, 160 Axis-fibrils, 157 Axolemma, 157 BAILLARGER'S striation, 421 Balsam, Canada, as mounting medium, 52 Bardeen's table for drawing of portions of sections to be reconstructed, 57 Bars of intercellular cement, 86 Bartholin's ducts, 253 glands, 360 - Basement membrane, 81, 88 of small intestine, 278 Basic stains, 41 Basichromatin granules, 62 Basilar membrane, 488 Basket cells, 254 Baskets, fiber, of retina, 462 Basophile granules, 193 cells with, 209 technic for, 228 Bechtereff and Kaes' striation, 421 Benda's chromatoid accessory nucleus, 377 method for demonstration of medul- lary sheath, 442 selective neuroglia staining method, 445 Berkley's method of demonstrating nerves of liver, 308 Berlin blue as injection fluid, 55 Bertini, columns of, 324, 330 Bethe's method of fixing methylene-blue for nerve-fibers, 184 of staining neuro fibrils and Golgi- nets, 443 Bile capillaries, 290, 291, 292 demonstration of, 306 impregnation of, 307 effect on red blood-corpuscles, 188 Bile-ducts, 296 Bioblasts, 60 Biondi-Heidenhain triple stain, 46 Bipolar cells of cone-visual cells, 463 of rod-visual cells, 463 Bismarck brown as stain, 44 Bladder, 336 nerves of, 339 technic of, 343 Blastema, 64 Blastodermic layers, primary, 79 Blastomeres, 70, 79 Blood, 1 86 coagulation of, 195 Blood, cover-glass preparations, 227 current, behavior of blood-cells in, 196 demonstration of, through vessels, 231 elements of, method of examining, 227 films, Wright's method of staining, 229 formation of, 186 islands, 186 plasma, 187 platelets, 194 fixation of, 227 shadows, 188 sinus, 222 supply of bronchi, 316 of Fallopian tubes, 355 of heart, 214 of intestine, 283 of lymph -glands, 200 of salivary glands, 259 of spleen, 203 of thymus gland, 212 of thyroid gland, 320 of uterus, 357 technic of, 226 Blood-cells, behavior of, in blood current, 196 counting, 232 red, nucleated, containing hemo- globin, 208 staining of, 227 Blood-corpuscles, cover-glass prepara- tions, 226 red, 187. See also Erythrocytes. technic of, 226 white, 191. See also Leucocytes. Blood-counting apparatus, Thoma-Zeiss, 232 Blood-forming organs, 186 technic of, 226 Blood-placques, 194 Blood-vessels, 186, 216 fetal, of eye, 468 in striated muscular tissue, 143 nerve supply of, 223 of bone-marrow, 210 of central nervous system, 439 of eyelid, 473 of kidney, 332 of liver, distribution of, demonstration, 306 examination of, 343 INDEX. 503 Blood-vessels of lung, 316 of membranous labyrinth, 494 of mucosa of large intestine, 284 of pelvis of kidney, 338 of small intestine, 284 of nasal cavity, 499 of optic nerve, 465 of ovary, 354 of pancreas, 302 of prostate, 370 of retina, 465 of sclera, 449 of stomach, 284 of suprarenal glands, 341 of teeth, 242 of testis, 367 Blutlymphdriisen, 200 Body-cell, 71 Bohmer's hematoxylin as stain for bulk, 46 hematoxylon, 42 Bone, 112 breakers, 120 calcium carbonate in, alkaline purpurin as stain for, 132 canaliculi, 113 compact, of shaft, development of, 124 corpuscles, 113 Schmorl's method of staining, 133 Virchow's method of isolating, 134 decalcification of, 132 fluids used for, 132 v. Ebner's method, 133 development of, 116 endochondral, 116 intramembranous, 122 endochondral , 1 1 6 intracartilaginous, 116 intramembranous, 116, 122 lacunae of, 112, 113 lamellae, 113 composition, 114 method of examining, 131 lime-salts in, hematoxylin as stain for, 132 isolation of, 132 soft and hard parts, relation of, method of studying, 132 spaces in, Ranvier's method for dem- onstrating, 132 structure of, 112 undecalcified, microscopic preparation of, 131 Bone-cells, 112, 115 Bone-marrow, 207 blood-vessels of, 210 gelatinous, 210 red, 207 technic of, 234 yellow, 207, 210 Bony cochlea, 484 labyrinth, 480 Borax-carmin, alcoholic, 41 in bulk, 46 aqueous, 41 Bern's method of construction by plates, 56 Bottcher's cells, 491 Boundary zone, choroid, 453 Bowman's capsule, 323 glands, 499 membrane, 449 Box for imbedding tissues, 28 Bronchi, 311 blood supply of, 316 branches of, 311 nerves of, 317 terminal branches of, 313 Bronchioles, 311 respiratory, 313 terminal, 314, 315 Brownian movement of cells, 61 Briicker's lines, 137 Brunner's glands, 265, 277 Budding, 64 Bulb hairs, 393 Bulbus oculi, 446 Burdach's column, 411 Butschli's foam-structure, staining for, 79 C^CUM CUPOLARE, 485 vestibulare, 485 Calcification of cartilages, in Camera lucida, 20 Canada balsam as mounting medium, 52 Canalicular system in cartilage, method of demonstrating, 131 lymph, 102 Canaliculi of bone, 113 Canalis communis, 480 Capillaries, 220 bile, 290, 291, 292 demonstration of, 306 impregnation of, 307 demonstrating distribution of, 235 lymph, 224 of cerebellar cortex, 440 of cerebral cortex, 440 of sweat-glands, 397 Capsule, Bowman's, 323 lens, 468 of cartilage, gold chlorid as stain for, J31 of glands, 92 of Glisson, 289 of lymph-glands, 198 of Tenon, 448 suprarenal, demonstration of, 343 Carmin as stain, 41 mass, cold, as injection fluid, =5/4 Carmin-bleu de Lyon, 45 Carnoy's acetic acid -alcohol-chloroform mixture, 23 acetic-alcohol mixture, 23 Carotid gland, 225 Cartilage, 108 calcification of, in canalicular system in, method of demonstrating, 131 504 INDEX. Cartilage, capsules of, gold chlorid as stain for, 131 connective tissue in, picrocarmin as stain for, 131 corrosive sublimate as fixative for, 130 cuneiform, 310 elastic fibers in, picrocarmin as stain for, 131 fibre-elastic, no glycogen in, iodo-iodid of potassium stain to demonstrate, 131 ground-substance of, change in, in hyaline, 108 of larynx, 310 of Wrisberg, 310 osmic acid a fixative for, 130 ossification of, in Caustic potash as macerating solution, 22 Cell, 58 absence of membrane, 62 air-, 314 amacrine, 464 auditory, outer, 491 basket, 254 blood, behavior of, in blood current, 196 counting of, 232 red, nucleated, containing hemoglo- bin, 208 staining of, 227 body-, 71 bone-, 112, 115 t Brownian movement of, 61 centro-acinal, 300 chief, of acini of thyroid gland, 320 of hypophysis, 423 chromophilic, of hypophysis, 423 ciliated, 60 colloid, of acini of thyroid gland, 320 commissural, 408 cone-visual, 459 bipolar cell of, 463 connective-tissue, fat producing, 97 fixed, 103 cortical, small, of cerebellar cortex, 415 crystals of, 61 cuneate, 301 definition of, 58 Deiter's, 491 demilunar, 257 diagram of, 59 diffuse, of retina, 464 double staining of, 76 enamel, 243 endothelial, 80, 94 and mesothelial, method of studying relations, 95 demonstration of, 95 technic for, 233 epithelial, in small intestine, 275 isolated, examination of, 95 fat of, 6 1 fat-, scheme of, 107 fixing of chromic acid for, 75 corrosive sublimate for, 75 Cell, Flemming's solution for, 75 picric acid for, 75 flagellated, 60 follicular, 372 ganglion, 149 demonstration of, 182 of Dogiel, in spinal ganglia, 426 giant, 209 glandular, 61 glycogen of, 61 goblet, 87, 265 granular, of cerebellar cortex, 416 granules in, Altmann's method of demonstrating, 77, 78 hair-, of utriculus, 483 hepatic, cords of, 290 horizontal, of retina, 464 liver-, examination of, 306 glycogen in, demonstration of, 306 lutein, 353 marrow-, 208 mast-, 104 granules of, technic for, 228 mesameboid, 80 mesothelial, 80 and endothelial, method of studying relations, 95 migratory, 103, 104, 193 mitosis of, demonstration of, 75 mitral, 421 of olfactory bulb, 421 molecular movement of, 61 monostratified, of retina, 464 mother, 374 mucus-secreting, 87 muscle-, cardiac, demonstration of, 148 nonstriated, 134 of fibers of Purkinje, 147 nerve-, 149. See also Ganglion cell. neuro-epithelial, 92 neurogliar, 434 of Bottcher, 492 of Cladius, 492 of column of Clark, 408 of Golgi, 408, 418, 419 of Hensen, 491, 492 of Langerhans, 300 of Leydig, 470 of Martinotti, 418, 419 of pancreas, inner and outer zones, methods of differentiating, 308 of Purkinje, 153 of cerebellar cortex, 415 of reticular connective tissue, 100 of Sertoli, 364 olfactory, 498 parareticular, 464 pigment, 61, 77, 104 pillar, 490 heads of, 490 inner, 490 outer, 490 plasma, 104 plurifunicular, 408 polarity of, 81 INDEX. 505 Cell, polygonal, of cerebral cortex, 417 polymorphous, of cerebral cortex, 418 polynuclear, 70 polystratified, of retina, 464 pyramidal, large, of cerebral cortex, 4i7 of cerebral cortex, 153 small, of cerebral cortex, 417 rod-visual, 458 bipolar cell of, 463 seminal, primitive, 372 sense, 81 sexual, fertilization of, 71 male, development of, 72 matured, 71 somatic, 71 spider, 435 spindle-shaped, of cerebral cortex, 417 staining of, 76 stellate, large, of cerebellar cortex, 416 of cerebellar cortex, 415 of cerebral cortex, 417 of liver, 295 sustentacular, 92, 250, 372, 483 tendon, from tail of rat, 107 visual, 458 wandering, 60, 103, 104 with basophilic granules, 209 with eosinophile granules, 209 Cell-bodies of neurones, 149 Cell-body, 59 Cell-division, 64 amitotic, 70 direct, 64, 70 indirect, 64 karyokinetic, heterotypic, 374 homeotypic, 374 mitotic, of fertilized whitefish eggs, 66, 67 ten stages of, 65 Cell-masses, intertubular, of pancreas, 301 Cell-microsomes, 59 Celloidin imbedding, 30 diagram for, 32 infiltration, 30 diagram for, 32 sections, cutting of, with sliding micro- tome, 36 dextrin method of fixing, 40 Celloidin-paraffin imbedding, 32 infiltration, 32 Cell-plate, 70 Cell-spaces of areolar connective tissue, 102 Cellular elements of areolar connective tissue, 103 Cement lines, 146 Cementum, 241, 246 Centers of ossification, 116 Central artery of retina, 465 gray nuclei of cerebellar cortex, 416 nervous system, 406 blood-vessels of, 439 fibrillar elements of, Apathy's method of demonstrating, 442 Central nervous system, lymph-vessels of, 440 membranes of, 436 technic of, 440 spindle, 68 vein of retina, 465 Centripetal fibers of cerebral cortex, 420 Centro-acinal cells, 300 Centrosomes, 62, 427 Centrospheres, 62, 427 Cerebellar columns, direct, 411 . cortex, 413 capillaries of, 440 central gray nuclei of, 416 granular layer of, 416 granular cells of, 416 large stellate cells of, 416 medullary substance of, 416 climbing fibers of, 416 mossy fibers of, 416 molecular layer of, 413 cells of Purkinje of, 415 small cortical cells of, 415 stellate cells of, 415 Cerebral cortex, 416 capillaries of, 440 medullary substance of, 419 association fibers of, 420 centripetal fibers of, 420 commissural fibers of, 420 projection fibers of, 419 stellate cells of, 417 molecular layer of, 417 polygonal cells of, 417 spindle-shaped cells of, 417 polymorphous cells of, 418 pyramidal cell of, 153 large, 417 small, 417 Ceruminous glands, 398, 476 Cervical canal, islands of ciliated epithe- lium in, 356 Chemotaxis, 61, 276 Chemotropism, 61 Chief cells of acini of thyroid gland, 320 of hypophysis, 423 Chlorate of potassium and nitric acid as macerating solution, 23 Chondrin, method of obtaining, 112 Choroid, 446, 452 arteries of, 455 boundary zone of, 453 glassy layer of, 452, 453 lamina vasculosa Halleri of, 452 plexus, 439 Choroidal fissure, 447 Chromatin, 63 Chromatoid accessory nucleus of proto- plasm of spermatid, 377 Chromatolysis, 74 technic, 74 ' Chromatophile granules, 149 i Chromic acid as fixing solution, 26 as macerating solution, 22 for fixing cells, 75 Chromophilic cells of hypophysis, 423 506 INDEX. Chromosomes, 67 daughter, 68 Chrzonszczewsky's physiologic auto-in- jection, 306 Chyle-vessels, 285 Cilia, 81, 470 movement of, method of observing, 95 Ciliary body, 446, 452, 453 nerve supply of, 456 glands, 398, 454 of Moll, 470 muscle, 454 meridional division, 454 middle division, 454 third or inner division, 454 processes, 453 Ciliated cells, 60 epithelium, islands of, in cervical canal, 356 Circulation of hypophysis, 424 Circulatory system, 212 technic of, 235 Circulus arteriosus iridis major, 455 minor, 456 Circumanal glands, 398 of Gay, 282 Circumferential lamellae, inner, 113 outer, 113 Circumvallate papillae, 249 Clark's column, 408 cells of, 408 Claudius, cells of, 492 Clearing fluids, 52 Climbing fibers of cerebellar cortex, 416 Clitoris, 360 Cloquet's canal, 468 Club hairs, 393 Coagulation of blood, 195 Coal-tar stains, 44 Cochlea, 484 bony, 484 perilymph of, 496 spiral ganglion of, 494 technic for, 297 Cochlear duct, 484, 485 Cohnheim's fields, 140 method of impregnation, 48 Coil-glands, 396 Collective lens, 19 Colloid cells of acini of thyroid gland, 320 Colostrum, 402 corpuscles, 402 Columns, anterolateral, ascending, 411 descending, 411 cerebellar, direct, 411 lateral, 408 mixed, 411 of Bertini, 324, 330 of Burdach, 411 of Clark, 408 cells of, 408 of Goll, 411 of Gower, 411 of Sertoli, 364 pyramidal, crossed, 411 ventrolateral, 408 Columns, ventromesial, 408 Columns rectales Morgagni, 282 Commissural cells, 408 fibers of cerebral cortex, 420 Commissures of spinal cord, 412 Compound microscope, 17 Concentric lamellae, 113 Concretions of prostate, 370 Condensers, 19 Cone-fibers of retina, 459 Cone-visual cells, 459 bipolar cells of, 463 Coni vasculosi Halleri, 364 Conjunctiva, 469 scleral, 448 Conjunctival portion of eyelids, 470 Connective tissue, 96 . action of acetic acid on, 128 of hydrochloric acid on, 128 of potassium hydrate on, 128 adenoid, 196 areolar, 101 cell-spaces of, 102 cellular elements of, 103 ground-substance of, 102 matrix of, 102 development of, schematic diagram of, 98 effect of pepsin on, 128 of trypsin digestion on, 127 fibrous, 101 white, 99 in cartilage, picrocarmin as stain for, J31 magenta red as stain for, 128 mucous, 100 of liver, 294 orcein as stain for, 128 Ranvier's method for examination of, 126 reticular, 100 cells of, TOO slide digestion of, 129 technic of, 126 Connective-tissue cells, fat producing, 97 fixed, 103 corpuscles, 103 fibrillae and reticulum, differential stain for, 128 framework of organs and tissues, digestion method for demonstrating, 129 Contraction-ring, 158 Conus medullaris, 406 Convoluted tubules of testes, 363 Cord, spinal, 406 See also Spinal cord. Cords, axial, 157, 160 fibrils of, demonstration of, 181 hepatic, 290 medullary, 199 of hepatic cells, 290 pulp, 204 Corium, 379, 382 Cornea, 446, 449 anterior elastic membrane of, 449 arcuate fibers of, 450 INDEX. 507 Cornea, epithelium of, 449 ground plexus of, 451 nerves of, 451 technic, 474 perforating fibers of, 450 posterior elastic membrane of, 450 subepithelial plexus of, 45 1 substantia propria of, 449 technic, 474 superficial plexus of, 451 Corneal corpuscles, 450 epithelium, technic of, 474 spaces, 450 technic of, 474 Corona radiata, 347 Coronary arteries, 214 Corpora amylacea of prostate, 370 lutea spuria, 353 Corpus albicans, 353 Highmori, 363 luteum, 353 verum, 353 Corpuscles, blood-, red, 187. See also Erythrocytes. blood-, white, 191. See also Leu- cocytes. bone, 113 SchmorPs method of staining, 133 Virchow's method of isolating, 134 colostrum, 402 connective-tissue, 103 corneal, 450 genital, 171 Golgi-Mazzoni, 388 Grandry's technic of, 405 Hassal's, 212 Herbst's, 174, 389 technic of, 405 Malpighian, 202, 203, 323, 324 Meissner's, 170 technic of, 405 Pacinian, 388 technic of, 405 tactile, 387 Vater-Pacinian, 173 distribution of, 174 Corrosive sublimate as fixative for car- tilage, 130 as fixing solution, 24 for fixing cells, 75 Cortex, cerebellar, 413. See also Cere- Cellar cortex. cerebral, 416. See also Cerebral cortex. of ovary, 344 Cortical cells, small, of cerebellar cortex, 4i5 layer of hair, 389 nodules, 198 substance of kidney, 323, 324 Corti's arches, 490 membrane, 489, 493 organ, 481, 489 spiral organ, 489 Cover-slips, 20 fixing of large number of paraffin sections to, 39 Cowper's glands, 370 Cox's method of impregnation, 51 Crescents of Gianuzzi, 256 Crista basilaris, 488 Cristae, 481 Crossed pyramidal columns, 411 Crosses, Ranvier's, demonstration of, 180 Crypts, Lieberkiihn's, 276 of stomach, 266 Crystalline lens, 467 anterior epithelium of, 468 Crystals, hematoidin, 231 hemin, method of obtaining, 230 hemoglobin, method of obtaining, 230 of cell, 61 Teichmann's, 188 method of obtaining, 230 Cuneate cells, 301 Cuneiform cartilages, 310 Cup, optic, 447 Cupola, 484 Cupula, 484 Currents of diffusion, 29 Cutaneous layer of tympanic membrane, 476 epidermis of, 476 Cuticle, 379 of hair, 389 inner, 389 Cuticula, 62, 81, 274 dentis, 238 Cuticular portion of eyelids, 470 ridge, 477 structures, 81 Cutis, 379. See also Skin. Cylindric end-bulb of Krause, 172 Czermak's interglobular spaces, 241 Czocor's cochineal solution, 42 DAMAR as mounting medium, 52 Daughter chromosomes, 68 nuclei, 64 stars, 374 Decalcification, 132 v. Ebner's method, 133 Decalcifying fluids, 132 aqueous solution of nitric acid, 133 hydrochloric acid, 132 Deiter's cells, 491 Delafield's hematoxylin, 43 Demilunar cells, 257 Demilunes of Heidenhain, 256 Dendrites, 149, 150 function of, 154 Dendritic fibrous structures of Gruber, 478 Dental sac, 244 Dentin, 239 development of, 244 fibrils of, demonstration of, 303 Dentinal fibers, 240 papillae, 243 tubules, 240 Dermis, 379, 382 508 INDEX. Descemet's membrane, 450 endothelium of, 451 technic of, 474 Descending anterolateral columns, 411 limb of Henle's loop, 327 Deutoplastic granules, 349 Dextrin method of fixing celloidin sec- tions, 40 paraffin sections, 40 Diapedesis, 193 Diaphragm, 17 iris, 1 8 Diaster, 69, 374 Diffuse cells of retina, 464 spongiblasts, 464 Diffusion, currents of, 29 Digestion method for demonstrating connective-tissue framework of organs and tissues, 129 slide, for connective tissue, 129 Digestive organs, 235 technic of, 303 tract, glands of, technic for, 304 Dilator muscle of pupil, 455 Direct cerebellar columns, 411 pyramidal tract, 411 Discus proligerus, 347 Dispirem, 69 Distilled water for fixing paraffin sections to slide, 39 Dorsal utriculus, 496 Double knife, 21 staining, 44 of cells, 76 Doyere's elevation, 162 Duct, alveolar, 314, 315 Bartholin's, 253 bile-, 296 cochlear, 484, 485 ejaculatory, 368 excretory, 367 Gartner's, 360 intralobular, of pancreas, 300 nasal, 474 pancreatic, 298 Steno's, 253 utriculosaccular, 481 Wharton's, 253 Wirsungian, 298 Wolffian, 360 Ductus endolymphaticus, 476 Dura mater, nerves of, 437 spinal, 436 EAR, 476 external, 476 technic for, 497 internal, 480 middle, 478 technic for, 497 technic for, 497 vestibule of, 480 Ectoderm, 79 tissues derived from, 79 Egg tubes, primary, of Pfluger, 345 Ehrlich-Biondi-Heidenhain three-color mixture, 229 Ehrlich's granulations, 227 hematoxylin, 43 for nuclei and granules, 228 leucocytic granules, 192 methylene-blue stain for nervous tissues, 182 neutrophile mixture, 229 Ejaculatory ducts, 368 Elastic elements, technic for, 235 fibers, 100 in cartilage, picrocarmin as stain for, 131 respiratory, demonstration of, 322 membrane, anterior, of cornea, 449 posterior, of cornea, 450 tissue, effect of trypsin digestion on, 127 method of obtaining, 127 Enamel, 238 cells, 243 germs, 243 prisms, 238 technic of, 303 Encoche d'ossification, 121 End-brush, 162, 167, 237 End-bulbs of Krause, 170, 388 . cylindric, 172 Endocardium, 213 lymphatic networks in, 215 Endochondral bone, 116 bone-development, 116 Endolymph, 495 Endomysium, 143 Endoneurium, 160 Endoplasm, 62, 98, 210 End-organ of Ruffini, 388 Endosteum, 207 Endothelial cells, 80, 94 and mesothelial cells, method of studying relations, 95 demonstration of, 95 technic for, 233 Endothelium, 92 anterior, of iris, 455 of Descemet's membrane, 451 of intima, technic for, 235 End-piece of Retzius, 361 End-plate, motor, 163 Engelman, accessory disc of, 139 Entoderm, 58, 79 tissues derived from, 80 Eosin as stain for blood-cells, 227 Eosinophile granules, 193 cells with, 209 technic for, 227 Epicardium, 214 Epidermis, 379 nerves of, technic of, 405 of cutaneous layer of tympanic mem- brane, 476 technic of, 403 Epidural space, 437 Epilamellar plexus, 261, 397 E pi my sium, 143 Epiphyses, development of, 121 INDEX. 509 Epiphysis, 422 Epithelial cells in small intestine, 275 isolated, examination of, 95 processes, interpapillary, 85 Epithelium, anterior, of crystalline lens, 468 ciliated, islands of, in cervical canal, 356 classification, 81, 82 columnar, pseudostratified, 83 simple, 83 stratified, 85 conical technic of, 474 germinal, examination of, 378 of ovary, 345 glandular, 87 neuro-, 92 of alveoli of mammary gland, 401 of cornea, 449 of kidney, demonstration of, 343 of mucous membrane of intestine, 274 leucocytes in, 275 of vagina, 358 of olfactory region, 498 of urethra, 371 of vestibule of vagina, 360 posterior, of iris, 455 respiratory, 315 examination, of, 322 simple, 82 columnar, 83 cubic, 82 squamous, 82 stratified, 83 columnar, 85 squamous, 84 technic of, 94 transitional, 85 Eponychium, 395 Epoophoron, 344, 360 Erlicki's fluid, 26 F.rythroblasts, 208 Erythrocytes, 187 diameter of, 190 effect of acetic acid on, 188 of alkalies on, 189 of bile on, 188 of fluids on, 188 of tannic acid on, 189 of water on, 188 examination of, 226 fresh, fixation of, 226 method of counting, 232, 233 size of, 190 stroma of, 187 technic for, 226 Esophagus, 262 method of examining, 305 Eustachian tube, 470 mucous membrane of, 479 Excavation, physiologic, of retina, 460 Excretory ducts, 367 Exoplasm, 62, 98, 210 External ear, 476 technic for, 497 External limiting membrane of retina, 459, 462 External semicircular canal, 480 Extra-epithelial glands, 88 Eye, 446 anterior lymph-channels of, 469 development of, 446 fetal blood-vessels of, 468 general structure of, 446 pigment membrane of, 446, 447, 457 protective organs of, 469 technic for, 474 tunica externa of, 446 fibrosa of, 446, 448 interna of, 446, 457 vasculosa of, 446, 452 tunics of, 446 Eyeball, 446 interchange of fluids in, 469 Eyelids, 469 blood-supply of, 473 conjunctiva! portion of, 470 cuticular portion of, 470 middle layer of, 471 third, 473 FALLOPIAN tubes, 354 blood-supply of, 355 mucous membrane of, 354 muscular coat of, 355 treatment of, 378 Farrant's gum glycerin, 53 Fasciculus gracilis, 411 Fat, absorption of, by intestine, 288 method of studying, 306 lobules, 107 of cells, 6 1 Sudan III as stain for, 130 Fat-cell, scheme of, 107 Fat-marrow, 207 Female genital organs, 344 pronucleus, 74 Fenestra cochleae, 479 rotunda, 479 vestibuli, 478 Fenestrated membranes, 107 Ferrein, pyramids of, 324 Fertilization, process of, 71 diagrams of, 72, 73 Fetal blood-vessels of eye, 468 Fiber-baskets of retina, 462 Fiber-layer, Henle's, 461 outer of, retina, 461 Fibers, arcuate, of cornea, 450 association, of cerebral cortex, 420 centripetal, of cerebral cortex, 420 climbing, of cercbellar cortex, 416 commissural, of cerebral cortex, 420 cone-, of retina, 459 dentinal, 240 elastic, 100 in cartilage, picrocarmin as stain for, 131 respiratory, demonstration of, 322 heart-muscle, MacCallum's nitric acid mixture for isolating, 23 5io INDEX. Fibers, lens, 468 mantle, 69 mossy, of cerebellar cortex, 416 motor, 162 Miiller's, 454, 462 muscle-, intrafusal, 17^ nonstriated, demonstration of, 148 striated, technic of, 147 striped, 136 voluntary, development of, 144 nerve-, 157 ending in muscular tissue, 162 medullated, demonstration of. 180 of teeth, 242 methylene-blue stain for, 184 nonmedullated, 160 demonstration of, 182 of hair follicles, 393 of utriculus, 483 neuroglia, Benda's method of staining, | 445 Mallory's methods of staining, 445 of olfactory nerve, staining of, 182 perforating, of cornea, 450 peripheral, of olfactory bulb, 421 projection, of cerebral cortex, 419 Purkinje's 213 isolated demonstration of, 148 muscle-cells of, 147 Remak's, 160 demonstration of, 182 reticular, of liver, demonstration of, 308 rod-, of retina, 458 Sharpey's, 115 method of isolating, 134 sustentacular, 492 terminal, of cerebral cortex, 420 tunnel-, 494 white, 99 rami, 429, 456 Fibrae circulares, 454 Fibril bundles, 140 Fibrillar elements of nervous system, | Apathy's method for demonstration of, 442 Fibrils, axis-, 1^7 of axial cord, demonstration of, 181 of dentin, demonstration of, 303 Fibrin, demonstration of, 231 Fibrocartilage, white, no Fibro-elastic cartilage, no Fibrous connective tissue, 101 tissue elastic, 106 Filiform papillae, 248 Films, blood, Wright's method of stain- ing, 229 Filum terminale, 406 Fimbrise linguae, 249 Fissure, choroidal, 447 Fixing methods, 23 solutions, 23 acetic sublimate, 25 alcohol, 23 Biitschli's foam-structure, for cells, 79 Fixing solutions, Carnoy's acetic-alcohol, 23 Carnoy's acetic acid-alcohol-chlo- roform, 23 chromic acid, 26 for cells, 75 corrosive sublimate, 24 for cartilage, 130 for cells, 75 Erlicki's, 26 Flemming's 24 for cells, 75 Fol's, 24 formalin, 27 formol, 2 7 Hayem's, 226 Hermann's, 24 Miiller's, 26 nitric acid, 26 osmic acid, 24 for cartilage, 130 picric acid, 25 for cells, 75 picric-nitric acid, 25 picric-osmic-acetic acid, 25 picric-sublimatc-osmic acid, 25 picrosulphuric acid, 25 potassium bichromate and formalin Raid's, 25 Tellyesnicky's, 26 vom Rath's, 25 Zenker's, 26 Flagellate cells, 60 Flagellum of spermatosome, 361 Flemming's germ centers, 194 solution, 24 for fixing cells, 75 Flower-like nerve-ending, 178 Fold, semilunar, 484 Foliate papillae, 249 Follicles, Graafian, 347 bursting of, 352 hair, 389 nerve-fibers of, 393 lymph-, germ centers of, technic for, 234 ' of mucosa of vermiform appendix, 281 of tongue, 251 of tonsils, 251 solitary, 197 technic for, 306 ovarian, atresia of, 3^3 simple, 197 Follicular cells, 372 glands, 91 Folliculi linguales, 251 Fol's solution, 24 Fontana's spaces, 455 Foramen apicis dentis, 238 'mina nervosa, 488 papillaria, 330 Formalin as fixing solution, 27 Formol as fixing solution, 27 'tralis, 460 INDEX. Foveolae of stomach, 266 Fragmentation, direct, of nucleus, 71 Freezing apparatus for sliding microtome, 36 Friedlander's glycerin-hematoxylin, 43 Front lens, 19 Fuchsin-resorcin elastic fibers stain, 128 Fundamental lamellae, 113 Fundus glands of stomach, 268 of fovea centralis, 460 Fungiform papillae, 248 Funiculi, of nerve-trunk, 160 compound, 162 Funnels, pial, 439 Future periosteum, 116 GANGLIA, 424 spinal, 424 sympathetic, 427 Ganglion cell, 149 demonstration of, 182 layer of retina, 459, 464 of Dogiel, in spinal ganglia, 426 spiral, of cochlea, 494 Gartner's duct, 360 Gastric mucous membrane, 266 Gastrulation, 79 Gay's circumanal glands, 282, 398 Gelatin-Berlin blue as injection fluid, 54 Gelatin-carmin as injection fluid, 54 Gelatinous bone-marrow, 210 substance of Rolando, 408 Genital corpuscles, 171 organs, female, 344 male, 361 Genito-urinary organs, 323 technic of, 342 Gerlach's method of impregnation, 48 Germ center, 197 of Flemming, 194 of lymph-follicles, technic for, 234 enamel, 243 hair, 389 layers, 58 primary, 79 Germinal epithelium, examination of, 378 of ovary, 345 vesicle, 71 Giant cells, 209 Gianuzzi, crescents of, 256 Giraldbs, organ of, 367 Gittcrfasern, 301 Glands, alveolar, 91. See also Alveolar glands. axillary, 398 capsule of, 92 carotid, 225 ceruminous, 389, 476 ciliary, 398, 454 Moll's, 470 circumanal, 282, 398 coil-, 396 extra-epithelial, 88 follicular, 91 hemal, 200 Glands, hemolymph, 200 structure of, 201 injection of, 55 intra-epithelial, 88 lacrimal, 473 accessory, 470 nerve supply of, 473 lenticular, 271 lymph-, 196, 197 blood supply of, 200 capsule of, 198 hilum of, 197 lymph-sinuses of, 199 marrow, 201, 202 technic for, 233 trabeculae of, 198 with blood-sinuses, 200 mammary, 400. See also Mammary gland. Meibomian, 472 mixed, 258 mucous, 255 multicellular, 88 classification, 91 of Bartholin, 360 of Bowman, 499 of Brunner, 265, 277 of Cowper, 370 of digestive tract, technic for, 304 of Gay, 282, 398 of Lieberkuhn's, 88, 276 of Moll, 398, 470 of Montgomery, 402 of mouth, small, 259 of oral cavity, 253 of skin, 396 of stomach, 267 cardiac, 267 fundus, 268 of Tyson, 372 parathyroid, 321 parotid, 255 peptic, 268 pineal, 422 prostate, 368. See also Prostate. pyloric, 269 salivary, 253, 255 blood supply of, 259 nerve supply of, 260 sebaceous, 398 serous, 255 splenolymph, 201 structure and classification, 88 sublingual, 255 submaxillary, 258 sudoriparous, 396. See also Sweat- glands. suprarenal, 339. See also Suprarenal glands. sweat-, 396. See also Sweat-glands. tarsal, 472 thymus, 210 blood supply of, 212 thyroid, 319. See also Thyroid gland. tubular, 89. See also Tubular glands. tubulo-alveolar, 90 512 INDEX. Glands, unicellular, 87 Glandula carotica, 225 Glandulas buccales, 236 duodenales, 265 labiales, 236 Glandular cells, 61 epithelium, 87 Glassy layer of choroid, 452, 453 membrane of hair, 391 Glisson's capsule, 289 Glomerular layer of olfactory bulb, 421 Glomeruli arteriosi cochleae, 495 Glomerulus, 323 Glomus caroticum, 225 Glycerin, Farrant's gum, mounting in, 53 mounting in, 53 Glycerin-albumen for fixing paraffin sec- tions to slide, 38 Glycogen in cartilage, iodo-iodid of potassium stain to demonstrate, 131 in liver-cells, demonstration of, 306 of cells, 6 1 Goblet cells, 87, 265 Gold chlorid as stain for capsules of car- tilage, 131 method of impregnation, 48 Golgi-Mazzoni corpuscle, 388 Golgi-nets, Bethe's method of staining, 443 Golgi's cells,- 408, 418, 419 chromsilver or chromsublimate method of impregnation, 49 gold chlorid method of impregnation, 48 methods of impregnation, 49, 50 mixed method of impregnation, 50 potassium bichromate and bichlorid of mercury method of impregna- tion, 50 method of impregnation, 49 preparations, Huber's method of per- manently mounting under cover- glass, 51 rapid method of impregnation, 50 slow method of impregnation, 50 Coil's column, 411 Gower's column, 411 Graafian follicle, 347 bursting of, 352 Grandry's corpuscles, technic of, 405 Granular cells of cerebellar cortex, 416 layer of cerebellar cortex, 416 of olfactory bulb, 421 Tomes', 246 sole plate, 163 Granulations of leukocytes, 227 Granules, acidophile, technic for, 227 amphophile, technic for, 228 basichromatin, 63 basophile, 193 cells with, 209 technic of, 228 chromatophile, 149 deutoplastic, 349 eosinophile, 193 cells with, 209 technic for, 227 Granules, in cells, Altmann's method of demonstrating, 77, 78 indulinophile, technic for, 228 interstitial, Kolliker's, 141 leukocytic, Ehrlich's, 192 neutrophile, 193 technic of, 228 oxychromatin, 63 Schron's, 344 tigroid, 149 zymogen, in pancreas, demonstration of, 308 Gray nuclei, central, of cerebellar cortex, 416 substance of spinal cord, 406, 409 Grenacher's alum-carmin, 42 as stain for bulk, 46 Ground bundle, anterior, 411 plexus of cornea, 451 Ground-substance interfascicular, 105 of areolar connective tissue, 102 of cartilage, changes, in, in Gruber's dendritic fibrous structure, 478 Gscheidtlen's method of obtaining hemo- globin crystals, 230 Gum glycerin, Farrant's mounting in, 53 HAIR, 389 auditory, 490 bulb, 389, 393 club, 393 cortical layer of, 389 cuticle of, 389 inner, 390 follicle, 389 nerve-fibers of, 393 germ, 389 glassy membrane of, 391 growth of, 392 medullary substance of, 390 olfactory, 499 papilla, 389 root, 389 root-sheaths of, 389 inner, 390 outer, 390 shaft, 389 shedding of, 393 technic for, 404 Hair-cells of utriculus, 483 Hamulus, 484 Hassal's corpuscles, 212 Haversian canals, 112 spaces, 120 Hayem's solution, 226 for diluting blood, 232 Hearing, organ of, 476. See also Ear. Heart, 186, 213 auriculoventricular valves of, 213 blood supply of, 214 elastic tissue of, 214 muscle, 145 motor nerve-supply of, 166 muscle-cells, demonstration of, 148 muscle-tissue, development of, 146 INDEX. 513 Heart, nerve supply of, 215 Heart-muscle fibers, MacCallum's nitric acid mixture for isolating, 23 Heidenhain's demilunes, 256 iron hematoxylin, 43 as stain for bulk, 46 median membrane, 137 Helicotrema, 485 Heliotropism, 61 Heller's plexus, 283 Hemal glands, 200 Hemalum, acid, as stain, 43 as stain, 43 in bulk, 46 Hematin, 187 Hematoblasts, 194 Hematoidin crystals, 231 Hematoxylin as stain, 42. See also Stains. Hematoxylin-eosin as stain, 45 Hematoxylin-safranin as stain, 46 Hemin, 188 crystals, method of obtaining, 230 Hemocytometer, Thoma-Zeiss, 232 Hemoglobin, 187 crystals, method of obtaining, 230 demonstration of, 230 nucleated red blood -cells containing, 208 Hemokonia, 195 Hemolymph glands, 200 structure of, 201 Henle's fiber layer, 461 layer, 390 loop, 323 descending limb of, 327 sheath, 162 Hensen's cells, 491, 492 median disc, 137 Hepatic artery, 293 cells, cords of, 290 cords, 290 Herbst's corpuscles, 174, 389 technic of, 405 Hermann's solution, 24 Heterotypic mitosis, 70 Hilum of lymph-glands, 197 Histology, general, 58 special, 186 Homeotypic mitosis, 70 Honing microtome knife, 37 Horizontal cells of retina, 464 semicircular canal, 480 Horns of spinal cord, 408 Howship's lacunae, 120 Hoyer's yellow gelatin mass, 54 Huber's method of permanently mount- ing Golgi's preparations under cover- glass, 51 Humor, aqueous, 446 Huschke's auditory teeth, 488 Huxley's layer, 390 Hyaline cartilage, 108 Hyaloid arteries, anterior, 468 posterior, 468 canal, 468 33 Hyaloid membrane of vitreous body, 467 Hydatids of Morgagni, 360 Hydrochloric acid, action of, on con- nective tissue, 128 as decalcifying fluid, 132 as macerating solution, 23 Hydrotropism, 61 Hymen, 359 Hypolamellar plexus, 261 Hypophysis, 423 chief cells of, 423 chromophilic cells of, 423 circulation of, 424 IMBEDDING, 27 celloidin, 30 diagram for, 32 celloidin-paraffin, 32 paraffin, 27 diagram for, 30 Immersion lens, 19 Impregnation, Cohnheim's method, 48 Cox's method 51 Gerlach's method, 48 gold chlorid method, 48 Golgi's chromsilver or chromsubli- mate method, 49 gold chlorid method, 48 methods, 49 mixed method, 50 potassium bichromate and bichlorid of mercury method, 50 potassium bichromate method, 49 rapid method, 50 slow method, 50 Kopsch's method, 52 Kuhne's method, 48 Lowit's method, 48 methods of, 47 of bile capillaries, 307 Ranvier's method, 48 silver nitrate method, 47 Indifferent fluids, 21, 22 Kronecker's, 22 physiologic saline solutions, 22 Ranvier's solution of iodin and po- tassium iodid, 22 Ripart and Petit's, 22 Schultze's iodized serum, 22 Indulinophile granules, technic for, 228 Inferior nasal artery of retina, 466 vein of retina, 466 papillary artery of retina, 466 vein of retina, 466 vertical semicircular canal, 480 I Infiltration, 27 celloidin, 30 diagram for, 32 celloidin-parafnn, 32 paraffin, 27 diagram for, 30 Injection fluids, 53 Altman's, 55 Berlin blue, 53 carmin mass, cold, 54 INDEX. Injection fluids, gelatin-Berlin blue, 54 gelatin-carmin, 54 silver nitrate, 55 yellow gelatin mass, 54 Injection method for demonstration of bile capillaries, 306 of distribution of hepatic blood- vessels, 306 methods of, 53 of animals, 54 of glands, 55 of lymph-channels, 55 of lymph-spaces, 55 of lymph-vessels, 55 of organs, 55 Inner molecular layer of retina, 464 nuclear layer of retina, 459, 462 scleral sulcus, 449 Intercellular bridges, 81, 380 demonstration of, 96 spaces, 81 substance, 79 Interfascicular ground-substance, 105 Interglobular spaces of Czermak, 241 Interlobular arteries of kidney, 332 veins of kidney, 334 of liver, 293 Intermediate tubule of pancreas, 300 Internal auditory artery, 494 ear, 480 limiting membrane of retina, 462 Interpapillary epithelial processes, 85 Interstitial granules of Kolliker, 141 Intertubular cell-masses of pancreas, 301 Intestine, 264 absorption of fat by, 288 blood supply of, 283 large, 281 blood-vessels of mucosa of, 284 lymph-vessels of, 284 lymph supply of, 283 mucous membrane of, general struc- ture, 264 nerves of, demonstration of, 306 muscularis mucosse of, 265 nerve supply of, 283 secretion of, 288 small, 274 axial canals of, 285 basement membrane of, 278 epthelial cells in, 275 lymphatics of, 285 lymph-nodules of, 279 mucous membrane of, 274, 277 blood-vessels of, 284 epithelium of, 274 leucocytes in, 275 lymph -nodules of, 279 villi of, 274 muscularis mucosje of, 279 villi of, lacteals of, 285 with villi, fixation of, 305 stratum circulare of, 266 fibrosum of, 265 longitudinale of, 266 submucosa of, 265 Intestine, tunica mucosa of, 265 Intima, endothelium of, technic for, 235 Intracapsular plexuses, 429 Intracartilaginous bone, 116 Intra-epithelial glands, 88 Intrafusal muscle-fibers, 175 Intralobular duct of pancreas, 300 Intramembranous bones, 116, 122 bone-development, 122 lodo-iodid of potassium stain to demon- strate glycogen in cartilage, 131 Iris, 446, 452, 455 anterior endothelium of, 455 diaphragm, 18 nerve supply of, 456 posterior epithelium of, 455 stroma of, 455 Islands of ciliated epithelium in cervical canal, 356 Isotropic intermediary disc, 138 JACOBSON'S organ, 499 Japanese method of fixing paraffin sec- tions to slide, 39 Jelly, Wharton's, 100 KAES and Bechtereff's striation, 421 Karyokinesis, 64 Karyokinetic cell-division, heterotypic, 374 homeotypic, 374 Karyolysis, 74 Karyosomes, 63 Keratohyalin, 380 technic for, 403 Kidney, 323 arched collecting portion of tubules, 323 329 blood-vessels of, 332 cortical substance of, 323, 324 distal convoluted portion of tubules, 323. 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 Lacunae, 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 apochromati c, 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 nuchse of ox, structure of, 1 06 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 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 trabeculae of, 198 with blood-sinuses, 200 Lymph-nodules, 196 agminated, 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 fibrillae 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 capsulopupfllaris, 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, 17 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, honin? 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, 421 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 Mononuclear 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 5i8 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, IDC 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, 178 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 cerebella>- cortex, 416 520 INDEX. Nucleus, leucocyte-, polymorphism of, 193 mother, 64 of spermatid, 377 resting, 63 segmentation, 71 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, 344 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 fungi form, 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 retinae, 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 Payer'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 HeDer's, 283 hypolamellar, 261 intracapsular, 429 myentericus, 286 of Auerbach, 286 of Meissner, 287 pericellular, 428 subepithelial, of cornea, 451 superficial, of cornea, 451 Plicae palmatae, 356 semilunares, 282, 473 sigmoideas, 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, 4'i8 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 CajaFs 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 camerse posterioris, 467 cochleae, 496 Rectum, 281 Red bone-marrow, 207 muscles, 141 Red-blood corpuscles, 187. See also Rrythrocytes. 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 Miiller's fibers of, 462 INDEX. 523 Retina, nerve-fiber layer of, 464 optic papilla of, 460 ora 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- brillae 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, 21 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, 71 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, 376 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, 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 n- 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, I3I 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, 41 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 Stellulae 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 mucosse 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 Taeniae 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 papillfe 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. 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 Solera. 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 Ufriculus, 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 Valvulae 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, 173 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 Virchov/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, X33 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 Born's method, 56 cutting out parts to be recon- structed, and completing model, 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, 81 Zonula ciliaris, 446, 467 Zonule of Zinn, 467 Zymogen, 255 granules in pancreas, demonstrating, 308 THIS BOOK IS DUE ON THE LAST DATE STAMPED BELOW AN INITIAL FINE OF 25 CENTS WILL BE ASSESSED FOR FAILURE TO RETURN THIS BOOK ON THE DATE DUE. THE PENALTY WILL INCREASE TO SO CENTS ON THE FOURTH DAY AND TO $1.OO ON THE SEVENTH DAY OVERDUE. _ 10LOGY LISRARt FEB 2 8 1956 NOVS7I965 THE UNIVERSITY OF CALIFORNIA LIBRARY